两个与棉纤维发育相关基因的克隆与鉴定
摘要
棉纤维是重要的纺织工业原料,在国民经济和人民生活中占有重要的位置。棉纤维是由棉花胚珠外珠被表皮层的单细胞发育而成,在受精后约16天,棉纤维细胞可延伸至2.5-3.0cm。棉纤维发育过程中合成大量的纤维素与非纤维素多糖参与纤维形态的建成,这一发育过程有众多的糖类物质参与,进行了复杂的生化代谢反应,因此棉花纤维发育相关基因的克隆既具有巨大的潜在经济价值,也有助于植物细胞发育以及纤维素合成分子机理的阐明。
本研究从陆地长绒种质系7235不同发育时期的棉纤维混合cDNA文库中分离出两个cDNA克隆:(1)水通道蛋白(aquaporin)我们将其命名为GhAQP。该cDNA克隆插入片段长度为1207bp,最大的ORF为837bp,编码279个氨基酸其理论上的等电点Theoretical pI=9.13,分子量Mw=29.7KD。Blastx结果表明,该cDNA与已报道的多种质膜内在蛋白(plasma membrane intrinsic protein)和水通道蛋白(aquaporin)cDNA克隆相似性均极高。(2)小泡相关膜蛋白(VESICLE-ASSOCIATED MEMBRANE PROTEIN),我们将其命名为GhVAMP。该cDNA克隆插入片段长度为1080bp,开放读码框长度为549bp,编码183个氨基酸,其理论上的等电点Theoretical pI=9.13,分子量Mw=20.4KD。Blastx结果表明,该cDNA序列与若干synptobrevin-related protein相似程度很高;(3)其余5个cDNA克隆经过BLAST比对,与已知的基因同源性很低。
我们采用signalP程序对这两个基因进行了N端信号肽预测,GhAQP没有典型的信号肽,GhVAMP有典型的信号肽,说明这个基因可能是分泌蛋白。采用NetPhos 2.0程序对这两个基因进行了磷酸化位点预测,GhAQP有12个磷酸化位点,GhVAMP有6个磷酸化位点。采用NetNGlyc1.0预测糖基化位点,都有一个糖基化位点。并且对这两个基因进行了保守区域及进化树分析等。
从表达特征来看,GhAQP与GhVAMP在根、茎、叶中都表达,在茎中优势表达,在胚珠和纤维细胞中表达,GhAQP在开花后3～14天的纤维中持续高效表达,17天后减弱;GhVAMP在开花后3～11天的纤维中持续高效表达,17天后减弱。
我们对GhAQP构建了pBI121-35s启动子正义载体和反义载体,对GhVAMP构建了pBI121-35s启动子反义载体,现在正在进行棉花转基因功能验证。
Cotton fibers are important textile materials that play important role in national economy and people's lives. Each cotton fiber is a single cell of ovule epidermis that elongates to 2.5-3.0 cm within approximately 16 days post anthesis (DPA). There are a great deal of cellulosic and noncellulosic polysaccharide been synthesized during cotton fiber development to participate in the formation of fiber configuration, and this developmental process consists of myriads of saccharide progressing complicated biochemical reactions. So cloning the developmentally regulated genes from cotton fibers may play an important role not only in improving the quality of cotton fiber, but also in studying plant cell development and cellulose biosynthesis.
Two cDNA clones were separated from developmentally different cotton fiber pool of premium material 7235 library:(1) plasma membrane intrinsic protein/aquaqporins.we named it GhAQP.The insert fragment of the cDNA clone was 1207bp,its open reading frame was 837bp,and encoded a polypeptide containing 249 amino acids and the Mw of the deduced amino acid is 29.7kDa, and pI is predicted to be 9.13. Blastx analysis indicated GhAQP had substantive homologies with some reported plasma membrane intrinsic protein/aquaqporins;(2) Vesicle-Associated Membrane Protein, We named it GhVAMP .The insert fragment of the cDNA clone was 1080bp,its open reading frame was 549bp,and encoded a polypeptide containing 183 amino acids ,and the Mw of the deduced amino acid is 20.4kDa, and pI is predicted to be 9.13. Blastx analysis indicated GhVAMP had some identity with some reported VAMP (3)The rest of the seven cDNA clones had little identity with the reported genes.
We predicted their signal peptide via signalP process.GhVAMP had typical signal peptide but GhAQP had not. We predicted their phosphorylation sites via NetPhos process ,they had 6 and 12 sites each. We predicted their N-glyoosylation sites via NetNGlyc process ,They had one site both.We also analyzed their conserved domain and phylogenetic tree.
Judging from their expression characters,GhAQP and GhVAMP were expressed in fiber cells and in root, leaf and had a preferential expression in stem, while it could be detected in ovules and fiber cells of different developing period, especially in elongating fibers; GhAQP was preferential expressed in fiber cells from 3DPA to 14DPA,and its expression was decreased after 17DPA in fiber developmental stages, GhVAMP was preferential expressed in fiber cells from 3DPA to 11DPA,and its expression was decreased after 17DPA in fiber developmental stages.
