早酥梨红皮芽变抑制消减cDNA文库构建及EST序列分析
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摘要
红皮梨育种是国内外梨育种的一个主要目标。与梨果实红皮性状形成的相关基因的分离和鉴定是红皮梨分子育种的基础。本研究以特有的早酥梨红皮芽变及其母株为材料,利用抑制消减杂交(Suppression subtractive hybridization, SSH)技术构建消减cDNA文库,筛选差异表达克隆,进行序列测定,对差异表达基因进行鉴定和功能分析,主要研究结果如下:
1.以早酥梨红皮芽变和母株果实为试验材料,以芽变果实为试验方(tester),早酥梨果实为驱动方(driver),利用抑制性消减杂交(Suppression Subtractive Hybridization,SSH)技术构建了消减cDNA文库,从消减文库中挑取了1039个克隆,获得了960个阳性克隆,插入片段长度经PCR鉴定,主要分布于250-1000bp之间。
2.去除污染、重复、低质量(﹤100 bp)的序列后,再进行序列测定,然后在GenBank中进行BLASTX分析比较,有314条特异的基因片段,其中203条(占64.7%)与数据库中已知功能基因有较高的同源性,49条(占15.6%)为功能未知基因,62条(占19.7%)比对没有同源性结果。推测这些没有任何同源性的基因可能是新的突变相关基因,或变异度较高的cDNA非编码区。已登录GenBank 212条ESTs,登录号为:HO774921-HO774955、HO840420-HO840592和HS573389-HS573392。
3.经过初步分析这些ESTs的功能,主要涉及花青素合成相关的基因、信号转导、能量和代谢、转录因子、抗病防卫以及导致芽变的基因。就BLASTX对比同源性最高的基因的来源看,大部分来源于梨(Pyrus)、苹果属(Malus x domestica)、拟南芥(Arabidopsis thaliana)、烟草(Nicotiana tabacum)、玉米(Maize)、蓖麻(Ricinus communis)和葡萄(Vitis vinifera)等。
4.获得3个与芽变相关的重要基因有:BEL1-like同源转录因子、逆转录转座子和核染色体重组亚基。
5.获得与花青素合成相关的基因主要有:MYBR转录因子、bHLH转录因子、细胞色素P450家族基因、黄烷酮-3-羟化酶(F3H)基因、锌指蛋白、花青素-O-甲基转移酶和ARF的同源基因等。
6.获得的抗性防卫的基因主要有:泛素蛋白连接酶、脱水素、热激蛋白、富含亮氨酸重复结构、半胱氨酸蛋白酶、多酚氧化酶、防卫素和诱导子等。
Red pear breeding is one of the major domestic and international pear breeding projects. Separation and identification about red skin development-related genes is the basic molecular breeding.In this experiment, red mutant of‘Zaosu’and its mother plant were used as the materials, we construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method.we got differentially expressed clonings and sequenced. These differentially expressed genes were identified and functional analysis. Main conclusions of this paper are as follows:
1.In this study, we used red mutant of‘Zaosu’and its mother plant as the materials , green pear‘Zaosu’as driver, and the red mutant as tester to construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method. 1039 cDNA library clones were selected, 970 positive clones were chosen from the library,the inserted segments were 250-1000 bp in length.
2. After clearing pollution, redundant, low quality(﹤100bp)sequence, according to blast screening and functional annotation in GenBank database, 314 unique genes were obtained. Among the obtained fragments, 203 (64.7%)ESTs were found to have homologous genes with known functions,49(15.6%) ESTs with unknown functions, and 62(19.7%) ESTs with no homologous genes. ESTs were found with no significant similarity which may represent new genes or high variant non-coding regions of cDNAs. 212 ESTs of them had been submitted to GenBank,and the accession number is HO774921-HO774955,HO840420-HO840592 and HS573389-HS573392.
3. Results showed that the differentially expressed genes are involved in anthocyanin biosynthesis, signal transduction, energy and metabolism, transcription factors, disease resistance and bud mutation. What’s more, those best matched protein were from many plant species, such as pear(Pyrus),apples(Malus x domestica),arabidopsis thaliana (Arabidopsis thaliana),tobacco(Nicotiana tabacum),maize (Maize), ricinus communis (Ricinus communis)and grape(Vitis vinifera ).
4.We have found 3 genes related to bud mutation,which are BEL1-like homeodomain transcription factor, retrotransposon protein and chromatin remodeling complex subunit.
5. We have found expressed genes which are involved in anthocyanin biosynthesis mainly have MYBR domain class transcription factor ,bHLH ranscription factor, Cytocrome P450 like protein, flavanone 3-hydroxylase, anthocyanin-O-methyltransferas, zinc finger and ARF domain class transcription factor.
