草原龙胆抗盐相关基因表达谱分析及BADH基因的遗传转化
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摘要
草原龙胆(Eustoma grandiflorum)属龙胆科草本植物,是流行的鲜切花材料,具有一定的耐盐性,是一种具有重要经济价值的研究材料。
为了研究草原龙胆的抗盐机理,分离抗盐相关基因,提高其抗盐水平,进行了以下三方面的研究:
1草原龙胆盐抗性相关基因的筛选
为了分离草原龙胆盐抗性相关基因,以NaCl 400 mmol/L处理的草原龙胆叶片为tester,清水处理的对照为driver,制作了盐胁迫抑制差减(SSH)文库。经过序列测定以及序列分析,获得了658条非重复序列。通过GO(Gene Ontology)的分子功能、生物学途径及细胞组件三方面的分析,获得了123条序列的注释,占总序列的19%。在生物学途径中分为:有丝分裂检查点、类泛素化蛋白修饰、氧化胁迫反应、转运、电子传递等14类;分子功能中分为过氧化氢酶活性、催化活性、环氧化物酶活性、单氧化酶活性、谷胱甘肽脱氢酶活性等21类;在细胞组件中分为线粒体、叶绿体、过氧化物酶体、乙醛酸循环体、细胞核等10类。
2草原龙胆盐胁迫基因表达分析
为了研究草原龙胆在盐胁迫条件下的基因表达谱,以构建的SSH文库制作了基因芯片。提取NaCl 600 mmol/L处理3 h草原龙胆的叶片以及清水处理的对照mRNA,经反转录后分别进行了Cy3和Cy5的标记,进行了芯片杂交。杂交数据利用limma软件包进行了数据标准化,分析获得了差异表达基因。结果显示,显著上调表达的基因为14条,显著下调的基因为16条。其中功能已知的序列为5条,分别为硫氧还蛋白、类泛素化蛋白、苏氨酸转运蛋白、脱氢抗坏血酸还原酶、细胞色素P450。
3草原龙胆BADH基因的遗传转化
为了提高草原龙胆的盐抗性,对草原龙胆进行了BADH基因的遗传转化。从拟南芥中克隆了BADH基因的编码区全长,经过测序验证正确后,将其构建到了植物表达载体pH2GW7,0上。表达载体转化农杆菌LBA4404后制备了农杆菌侵染菌株。通过农杆菌介导法转化草原龙胆,获得了1个转化株系。PCR和Southern杂交检测表明,目的基因已经整合到了草原龙胆基因组中。
Eustoma grandiflorum,a popular fresh cut flower,and a Gentianaceae herbaceous plant,is tolerant to salt to some degree.It is an important research material with economic value.
To analyse its' molecular mechanism of salt tolerance,to isolate its' salt tolerant genes,and to enhance its' salt tolerant ability,three programs were done.
1.Selection of salt tolerant genes from Eustoma grandiflorum
To get the salt tolerant genes of it,a suppression subtractive library was constructed, useing the leaves of plants watered with 400 mmol/L NaCl solution as tester and with pure water as driver.After sequencing and blasting,658 non-repeat sequences were gotton.These 658 sequences were classified by gene ontology(GO),and 123 of them got annotation,which are 19%of the total sequences.They were sorted to 14 classes in Biological Process classification,including mitotic spindle checkpoint,response to oxidative stress,protein sumoylation,transportation,electron transportation,etc.;21 classes in Molecular Function classification,including catalase activity,catalytic activity,epoxide hydrolase activity, monooxygenase activity,glutathione dehydrogenase(ascorbate) activity etc.;and 10 classes in Cell Component classification,including mitochondrion,chloroplast,peroxisome,glyoxysome, nucleus etc..
2.Gene expression analysis of salt stressed Eustoma grandiflorum
To analyse the gene expression profile of salt stressed Eustoma grandiflorum,microarrays were manufactured with that library.The mRNA from leaves of the plants watered with 600 mmol/L NaCl solution and pure water were first reverse transcripted,then labeled with Cy3 and Cy5 seperatelly,finally hybridized with microarrays.The differently expressed genes were got by limma software package.We got 14 up-regulated genes,16 down-regulated genes,5 of them with known functions,including ubiquitin-like protein SUMO,peptide transport protein, cytochrome P450,thioredoxin,and dehydroascorbate reductase.
3.Transformation of Eustoma grandiflorum with BADH gene
To enhance its' salt tolerant ability,Eustoma grandiflorum was transformed with BADH gene.The full length of Arabidopsis Arabidopsis BADH CDS was first cloned and verified by sequencing,then constructed into plant expression vector pH2GW7.The expression vector was transformed into Agrobacterium LBA4404.The resistance plants were got via agrobacterium-mediated transformation,and identified by PCR and southern blotting.The results indicated that the BADH gene has been inserted into the genome of Eustoma grandiflorum.
