野生型与耐盐型短芒大麦基因序列差异和盐碱胁迫条件下基因表达差异的研究
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摘要
土壤盐碱化的危害日益严重,因此,研究植物耐盐胁迫机理具有重要意义。
本文选择了中度耐盐碱的短芒大麦为实验材料,比较分析了野生型和耐盐型短芒大麦耐盐机制的差异。首先使用不同浓度梯度的NaHCO3分别处理野生型短芒大麦,最终确定了最适的处理浓度:100mM NaHCO3.在后续的实验中,运用AFLP技术分析出野生型和耐盐型短芒大麦基因序列的差异,得到4条与非生物胁迫相关的基因片段;说明通过AFLP技术是可以筛选到野生型和耐盐型短芒大麦中与盐碱相关的差异基因片段的;同时利用cDNA-AFLP以及Blast软件等技术分析,找到了在野生型和耐盐型短芒大麦有差异的7个转录片段,下一步,可将此片段连接到表达载体,通过转基因等手段在植物中过表达来验证植物的耐盐性是否有所提高。通过本研究,我们分别在基因组水平和基因转录水平找到了再野生型和耐盐型短芒大麦中有差异的盐碱相关片段,为盐碱调控网络的完善奠定了基础,同时为盐碱胁迫条件下植物分子育种提供了可以利用的基因资源。
实验过程中采用了生理实验确定植物合适的处理浓度,反转录半定量PCR、AFLP、巢式PCR、cDNA-AFLP等实验技术和手段解析出野生型和耐盐型短芒大麦在盐碱胁迫条件下响应机制的异同,同时筛选得到了在盐碱胁迫条件下有明显应答反应的基因序列,为今后植物耐盐碱机理研究和耐盐碱植物育种工作提供了理论依据。
The soil salinization is the most urgent problem for agriculture. It can influence most plants’normal growth. But we also find some plants can grow and breed for community under this extreme environment. Hordeum brevisubulatium (Trin.) link is a kind of perennial forage grass belonging to gramineous type barley genus, which salt-tolerance capacity is strong. The mechanism of adapting salinity environment made shortsubulate barley as an ideal plant for researching of anti-adversity.
In order to know the salt-tolerance mechanism and utilize key salt-tolerance gene, this paper chose wild type and salt-tolerance type barley as material, analyzed the physiological response to NaHCO3 stress, determined the suitable concentration to deal with the plants. At the same time, we analyzed the difference of gene expression by RT-PCR and cDNA-AFLP technonlgies, then acquired the relative important genes by nested PCR. The results were as follows:
Through physiological experiment to determine the suitable treeting concentration for the plants: 100mM.
Using RT-PCR to compare DREB1 and CIPK gene that expressed differently between wild type and salt-tolerance type barley at different time. We chose a-tubulin as the internal parameters. The results showed that in the plants which treeting with alkaline salt, its DREB1 expressed highest after 6hrs, and while treeting with neutral salt, DREB1 expressed highest at 12hrs later. Anyway CIPK expressed highest after 24hrs. Alkaline condition has little influence on CIPK, but under NaHCO3 stress, the response of wild type and salt-tolerance type barley are consistent.
Under alkaline salt stress, Wild type and salt-tolerance type Barley have obvious difference in phenotype. So this study analyzed sequence difference by the AFLP technology. The result shows the difference is 1% when stating 1400 sequences.
Using nested-PCR obtained the gene sequence of CIPK、NHX3、DREB1 The experiment respectively cloned the cDNA sequence of CIPK、NHX3、DREB1 from the normal type and the anti type of barley. At the same time, BLAST aligned the sequence. Analyzed the difference between the two type found the proteins has no difference. So we can not deduced, transcription has correlation with anti-salt.
Analyse the expression difference by cDNA-AFLP. The normal type and the anti type of barley displayed different expression profile. By BLAST aligning, we chose one TDF which is a relative gene about salt stress. Then we designed primers for the sequence amplification. The sequence of the gene is the same in both wild type and tolerance type. But the expression level has difference in the two tyoe barley. Therefore the next step we may prove the tolerance of the gene through transgenic technology.
