毛尖紫萼藓（Grimmia pilifera）cDNA文库构建及抗旱相关基因克隆与分析
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摘要
干旱胁迫是影响植物生长发育的主要环境因素,严重影响农作物的产量。解决这个问题的有效途径是培育和利用优良的抗旱品种。应用比较功能基因组学方法筛选抗旱相关基因,并通过基因工程培育抗旱品种已成为植物遗传资源与品种改良研究的重要内容。毛尖紫萼藓(Grimmia pilifera)是典型旱生藓类,生长在向阳的裸岩上,具有很强的抗旱能力,是很好的抗旱基因资源。
为研究毛尖紫萼藓响应干旱的分子机制,本研究采用SMART技术构建了毛尖紫萼藓干旱cDNA文库,文库滴度2.8×105pfu/mL,菌落PCR检测表明,插入片段分布在500-2000bp之间,平均大小约800bp,重组率约为91.7%。通过文库测序共获得1045条有效序列,其中高质量的996条,GenBank接收号为GR307103-GR308098。这些ESTs代表875个Unigenes,其中contig62个,singlet 813个。Blast N及Blast X分析表明,625个Unigenes与Nr数据库中已知基因序列具有相似性,250个Unigenes没有相似性。与已知序列具有相似性的Unigene依据GO注释分为16类,其中生理学进程占(21.17%),细胞进程占(14.60%),细胞占(13.63%),催化占(13.14%),结合占(10.71%),细胞器占(10.46%),蛋白复合体占(7.54%),结构分子占(3.41%),应激响应占(1.95%),转运蛋白占(1.22%),抗氧化剂占(0.97%),翻译调节占(0.24%),酶调节占(0.24%),运动活性占(0.24%),转录调节占(0.24%),生物学途径调控占(0.24%)
根据基因功能注释,共获得参与信号转导与基因表达调控、活性氧清除、光合系统保护及修复、蛋白质合成与降解、代谢、物质转运及离子平衡、细胞壁及细胞救援防御、抗胁迫8类68个抗旱相关基因。
根据生物信息学分析结果,选择了9条毛尖紫萼藓抗旱相关的基因片段,采用荧光定量PCR进行干旱及复水条件下的表达分析。结果表明这些基因的表达都在一定程度上受干旱或复水的诱导,但不同基因的表达模式不同。
通过Blast X分析,获得了毛尖紫萼藓抗坏血酸过氧化物酶、铜锌超氧化物歧化酶、谷胱甘肽转移酶及半胱氨酸过氧化物酶基因全长序列,命名为GpAPX、GpSOD、GpGST和GpCysPrx, GenBank接受号分别为GU989311、GU989312、GU989313和GU989314,通过生物信息学对其所编码的蛋白进行分析,同时利用荧光定量PCR技术分析干旱及复水条件下基因表达情况。
本研究筛选获得部分抗旱相关基因,为植物抗旱基因工程研究提供了基因资源,同时也为毛尖紫萼藓抗旱分子机制研究奠定了基础。
Drought is one of the major environmental factors that affect the growth of plant and account for significant reduction in the yields of crops. An effective approach to solve this problem is to produce drought-tolerant crops. Therefore, comparative functional genomics studies are required to identify key genes responsible for dehydration and drought stress tolerance as well as candidate genes for genetic engineering of drought stress tolerance in crop plants. Grimmia pilifera, as a common drought tolerant moss, grows in habitats of rocky outcrops, having a high ability in resisting drought stress. Grimmia pilifera may serve as a rich genetic resource for the identification of novel genes associated with environmental stress and dehydration tolerance
To further understand the mechanisms of Grimmia pilifera responding to drought stress, we constructed a cDNA library from the gametophyte of Grimmia pilifera, and the titer of the library was 2.8×105 pfu/mL. PCR amplification revealed that the insert cDNA fragments ranged mostly from 500 to 2000bp, with an average length of 800 bp, and the percentage of recombinants was 91.7%. A total of 1045 clones were randomly picked and subjected to 5'end single-pass sequencing from the library. After trimming of vector/adaptor sequences and discarding of low quality or short clones,996 high-quality ESTs were generated with GenBank accession numbers from GR307103 to GR308098. The total 996 ESTs represented 875 non-redundant unique transcripts, including 62 contigs and 813 singletons. Among them,625 Unigenes showed similarities to gene sequences in the non-redundant (Nr) GenBank database, and the other 250 had no similarities. The database-matched Unigene were further grouped into 16 functions according to GO assignments, i.e, physiological process(21.17%), cellular process(14.60%), cell(13.63%), catalytic activity(13.14%), binding(10.71%), organelle(10.46%), protein complex(7.54%), structural molecule activity(3.41%), response to stimulus(1.95%), transporter activity(1.22%), antioxidant activity(0.97%), translation regulator activity(0.24%), enzyme regulator activity(0.24%), motor activity(0.24%), transcription regulator activity(0.24%), and regulation of biological process(0.24%).
In total,68 Unigenes involved in signal transduction, reactive oxygen scavengers, protection and repair of photosynthesis system, protein synthesis and degradation, metabolism, materials transport and ionic equilibrium, cell wall and cell defense, and tress response were identified.
Real-time quantitative PCR was used for identification of expression profiles of 9 drought stress induced genes from Grimmia pilifera. The results showed that the expressions of the 9 genes were all induced by dehydration or rehydration, and the expression patterns of those genes were different.
Four genes including ascorbate peroxidase, copper/zinc-superoxide dismutase, glutathione S-transferase and cysperoxiredoxin were obtained, and named GpAPX、GpSOD、GpGST and GpCysPrx. They were submitted to the GenBank databases with accession numbers from GU989311 to GU989314. Protein analysis was performed using bioinformatics software, and QRT-PCR was used to study the expression profile during dehydration and rehydration.