为了研究草原龙胆的抗盐机理,分离抗盐相关基因,提高其抗盐水平,进行了以下三方面的研究:
1草原龙胆盐抗性相关基因的筛选
为了分离草原龙胆盐抗性相关基因,以NaCl 400 mmol/L处理的草原龙胆叶片为tester,清水处理的对照为driver,制作了盐胁迫抑制差减(SSH)文库。经过序列测定以及序列分析,获得了658条非重复序列。通过GO(Gene Ontology)的分子功能、生物学途径及细胞组件三方面的分析,获得了123条序列的注释,占总序列的19%。在生物学途径中分为:有丝分裂检查点、类泛素化蛋白修饰、氧化胁迫反应、转运、电子传递等14类;分子功能中分为过氧化氢酶活性、催化活性、环氧化物酶活性、单氧化酶活性、谷胱甘肽脱氢酶活性等21类;在细胞组件中分为线粒体、叶绿体、过氧化物酶体、乙醛酸循环体、细胞核等10类。
2草原龙胆盐胁迫基因表达分析
为了研究草原龙胆在盐胁迫条件下的基因表达谱,以构建的SSH文库制作了基因芯片。提取NaCl 600 mmol/L处理3 h草原龙胆的叶片以及清水处理的对照mRNA,经反转录后分别进行了Cy3和Cy5的标记,进行了芯片杂交。杂交数据利用limma软件包进行了数据标准化,分析获得了差异表达基因。结果显示,显著上调表达的基因为14条,显著下调的基因为16条。其中功能已知的序列为5条,分别为硫氧还蛋白、类泛素化蛋白、苏氨酸转运蛋白、脱氢抗坏血酸还原酶、细胞色素P450。
3草原龙胆BADH基因的遗传转化
为了提高草原龙胆的盐抗性,对草原龙胆进行了BADH基因的遗传转化。从拟南芥中克隆了BADH基因的编码区全长,经过测序验证正确后,将其构建到了植物表达载体pH2GW7,0上。表达载体转化农杆菌LBA4404后制备了农杆菌侵染菌株。通过农杆菌介导法转化草原龙胆,获得了1个转化株系。PCR和Southern杂交检测表明,目的基因已经整合到了草原龙胆基因组中。
Eustoma grandiflorum,a popular fresh cut flower,and a Gentianaceae herbaceous plant,is tolerant to salt to some degree.It is an important research material with economic value.
To analyse its' molecular mechanism of salt tolerance,to isolate its' salt tolerant genes,and to enhance its' salt tolerant ability,three programs were done.
1.Selection of salt tolerant genes from Eustoma grandiflorum
To get the salt tolerant genes of it,a suppression subtractive library was constructed, useing the leaves of plants watered with 400 mmol/L NaCl solution as tester and with pure water as driver.After sequencing and blasting,658 non-repeat sequences were gotton.These 658 sequences were classified by gene ontology(GO),and 123 of them got annotation,which are 19%of the total sequences.They were sorted to 14 classes in Biological Process classification,including mitotic spindle checkpoint,response to oxidative stress,protein sumoylation,transportation,electron transportation,etc.;21 classes in Molecular Function classification,including catalase activity,catalytic activity,epoxide hydrolase activity, monooxygenase activity,glutathione dehydrogenase(ascorbate) activity etc.;and 10 classes in Cell Component classification,including mitochondrion,chloroplast,peroxisome,glyoxysome, nucleus etc..
2.Gene expression analysis of salt stressed Eustoma grandiflorum
To analyse the gene expression profile of salt stressed Eustoma grandiflorum,microarrays were manufactured with that library.The mRNA from leaves of the plants watered with 600 mmol/L NaCl solution and pure water were first reverse transcripted,then labeled with Cy3 and Cy5 seperatelly,finally hybridized with microarrays.The differently expressed genes were got by limma software package.We got 14 up-regulated genes,16 down-regulated genes,5 of them with known functions,including ubiquitin-like protein SUMO,peptide transport protein, cytochrome P450,thioredoxin,and dehydroascorbate reductase.
3.Transformation of Eustoma grandiflorum with BADH gene
To enhance its' salt tolerant ability,Eustoma grandiflorum was transformed with BADH gene.The full length of Arabidopsis Arabidopsis BADH CDS was first cloned and verified by sequencing,then constructed into plant expression vector pH2GW7.The expression vector was transformed into Agrobacterium LBA4404.The resistance plants were got via agrobacterium-mediated transformation,and identified by PCR and southern blotting.The results indicated that the BADH gene has been inserted into the genome of Eustoma grandiflorum.
引文
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