本文选择了中度耐盐碱的短芒大麦为实验材料,比较分析了野生型和耐盐型短芒大麦耐盐机制的差异。首先使用不同浓度梯度的NaHCO3分别处理野生型短芒大麦,最终确定了最适的处理浓度:100mM NaHCO3.在后续的实验中,运用AFLP技术分析出野生型和耐盐型短芒大麦基因序列的差异,得到4条与非生物胁迫相关的基因片段;说明通过AFLP技术是可以筛选到野生型和耐盐型短芒大麦中与盐碱相关的差异基因片段的;同时利用cDNA-AFLP以及Blast软件等技术分析,找到了在野生型和耐盐型短芒大麦有差异的7个转录片段,下一步,可将此片段连接到表达载体,通过转基因等手段在植物中过表达来验证植物的耐盐性是否有所提高。通过本研究,我们分别在基因组水平和基因转录水平找到了再野生型和耐盐型短芒大麦中有差异的盐碱相关片段,为盐碱调控网络的完善奠定了基础,同时为盐碱胁迫条件下植物分子育种提供了可以利用的基因资源。
实验过程中采用了生理实验确定植物合适的处理浓度,反转录半定量PCR、AFLP、巢式PCR、cDNA-AFLP等实验技术和手段解析出野生型和耐盐型短芒大麦在盐碱胁迫条件下响应机制的异同,同时筛选得到了在盐碱胁迫条件下有明显应答反应的基因序列,为今后植物耐盐碱机理研究和耐盐碱植物育种工作提供了理论依据。
The soil salinization is the most urgent problem for agriculture. It can influence most plants’normal growth. But we also find some plants can grow and breed for community under this extreme environment. Hordeum brevisubulatium (Trin.) link is a kind of perennial forage grass belonging to gramineous type barley genus, which salt-tolerance capacity is strong. The mechanism of adapting salinity environment made shortsubulate barley as an ideal plant for researching of anti-adversity.
In order to know the salt-tolerance mechanism and utilize key salt-tolerance gene, this paper chose wild type and salt-tolerance type barley as material, analyzed the physiological response to NaHCO3 stress, determined the suitable concentration to deal with the plants. At the same time, we analyzed the difference of gene expression by RT-PCR and cDNA-AFLP technonlgies, then acquired the relative important genes by nested PCR. The results were as follows:
Through physiological experiment to determine the suitable treeting concentration for the plants: 100mM.
Using RT-PCR to compare DREB1 and CIPK gene that expressed differently between wild type and salt-tolerance type barley at different time. We chose a-tubulin as the internal parameters. The results showed that in the plants which treeting with alkaline salt, its DREB1 expressed highest after 6hrs, and while treeting with neutral salt, DREB1 expressed highest at 12hrs later. Anyway CIPK expressed highest after 24hrs. Alkaline condition has little influence on CIPK, but under NaHCO3 stress, the response of wild type and salt-tolerance type barley are consistent.
Under alkaline salt stress, Wild type and salt-tolerance type Barley have obvious difference in phenotype. So this study analyzed sequence difference by the AFLP technology. The result shows the difference is 1% when stating 1400 sequences.
Using nested-PCR obtained the gene sequence of CIPK、NHX3、DREB1 The experiment respectively cloned the cDNA sequence of CIPK、NHX3、DREB1 from the normal type and the anti type of barley. At the same time, BLAST aligned the sequence. Analyzed the difference between the two type found the proteins has no difference. So we can not deduced, transcription has correlation with anti-salt.
Analyse the expression difference by cDNA-AFLP. The normal type and the anti type of barley displayed different expression profile. By BLAST aligning, we chose one TDF which is a relative gene about salt stress. Then we designed primers for the sequence amplification. The sequence of the gene is the same in both wild type and tolerance type. But the expression level has difference in the two tyoe barley. Therefore the next step we may prove the tolerance of the gene through transgenic technology.
引文
[1] Kenneth K T. Agricultural Salinity Assessment and Management [J]. New York: American Society of Civil Engineers, 1990, 1-17.
