小麦穗发芽抗性相关Vp-1B和AIP2基因的克隆及功能分析
|
摘要
小麦成熟期穗发芽不仅影响产量,而且严重影响小麦品质,是世界性的自然灾害。本研究以三种不同穗发芽抗性小麦:中优9507(感穗发芽,Vp-1Ba)、西农979(中抗,Vp-1Bc,83 bp缺失)、永川白麦(高抗,Vp-1Bb,193 bp插入)为研究材料,分离得到小麦中与穗发芽抗性相关的Vp-1B和AIP2基因,并对其表达特性和基因功能进行了初步分析和鉴定。
Vp-1(Viviparous-1)是调节胚发育,促进胚成熟和休眠的主要转录调节因子。利用三种抗性不同的小麦材料,分离了Vp-1B三个等位基因的基因组及cDNA序列,在进行表达分析、核定位分析的基础上,采用Gateway技术分别构建了含有三种DNA序列、永川白麦cDNA序列的pLeela高效表达载体,通过农杆菌介导法,分别将其转入拟南芥突变体abi3-1中。结果表明:转基因各株系种子的GI值、ABA敏感性、转基因株系的花期时间及叶数与对照相比均有变化,结合Real-time方法检测ABA传导途径中基因表达的变化,结果说明小麦Vp-1B基因的导入抑制了abi3-1突变体中ABI1、ABI2基因的表达,同时激活了ABI5基因的表达;转基因株系表现出营养生长期延长,花期推迟;同时,对ABA的敏感性增强,转基因种子的GI值降低。转入的四种序列中,功能最强的是YCCVp-1B基因;三种基因组序列中,功能较强的是YCGVp-1B序列,说明插入和缺失影响了小麦Vp-1B基因的功能。
AIP2(ABI3-interacting protein 2)基因编码蛋白可在转录后水平降解Vp-1蛋白,从而间接影响穗发芽抗性。本文采用RACE技术结合电子序列拼接的方法首次从小麦中分离得到AIP2基因的两种cDNA序列,命名为:AIP2-1、AIP2-2。表达特性分析表明两种AIP2基因表达量均随胚的成熟而呈下降趋势。在穗发芽敏感时期,AIP2-2基因在感性品种的表达量明显高于抗性品种,说明AIP2-2基因可能与穗发芽抗性相关。通过农杆菌介导法分别将其转入拟南芥突变体aip2-1中,结果表明小麦AIP2基因的转入激活了拟南芥ABI1、ABI2基因的表达,使转基因种子对ABA的敏感性减弱,GI值分析表明:转ZYAIP2-1、ZYAIP2-2基因种子发芽指数均高于突变体,与野生型相近;转ZYAIP2-2基因的种子发芽指数最高,比突变体材料提高近10%。
在此基础上,以单子叶植物表达载体pAHC25为基础,构建了Vp-1B、AIP2基因的过表达,Vp-1、AIP2基因干扰表达载体,通过基因枪法分别将其转入小麦受体材料新春9号。利用Bar基因引物和基因特异引物对再生植株进行PCR检测,得到了转基因植株。通过检测T0代转基因种子GI值变化,对Vp-1B基因、AIP2基因在小麦中的功能进行了初步分析和鉴定。
结合拟南芥中的分析结果,说明小麦Vp-1B、AIP2基因调节种子发育具有重要作用,对小麦品种穗发芽抗性具有重要影响。本研究将为阐明小麦穗发芽抗性机理,进而为通过分子育种进行小麦品种改良提供一定的依据和借鉴。
Pre-harvest sprouting (PHS) of grain while it is still in the ear, usually in response to damp conditions, is due to an early disruption of seed dormancy. As a worldwide natural disaster, PHS reduces the quality of wheat and the value of the grain, seriously affects the yield of wheat. In particular, the synthesis ofα-amylase results in starch breakdown and the production of breads with a lower volume and a compact, sticky crumb structure. Producers suffer great losses when wheat damaged by sprouting is sold at a discount; millers are faced with reduced flour yields and bakers encounter problems in processing and quality due to starch damage. In China, accounting for about 83% of the cultivating area of wheat was threatened by Sprouting. Moreover, since the over emphasis on the yield ability of wheat, the quality was often more or less overlooked in the past two decades. This directly leads to a number of wheat varieties which are sensitive to PHS, and great threat to food security.
PHS is, generally speaking, a multi-factor induced phenomenon. It has a strong relationship with the environmental conditions, which makes things more complex. At present, it has been reported that PHS was mainly controlled by hereditary factors. But due to the huge hexaploid wheat genome and multi-location of the PHS-related genes (all seven chromosome groups except Group 1), the reason why some species possess resistance ability to PHS is still not fully explained. Thus, the problem of finding out a number of genes involved in PHS tolerance and clarifying the mechanism of pre-harvest sprouting resistance is not only a world wide important topic in wheat variety improvement, but also one of the most important traits in Chinese wheat breeding.
Abscisic acid (ABA) has broad influence on many complex reactions during plant development, including seed maturation, dormancy and germination. ABA sensitivity in embryos is one of the key factors affecting seed dormancy. Maize Vp-1(Viviparous-1) and Arabidopsis ABI3 (Abscisic acid-insensitive 3) are orthologous transcription factors that regulate key aspects of plant seed development and ABA signal transduction. Vp-1 gene encodes a seed-specific transcription factor and plays an important role in wheat PHS resistance. Three orthologues of Vp-1 genes are present in bread wheat, which are located on the long arms of chromosomes 3A, 3B and 3D, respectively. Semi-quantitative RT-PCR analysis showed that there were difference in the splicing patterns or abundances of Vp-1B between PHS-tolerant and susceptible cultivars. This result indicated that the expression of the Vp-1B gene may affect its sensitivity to ABA, and thus its resistance to PHS. ABI3 is a central regulator in ABA sigaling, but little is known of how this factor is regulated. AIP2 (ABI3-interacting protein 2), which encodes a E3 ligase, can polyubiquitinate ABI3 and negatively regulate ABA signaling by targeting ABI3 in seed of Arabidopsis. This result indicates that AIP2 gene may affect embryo’s sensitivity to ABA, and thus its tolerance to PHS, indirectly.
In this study, three typical genotypes with different levels of PHS tolerance were selected and used for PCR amplification. Zhongyou 9507, a PHS susceptible cultivar, its GI (Germinated index) value was 0.84; Xinong 979, a PHS tolerant cultivar, its GI value was 0.50; Yongchuan white wheat, a typical PHS tolerant Chinese landrace, its GI value was 0.14. Moreover, two alleles were located in wheat genotypes with distinct PHS tolerance and ABA responsiveness and designated as Vp-1Bb (Yongchuan white wheat, YC) and Vp-1Bc (Xinong 979, XN). Compared with Vp-1Ba (Zhongyou 9507, ZY), these alleles had an insertion of 193-bp and a deletion of 83-bp in the third intron in the region of B3 domain, respectively. Further analysis indicated that different Vp-1B alleles showed different expression and responsiveness to ABA exposure at transcriptional level. We inferred that the insertion or deletion in the third intron region may affect the structure of pre-mRNA and thus the resistance to PHS. Therefore, in this experiment, we analyzed the characters and functions of Vp-1B and AIP2 gene with three typical materials of different levels of PHS tolerant wheat.
Different cDNA clones of Vp-1B alleles were isolated from seeds of three typical wheats and named them as YCCVp-1B (GenBank accession no. FJ640558), XNCVp-1B and ZYCVp-1B (FJ640559), respectively. The sequence analysis revealed that the sequence homology between YCCVp-1B and XNCVp-1B is 100%. YCCVp-1B and XNCVp-1B comprised a 2,064 bp complete open reading frame (ORF), encoding a polypeptide of 72.66 kDa, consist of 687 amino acid residues, with its pI 8.23. The sequence homology of ZYCVP-1B with them is 99.85%. The cDNA sequence of ZYCVP-1B was 2,067 bp in length, encoding a protein of 688 amino acids with a predicted molecular mass of 72.79 kDa and a predicted pI of 8.46. The deduced amino acid sequence of YCCVp-1B contains a highly conserved B3 domain. Phylogenetic tree analysis showed that YCCVp-1B shares greater similarity with the other plant ABI3/Vp-1(25%-87%). Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that YCCVp-1B was a typical cytoplasmic protein.To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5’- and 3’-ends of the YCGVp-1B cDNA and named them as YCGVp-1B(FJ643534), XNGVp-1B(FJ517258) and ZYGVp-1B(FJ643535), respectively. The sequence analysis revealed that the DNA sequence of YCGVp-1B ,XNGVp-1B and ZYCVP-1B were 4244 bp, 3965 bp , 4051 bp in length, respectively.
To investigate the biological activity of the YCCVp-1B protein, the full-length cDNAs of YCCVp-1B was fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into onion epidermal cells for analysis of their subcellular localizations. Fluorescence of YCCVp-1B:hGFP occurred predominantly in the cell nucleus and also distributed a little in the plasma membrane.
In order to define the expression patterns of the three Vp-1B alleles and their relationship with PHS tolerance, semi-quantitative RT-PCR analysis was carried out to determine the expression levels of Vp-1B alleles at different seed developmental stages, Under H2O treatment and upon ABA treatment conditions in the three wheat cultivars differing in PHS tolerance. The result of semi-quantitative RT-PCR shown that during seed developmental stages, especially in 55 days after pollination (DAP) embryos, the Vp-1Bb and Vp-1Bc alleles had a higher abundances of transcripts than Vp-1Ba, with the level being highest in the Vp-1Bb allele and lowest in Vp-1Ba. Under H2O treatment,the transcript levels of Vp-1Ba ,Vp-1Bb and Vp-1Bc reached a maximum at 24 h after treatment then decreased, at 96 h, no Vp-1Ba allele was detected but the transcript levels of Vp-1Bb and Vp-1Bc were still maintained highly. The influences of exogenous ABA on transcription of three Vp-1B alleles in embryos were also investigated. Vp-1Bb was rapidly induced following treatment with 50μM ABA solution, the transcript levels of Vp-1Bb reached a maximum level at 12 h. However, Vp-1Ba and Vp-1Bc mRNA levels were upregulated in a time-dependent manner, reaching peak levels after 24 h. These results suggested that the three alleles differed in their response to ABA exposure. In conclusion, the analysis revealed that the expression level of three Vp-1B alleles have a positive correlation between embryo’s sensitivity to ABA and seed dormancy, thus resistance to PHS.
To further investigate the function of VP-1B gene, Using Gateway cloning technology, four plant ovexpression vectors of Vp-1B gene including ZYGVp-1B, XNGVp-1B, YCGVp-1B and YCCVp-1B sequence which were driven by the double 35S promoter were successfully constructed and induced into abi3-1, a deletion mutant allele of abi3, via agrobacterium. The results of experiments showed that expression of Vp-1B can partially complement abi3-1. ABI5, a positive regulator of ABA signaling, was activated by Vp-1B. ABI1 and ABI2, negative regulators of ABA signaling were strongly inhibited. These results indicated that Vp-1B strongly modified ABA signaling through feed forward regulation of ABI1 / ABI5-related genes. Furthermore, the wheat VP-1B fully restores ABA sensitivity of abi3-1 during seed germination and suppresses the early flowering phenotype of abi3-1. The expression of YCCVp-1B could perfectly compensate the inability of mutated genes in abi3-1.The complement ability of YCGVp-1B was the highest among three genomic sequences. This result showed that the sequence insertion and deletion in B3 domain affected the expression level of the Vp-1B and thus PHS tolerance..