The identification of the drought-resistance genes provided genetic resources for plant breeding and genetic engineering of crop plants. This work represented an important step in understanding the molecular mechanism of drought tolerance in Grimmia pilifera.
为研究毛尖紫萼藓响应干旱的分子机制,本研究采用SMART技术构建了毛尖紫萼藓干旱cDNA文库,文库滴度2.8×105pfu/mL,菌落PCR检测表明,插入片段分布在500-2000bp之间,平均大小约800bp,重组率约为91.7%。通过文库测序共获得1045条有效序列,其中高质量的996条,GenBank接收号为GR307103-GR308098。这些ESTs代表875个Unigenes,其中contig62个,singlet 813个。Blast N及Blast X分析表明,625个Unigenes与Nr数据库中已知基因序列具有相似性,250个Unigenes没有相似性。与已知序列具有相似性的Unigene依据GO注释分为16类,其中生理学进程占(21.17%),细胞进程占(14.60%),细胞占(13.63%),催化占(13.14%),结合占(10.71%),细胞器占(10.46%),蛋白复合体占(7.54%),结构分子占(3.41%),应激响应占(1.95%),转运蛋白占(1.22%),抗氧化剂占(0.97%),翻译调节占(0.24%),酶调节占(0.24%),运动活性占(0.24%),转录调节占(0.24%),生物学途径调控占(0.24%)
根据基因功能注释,共获得参与信号转导与基因表达调控、活性氧清除、光合系统保护及修复、蛋白质合成与降解、代谢、物质转运及离子平衡、细胞壁及细胞救援防御、抗胁迫8类68个抗旱相关基因。
根据生物信息学分析结果,选择了9条毛尖紫萼藓抗旱相关的基因片段,采用荧光定量PCR进行干旱及复水条件下的表达分析。结果表明这些基因的表达都在一定程度上受干旱或复水的诱导,但不同基因的表达模式不同。
通过Blast X分析,获得了毛尖紫萼藓抗坏血酸过氧化物酶、铜锌超氧化物歧化酶、谷胱甘肽转移酶及半胱氨酸过氧化物酶基因全长序列,命名为GpAPX、GpSOD、GpGST和GpCysPrx, GenBank接受号分别为GU989311、GU989312、GU989313和GU989314,通过生物信息学对其所编码的蛋白进行分析,同时利用荧光定量PCR技术分析干旱及复水条件下基因表达情况。
本研究筛选获得部分抗旱相关基因,为植物抗旱基因工程研究提供了基因资源,同时也为毛尖紫萼藓抗旱分子机制研究奠定了基础。
Drought is one of the major environmental factors that affect the growth of plant and account for significant reduction in the yields of crops. An effective approach to solve this problem is to produce drought-tolerant crops. Therefore, comparative functional genomics studies are required to identify key genes responsible for dehydration and drought stress tolerance as well as candidate genes for genetic engineering of drought stress tolerance in crop plants. Grimmia pilifera, as a common drought tolerant moss, grows in habitats of rocky outcrops, having a high ability in resisting drought stress. Grimmia pilifera may serve as a rich genetic resource for the identification of novel genes associated with environmental stress and dehydration tolerance
To further understand the mechanisms of Grimmia pilifera responding to drought stress, we constructed a cDNA library from the gametophyte of Grimmia pilifera, and the titer of the library was 2.8×105 pfu/mL. PCR amplification revealed that the insert cDNA fragments ranged mostly from 500 to 2000bp, with an average length of 800 bp, and the percentage of recombinants was 91.7%. A total of 1045 clones were randomly picked and subjected to 5'end single-pass sequencing from the library. After trimming of vector/adaptor sequences and discarding of low quality or short clones,996 high-quality ESTs were generated with GenBank accession numbers from GR307103 to GR308098. The total 996 ESTs represented 875 non-redundant unique transcripts, including 62 contigs and 813 singletons. Among them,625 Unigenes showed similarities to gene sequences in the non-redundant (Nr) GenBank database, and the other 250 had no similarities. The database-matched Unigene were further grouped into 16 functions according to GO assignments, i.e, physiological process(21.17%), cellular process(14.60%), cell(13.63%), catalytic activity(13.14%), binding(10.71%), organelle(10.46%), protein complex(7.54%), structural molecule activity(3.41%), response to stimulus(1.95%), transporter activity(1.22%), antioxidant activity(0.97%), translation regulator activity(0.24%), enzyme regulator activity(0.24%), motor activity(0.24%), transcription regulator activity(0.24%), and regulation of biological process(0.24%).
In total,68 Unigenes involved in signal transduction, reactive oxygen scavengers, protection and repair of photosynthesis system, protein synthesis and degradation, metabolism, materials transport and ionic equilibrium, cell wall and cell defense, and tress response were identified.
Real-time quantitative PCR was used for identification of expression profiles of 9 drought stress induced genes from Grimmia pilifera. The results showed that the expressions of the 9 genes were all induced by dehydration or rehydration, and the expression patterns of those genes were different.
Four genes including ascorbate peroxidase, copper/zinc-superoxide dismutase, glutathione S-transferase and cysperoxiredoxin were obtained, and named GpAPX、GpSOD、GpGST and GpCysPrx. They were submitted to the GenBank databases with accession numbers from GU989311 to GU989314. Protein analysis was performed using bioinformatics software, and QRT-PCR was used to study the expression profile during dehydration and rehydration.
The identification of the drought-resistance genes provided genetic resources for plant breeding and genetic engineering of crop plants. This work represented an important step in understanding the molecular mechanism of drought tolerance in Grimmia pilifera.
引文
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