[2]柴凤九,郭宝华.野大麦的特性及其栽培利用[J].内蒙古畜牧业,1992(4):14-15.
[3]王传东.抗盐碱植物-野大麦[J].吉林畜牧兽医,1986(1):7-8.
[4]董卫民等.野大麦人工栽培驯化实验室[J].草业科学,1992,9(5):38-39.
[5]贾慎修主编《.中国饲用植物志》第一卷[M].北京:农业出版社,1987,121-124.
[6]徐恒刚.禾本科牧草发芽期和苗期耐盐性的初步研究[J].中国草地,1988(4):53-55.
[7] Peng Z, et al. dehydrogenase Reciprocal regulation of DELTA 1-pyrroline-5- carboxylate synthetase and proline genes controls proline levels during and after osmotic stress in plants[J]. Molecular and General Genetics, 1997, 253(3): 334-341.
[8] Igarasbi Y, Yoshiba Y, et al. Characterization of the gene for pyrroline-5- carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L [J]. Plant Molecular Biology, 1997,33(5): 857-865.
[9] Ishitani M, et al. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid [J]. Plant Molecular Biology, 1995, 27 (2): 307-315.
[10]郭北海,张艳敏等甜菜碱醛脱氢酶BADH基因转化小麦及其表达[J].植物学报,2000,42(3):279-283.
[11]梁峥,马佳,汤岚.菠菜甜菜碱醛脱氢酶基因在烟草中的表达[J].生物工程学报,1997,13(3):236-240.
[12]郭房庆,周建明,汤章城.NaCI胁迫下小麦突变体和野生型叶片中一些有机溶质累积和基因表达差异[J].植物生理学报,1999,25:263-268.
[13] Tarczynski M C, Jensen R G, Bohnert H J. Stress protection of transgenic tobacco by production of the osmolyte mannitol [J]. Science, 1993, 259:509-510.
[14] Singh N K. Characterization of osmoin [J]. Plant Physiol, 1987, 90:1096.
[15] Yamaguchi S K, et al. Characterization of the expression ora desiccation- responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants [J]. Molecular and General Genetics, 1993, 236 (2-3): 331-340.
[16] Niu Xiaomu, Damsz B, Kononowicz A K, et al. NaCI-induced alteration in both cell structure and tissue specific plasma membrand H+-ATPase gene expression [J]. Plant Physiol, 1996, 111:679-689.
[17] Niu Xiaomu, Ziu Jingkang, Narasimhan M I, et al. Plasma membrane H+-ATPase gene expression is regulated by NaCI in cell of the halophyte Atriplex nummularia L [J]. Planta 1993b, 190:433-438.
[18] Perez-Prat E, Narasimhan Iv L, Niu et al. Growth cycle stage dependent NaCI induction of plasma membrane H+-ATPase mRNA accumulation in adapted tobacco cell [J]. Plant Cell Environ, 1994, 17:327-333.
[19] Wu S J, et al. SOS1, a Genetic locus essential for salt tolerance and potaasium acquisition [J]. Plant Cell, 1996, 8: 617- 662.
[20] Nancy R F, et al. A salinity-induced gene from the halophyte M.encodes a glucolytic enzyme, cofactor-independent phosphoglyceromutase [J]. Plant Molecular Biology, 1995, 29:213-226.
[21] Knight H, Trewavas A J, Knight M R. Calcium signalling in Arabidopsis thaliana responding to drought and salinity [J]. Plant Journal, 1997,12(5): 1067-1078.
[22] Liu J, Zhu J K. A calcium sensor homolog required for plant salt tolerance [J]. Science, 1998, 280: 1943-1945.
[23] Albrecht V, Weinl S, Blazevic D. The calcium sensor CBL1 integrates plant responses to abiotic stresses [J].Plant J, 2003, 36(4):457-470.
[24] Kim K N, Cheong Y H, Grant J J. CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis [J]. Plant Cell, 2003, 15(2):411-423.