To further investigate the function of VP-1B gene, according to YCVp-1B sequence, the overexpression vector of YCVp-1B gene named pAHC-YCCVp-1B was successfully constructed and transformed into wheat variety Xinchun 9. Finally, three transgenic lines both with Bar and YCVp-1B gene were identified, with the transformation frequency of 0.17%. GI analysis showed that the value in transgenic seeds was significantly lower compared with control. It was suggested that overexpression of YCVp-1B gene could inhibit seed germination.To further confirm the function of VP-1 gene, according to the Vp-1 sequence from wheat, the RNAi expression vector of Vp-1 gene named pAHC-WVpRi was successfully constructed and transformed into wheat variety Xinchun 9. In total, 1825 embryos were bombarded. 34 regenerated plants with resistance to Bialaphos were obtained. Among them, four transgenic lines both with the Bar gene and interference fragment were identified, with the transformation frequency of 0.16%. The transgenic plants showed a higher GI value due to Vp-1 interference phenomena.
In conclusion, our results suggest that Vp-1B proteins are important transcription factors that influence resistance to PHS by regulating embryo development and repressing germination of seed.
A full-length cDNA sequence of AIP2-1 was isolated using RACE and DNA fragment assembly method from three typical wheats and named them as ZYCAIP2-1 (GenBank accession no. FJ640556), XNCAIP2-1 and YCCAIP2-1, respectively. The sequence analysis revealed that the sequence homology between them is 100%.The cDNA sequence of ZYCAIP2-1 was 1,111 bp in length, including a complete open reading frame of 972 bp with a 5’-UTR of 69 bp and a 3’-UTR of 70 bp, encoding a protein of 323 amino acids with a predicted molecular mass of 36.16 kDa and a predicted pI of 4.83. The deduced amino acid sequence of ZYCAIP2-1 contains a highly conserved Ring motif.
Another cDNA copy of AIP2 gene were isolated from seeds of three typical wheat and named them as ZYCAIP2-2 (GenBank accession no. FJ640557), XNCAIP2-2 and YCCAIP2-2, respectively. The sequence analysis revealed that the sequence homology between them is 100%. ZYCAIP2-2 comprised a 972 bp complete open reading frame (ORF), encoding a polypeptide of 36.10 kDa, consist of 323 amino acid residues, with its pI 4.83. The cDNA sequence homology between ZYCAIP2-1 and ZYCAIP2-2 is 98.56%. Although there were 14 single-base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was almost completely conserved (99.69%, V76A).Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that ZYCAIP2-1 was a typical cytoplasmic protein. Homology alignment analysis showed that ZYCAIP2-1 and ZYCAIP2-2 shares greater similarity with the other plant AIP2 (48%-82%).
To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5’- and 3’-ends of the AIP2 cDNA and named them as ZYGAIP2-1(3298 bp, FJ643532),ZYGAIP2-2 (3597 bp, FJ643533), respectively. Sequence alignment of the cDNA with their genomic counterparts revealed that both AIP2-1 and AIP2-2 were composed of 5 exons and 4 introns.
AIP2 protein does not have a recognizable NLS. Fluorescence of AIP2-1:hGFP and AIP2-2:hGFP uniformly distributed throughout the onion epidermal cell just like control hGFP. These results suggest that AIP2-1 and AIP2-2 can’t localize into the nuclei, they may depends on its complexation with other proteins into the nucleus. AIP2-1 and AIP2-2 genes were assigned respectively to chromosomes 5A and 5B by PCR using nullisomic–tetrasomic series of T. aestivum L.cv. Chinese Spring as materials with specific primers. Using three diploid relative-species (AA: Triticum urartu; BB: Aegilops speltoides; DD: Aegilops tauschii) as materials, further analysis suggested that AIP2-1 gene may be derived from the evolution of future generations but AIP2-2 gene may be derived from ancestral species- Aegilops speltoides.
The result of semi-quantitative RT-PCR shown that during seed developmental stages, the expression of AIP2-1 and AIP2-2 genes had a gradually downward trend which reach the minimum at 55 DAP in three varieties. But the two transcripts indicated different expression pattern. The expression of AIP2-1 gene had a higher abundance of transcripts in tolerant cultivars than susceptible ones. Under H2O treatment,the transcript levels of AIP2-1 and AIP2-2 genes reached a maximum at 24 h after treatment, then decreased, at 96 h, the transcript levels of AIP2-1 and AIP2-2 genes were no significant difference among three varieties. But at 24 h, the expression of AIP2-1 and AIP2-2 genes had a higher abundance of transcripts in the susceptible cultivars than tolerant ones. The influences of exogenous ABA on transcription of AIP2-1 and AIP2-2 genes in embryos of three varieties were also investigated. The susceptible and tolerant cultivars had a greater sensitivity to ABA than resistant ones. From these results we conclude that the expression levels of AIP2-2 gene has a positive correlation with the resistance to PHS during seed developmental stages. Furthermore, the AIP2-1 and AIP2-2 genes were induced by exogenous ABA.
Two overexpression vectors of ZYAIP2-1 and ZYAIP2-2 were constructed from the plant expression vector pLeela and transformed into the Arabidopsis mutant aip2-1 by Agrobacterium-mediated method. Seeds of T2 generation plants were tested by ABA. The results showed that transformed plants tended to have a higher germination rate and earlier flowering time compared with control. This indicated that the introduced wheat gene AIP2 was able to activate the endogenous genes ABI1, ABI2 in Arabidopsis thaliana and led to less sensitivity to ABA. Comparing two different copies of AIP2, we observed stronger activation ability of ZYAIP2-2 than ZYAIP2-1. Based on the conclusion above, another two vectors were constructed for further function analysis in wheat. pAHC-ZYAIP2-2 was built for overexpression of ZYAIP2-2 and pAHC- Vp-AIPRi was a RNAi vector with a Vp-1 promoter. Two plasmids were transformed separately into wheat by microprojectile bombardment. Ten transformed wheat lines were finally obtained in total: 7 lines in the overexpression population while 3 lines in the RNAi population. Lines with alteration of ear type only accounted for a small part of these two populations. These lines had a GI value as we expected before.
Combining results of the above two functional analysis experiment in model plant Arabidopsis thaliana and common wheat, we believe that wheat AIP2 gene plays an important role in seed germination acceleration and embryo dormancy inhibition.
Vp-1(Viviparous-1)是调节胚发育,促进胚成熟和休眠的主要转录调节因子。利用三种抗性不同的小麦材料,分离了Vp-1B三个等位基因的基因组及cDNA序列,在进行表达分析、核定位分析的基础上,采用Gateway技术分别构建了含有三种DNA序列、永川白麦cDNA序列的pLeela高效表达载体,通过农杆菌介导法,分别将其转入拟南芥突变体abi3-1中。结果表明:转基因各株系种子的GI值、ABA敏感性、转基因株系的花期时间及叶数与对照相比均有变化,结合Real-time方法检测ABA传导途径中基因表达的变化,结果说明小麦Vp-1B基因的导入抑制了abi3-1突变体中ABI1、ABI2基因的表达,同时激活了ABI5基因的表达;转基因株系表现出营养生长期延长,花期推迟;同时,对ABA的敏感性增强,转基因种子的GI值降低。转入的四种序列中,功能最强的是YCCVp-1B基因;三种基因组序列中,功能较强的是YCGVp-1B序列,说明插入和缺失影响了小麦Vp-1B基因的功能。
AIP2(ABI3-interacting protein 2)基因编码蛋白可在转录后水平降解Vp-1蛋白,从而间接影响穗发芽抗性。本文采用RACE技术结合电子序列拼接的方法首次从小麦中分离得到AIP2基因的两种cDNA序列,命名为:AIP2-1、AIP2-2。表达特性分析表明两种AIP2基因表达量均随胚的成熟而呈下降趋势。在穗发芽敏感时期,AIP2-2基因在感性品种的表达量明显高于抗性品种,说明AIP2-2基因可能与穗发芽抗性相关。通过农杆菌介导法分别将其转入拟南芥突变体aip2-1中,结果表明小麦AIP2基因的转入激活了拟南芥ABI1、ABI2基因的表达,使转基因种子对ABA的敏感性减弱,GI值分析表明:转ZYAIP2-1、ZYAIP2-2基因种子发芽指数均高于突变体,与野生型相近;转ZYAIP2-2基因的种子发芽指数最高,比突变体材料提高近10%。
在此基础上,以单子叶植物表达载体pAHC25为基础,构建了Vp-1B、AIP2基因的过表达,Vp-1、AIP2基因干扰表达载体,通过基因枪法分别将其转入小麦受体材料新春9号。利用Bar基因引物和基因特异引物对再生植株进行PCR检测,得到了转基因植株。通过检测T0代转基因种子GI值变化,对Vp-1B基因、AIP2基因在小麦中的功能进行了初步分析和鉴定。
结合拟南芥中的分析结果,说明小麦Vp-1B、AIP2基因调节种子发育具有重要作用,对小麦品种穗发芽抗性具有重要影响。本研究将为阐明小麦穗发芽抗性机理,进而为通过分子育种进行小麦品种改良提供一定的依据和借鉴。
Pre-harvest sprouting (PHS) of grain while it is still in the ear, usually in response to damp conditions, is due to an early disruption of seed dormancy. As a worldwide natural disaster, PHS reduces the quality of wheat and the value of the grain, seriously affects the yield of wheat. In particular, the synthesis ofα-amylase results in starch breakdown and the production of breads with a lower volume and a compact, sticky crumb structure. Producers suffer great losses when wheat damaged by sprouting is sold at a discount; millers are faced with reduced flour yields and bakers encounter problems in processing and quality due to starch damage. In China, accounting for about 83% of the cultivating area of wheat was threatened by Sprouting. Moreover, since the over emphasis on the yield ability of wheat, the quality was often more or less overlooked in the past two decades. This directly leads to a number of wheat varieties which are sensitive to PHS, and great threat to food security.
PHS is, generally speaking, a multi-factor induced phenomenon. It has a strong relationship with the environmental conditions, which makes things more complex. At present, it has been reported that PHS was mainly controlled by hereditary factors. But due to the huge hexaploid wheat genome and multi-location of the PHS-related genes (all seven chromosome groups except Group 1), the reason why some species possess resistance ability to PHS is still not fully explained. Thus, the problem of finding out a number of genes involved in PHS tolerance and clarifying the mechanism of pre-harvest sprouting resistance is not only a world wide important topic in wheat variety improvement, but also one of the most important traits in Chinese wheat breeding.