6. The disease and defence genes obtained as flows: ubiquitin-protein ligase,dehydrin, heat shock protein,leucine rich repeat protein,cysteine proteinase, polyphenol oxidase, defensin protein and elicitors et al.
1.以早酥梨红皮芽变和母株果实为试验材料,以芽变果实为试验方(tester),早酥梨果实为驱动方(driver),利用抑制性消减杂交(Suppression Subtractive Hybridization,SSH)技术构建了消减cDNA文库,从消减文库中挑取了1039个克隆,获得了960个阳性克隆,插入片段长度经PCR鉴定,主要分布于250-1000bp之间。
2.去除污染、重复、低质量(﹤100 bp)的序列后,再进行序列测定,然后在GenBank中进行BLASTX分析比较,有314条特异的基因片段,其中203条(占64.7%)与数据库中已知功能基因有较高的同源性,49条(占15.6%)为功能未知基因,62条(占19.7%)比对没有同源性结果。推测这些没有任何同源性的基因可能是新的突变相关基因,或变异度较高的cDNA非编码区。已登录GenBank 212条ESTs,登录号为:HO774921-HO774955、HO840420-HO840592和HS573389-HS573392。
3.经过初步分析这些ESTs的功能,主要涉及花青素合成相关的基因、信号转导、能量和代谢、转录因子、抗病防卫以及导致芽变的基因。就BLASTX对比同源性最高的基因的来源看,大部分来源于梨(Pyrus)、苹果属(Malus x domestica)、拟南芥(Arabidopsis thaliana)、烟草(Nicotiana tabacum)、玉米(Maize)、蓖麻(Ricinus communis)和葡萄(Vitis vinifera)等。
4.获得3个与芽变相关的重要基因有:BEL1-like同源转录因子、逆转录转座子和核染色体重组亚基。
5.获得与花青素合成相关的基因主要有:MYBR转录因子、bHLH转录因子、细胞色素P450家族基因、黄烷酮-3-羟化酶(F3H)基因、锌指蛋白、花青素-O-甲基转移酶和ARF的同源基因等。
6.获得的抗性防卫的基因主要有:泛素蛋白连接酶、脱水素、热激蛋白、富含亮氨酸重复结构、半胱氨酸蛋白酶、多酚氧化酶、防卫素和诱导子等。
Red pear breeding is one of the major domestic and international pear breeding projects. Separation and identification about red skin development-related genes is the basic molecular breeding.In this experiment, red mutant of‘Zaosu’and its mother plant were used as the materials, we construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method.we got differentially expressed clonings and sequenced. These differentially expressed genes were identified and functional analysis. Main conclusions of this paper are as follows:
1.In this study, we used red mutant of‘Zaosu’and its mother plant as the materials , green pear‘Zaosu’as driver, and the red mutant as tester to construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method. 1039 cDNA library clones were selected, 970 positive clones were chosen from the library,the inserted segments were 250-1000 bp in length.
2. After clearing pollution, redundant, low quality(﹤100bp)sequence, according to blast screening and functional annotation in GenBank database, 314 unique genes were obtained. Among the obtained fragments, 203 (64.7%)ESTs were found to have homologous genes with known functions,49(15.6%) ESTs with unknown functions, and 62(19.7%) ESTs with no homologous genes. ESTs were found with no significant similarity which may represent new genes or high variant non-coding regions of cDNAs. 212 ESTs of them had been submitted to GenBank,and the accession number is HO774921-HO774955,HO840420-HO840592 and HS573389-HS573392.
3. Results showed that the differentially expressed genes are involved in anthocyanin biosynthesis, signal transduction, energy and metabolism, transcription factors, disease resistance and bud mutation. What’s more, those best matched protein were from many plant species, such as pear(Pyrus),apples(Malus x domestica),arabidopsis thaliana (Arabidopsis thaliana),tobacco(Nicotiana tabacum),maize (Maize), ricinus communis (Ricinus communis)and grape(Vitis vinifera ).
4.We have found 3 genes related to bud mutation,which are BEL1-like homeodomain transcription factor, retrotransposon protein and chromatin remodeling complex subunit.
5. We have found expressed genes which are involved in anthocyanin biosynthesis mainly have MYBR domain class transcription factor ,bHLH ranscription factor, Cytocrome P450 like protein, flavanone 3-hydroxylase, anthocyanin-O-methyltransferas, zinc finger and ARF domain class transcription factor.
6. The disease and defence genes obtained as flows: ubiquitin-protein ligase,dehydrin, heat shock protein,leucine rich repeat protein,cysteine proteinase, polyphenol oxidase, defensin protein and elicitors et al.
引文
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