[25] Hicham Zegzouti. Ethylene-responsive ABA and wounding.Brian Jones, Christel Many et al. ERS, a tomato cDNA encoding an LEA-like protein: characterization and expression in response to drought [J]. Plant Molecular Biology, 1997, 35: 847-854.
[26] Takeshi Urao, Bakhtiyor Yakubov, Rie Satoh. A Transmembrane Hybrid-type Histidine Kinase in Arabidopsis Functions as an Osmosensor [J]. The Plant Cell,1999, 11:1743-1754.
[27]刘强,张勇,陈受宜.干旱、高盐及低温诱导的植物蛋白激酶基因[J].科学通报,2000,45:561-566.
[28] Michael F. Thomashow. Plant cold acclimation: Freezing tolerance genes and regulatory mechanism [J]. Plant Physiol, Plant Mot. 1999, 50: 71-78.
[29] Yamaguchi SK, et al. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress [J]. Plant Cell, 1994, 6(2): 251-264.
[30] Hai L J, Martin C. Multifunctionality and diversity within the plant MYB-gene family [J]. Plant Molecular Biology, 1999, 41:577-585.
[31] Bachem C W B, Vander Hoeven R S, de Bruijn S M, et al. Visualization of differential gene expression using a novel method RNAf fingerprinting based on AFLP: analysis of gene express during potato tuber development [J]. The Plant Journal,1996, 9(5)745-753.
[32] Bachem C W B, Oomen R J F J, Visser R G F. Transcript imaging with cDNA-AFLP: a step-by-step protocol [J]. Plant Molecular Biology Report.1998, 16:157-173.
[33] Donson J, Fang Y, Espiritu-Santo G, et al. Comprehensive gene expression analysis by transcript profiling [J]. Plant Molecular Biology, 2002, 48:75-97.
[34] Kuhn E. From library screening to microarray technology: strategies to determine gene expression profiles and to identify differentially regulated genes in plants [J]. The Plant Journal, 1999, 41: 1070-1073.
[35] Bachem C W B, Vander Hoeven R S, de Bruijn S M, et al. Visualization of differential geneexpression using a novel method RNAf fingerprinting based on AFLP: analysis of gene express during potato tuber development [J]. The Plant Journal, 1996, 9(5)745-753.
[36] Gellatly K S, Ash G J, Taylor J L. Development of a method for mRNA differential display in filamentous fungi: comparison of mRNA differential display reverse transc-ription polymerase chain reaction and cDNA amplified fragment length polymerphism in Leptosphaeria maculans [J].Canada Journal of Microbiology. 2001, 47:955-960.
[37] Eckey C, Korell M, Leib K, Biedenkopf D, Jansen C, Langen G,Kogel K H. Identification of powdery mildew-induced barley genes by cDNA-AFLP:functional assessment of an early expressed MAP kinase [J]. Plant Mol Biol, 2004, 55(1):1-15.
[38] SANTA-CRUZ A, ACOSTA M, RUS A, et al. Short-term salt toleraace mechanisms in differentially salt tolerant tomato species [J]. Plant Physiol Biochem.1999, 37(1):65-71.
[39] Aoki A, Kanegami A, Mihara M,et a1. Molecular cloning and characterization of a novel soybean gene encoding a leucine-zipper-like protein induced to salt stress [J].Gene.2005,356:135-145.
[40] Chen G P, Ma W S, Huang Z J. Isolation and characterization of TaGSKl in wheat: salt tolerance [J]. Plant Science, 2003, 1369-1375.
[41] Yang L, Zheng B S, Mao C Z, Yi K K, et al. cDNA-AFLP analysis of inducible gene espresion in rice seminal root tips under a water deficit [J].Gene.2003, 314:141-148.
[42] Shi D C, et al. Stress effects of mixed salts with various salinities on the seedlings of Aneurolepidium chinense [J]. Acta Botanica Sinica, 1998, 40 (12): 1136-1142.
[43] Tewary P K, et al. In vitro response of promising mulberry (Morus sp.) genotypes for tolerance to salt and osmotic stresses [J]. Plant Growth Reguation, 2000, 30(1): 17-21.