Abscisic acid (ABA) has broad influence on many complex reactions during plant development, including seed maturation, dormancy and germination. ABA sensitivity in embryos is one of the key factors affecting seed dormancy. Maize Vp-1(Viviparous-1) and Arabidopsis ABI3 (Abscisic acid-insensitive 3) are orthologous transcription factors that regulate key aspects of plant seed development and ABA signal transduction. Vp-1 gene encodes a seed-specific transcription factor and plays an important role in wheat PHS resistance. Three orthologues of Vp-1 genes are present in bread wheat, which are located on the long arms of chromosomes 3A, 3B and 3D, respectively. Semi-quantitative RT-PCR analysis showed that there were difference in the splicing patterns or abundances of Vp-1B between PHS-tolerant and susceptible cultivars. This result indicated that the expression of the Vp-1B gene may affect its sensitivity to ABA, and thus its resistance to PHS. ABI3 is a central regulator in ABA sigaling, but little is known of how this factor is regulated. AIP2 (ABI3-interacting protein 2), which encodes a E3 ligase, can polyubiquitinate ABI3 and negatively regulate ABA signaling by targeting ABI3 in seed of Arabidopsis. This result indicates that AIP2 gene may affect embryo’s sensitivity to ABA, and thus its tolerance to PHS, indirectly.
In this study, three typical genotypes with different levels of PHS tolerance were selected and used for PCR amplification. Zhongyou 9507, a PHS susceptible cultivar, its GI (Germinated index) value was 0.84; Xinong 979, a PHS tolerant cultivar, its GI value was 0.50; Yongchuan white wheat, a typical PHS tolerant Chinese landrace, its GI value was 0.14. Moreover, two alleles were located in wheat genotypes with distinct PHS tolerance and ABA responsiveness and designated as Vp-1Bb (Yongchuan white wheat, YC) and Vp-1Bc (Xinong 979, XN). Compared with Vp-1Ba (Zhongyou 9507, ZY), these alleles had an insertion of 193-bp and a deletion of 83-bp in the third intron in the region of B3 domain, respectively. Further analysis indicated that different Vp-1B alleles showed different expression and responsiveness to ABA exposure at transcriptional level. We inferred that the insertion or deletion in the third intron region may affect the structure of pre-mRNA and thus the resistance to PHS. Therefore, in this experiment, we analyzed the characters and functions of Vp-1B and AIP2 gene with three typical materials of different levels of PHS tolerant wheat.
Different cDNA clones of Vp-1B alleles were isolated from seeds of three typical wheats and named them as YCCVp-1B (GenBank accession no. FJ640558), XNCVp-1B and ZYCVp-1B (FJ640559), respectively. The sequence analysis revealed that the sequence homology between YCCVp-1B and XNCVp-1B is 100%. YCCVp-1B and XNCVp-1B comprised a 2,064 bp complete open reading frame (ORF), encoding a polypeptide of 72.66 kDa, consist of 687 amino acid residues, with its pI 8.23. The sequence homology of ZYCVP-1B with them is 99.85%. The cDNA sequence of ZYCVP-1B was 2,067 bp in length, encoding a protein of 688 amino acids with a predicted molecular mass of 72.79 kDa and a predicted pI of 8.46. The deduced amino acid sequence of YCCVp-1B contains a highly conserved B3 domain. Phylogenetic tree analysis showed that YCCVp-1B shares greater similarity with the other plant ABI3/Vp-1(25%-87%). Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that YCCVp-1B was a typical cytoplasmic protein.To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5’- and 3’-ends of the YCGVp-1B cDNA and named them as YCGVp-1B(FJ643534), XNGVp-1B(FJ517258) and ZYGVp-1B(FJ643535), respectively. The sequence analysis revealed that the DNA sequence of YCGVp-1B ,XNGVp-1B and ZYCVP-1B were 4244 bp, 3965 bp , 4051 bp in length, respectively.
To investigate the biological activity of the YCCVp-1B protein, the full-length cDNAs of YCCVp-1B was fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into onion epidermal cells for analysis of their subcellular localizations. Fluorescence of YCCVp-1B:hGFP occurred predominantly in the cell nucleus and also distributed a little in the plasma membrane.
In order to define the expression patterns of the three Vp-1B alleles and their relationship with PHS tolerance, semi-quantitative RT-PCR analysis was carried out to determine the expression levels of Vp-1B alleles at different seed developmental stages, Under H2O treatment and upon ABA treatment conditions in the three wheat cultivars differing in PHS tolerance. The result of semi-quantitative RT-PCR shown that during seed developmental stages, especially in 55 days after pollination (DAP) embryos, the Vp-1Bb and Vp-1Bc alleles had a higher abundances of transcripts than Vp-1Ba, with the level being highest in the Vp-1Bb allele and lowest in Vp-1Ba. Under H2O treatment,the transcript levels of Vp-1Ba ,Vp-1Bb and Vp-1Bc reached a maximum at 24 h after treatment then decreased, at 96 h, no Vp-1Ba allele was detected but the transcript levels of Vp-1Bb and Vp-1Bc were still maintained highly. The influences of exogenous ABA on transcription of three Vp-1B alleles in embryos were also investigated. Vp-1Bb was rapidly induced following treatment with 50μM ABA solution, the transcript levels of Vp-1Bb reached a maximum level at 12 h. However, Vp-1Ba and Vp-1Bc mRNA levels were upregulated in a time-dependent manner, reaching peak levels after 24 h. These results suggested that the three alleles differed in their response to ABA exposure. In conclusion, the analysis revealed that the expression level of three Vp-1B alleles have a positive correlation between embryo’s sensitivity to ABA and seed dormancy, thus resistance to PHS.
To further investigate the function of VP-1B gene, Using Gateway cloning technology, four plant ovexpression vectors of Vp-1B gene including ZYGVp-1B, XNGVp-1B, YCGVp-1B and YCCVp-1B sequence which were driven by the double 35S promoter were successfully constructed and induced into abi3-1, a deletion mutant allele of abi3, via agrobacterium. The results of experiments showed that expression of Vp-1B can partially complement abi3-1. ABI5, a positive regulator of ABA signaling, was activated by Vp-1B. ABI1 and ABI2, negative regulators of ABA signaling were strongly inhibited. These results indicated that Vp-1B strongly modified ABA signaling through feed forward regulation of ABI1 / ABI5-related genes. Furthermore, the wheat VP-1B fully restores ABA sensitivity of abi3-1 during seed germination and suppresses the early flowering phenotype of abi3-1. The expression of YCCVp-1B could perfectly compensate the inability of mutated genes in abi3-1.The complement ability of YCGVp-1B was the highest among three genomic sequences. This result showed that the sequence insertion and deletion in B3 domain affected the expression level of the Vp-1B and thus PHS tolerance..
To further investigate the function of VP-1B gene, according to YCVp-1B sequence, the overexpression vector of YCVp-1B gene named pAHC-YCCVp-1B was successfully constructed and transformed into wheat variety Xinchun 9. Finally, three transgenic lines both with Bar and YCVp-1B gene were identified, with the transformation frequency of 0.17%. GI analysis showed that the value in transgenic seeds was significantly lower compared with control. It was suggested that overexpression of YCVp-1B gene could inhibit seed germination.To further confirm the function of VP-1 gene, according to the Vp-1 sequence from wheat, the RNAi expression vector of Vp-1 gene named pAHC-WVpRi was successfully constructed and transformed into wheat variety Xinchun 9. In total, 1825 embryos were bombarded. 34 regenerated plants with resistance to Bialaphos were obtained. Among them, four transgenic lines both with the Bar gene and interference fragment were identified, with the transformation frequency of 0.16%. The transgenic plants showed a higher GI value due to Vp-1 interference phenomena.
In conclusion, our results suggest that Vp-1B proteins are important transcription factors that influence resistance to PHS by regulating embryo development and repressing germination of seed.
A full-length cDNA sequence of AIP2-1 was isolated using RACE and DNA fragment assembly method from three typical wheats and named them as ZYCAIP2-1 (GenBank accession no. FJ640556), XNCAIP2-1 and YCCAIP2-1, respectively. The sequence analysis revealed that the sequence homology between them is 100%.The cDNA sequence of ZYCAIP2-1 was 1,111 bp in length, including a complete open reading frame of 972 bp with a 5’-UTR of 69 bp and a 3’-UTR of 70 bp, encoding a protein of 323 amino acids with a predicted molecular mass of 36.16 kDa and a predicted pI of 4.83. The deduced amino acid sequence of ZYCAIP2-1 contains a highly conserved Ring motif.
Another cDNA copy of AIP2 gene were isolated from seeds of three typical wheat and named them as ZYCAIP2-2 (GenBank accession no. FJ640557), XNCAIP2-2 and YCCAIP2-2, respectively. The sequence analysis revealed that the sequence homology between them is 100%. ZYCAIP2-2 comprised a 972 bp complete open reading frame (ORF), encoding a polypeptide of 36.10 kDa, consist of 323 amino acid residues, with its pI 4.83. The cDNA sequence homology between ZYCAIP2-1 and ZYCAIP2-2 is 98.56%. Although there were 14 single-base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was almost completely conserved (99.69%, V76A).Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that ZYCAIP2-1 was a typical cytoplasmic protein. Homology alignment analysis showed that ZYCAIP2-1 and ZYCAIP2-2 shares greater similarity with the other plant AIP2 (48%-82%).
To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5’- and 3’-ends of the AIP2 cDNA and named them as ZYGAIP2-1(3298 bp, FJ643532),ZYGAIP2-2 (3597 bp, FJ643533), respectively. Sequence alignment of the cDNA with their genomic counterparts revealed that both AIP2-1 and AIP2-2 were composed of 5 exons and 4 introns.
AIP2 protein does not have a recognizable NLS. Fluorescence of AIP2-1:hGFP and AIP2-2:hGFP uniformly distributed throughout the onion epidermal cell just like control hGFP. These results suggest that AIP2-1 and AIP2-2 can’t localize into the nuclei, they may depends on its complexation with other proteins into the nucleus. AIP2-1 and AIP2-2 genes were assigned respectively to chromosomes 5A and 5B by PCR using nullisomic–tetrasomic series of T. aestivum L.cv. Chinese Spring as materials with specific primers. Using three diploid relative-species (AA: Triticum urartu; BB: Aegilops speltoides; DD: Aegilops tauschii) as materials, further analysis suggested that AIP2-1 gene may be derived from the evolution of future generations but AIP2-2 gene may be derived from ancestral species- Aegilops speltoides.
The result of semi-quantitative RT-PCR shown that during seed developmental stages, the expression of AIP2-1 and AIP2-2 genes had a gradually downward trend which reach the minimum at 55 DAP in three varieties. But the two transcripts indicated different expression pattern. The expression of AIP2-1 gene had a higher abundance of transcripts in tolerant cultivars than susceptible ones. Under H2O treatment,the transcript levels of AIP2-1 and AIP2-2 genes reached a maximum at 24 h after treatment, then decreased, at 96 h, the transcript levels of AIP2-1 and AIP2-2 genes were no significant difference among three varieties. But at 24 h, the expression of AIP2-1 and AIP2-2 genes had a higher abundance of transcripts in the susceptible cultivars than tolerant ones. The influences of exogenous ABA on transcription of AIP2-1 and AIP2-2 genes in embryos of three varieties were also investigated. The susceptible and tolerant cultivars had a greater sensitivity to ABA than resistant ones. From these results we conclude that the expression levels of AIP2-2 gene has a positive correlation with the resistance to PHS during seed developmental stages. Furthermore, the AIP2-1 and AIP2-2 genes were induced by exogenous ABA.