Sense and antisense expression vector containing 35S promoter e using pBI121 plasmid were constructed for GhAQP; Antisense expression vectors containing 35S promoter using pBI121 plasmid were constructed for GhVAMP.The work of transferring these recombined vectors into Gossypium hirsutum L. by Agrobacterium tumefaciens is ongoing.
本研究从陆地长绒种质系7235不同发育时期的棉纤维混合cDNA文库中分离出两个cDNA克隆:(1)水通道蛋白(aquaporin)我们将其命名为GhAQP。该cDNA克隆插入片段长度为1207bp,最大的ORF为837bp,编码279个氨基酸其理论上的等电点Theoretical pI=9.13,分子量Mw=29.7KD。Blastx结果表明,该cDNA与已报道的多种质膜内在蛋白(plasma membrane intrinsic protein)和水通道蛋白(aquaporin)cDNA克隆相似性均极高。(2)小泡相关膜蛋白(VESICLE-ASSOCIATED MEMBRANE PROTEIN),我们将其命名为GhVAMP。该cDNA克隆插入片段长度为1080bp,开放读码框长度为549bp,编码183个氨基酸,其理论上的等电点Theoretical pI=9.13,分子量Mw=20.4KD。Blastx结果表明,该cDNA序列与若干synptobrevin-related protein相似程度很高;(3)其余5个cDNA克隆经过BLAST比对,与已知的基因同源性很低。
我们采用signalP程序对这两个基因进行了N端信号肽预测,GhAQP没有典型的信号肽,GhVAMP有典型的信号肽,说明这个基因可能是分泌蛋白。采用NetPhos 2.0程序对这两个基因进行了磷酸化位点预测,GhAQP有12个磷酸化位点,GhVAMP有6个磷酸化位点。采用NetNGlyc1.0预测糖基化位点,都有一个糖基化位点。并且对这两个基因进行了保守区域及进化树分析等。
从表达特征来看,GhAQP与GhVAMP在根、茎、叶中都表达,在茎中优势表达,在胚珠和纤维细胞中表达,GhAQP在开花后3～14天的纤维中持续高效表达,17天后减弱;GhVAMP在开花后3～11天的纤维中持续高效表达,17天后减弱。
我们对GhAQP构建了pBI121-35s启动子正义载体和反义载体,对GhVAMP构建了pBI121-35s启动子反义载体,现在正在进行棉花转基因功能验证。
Cotton fibers are important textile materials that play important role in national economy and people's lives. Each cotton fiber is a single cell of ovule epidermis that elongates to 2.5-3.0 cm within approximately 16 days post anthesis (DPA). There are a great deal of cellulosic and noncellulosic polysaccharide been synthesized during cotton fiber development to participate in the formation of fiber configuration, and this developmental process consists of myriads of saccharide progressing complicated biochemical reactions. So cloning the developmentally regulated genes from cotton fibers may play an important role not only in improving the quality of cotton fiber, but also in studying plant cell development and cellulose biosynthesis.
Two cDNA clones were separated from developmentally different cotton fiber pool of premium material 7235 library:(1) plasma membrane intrinsic protein/aquaqporins.we named it GhAQP.The insert fragment of the cDNA clone was 1207bp,its open reading frame was 837bp,and encoded a polypeptide containing 249 amino acids and the Mw of the deduced amino acid is 29.7kDa, and pI is predicted to be 9.13. Blastx analysis indicated GhAQP had substantive homologies with some reported plasma membrane intrinsic protein/aquaqporins;(2) Vesicle-Associated Membrane Protein, We named it GhVAMP .The insert fragment of the cDNA clone was 1080bp,its open reading frame was 549bp,and encoded a polypeptide containing 183 amino acids ,and the Mw of the deduced amino acid is 20.4kDa, and pI is predicted to be 9.13. Blastx analysis indicated GhVAMP had some identity with some reported VAMP (3)The rest of the seven cDNA clones had little identity with the reported genes.
We predicted their signal peptide via signalP process.GhVAMP had typical signal peptide but GhAQP had not. We predicted their phosphorylation sites via NetPhos process ,they had 6 and 12 sites each. We predicted their N-glyoosylation sites via NetNGlyc process ,They had one site both.We also analyzed their conserved domain and phylogenetic tree.
Judging from their expression characters,GhAQP and GhVAMP were expressed in fiber cells and in root, leaf and had a preferential expression in stem, while it could be detected in ovules and fiber cells of different developing period, especially in elongating fibers; GhAQP was preferential expressed in fiber cells from 3DPA to 14DPA,and its expression was decreased after 17DPA in fiber developmental stages, GhVAMP was preferential expressed in fiber cells from 3DPA to 11DPA,and its expression was decreased after 17DPA in fiber developmental stages.
Sense and antisense expression vector containing 35S promoter e using pBI121 plasmid were constructed for GhAQP; Antisense expression vectors containing 35S promoter using pBI121 plasmid were constructed for GhVAMP.The work of transferring these recombined vectors into Gossypium hirsutum L. by Agrobacterium tumefaciens is ongoing.
引文
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