[2]柴凤九,郭宝华.野大麦的特性及其栽培利用[J].内蒙古畜牧业,1992(4):14-15.
[3]王传东.抗盐碱植物-野大麦[J].吉林畜牧兽医,1986(1):7-8.
[4]董卫民等.野大麦人工栽培驯化实验室[J].草业科学,1992,9(5):38-39.
[5]贾慎修主编《.中国饲用植物志》第一卷[M].北京:农业出版社,1987,121-124.
[6]徐恒刚.禾本科牧草发芽期和苗期耐盐性的初步研究[J].中国草地,1988(4):53-55.
[7] Peng Z, et al. dehydrogenase Reciprocal regulation of DELTA 1-pyrroline-5- carboxylate synthetase and proline genes controls proline levels during and after osmotic stress in plants[J]. Molecular and General Genetics, 1997, 253(3): 334-341.
[8] Igarasbi Y, Yoshiba Y, et al. Characterization of the gene for pyrroline-5- carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L [J]. Plant Molecular Biology, 1997,33(5): 857-865.
[9] Ishitani M, et al. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid [J]. Plant Molecular Biology, 1995, 27 (2): 307-315.
[10]郭北海,张艳敏等甜菜碱醛脱氢酶BADH基因转化小麦及其表达[J].植物学报,2000,42(3):279-283.
[11]梁峥,马佳,汤岚.菠菜甜菜碱醛脱氢酶基因在烟草中的表达[J].生物工程学报,1997,13(3):236-240.
[12]郭房庆,周建明,汤章城.NaCI胁迫下小麦突变体和野生型叶片中一些有机溶质累积和基因表达差异[J].植物生理学报,1999,25:263-268.
[13] Tarczynski M C, Jensen R G, Bohnert H J. Stress protection of transgenic tobacco by production of the osmolyte mannitol [J]. Science, 1993, 259:509-510.
[14] Singh N K. Characterization of osmoin [J]. Plant Physiol, 1987, 90:1096.
[15] Yamaguchi S K, et al. Characterization of the expression ora desiccation- responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants [J]. Molecular and General Genetics, 1993, 236 (2-3): 331-340.
[16] Niu Xiaomu, Damsz B, Kononowicz A K, et al. NaCI-induced alteration in both cell structure and tissue specific plasma membrand H+-ATPase gene expression [J]. Plant Physiol, 1996, 111:679-689.
[17] Niu Xiaomu, Ziu Jingkang, Narasimhan M I, et al. Plasma membrane H+-ATPase gene expression is regulated by NaCI in cell of the halophyte Atriplex nummularia L [J]. Planta 1993b, 190:433-438.
[18] Perez-Prat E, Narasimhan Iv L, Niu et al. Growth cycle stage dependent NaCI induction of plasma membrane H+-ATPase mRNA accumulation in adapted tobacco cell [J]. Plant Cell Environ, 1994, 17:327-333.
[19] Wu S J, et al. SOS1, a Genetic locus essential for salt tolerance and potaasium acquisition [J]. Plant Cell, 1996, 8: 617- 662.
[20] Nancy R F, et al. A salinity-induced gene from the halophyte M.encodes a glucolytic enzyme, cofactor-independent phosphoglyceromutase [J]. Plant Molecular Biology, 1995, 29:213-226.
[21] Knight H, Trewavas A J, Knight M R. Calcium signalling in Arabidopsis thaliana responding to drought and salinity [J]. Plant Journal, 1997,12(5): 1067-1078.
[22] Liu J, Zhu J K. A calcium sensor homolog required for plant salt tolerance [J]. Science, 1998, 280: 1943-1945.
[23] Albrecht V, Weinl S, Blazevic D. The calcium sensor CBL1 integrates plant responses to abiotic stresses [J].Plant J, 2003, 36(4):457-470.
[24] Kim K N, Cheong Y H, Grant J J. CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis [J]. Plant Cell, 2003, 15(2):411-423.