Two overexpression vectors of ZYAIP2-1 and ZYAIP2-2 were constructed from the plant expression vector pLeela and transformed into the Arabidopsis mutant aip2-1 by Agrobacterium-mediated method. Seeds of T2 generation plants were tested by ABA. The results showed that transformed plants tended to have a higher germination rate and earlier flowering time compared with control. This indicated that the introduced wheat gene AIP2 was able to activate the endogenous genes ABI1, ABI2 in Arabidopsis thaliana and led to less sensitivity to ABA. Comparing two different copies of AIP2, we observed stronger activation ability of ZYAIP2-2 than ZYAIP2-1. Based on the conclusion above, another two vectors were constructed for further function analysis in wheat. pAHC-ZYAIP2-2 was built for overexpression of ZYAIP2-2 and pAHC- Vp-AIPRi was a RNAi vector with a Vp-1 promoter. Two plasmids were transformed separately into wheat by microprojectile bombardment. Ten transformed wheat lines were finally obtained in total: 7 lines in the overexpression population while 3 lines in the RNAi population. Lines with alteration of ear type only accounted for a small part of these two populations. These lines had a GI value as we expected before.
Combining results of the above two functional analysis experiment in model plant Arabidopsis thaliana and common wheat, we believe that wheat AIP2 gene plays an important role in seed germination acceleration and embryo dormancy inhibition.
引文
[1] GROOS C, GAY G, PERRETANT M R, et al. Study of the relationship between pre-harvest sprouting and grain color by quantitative trait loci analysis in a white×red grain bread-wheat cross [J]. Theoretical and Applied Genetics, 2002, 104: 39-47.
[2] MCMASTER G J. Pre-harvest sprouting in wheat-The Australian exprience. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1987.
[3] NILSSON-EHLE H. Zur Kenntnis der mit der Keimungs-physiologie des Weizens in Zusammenhang stehenden inneren Faktoren[J]. Z Pflanzenzuecht, 1914, 2: 153-157.
[4]赵同芳.几个小麦品种种子休眠期[J].植物生理通讯,1956,27:4-7.
[5]吴兆苏.小麦品种种子休眠特性的研究[J].农业学报,1959,10(6):461-474.
[6]肖世和.小麦穗发芽研究[M].北京:中国农业科学技术出版社,2004:4-10.
[7] DRAPRON R, ANH N X, LAUNAY B, et al. Development and distribution of wheat lipase activity during the course of germination[J]. Cereal chem, 1969, 46: 647-655.
[8] GALE M D, FLINTHAM J E, ARTHUR E D. Alpha-Amylase production in the late stage of grain development—an early sprouting damage risk period? Third International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1983.
[9] GALE M D, SALTER A.M, LENTON J R. The induction of germination alpha-amylase during wheat grain development in unfavourable weather conditions. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1987.
[10] KRUGER J E. Changes in the amylases of hard red spring wheat during growth and maturation[J]. Cereal Chemistry, 1972, 49: 379-390.
[11] COHN M A. Chemical mechanisms of breaking seed dormancy[J]. Seed Science Research, 1996, 6: 95-99.
[12] MARCHYLO B A, KRUGER J E, MACGREGOR A W. Production of Multiple Forms of Alpha-Amylase in Germinated, Incubated, Whole, De-embryonated Wheat Kernels[J]. Cereal Chemistry, 1984, 61(4): 305-310.
[13] MARCHYLO R A, LACROIX L J, KRUGER J E. Synthesis of a-amylase in specific tissues of the immature wheat kernel[J]. Cereal Res. Commun., 1980, 8: 61-68.
[14] MACLEOD A M, PALMER G H. Gibberellin from Barley Embryos[J]. Nature, 1967, 216: 1342-1343.
[15] DERERA N F. Preharvest Field Sprouting in Cereals[M]. Boca Raton: CRC Press Inc., 1989.
[16] LANG G A. Plant Dormancy: physiology, Biochemistry and Molecular Biology [M]. Wallington: CAB International., 1996.
[17] MARCHYLO B A, KRUGER J E, IRVINE G N. a-Amylase from immature hard red springwheat.I. Purification and some chemical and physical properties[J]. Cereal Chemistry, 1976, 53: 157-173.
[18] MARES D J, EILLISON F W, DERERA N F. Gibberellic acid insensitivity genes and pre-harvest sprouting resistance. Third International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1983.
[19] MARES D J, GALE M D. Control of alpha-amylase synthesis in wheat grains. Fifth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1990.
[20] BHATTACHARYA M, CORKE H. Selection of desirable starch pasting properties in wheat for use in white salted or yellow alkaline noodles[J]. Cereal Chemistry, 1996, 73(6): 721-728.
[21] WU J, CARVER B F. Sprout damage and preharvest sprout resistance in hard white winter wheat[J]. Crop Sci, 1999, 39(6): 441-447.
[22] DERERA N F, A perspective of sprouting research. Fifth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1990.
[23]肖世和.抗收获前穗发芽小麦品种的初步研究[J].四川农业大学学报,1986,4(2): 219-224.
[24]何震天.白皮小麦抗穗发芽研究概况[J].种子,2000,(2):36-38.
[25]肖世和.国外小麦抗穗发芽研究概况[J].国外农学-麦类作物,1985,(6):13-16.
[26] KING R W, CHADIM H, KRUGER J E, et al. Ear wetting and pre-harvest sprouting of wheat. Third international symposium on pre-harvest sprouting in cereals[C]. Boulder: Westview Press, 1983.
[27] DAVIDSON S. Shot, sprung, and down-graded[J]. Rural Research, 1984, 123: 4-7.
[28] KING R W, WETTSTEIN–KNOWLES P V. Epicuticular waxes and regulation of ear wetting and pre-harvest sprouting in barley and wheat[J]. Euphytica, 2000, 112(2): 157-166.
[29]张海峰.冬小麦穗发芽抗性及其鉴定方法的研究[J].作物学报,1989,(2):116-121.
[30] COME D, LENOIR C, CORBINEAU F. La dormancy des aerials et son elimination[J]. Seed Science and Technology, 1984, 12: 633.
[31]兰秀锦.小麦抗穗发芽的鉴定及其灌浆过程中胚、种皮和颖壳三因素与籽粒发芽率的关系[J].四川农业大学学报,1996,14 (4):533-536.
[32] GATFORD K T, EASTWOOD R F, HALLORAN G M. Germination inhibitors in bracts surrounding the grain of Triticum tauschii[J]. Functional Plant Biology, 2002, 29(1): 881-890.
[33]刘小冰.春小麦穗发芽抑制剂的研制及效果[J].农业系统科学与综合研究,1999,15(2):98-100.
[34]张海峰.小麦穗发芽抗性机理与遗传研究[J].作物学报,1993,19(6):523-530.
[35]蒋国梁.白皮小麦收获前穗发芽及品种抗性机制探讨[J].作物学报,1998,24(6):793-798.
[36] BELDEROK B. Seed dormancy problems in cereals[J]. Field Crop Abstr, 1968, 21: 203-211.
[37] WELLINGTON P S. Studies of the germination of cereals. 2. Factors determining the germination behaviour of wheat grains during maturation[J]. Ann Bot. N.S., 1956, 20: 481-500.
[38] MIYAMOTO T, TOLBERT N E , EVERSON E H. Germination inhibitors related to dormancy in wheat seeds[J]. Plant Physiology, 1961, 36(6): 739-746.
[39]沈正兴.小麦品种抗穗发芽特性的研究[J].中国农业科学,1991,24(5):44-50.
[40]闫长生.中国北方冬小麦品种穗发芽抗性的遗传变异[D].北京:中国农业科学院,2002.
[41] HUANG G, VARRIANO-MARSTON E, PAULSON G M. Structural and imbibition characteristics of Kansas hard white wheat[J]. Cereal Foods World, 1979, 24(9): 458.
[42]毛伯韧.小麦种子休眠性的遗传及其机理的研究[J].中国农业科学,1983,(6):53-60.
[43]孙果忠.小麦穗发芽机制研究进展[J].中国农业科技导报,2003,5(6):13-18.
[44] DERERA N F, BHATT G M, MCMASTER G J. On the problem of pre-harvest sprouting of wheat[J]. Euphytica, 1977, 26: 299-308.
[45] DEPAUW R M, MCCAIG, T N. Recombining dormancy and white seed color in a spring wheat cross[J]. Can J Plant Sci, 1983, 63: 581-589.
[46] BEWLEY J D, BLACK M. Seed: physiology of development and germination[M]. New York: Plenum press, 1994.
[47] NODA K, MARES D J. Pre-harvest Sprouting in Cereals[M]. Osaka, Japan(CASJO):1996.
[48] HIMI E, NODA K. Red grain colour gene(R) of wheat is a Myb-type transcription factor[J]. Euphytica, 2005, 143(3): 239-242.
[49] FOLEY M E. Plant Dormancy: physiology, Biochemistry and Molecular Biology[M]. Wallington: CAB International, 1996.
[50] LI C D, NI P X. Genes controlling seed dormancy and pre-harvest sprouting in rice-wheat- barley comparison[J]. Functional & Integrative Genomics, 2004, 4(2): 84-93.
[51] WALKER-SIMMONS M. ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars[J]. Plant Physiology, 1987, 84: 61-66.
[52]张海萍.小麦胚休眠中ABA信号转导的蛋白质组分析[J].作物学报,2006,32(5) :690-697.
[53] PéREZ-FLORES L, CARRARI F, OSUNA-FERNáNDEZ R, et al. Expression analysis of a GA 20-oxidase in embryos from two sorghum lines with contrasting dormancy: possible participation of this gene in the hormonal control of germination[J]. Journal Of Experimental Botany, 2003, 54: 2071-2079.
[54] VILLIERS T A, WAREING P F. Dormancy in fruits of Fraxinus excelsior L.[J]. Journal Of Experimental Botany, 1964, 15(2): 359-367.
[55] UPADHYAY M P. Characterization of pre-harvest sprouting resistance in Clark’screamwhite winter wheat[J]. Euphytica, 1988, 37: 85-92.
[56] HOLDSWORTH M, KURUP S, MCKIBBIN R. Molecular and genetic mechanisms regulating the transition from embryo development to germination[J]. Trends Plant Science, 1997, 4: 275-280.
[57] CORBINEAU F, BENAMAR A, COME D. Changes in sensitivity to abscisic acid of the developing and maturing embryo of two wheat cultivars with different sprouting susceptibility[J]. Israel Journal of Plant Sciences, 2000, 48(3): 189-197.
[58] FRETZDORFF B, M K WALKER-SIMMONS, J L RIED. Lipolytic enzyme activities in germinating wheat rye and triticale. Sixth international symposium on pre-harvest sprouting in cereals[C]. St Paul, Minnesota: America Association of Cereal Chemists, 1993.
[59]蓝秀锦.小麦新材料RSP的人工合成及其穗发芽抗性研究[D].雅安:四川农业大学,2005.
[60]周苏玫.反义TrxS基因在小麦中的表达、生理生化作用与抗穗发芽特性的研究[D].郑州:河南农业大学,2006.
[61]吕国锋.小麦抗收获前穗发芽的遗传及与其它农艺性状的相关研究[D].南京:南京农业大学,2001.
[62] NEILSEN M T. Effects of weather variables during maturation on pre-harvests prouting of hard white winter wheat[J], Crop Science, 1984, 24: 779-782.
[63] REDDY L V, M ETZGER R J. Effect of temperature on seed do rmancy of wheat[J]. Crop Science, 1985, 25: 455-458.
[64] BLACK M, BUTLER J. Control and development of dormancy in cereals. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C], Boulder: Westview Press, 1987.
[65] MARES D J. Temperature dependence of germinability of wheat grain (triticum aestivum L.) in relation to pre-harvests prouting[J]. Aust. J. Agric. Res., 1984, 35: 115-128.
[66] UENO K. Effects of desiccation and a change in temperature on germination of immature grains of wheat (T riticum aestivuml)[J]. Euphytica, 2002, 126: 107-113.
[67] NYACHIRO J M, CLAKE F R, DEPAUW R M, et al. Temperature effects on seed germination and expression of seed dormancy in wheat[J]. Euphytica, 2002, 126: 123-127.
[68]秦代红.小麦抗穗发芽特性的研究[J].四川农业大学学报,1989,7(3):188-193.
[69]秦代红.小麦抗穗发芽生理[J].植物生理学通讯,1990,6:62-64.
[70] KARVONEN T, J PELTONEN, E KIVI. The effect of northern climate conditions on sprouting damage of wheat grains[J]. Acta Agriculturae Scandinavica, 1991, 41(I): 55-64.
[71] NIELSEN M T, MCCRATE A J, HEYNE E G, et al. Effect of weather variables during maturation on preharvest sprouting of hard white winter wheat[J]. Crop Science, 1984, 24: 779-782.
[72] STRAND E. Studies on seed dormancy in small grain species. II. Wheat[J]. NorwegianJournal of Agricultural Science, 1989, 3(1):10l-115.
[73] HUANG G, MARSTON E V.α-amylase activity and pre-harvest sprouting damage in Kansas red and white wheat[J]. J. Agric. Food Chem., 1980, 28: 509.
[74] CAIRNS A L P, KRITZINGER J H. The effect of molybdenum on seed dormancy in wheat[J]. Plant and Soil, 1992, 145(2): 295-297.
[75]徐成彬.小麦收获前穗发芽的生理生化特性研究[J].中国农业科学,1988,21 (3 ):14-20.
[76] HAGEMANN M G, CIHA A J. Evaluation of methods used in testing winter wheat susceptibility to preharvest sprouting[J]. Crop Science, 1984, 24: 249-254.
[77]原亚萍.小麦穗发芽的研究进展[J].麦类作物学报,2003,23 (3):136-139.
[78] CAMPBELL J A. A new method for detection of sprout damaged wheat using nephlometric determination of alpha-amylase activity[J]. Cereal Res.commun., 1980, 8: 107-112.
[79] MASOJ? P, MARTHA L R. Variations of the levels ofα-amylase and endogenousα-amylase inhibitor in Rye and tritical grain[J]. Swed. J. Agric. Res., 1991, 21: 3-9.
[80]胡汉桥.春小麦穗发芽抗性鉴定及机理研究[J].麦类作物学报,2001,2(13):13-17.
[81] GITTA OETTLER, MARES D J. Falling Number and Alpha-Amylase Activity in Developing Grain of Alloplasm is Hexaploid wheat[J]. Plant Breeding, 1994, 112: 47-52.
[82] ANDERSON J A, SORRELLS M E, TANKSLEY S D. RFLP analysis of genomic regions associated with resistance to preharvest sprouting in wheat[J]. Crop Science, l993, 33: 453-459.
[83]周苏玫.反义trxs基因小麦籽粒蛋白组分的巯基含量与α-淀粉酶活性的关系[J].作物学报,2006,32(6):821-826.
[84] MUNDY J, HEJGAARD J, SVENDSEN I. Characterization of a bifunctional wheat inhibitor of endogenous a-amylase and subtilisin[J]. FEBS, 1984, 167(2): 210-214.
[85] MUNDY J, SVENDSEN I, HEJGAARD J. Barley a-amylase/subtilisin inhibitor. I. Isolation and characterization[J]. Carlsberg Res. Commun, 1983, 48: 81-90.
[86] MASOJ? P, LAPI?SKI M. Polymorphism of amylases from rye endosperm. 2. Intra- and inter-cultivar variability of the electrophoretic pattern of amylases[J]. Genet Pol., 1984, 25: 17-27.
[87]原亚萍.大麦2H染色体上的α-淀粉酶抑制蛋白基因Isa-H1向普通小麦的导入、鉴定及表达[D].北京:中国农业科学院,2001.
[88] MASOJ??P, ZAWISTOWSKI J, HOWES N K, et al. Polymorphism and chromosomal location of endogenous a-amylase inhibitor genes in common wheat[J]. Theor. Appl. Genet., 1993, 85(8): 1043-1048.
[89]张莉.种子胎萌机制研究进展[J].细胞生物学杂志,2007,29:701-705.
[90] KIGEL J, GALILI G. Seed Development and Germination[M]. New York: Marcel Dekker Press, 1995.
[91] VIéMONT J D, CRABBéJ. Dormancy in Plants: from Whole Plant Behavior to CellularControl[M]. Wallingford UK: CABI Press, 2000.
[92] KING R W. Abscisic acid in developing wheat grains and its relationship to grain growth and maturation[J]. Planta, 1976, 132(1): 43-51.
[93] JACOBSEN J V, CLOSE T J. Control of transient of expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barely aleurone layers[J]. Plant molecular Biology, 1991, 16: 713-724.
[94]孙果忠.小麦胚的ABA敏感性及其与休眠基因表达的关系[D].北京:中国农业科学院,2004.
[95]孙果忠.不同穗发芽抗性的小麦胚对ABA敏感性及抗性机制研究[J].麦类作物学报.2005,25(2):27-32.
[96] LEUNG J, GIRAUDAT J. Abscisic acid signal transduction[J]. Atnnual Review of Plant Physiology and Plant molecular Biology, 1998, 49: 199-222.
[97] GARELLO G. PAGE-DEGIVRY M T L. Evidence for the role of abscisic acid in the genetic and environmental control of dormancy in wheat (Triticum aestivum L.)[J]. Seed Science Research, 1999, 9: 219-226.
[98] JULLIEN M, BROUINOT D, ALI-RACHEDI S. Dormancy in Plants: From Whole Plant Behavior to Cellular Contro[M]. Wallingford UK: CABI Press, 2000.
[99]杨燕.小麦Vp-1基因的表达特性和STS标记的开发与应用[D].呼和浩特:内蒙古农业大学,2007.
[100] FLINTHAM J E. Different genetic components control coat-imposed and embryo-imposed dormancy in wheat [J]. Seed Science Research, 2000, 10: 43-50.
[101]张春利.小麦抗穗发芽分子标记的发掘与验证[D].北京:中国农业科学院,2008.
[102] YANG Y, ZHAO X L, XIA L Q, et al. Develpoment and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats[J]. Theoretical and Applied Genetics, 2007, 115(7): 971-980.
[103]宋雪梅.比较基因组学及其应用[J].生命的化学,2006,26(5):425-426.
[104] WILKINSON M D, MCKIBBIN R S, BAILEY P C, et al. Use of comparative molecular genetics to study pre harvest sprouting in wheat[J]. Euphytica, 2002, 126(1): 27-33
[105]杨泽峰.利用比较分子遗传学鉴定小麦穗发芽基因的研究进展[J].中国农学通报,2004,20(5):50-54.
[106] MCCARTY D R, CARSON C B, STINARD P S, et al. Molecular Analysis of Viviparous-1: An Abscisic Acid-Insensitive Mutant of Maize[J]. The Plant cell, 1989, 1: 523-532.
[107] HOECKER U, VASIL I K, MCCARTY D R. Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize[J]. Genes & Development, 1995, 9: 2459-2469.
[108] ZHANG X R, GARRETON V, CHUA N H. The AIP2 E3 ligase acts as a novel negativereglator of ABA signaling by promoting ABI3 degradation[J]. Genes & Development, 2005, 19: 1532-1543.
[109] KURUP S, JONES H D, HOLDSWORTH M J. Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds[J]. The Plant Journal, 2000, 21(2): 143-155.
[110] SUZUKI M, KETTERLING M G, LI Q B, et al. Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling[J]. Plant Physiology, 2003, 132: 1664-1677.
[111] BAILEY P C, MCKIBBIN R S, LENTON J R, et al. Genetic map location for orthologous VP1 genes in wheat and rice[J]. Theoretical and Applied Genetics, 1999, 98(2): 281-284.
[112] MCKIBBIN R S, WILKINSON M D, BAILEY P C, et al. Transcripts of Vp-1 homologues are misspliced in modern wheat and ancestral species[J]. Proceedings of the National Academy of Science USA, 2002, 99: 10203-10208.
[113] NAKAMURA S, TOYAMA T. Isolation of a VP1 homologue from wheat and analysis of its expression in embryos of dormant and non-dormant cultivars[J]. Journal of Experimental Botany, 2001, 52: 875-876
[114] YANG Y, MA Y Z, XU Z S, et al. Isolation and characterization of Viviparous-1 genes in wheat cultivars with distinct ABA sensitivity and pre-harvest sprouting tolerance[J]. Journal of Experimental Botany, 2007, 58(11): 2863-2871.
[115] LOPEZ-MOLINA L, MONGRAND S, CHUA N H.A postgermination developmental arrest checkpoint is mediated by abscisicacid and requires the AB15 transcription factor in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the united states of America. 2001, 98: 4782-4787.
[116] LOPEZ-MOLINA L, MONGRAND S, MCLACHLIN D, et al. ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination[J]. The Plant Journal, 2002, 32(3): 317-323.
[117] ROHDE A, DE RYCKE R, BEECKMAN T, et al. ABI3 affects plastid differentiation in dark-grown Arabidopsis seedlings[J]. The Plant Cell, 2000, 12: 35–52.
[118] ROHDE A, KURUP S, HOLDSWOTH M. ABI3 emerges from the seed[J]. Trends in Plant Science, 2000, 5(10): 418-419.
[119] ROHDE A, PRINSEN E, DE RYCKE R, et al. PtABI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar[J]. The Plant Cell, 2002, 14: 1885-1901.
[120] RHODE A, VAN MONTAGU M, BOERJAN M. The ABSCISIC ACID-INSENSITIVE 3 (ABI3) gene is expressed during vegetative quiescence processes in Arabidopsis[J]. Plant Cell Environ., 1999, 22: 261-270.
[121] BRADY S M, SARKAR S F, BONETTA D, et al. The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signalling and lateral rootdevelopment in Arabidopsis[J]. The Plant Journal, 2003, 34(1): 67-75.
[122] GIRAUDAT J, HAUGE B M, VALON C, et al. Isolation of the Arabidopsis ABI3 gene by positional cloning[J]. Plant Cell, 1992, 4(10): 1251-1261.
[123] SUZUKI M, KAO C Y, MCCARTY D R. The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity[J]. Plant Cell, 1997, 9(5): 799-807.