[25] Hicham Zegzouti. Ethylene-responsive ABA and wounding.Brian Jones, Christel Many et al. ERS, a tomato cDNA encoding an LEA-like protein: characterization and expression in response to drought [J]. Plant Molecular Biology, 1997, 35: 847-854.
[26] Takeshi Urao, Bakhtiyor Yakubov, Rie Satoh. A Transmembrane Hybrid-type Histidine Kinase in Arabidopsis Functions as an Osmosensor [J]. The Plant Cell,1999, 11:1743-1754.
[27]刘强,张勇,陈受宜.干旱、高盐及低温诱导的植物蛋白激酶基因[J].科学通报,2000,45:561-566.
[28] Michael F. Thomashow. Plant cold acclimation: Freezing tolerance genes and regulatory mechanism [J]. Plant Physiol, Plant Mot. 1999, 50: 71-78.
[29] Yamaguchi SK, et al. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress [J]. Plant Cell, 1994, 6(2): 251-264.
[30] Hai L J, Martin C. Multifunctionality and diversity within the plant MYB-gene family [J]. Plant Molecular Biology, 1999, 41:577-585.
[31] Bachem C W B, Vander Hoeven R S, de Bruijn S M, et al. Visualization of differential gene expression using a novel method RNAf fingerprinting based on AFLP: analysis of gene express during potato tuber development [J]. The Plant Journal,1996, 9(5)745-753.
[32] Bachem C W B, Oomen R J F J, Visser R G F. Transcript imaging with cDNA-AFLP: a step-by-step protocol [J]. Plant Molecular Biology Report.1998, 16:157-173.
[33] Donson J, Fang Y, Espiritu-Santo G, et al. Comprehensive gene expression analysis by transcript profiling [J]. Plant Molecular Biology, 2002, 48:75-97.
[34] Kuhn E. From library screening to microarray technology: strategies to determine gene expression profiles and to identify differentially regulated genes in plants [J]. The Plant Journal, 1999, 41: 1070-1073.
[35] Bachem C W B, Vander Hoeven R S, de Bruijn S M, et al. Visualization of differential geneexpression using a novel method RNAf fingerprinting based on AFLP: analysis of gene express during potato tuber development [J]. The Plant Journal, 1996, 9(5)745-753.
[36] Gellatly K S, Ash G J, Taylor J L. Development of a method for mRNA differential display in filamentous fungi: comparison of mRNA differential display reverse transc-ription polymerase chain reaction and cDNA amplified fragment length polymerphism in Leptosphaeria maculans [J].Canada Journal of Microbiology. 2001, 47:955-960.
[37] Eckey C, Korell M, Leib K, Biedenkopf D, Jansen C, Langen G,Kogel K H. Identification of powdery mildew-induced barley genes by cDNA-AFLP:functional assessment of an early expressed MAP kinase [J]. Plant Mol Biol, 2004, 55(1):1-15.
[38] SANTA-CRUZ A, ACOSTA M, RUS A, et al. Short-term salt toleraace mechanisms in differentially salt tolerant tomato species [J]. Plant Physiol Biochem.1999, 37(1):65-71.
[39] Aoki A, Kanegami A, Mihara M,et a1. Molecular cloning and characterization of a novel soybean gene encoding a leucine-zipper-like protein induced to salt stress [J].Gene.2005,356:135-145.
[40] Chen G P, Ma W S, Huang Z J. Isolation and characterization of TaGSKl in wheat: salt tolerance [J]. Plant Science, 2003, 1369-1375.
[41] Yang L, Zheng B S, Mao C Z, Yi K K, et al. cDNA-AFLP analysis of inducible gene espresion in rice seminal root tips under a water deficit [J].Gene.2003, 314:141-148.
[42] Shi D C, et al. Stress effects of mixed salts with various salinities on the seedlings of Aneurolepidium chinense [J]. Acta Botanica Sinica, 1998, 40 (12): 1136-1142.
[43] Tewary P K, et al. In vitro response of promising mulberry (Morus sp.) genotypes for tolerance to salt and osmotic stresses [J]. Plant Growth Reguation, 2000, 30(1): 17-21.