[124] HATTORI T, TERADA T, HAMASUNA S T. Sequence and functional, analyses of the rice gene homologous to the maize Vp1[J]. Plant Molecular Biology, 1994, 24(5): 805-810.
[125] BOBB A J, EIBEN H G, BUSTOS M M. PvAlf, an embryo-specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters[J]. Plant Journal, 1995, 8(3): 331-343.
[126] JONES H D, PETERS N C B, HOLDSWORTH M J. Genotype and environment interact to control dormancy and differential expression of the VIVIPAROUS1 homologue in embryos of Avena fatus[J]. Plant Journal, 1997, 12(4): 911-920.
[127] CHANDLER J W, BARTELS D. Structure and function of the Vp1 gene homologue from the resurrection plant Craterostigma plantagineum Hochst[J]. Molecular & General Genetics, 1997, 256(5): 539-546.
[128] SHIOTA H, WATABE K. C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes[J]. Plant Cell Physiol, 1998, 39(11): 1184-1193.
[129] ROHDE A, ARDILES D W, VAN MONTAGU M. Isolation and expression analysis of an ABSCISIC ACID-INSENSITIVE3 (ABI3) homologue from Populus trichocarpa[J]. Journal of Experimental Botay, 1998, 49: 1059-1060
[130]李浩.高质量的小麦种子总RNA的快速提取方法[J].分子植物育种,2006,4(6):877-881.
[131] WILKINSON M, LENTON J. Transcripts of VP-1 homologues are alternatively spliced within the Triticeae tribe[J]. Euphytica, 2005, 143: 243-246.
[132] FERNANDO C, LAURA P F, DIEGO L, et al. Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance[J]. Plant Molecular Biology, 2001, 45: 631-640.
[133] MCCARTY D R, HATTORI T, CARSON C B, et al. The viviparous-1 developmental gene of maize encodes a novel transcriptional activator[J]. Cell, 1991, 66(5): 895-905.
[134] GILL R W, SANSEAU P. Rapid in silico cloning of genes using expressed sequence tags(ESTs)[J]. Biotechnology Annnual Review, 2000, 5: 25-44
[135] KOZAK M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[J]. Nucleic Acids Research, 1987, 15(20): 8125-8148.
[136]黄骥.水稻功能基因的电子克隆策略[J].中国水稻科学,2002,16(4):295-298.
[137] PATRON N J, SMITH A M, FAHY B F, et al. The altered pattern of amylose accumulationin the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5 -non-coding region[J]. Plant Physiology, 2002, 130(1): 190-198.
[138] DOMON E, FUIJITA M, ISHIKAWA N. The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia[J]. Theoretical and Applied Genetics, 2002, 104(1): 132-138.
[139]杨娜.泛素连接酶的结构与功能研究进展[J].生物化学与生物物理进展,2008,35(1):14-20.
[140]杨东叶.泛素连接酶E3[J ].细胞生物学杂志,2005,27:281-285.
[141]王恒英.绿色荧光蛋白(GFP)研究进展[J].生物技术,2004,14(3):70-73.
[142]邢浩然.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21(增刊):1-6.
[143]吴瑞.绿色荧光蛋白及其在植物分子生物学中的应用[J].分子植物育种, 2005,3(2) :240-244.
[144] ZHANG H, HUANG Z, XIE B, et al. Ethylene, jasmonate, abscisic acid and NaCl responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco[J]. Planta, 2004, 220: 262-270.
[145] SHUKLA R K, RAHA S, TRIPATHI V, et al. Expression of CAP2, an APETALA2-Family Transcription Factor from Chickpea, Enhances Growth and Tolerance to Dehydration and Salt Stress in Transgenic Tobacco[J]. Plant Physiology, 2006, 142(1): 113-123.
[146]李洁.植物转录因子与基因调控[J].生物学通报,2004,39(3):9-10.
[147] MIZUNO S, HIRASAWA Y, SONODA M, et al. Isolation and characterization of three DREB/ERF-type transcription factors from melon(Cucunis melo)[J]. Plant Science, 2006, 170(6): 1156-1163.
[148] HUANG B, JIN L, LIU J Y, et al. Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton(Gosssypium hirsutum)[J]. Plant Physiology, 2008, 165(2): 214-223.
[149] LANDY A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination[J]. Annual Review of Biochemistry. 1989, 58: 913-941.
[150] BERNARD P, COUTURIER M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomeraseⅡcomplexes[J]. Molecular Biology, 1992, 226(3): 735-745.
[151] CHEN Q J, ZHOU H M, CHEN J. A Gateway-based platform for multigene plant transformation[J]. Plant Molecular Biology, 2006, 62(6): 927-936.
[152]张恭.RNA干扰及其植物抗病毒应用[J].中国农学通报,2007,23(1):42-45.
[153]曾晓珊. dsRNA介导植物基因沉默及其应用[J].生命科学,2007,19(2):132-138.
[154]杨迎伍. VIGS技术在植物基因功能研究中的应用[J].植物生理学通讯,2007,43(2):379-383.
[155]刁文一. RNAi在植物功能基因组中的应用[J].现代生物医学进展,2006,6(2): 81-83.
[156] WESLEY S V, HELLIWELL C A, SMITH N A, et al. Construct design for efficient, effective and high-throughput gene silencing in plants[J]. Plant Journal, 2001, 27(6): 581-590.
[157] WIANNY F, ZERMICKA-GOETZ M. Specific interference with gene function by double-stranded RNA in early mouse development[J]. Nature Cell Biology, 2000, 2: 70-75.
[158] LEUNG J, BOUVIER-DURAND M, MORRIS P C, et al. Arabidopsis AbA response gene ABI1: Features of a calcium-modulated protein phosphatase[J]. Science, 1994, 264: 1448-1452.
[159] RODRIGUEZ P L, BENNING G, GRILL E. ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis[J]. FEBS Letters, 1998, 421: 185-190.
[160] BROCARD-GIFFORD I M., LYNCH T J, FINKELSTEIN R R. Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling[J]. Plant Physiology, 2003, 131(1): 78-92.
[161] FINKELSTEIN R R, LYNCH T J. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor[J]. Plant Cell, 2000, 12(4): 599-609.
[162] AMANO Y, TORADA A. Breeding of white-grained wheats for Japan[J]. Euphytica, 2002, 126(1): 83-88.
[163]聂丽娜.小麦Vp-1和W73基因启动子的克隆与功能鉴定[D].呼和浩特:内蒙古农业大学,2008.
[2] MCMASTER G J. Pre-harvest sprouting in wheat-The Australian exprience. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1987.
[3] NILSSON-EHLE H. Zur Kenntnis der mit der Keimungs-physiologie des Weizens in Zusammenhang stehenden inneren Faktoren[J]. Z Pflanzenzuecht, 1914, 2: 153-157.
[4]赵同芳.几个小麦品种种子休眠期[J].植物生理通讯,1956,27:4-7.
[5]吴兆苏.小麦品种种子休眠特性的研究[J].农业学报,1959,10(6):461-474.
[6]肖世和.小麦穗发芽研究[M].北京:中国农业科学技术出版社,2004:4-10.
[7] DRAPRON R, ANH N X, LAUNAY B, et al. Development and distribution of wheat lipase activity during the course of germination[J]. Cereal chem, 1969, 46: 647-655.
[8] GALE M D, FLINTHAM J E, ARTHUR E D. Alpha-Amylase production in the late stage of grain development—an early sprouting damage risk period? Third International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1983.
[9] GALE M D, SALTER A.M, LENTON J R. The induction of germination alpha-amylase during wheat grain development in unfavourable weather conditions. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1987.
[10] KRUGER J E. Changes in the amylases of hard red spring wheat during growth and maturation[J]. Cereal Chemistry, 1972, 49: 379-390.
[11] COHN M A. Chemical mechanisms of breaking seed dormancy[J]. Seed Science Research, 1996, 6: 95-99.
[12] MARCHYLO B A, KRUGER J E, MACGREGOR A W. Production of Multiple Forms of Alpha-Amylase in Germinated, Incubated, Whole, De-embryonated Wheat Kernels[J]. Cereal Chemistry, 1984, 61(4): 305-310.
[13] MARCHYLO R A, LACROIX L J, KRUGER J E. Synthesis of a-amylase in specific tissues of the immature wheat kernel[J]. Cereal Res. Commun., 1980, 8: 61-68.
[14] MACLEOD A M, PALMER G H. Gibberellin from Barley Embryos[J]. Nature, 1967, 216: 1342-1343.
[15] DERERA N F. Preharvest Field Sprouting in Cereals[M]. Boca Raton: CRC Press Inc., 1989.
[16] LANG G A. Plant Dormancy: physiology, Biochemistry and Molecular Biology [M]. Wallington: CAB International., 1996.
[17] MARCHYLO B A, KRUGER J E, IRVINE G N. a-Amylase from immature hard red springwheat.I. Purification and some chemical and physical properties[J]. Cereal Chemistry, 1976, 53: 157-173.
[18] MARES D J, EILLISON F W, DERERA N F. Gibberellic acid insensitivity genes and pre-harvest sprouting resistance. Third International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1983.
[19] MARES D J, GALE M D. Control of alpha-amylase synthesis in wheat grains. Fifth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1990.
[20] BHATTACHARYA M, CORKE H. Selection of desirable starch pasting properties in wheat for use in white salted or yellow alkaline noodles[J]. Cereal Chemistry, 1996, 73(6): 721-728.
[21] WU J, CARVER B F. Sprout damage and preharvest sprout resistance in hard white winter wheat[J]. Crop Sci, 1999, 39(6): 441-447.
[22] DERERA N F, A perspective of sprouting research. Fifth International Symposium on Pre-harvest Sprouting in Cereals[C]. Boulder: Westview Press, 1990.
[23]肖世和.抗收获前穗发芽小麦品种的初步研究[J].四川农业大学学报,1986,4(2): 219-224.
[24]何震天.白皮小麦抗穗发芽研究概况[J].种子,2000,(2):36-38.
[25]肖世和.国外小麦抗穗发芽研究概况[J].国外农学-麦类作物,1985,(6):13-16.
[26] KING R W, CHADIM H, KRUGER J E, et al. Ear wetting and pre-harvest sprouting of wheat. Third international symposium on pre-harvest sprouting in cereals[C]. Boulder: Westview Press, 1983.
[27] DAVIDSON S. Shot, sprung, and down-graded[J]. Rural Research, 1984, 123: 4-7.
[28] KING R W, WETTSTEIN–KNOWLES P V. Epicuticular waxes and regulation of ear wetting and pre-harvest sprouting in barley and wheat[J]. Euphytica, 2000, 112(2): 157-166.
[29]张海峰.冬小麦穗发芽抗性及其鉴定方法的研究[J].作物学报,1989,(2):116-121.
[30] COME D, LENOIR C, CORBINEAU F. La dormancy des aerials et son elimination[J]. Seed Science and Technology, 1984, 12: 633.
[31]兰秀锦.小麦抗穗发芽的鉴定及其灌浆过程中胚、种皮和颖壳三因素与籽粒发芽率的关系[J].四川农业大学学报,1996,14 (4):533-536.
[32] GATFORD K T, EASTWOOD R F, HALLORAN G M. Germination inhibitors in bracts surrounding the grain of Triticum tauschii[J]. Functional Plant Biology, 2002, 29(1): 881-890.
[33]刘小冰.春小麦穗发芽抑制剂的研制及效果[J].农业系统科学与综合研究,1999,15(2):98-100.
[34]张海峰.小麦穗发芽抗性机理与遗传研究[J].作物学报,1993,19(6):523-530.
[35]蒋国梁.白皮小麦收获前穗发芽及品种抗性机制探讨[J].作物学报,1998,24(6):793-798.
[36] BELDEROK B. Seed dormancy problems in cereals[J]. Field Crop Abstr, 1968, 21: 203-211.
[37] WELLINGTON P S. Studies of the germination of cereals. 2. Factors determining the germination behaviour of wheat grains during maturation[J]. Ann Bot. N.S., 1956, 20: 481-500.
[38] MIYAMOTO T, TOLBERT N E , EVERSON E H. Germination inhibitors related to dormancy in wheat seeds[J]. Plant Physiology, 1961, 36(6): 739-746.
[39]沈正兴.小麦品种抗穗发芽特性的研究[J].中国农业科学,1991,24(5):44-50.
[40]闫长生.中国北方冬小麦品种穗发芽抗性的遗传变异[D].北京:中国农业科学院,2002.
[41] HUANG G, VARRIANO-MARSTON E, PAULSON G M. Structural and imbibition characteristics of Kansas hard white wheat[J]. Cereal Foods World, 1979, 24(9): 458.
[42]毛伯韧.小麦种子休眠性的遗传及其机理的研究[J].中国农业科学,1983,(6):53-60.
[43]孙果忠.小麦穗发芽机制研究进展[J].中国农业科技导报,2003,5(6):13-18.
[44] DERERA N F, BHATT G M, MCMASTER G J. On the problem of pre-harvest sprouting of wheat[J]. Euphytica, 1977, 26: 299-308.
[45] DEPAUW R M, MCCAIG, T N. Recombining dormancy and white seed color in a spring wheat cross[J]. Can J Plant Sci, 1983, 63: 581-589.
[46] BEWLEY J D, BLACK M. Seed: physiology of development and germination[M]. New York: Plenum press, 1994.
[47] NODA K, MARES D J. Pre-harvest Sprouting in Cereals[M]. Osaka, Japan(CASJO):1996.
[48] HIMI E, NODA K. Red grain colour gene(R) of wheat is a Myb-type transcription factor[J]. Euphytica, 2005, 143(3): 239-242.
[49] FOLEY M E. Plant Dormancy: physiology, Biochemistry and Molecular Biology[M]. Wallington: CAB International, 1996.
[50] LI C D, NI P X. Genes controlling seed dormancy and pre-harvest sprouting in rice-wheat- barley comparison[J]. Functional & Integrative Genomics, 2004, 4(2): 84-93.
[51] WALKER-SIMMONS M. ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars[J]. Plant Physiology, 1987, 84: 61-66.
[52]张海萍.小麦胚休眠中ABA信号转导的蛋白质组分析[J].作物学报,2006,32(5) :690-697.
[53] PéREZ-FLORES L, CARRARI F, OSUNA-FERNáNDEZ R, et al. Expression analysis of a GA 20-oxidase in embryos from two sorghum lines with contrasting dormancy: possible participation of this gene in the hormonal control of germination[J]. Journal Of Experimental Botany, 2003, 54: 2071-2079.
[54] VILLIERS T A, WAREING P F. Dormancy in fruits of Fraxinus excelsior L.[J]. Journal Of Experimental Botany, 1964, 15(2): 359-367.
[55] UPADHYAY M P. Characterization of pre-harvest sprouting resistance in Clark’screamwhite winter wheat[J]. Euphytica, 1988, 37: 85-92.
[56] HOLDSWORTH M, KURUP S, MCKIBBIN R. Molecular and genetic mechanisms regulating the transition from embryo development to germination[J]. Trends Plant Science, 1997, 4: 275-280.
[57] CORBINEAU F, BENAMAR A, COME D. Changes in sensitivity to abscisic acid of the developing and maturing embryo of two wheat cultivars with different sprouting susceptibility[J]. Israel Journal of Plant Sciences, 2000, 48(3): 189-197.
[58] FRETZDORFF B, M K WALKER-SIMMONS, J L RIED. Lipolytic enzyme activities in germinating wheat rye and triticale. Sixth international symposium on pre-harvest sprouting in cereals[C]. St Paul, Minnesota: America Association of Cereal Chemists, 1993.
[59]蓝秀锦.小麦新材料RSP的人工合成及其穗发芽抗性研究[D].雅安:四川农业大学,2005.
[60]周苏玫.反义TrxS基因在小麦中的表达、生理生化作用与抗穗发芽特性的研究[D].郑州:河南农业大学,2006.
[61]吕国锋.小麦抗收获前穗发芽的遗传及与其它农艺性状的相关研究[D].南京:南京农业大学,2001.
[62] NEILSEN M T. Effects of weather variables during maturation on pre-harvests prouting of hard white winter wheat[J], Crop Science, 1984, 24: 779-782.
[63] REDDY L V, M ETZGER R J. Effect of temperature on seed do rmancy of wheat[J]. Crop Science, 1985, 25: 455-458.
[64] BLACK M, BUTLER J. Control and development of dormancy in cereals. Fourth International Symposium on Pre-harvest Sprouting in Cereals[C], Boulder: Westview Press, 1987.
[65] MARES D J. Temperature dependence of germinability of wheat grain (triticum aestivum L.) in relation to pre-harvests prouting[J]. Aust. J. Agric. Res., 1984, 35: 115-128.
[66] UENO K. Effects of desiccation and a change in temperature on germination of immature grains of wheat (T riticum aestivuml)[J]. Euphytica, 2002, 126: 107-113.
[67] NYACHIRO J M, CLAKE F R, DEPAUW R M, et al. Temperature effects on seed germination and expression of seed dormancy in wheat[J]. Euphytica, 2002, 126: 123-127.
[68]秦代红.小麦抗穗发芽特性的研究[J].四川农业大学学报,1989,7(3):188-193.
[69]秦代红.小麦抗穗发芽生理[J].植物生理学通讯,1990,6:62-64.
[70] KARVONEN T, J PELTONEN, E KIVI. The effect of northern climate conditions on sprouting damage of wheat grains[J]. Acta Agriculturae Scandinavica, 1991, 41(I): 55-64.
[71] NIELSEN M T, MCCRATE A J, HEYNE E G, et al. Effect of weather variables during maturation on preharvest sprouting of hard white winter wheat[J]. Crop Science, 1984, 24: 779-782.
[72] STRAND E. Studies on seed dormancy in small grain species. II. Wheat[J]. NorwegianJournal of Agricultural Science, 1989, 3(1):10l-115.
[73] HUANG G, MARSTON E V.α-amylase activity and pre-harvest sprouting damage in Kansas red and white wheat[J]. J. Agric. Food Chem., 1980, 28: 509.
[74] CAIRNS A L P, KRITZINGER J H. The effect of molybdenum on seed dormancy in wheat[J]. Plant and Soil, 1992, 145(2): 295-297.
[75]徐成彬.小麦收获前穗发芽的生理生化特性研究[J].中国农业科学,1988,21 (3 ):14-20.
[76] HAGEMANN M G, CIHA A J. Evaluation of methods used in testing winter wheat susceptibility to preharvest sprouting[J]. Crop Science, 1984, 24: 249-254.
[77]原亚萍.小麦穗发芽的研究进展[J].麦类作物学报,2003,23 (3):136-139.
[78] CAMPBELL J A. A new method for detection of sprout damaged wheat using nephlometric determination of alpha-amylase activity[J]. Cereal Res.commun., 1980, 8: 107-112.
[79] MASOJ? P, MARTHA L R. Variations of the levels ofα-amylase and endogenousα-amylase inhibitor in Rye and tritical grain[J]. Swed. J. Agric. Res., 1991, 21: 3-9.
[80]胡汉桥.春小麦穗发芽抗性鉴定及机理研究[J].麦类作物学报,2001,2(13):13-17.
[81] GITTA OETTLER, MARES D J. Falling Number and Alpha-Amylase Activity in Developing Grain of Alloplasm is Hexaploid wheat[J]. Plant Breeding, 1994, 112: 47-52.
[82] ANDERSON J A, SORRELLS M E, TANKSLEY S D. RFLP analysis of genomic regions associated with resistance to preharvest sprouting in wheat[J]. Crop Science, l993, 33: 453-459.
[83]周苏玫.反义trxs基因小麦籽粒蛋白组分的巯基含量与α-淀粉酶活性的关系[J].作物学报,2006,32(6):821-826.
[84] MUNDY J, HEJGAARD J, SVENDSEN I. Characterization of a bifunctional wheat inhibitor of endogenous a-amylase and subtilisin[J]. FEBS, 1984, 167(2): 210-214.
[85] MUNDY J, SVENDSEN I, HEJGAARD J. Barley a-amylase/subtilisin inhibitor. I. Isolation and characterization[J]. Carlsberg Res. Commun, 1983, 48: 81-90.
[86] MASOJ? P, LAPI?SKI M. Polymorphism of amylases from rye endosperm. 2. Intra- and inter-cultivar variability of the electrophoretic pattern of amylases[J]. Genet Pol., 1984, 25: 17-27.
[87]原亚萍.大麦2H染色体上的α-淀粉酶抑制蛋白基因Isa-H1向普通小麦的导入、鉴定及表达[D].北京:中国农业科学院,2001.
[88] MASOJ??P, ZAWISTOWSKI J, HOWES N K, et al. Polymorphism and chromosomal location of endogenous a-amylase inhibitor genes in common wheat[J]. Theor. Appl. Genet., 1993, 85(8): 1043-1048.
[89]张莉.种子胎萌机制研究进展[J].细胞生物学杂志,2007,29:701-705.
[90] KIGEL J, GALILI G. Seed Development and Germination[M]. New York: Marcel Dekker Press, 1995.
[91] VIéMONT J D, CRABBéJ. Dormancy in Plants: from Whole Plant Behavior to CellularControl[M]. Wallingford UK: CABI Press, 2000.
[92] KING R W. Abscisic acid in developing wheat grains and its relationship to grain growth and maturation[J]. Planta, 1976, 132(1): 43-51.
[93] JACOBSEN J V, CLOSE T J. Control of transient of expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barely aleurone layers[J]. Plant molecular Biology, 1991, 16: 713-724.
[94]孙果忠.小麦胚的ABA敏感性及其与休眠基因表达的关系[D].北京:中国农业科学院,2004.
[95]孙果忠.不同穗发芽抗性的小麦胚对ABA敏感性及抗性机制研究[J].麦类作物学报.2005,25(2):27-32.
[96] LEUNG J, GIRAUDAT J. Abscisic acid signal transduction[J]. Atnnual Review of Plant Physiology and Plant molecular Biology, 1998, 49: 199-222.
[97] GARELLO G. PAGE-DEGIVRY M T L. Evidence for the role of abscisic acid in the genetic and environmental control of dormancy in wheat (Triticum aestivum L.)[J]. Seed Science Research, 1999, 9: 219-226.
[98] JULLIEN M, BROUINOT D, ALI-RACHEDI S. Dormancy in Plants: From Whole Plant Behavior to Cellular Contro[M]. Wallingford UK: CABI Press, 2000.
[99]杨燕.小麦Vp-1基因的表达特性和STS标记的开发与应用[D].呼和浩特:内蒙古农业大学,2007.
[100] FLINTHAM J E. Different genetic components control coat-imposed and embryo-imposed dormancy in wheat [J]. Seed Science Research, 2000, 10: 43-50.
[101]张春利.小麦抗穗发芽分子标记的发掘与验证[D].北京:中国农业科学院,2008.
[102] YANG Y, ZHAO X L, XIA L Q, et al. Develpoment and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats[J]. Theoretical and Applied Genetics, 2007, 115(7): 971-980.
[103]宋雪梅.比较基因组学及其应用[J].生命的化学,2006,26(5):425-426.
[104] WILKINSON M D, MCKIBBIN R S, BAILEY P C, et al. Use of comparative molecular genetics to study pre harvest sprouting in wheat[J]. Euphytica, 2002, 126(1): 27-33
[105]杨泽峰.利用比较分子遗传学鉴定小麦穗发芽基因的研究进展[J].中国农学通报,2004,20(5):50-54.
[106] MCCARTY D R, CARSON C B, STINARD P S, et al. Molecular Analysis of Viviparous-1: An Abscisic Acid-Insensitive Mutant of Maize[J]. The Plant cell, 1989, 1: 523-532.
[107] HOECKER U, VASIL I K, MCCARTY D R. Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize[J]. Genes & Development, 1995, 9: 2459-2469.
[108] ZHANG X R, GARRETON V, CHUA N H. The AIP2 E3 ligase acts as a novel negativereglator of ABA signaling by promoting ABI3 degradation[J]. Genes & Development, 2005, 19: 1532-1543.
[109] KURUP S, JONES H D, HOLDSWORTH M J. Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds[J]. The Plant Journal, 2000, 21(2): 143-155.
[110] SUZUKI M, KETTERLING M G, LI Q B, et al. Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling[J]. Plant Physiology, 2003, 132: 1664-1677.
[111] BAILEY P C, MCKIBBIN R S, LENTON J R, et al. Genetic map location for orthologous VP1 genes in wheat and rice[J]. Theoretical and Applied Genetics, 1999, 98(2): 281-284.
[112] MCKIBBIN R S, WILKINSON M D, BAILEY P C, et al. Transcripts of Vp-1 homologues are misspliced in modern wheat and ancestral species[J]. Proceedings of the National Academy of Science USA, 2002, 99: 10203-10208.
[113] NAKAMURA S, TOYAMA T. Isolation of a VP1 homologue from wheat and analysis of its expression in embryos of dormant and non-dormant cultivars[J]. Journal of Experimental Botany, 2001, 52: 875-876
[114] YANG Y, MA Y Z, XU Z S, et al. Isolation and characterization of Viviparous-1 genes in wheat cultivars with distinct ABA sensitivity and pre-harvest sprouting tolerance[J]. Journal of Experimental Botany, 2007, 58(11): 2863-2871.
[115] LOPEZ-MOLINA L, MONGRAND S, CHUA N H.A postgermination developmental arrest checkpoint is mediated by abscisicacid and requires the AB15 transcription factor in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the united states of America. 2001, 98: 4782-4787.
[116] LOPEZ-MOLINA L, MONGRAND S, MCLACHLIN D, et al. ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination[J]. The Plant Journal, 2002, 32(3): 317-323.
[117] ROHDE A, DE RYCKE R, BEECKMAN T, et al. ABI3 affects plastid differentiation in dark-grown Arabidopsis seedlings[J]. The Plant Cell, 2000, 12: 35–52.
[118] ROHDE A, KURUP S, HOLDSWOTH M. ABI3 emerges from the seed[J]. Trends in Plant Science, 2000, 5(10): 418-419.
[119] ROHDE A, PRINSEN E, DE RYCKE R, et al. PtABI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar[J]. The Plant Cell, 2002, 14: 1885-1901.
[120] RHODE A, VAN MONTAGU M, BOERJAN M. The ABSCISIC ACID-INSENSITIVE 3 (ABI3) gene is expressed during vegetative quiescence processes in Arabidopsis[J]. Plant Cell Environ., 1999, 22: 261-270.
[121] BRADY S M, SARKAR S F, BONETTA D, et al. The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signalling and lateral rootdevelopment in Arabidopsis[J]. The Plant Journal, 2003, 34(1): 67-75.
[122] GIRAUDAT J, HAUGE B M, VALON C, et al. Isolation of the Arabidopsis ABI3 gene by positional cloning[J]. Plant Cell, 1992, 4(10): 1251-1261.
[123] SUZUKI M, KAO C Y, MCCARTY D R. The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity[J]. Plant Cell, 1997, 9(5): 799-807.
[124] HATTORI T, TERADA T, HAMASUNA S T. Sequence and functional, analyses of the rice gene homologous to the maize Vp1[J]. Plant Molecular Biology, 1994, 24(5): 805-810.
[125] BOBB A J, EIBEN H G, BUSTOS M M. PvAlf, an embryo-specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters[J]. Plant Journal, 1995, 8(3): 331-343.
[126] JONES H D, PETERS N C B, HOLDSWORTH M J. Genotype and environment interact to control dormancy and differential expression of the VIVIPAROUS1 homologue in embryos of Avena fatus[J]. Plant Journal, 1997, 12(4): 911-920.
[127] CHANDLER J W, BARTELS D. Structure and function of the Vp1 gene homologue from the resurrection plant Craterostigma plantagineum Hochst[J]. Molecular & General Genetics, 1997, 256(5): 539-546.
[128] SHIOTA H, WATABE K. C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes[J]. Plant Cell Physiol, 1998, 39(11): 1184-1193.
[129] ROHDE A, ARDILES D W, VAN MONTAGU M. Isolation and expression analysis of an ABSCISIC ACID-INSENSITIVE3 (ABI3) homologue from Populus trichocarpa[J]. Journal of Experimental Botay, 1998, 49: 1059-1060
[130]李浩.高质量的小麦种子总RNA的快速提取方法[J].分子植物育种,2006,4(6):877-881.
[131] WILKINSON M, LENTON J. Transcripts of VP-1 homologues are alternatively spliced within the Triticeae tribe[J]. Euphytica, 2005, 143: 243-246.
[132] FERNANDO C, LAURA P F, DIEGO L, et al. Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance[J]. Plant Molecular Biology, 2001, 45: 631-640.
[133] MCCARTY D R, HATTORI T, CARSON C B, et al. The viviparous-1 developmental gene of maize encodes a novel transcriptional activator[J]. Cell, 1991, 66(5): 895-905.
[134] GILL R W, SANSEAU P. Rapid in silico cloning of genes using expressed sequence tags(ESTs)[J]. Biotechnology Annnual Review, 2000, 5: 25-44
[135] KOZAK M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[J]. Nucleic Acids Research, 1987, 15(20): 8125-8148.
[136]黄骥.水稻功能基因的电子克隆策略[J].中国水稻科学,2002,16(4):295-298.
[137] PATRON N J, SMITH A M, FAHY B F, et al. The altered pattern of amylose accumulationin the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5 -non-coding region[J]. Plant Physiology, 2002, 130(1): 190-198.
[138] DOMON E, FUIJITA M, ISHIKAWA N. The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia[J]. Theoretical and Applied Genetics, 2002, 104(1): 132-138.
[139]杨娜.泛素连接酶的结构与功能研究进展[J].生物化学与生物物理进展,2008,35(1):14-20.
[140]杨东叶.泛素连接酶E3[J ].细胞生物学杂志,2005,27:281-285.
[141]王恒英.绿色荧光蛋白(GFP)研究进展[J].生物技术,2004,14(3):70-73.
[142]邢浩然.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21(增刊):1-6.
[143]吴瑞.绿色荧光蛋白及其在植物分子生物学中的应用[J].分子植物育种, 2005,3(2) :240-244.
[144] ZHANG H, HUANG Z, XIE B, et al. Ethylene, jasmonate, abscisic acid and NaCl responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco[J]. Planta, 2004, 220: 262-270.
[145] SHUKLA R K, RAHA S, TRIPATHI V, et al. Expression of CAP2, an APETALA2-Family Transcription Factor from Chickpea, Enhances Growth and Tolerance to Dehydration and Salt Stress in Transgenic Tobacco[J]. Plant Physiology, 2006, 142(1): 113-123.
[146]李洁.植物转录因子与基因调控[J].生物学通报,2004,39(3):9-10.
[147] MIZUNO S, HIRASAWA Y, SONODA M, et al. Isolation and characterization of three DREB/ERF-type transcription factors from melon(Cucunis melo)[J]. Plant Science, 2006, 170(6): 1156-1163.
[148] HUANG B, JIN L, LIU J Y, et al. Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton(Gosssypium hirsutum)[J]. Plant Physiology, 2008, 165(2): 214-223.
[149] LANDY A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination[J]. Annual Review of Biochemistry. 1989, 58: 913-941.
[150] BERNARD P, COUTURIER M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomeraseⅡcomplexes[J]. Molecular Biology, 1992, 226(3): 735-745.
[151] CHEN Q J, ZHOU H M, CHEN J. A Gateway-based platform for multigene plant transformation[J]. Plant Molecular Biology, 2006, 62(6): 927-936.
[152]张恭.RNA干扰及其植物抗病毒应用[J].中国农学通报,2007,23(1):42-45.
[153]曾晓珊. dsRNA介导植物基因沉默及其应用[J].生命科学,2007,19(2):132-138.
[154]杨迎伍. VIGS技术在植物基因功能研究中的应用[J].植物生理学通讯,2007,43(2):379-383.
[155]刁文一. RNAi在植物功能基因组中的应用[J].现代生物医学进展,2006,6(2): 81-83.
[156] WESLEY S V, HELLIWELL C A, SMITH N A, et al. Construct design for efficient, effective and high-throughput gene silencing in plants[J]. Plant Journal, 2001, 27(6): 581-590.
[157] WIANNY F, ZERMICKA-GOETZ M. Specific interference with gene function by double-stranded RNA in early mouse development[J]. Nature Cell Biology, 2000, 2: 70-75.
[158] LEUNG J, BOUVIER-DURAND M, MORRIS P C, et al. Arabidopsis AbA response gene ABI1: Features of a calcium-modulated protein phosphatase[J]. Science, 1994, 264: 1448-1452.
[159] RODRIGUEZ P L, BENNING G, GRILL E. ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis[J]. FEBS Letters, 1998, 421: 185-190.
[160] BROCARD-GIFFORD I M., LYNCH T J, FINKELSTEIN R R. Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling[J]. Plant Physiology, 2003, 131(1): 78-92.
[161] FINKELSTEIN R R, LYNCH T J. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor[J]. Plant Cell, 2000, 12(4): 599-609.
[162] AMANO Y, TORADA A. Breeding of white-grained wheats for Japan[J]. Euphytica, 2002, 126(1): 83-88.
[163]聂丽娜.小麦Vp-1和W73基因启动子的克隆与功能鉴定[D].呼和浩特:内蒙古农业大学,2008.