五个与棉纤维发育相关基因的克隆与鉴定
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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205-300cm。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。
本研究从优质材料7235不同发育时期的棉纤维混合cDNA文库中分离出五个cDNA克隆：(1)陆地棉纤维表达蛋白(cotton fiber expressed protein，GhCFE)(GenBank登录号：DQ073045)，该cDNA克隆插入片段长度为1274bp，开放读码框长度为996bp，编码331个氨基酸，Blastx结果表明，该cDNA序列与已报道的3个陆地棉纤维表达蛋白cDNA克隆相似性均很高，与CFE1，CFE2和CFE3分别达到了95％，98％和96％；(2)2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase，HGD)，该cDNA克隆插入片段长度为1697bp，开放读码框长度为1422bp，编码473个氨基酸，Blastx结果表明，该cDNA序列与已报道的番茄(Lycopersicon esculentum)的2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase)的相似性为84％，与拟南芥(Arabidopsis thaliana)的2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase)(At5g54080)相似性为81％；(3)两个过氧化物酶(peroxidase，POD)，其中一个是利用5’RACE技术得到的长度为1489bp的完整cDNA序列，开放读码框长度为816bp，编码271个氨基酸，Blastx结果表明，该基因最大的ORF编码的氨基酸和拟南芥中一个过氧化物酶基因的氨基酸相似性为70％，我们将其命名为GhPOD1；另一个cDNA克隆插入片段长度为1355bp，开放读码框长度为999bp，编码332个氨基酸，Blastx结果表明，该cDNA序列与已报道的陆地棉纤维过氧化物酶基因(pod8，L08199)相似性很高，达到了99％，并且氨基酸比较也基本相似，将其命名为GhPOD2；(4)果胶裂解酶(pectate lyase，PL)，该cDNA克隆插入片段长度为1539bp，开放读码框长度为1236bp，编码411个氨基酸，Blastx比较结果显示，我们克隆的这个cDNA序列与苹果(Malus x domestica)的一个果胶裂解酶相似性最高，达到85％，与辣椒(Capsicum annuum)、草莓(Fragaria x ananassa)、百日菊(Zinnia elegans)、葡萄(Vitis vinifera)等物种的果胶裂解酶也都有一定的相似性。因此，我们将这个基因命名为GhPL，它在棉花中是首次报道(GenBank登录号：DQ073046)。
我们采用SignalP程序对这五个基因进行了N端信号肽预测，除了GhHGD没有典型的信号肽，其余四个基因都有典型的信号肽，说明这四个基因可能是分泌蛋白。并且对这五个基因进行了保守区域及进化树分析等。
从表达特征来看，GhCFE在根、茎中都不表达，在叶中微弱表达，在胚珠和纤维细胞中表达，并在开花后5～20天的纤维中持续高效表达，20天后减弱，是一个棉纤维优势表达基因：GhHGD在根、茎、叶、胚珠和纤维细胞中都表达，但是在叶、开花后1天和3天的胚珠中表达较弱，在开花后5天的纤维中表达增强，在17天的纤维中表达又减弱，所以该基因是一个在开花后5天到14天的纤维中优势表达的基因；GhPOD2在根中不表达，在茎、叶、胚珠和纤维细胞中表达，并在开花后14天的纤维中表达开始逐渐减弱；GhPL在根、茎、叶中都不表达，在纤维和胚珠中表达，是一个棉纤维特异表达基因；GhPOD1是组成性表达的基因。
Southern杂交结果表明这五个基因在陆地棉基因组中都存在两个拷贝，其中A，D亚组中各有一个拷贝。
我们对GhCFE，GhPL构建了pBI121-35s启动子正义载体及E6启动子正义和反义载体，对GhPOD2构建了pBI121-35s启动子正义和反义载体，并对GhPL构建了RNAi载体，现在正在进行棉花转基因功能验证。
将GhCFE，GhHGD和GhPL转入酵母表达载体pREP5N，通过电击转化法转化酵母细胞，结果表明GhHGD的导入使酵母细胞在长度及长／宽上有显著差异，GhPL的导入使酵母细胞在宽度及长／宽上有显著差异，而GhCFE的导入对酵母细胞的长度、宽度及长／宽都没有显著变化，说明该基因的表达与细胞的极性生长无关。
利用本实验室陆地棉遗传标准系TM-1和海岛棉海7124培育的140个BC_1作图群体，将GhCFE和GhPL分别定位在第18号染色体和第3号染色体上。
Cotton fibers are important textile materials that play important role in nationaleconomy and people's lives. Each cotton fiber is a single cell of ovule epidermis thatelongates to 2.5-3.0 cm within approximately 16 days post anthesis (DPA). There are agreat deal of cellulosic and noncellulosic polysacchafide been synthesized during cottonfiber development to participate in the formation of fiber configuration, and thisdevelopmental process consists of myriads of saccharide progressing complicatedbiochemical reactions. So cloning the developmentally regulated genes from cotton fibersmay play an important role not only in improving the quality of cotton fiber, but also instudying plant cell development and cellulose biosynthesis.
Five cDNA clones were separated from developmentally different cotton fiber pool ofpremium material 7235 library:(1)cotton fiber expressed protein (GhCFE, GenBankAccession numbe:DQ073045), the insert fragment of the cDNA clone was 1274bp,its openreading frame was 996bp, and encoded a polypeptide containing 331 amino acids. Blastxanalysis indicated GhCFE had 95%,98% and 96% homologies with CFE1,CFE2 and CFE3respectively;(2)homogentisate 1,2-dioxygenase (HGD), the insert fragment of the cDNAclone was 1697bp,its open reading frame was 1422bp,and encoded a polypeptidecontaining 473 amino acids. Blastx analysis indicated this gene had a 84% identity withhomogentisate 1,2-dioxygenase of Lycopersicon esculentum and 81% with homogentisate1,2-dioxygenase of Arabidopsis thaliana(At5g54080);(3)two peroxidases (POD), one wasa 1489bp cDNA sequence obtained via 5'RACE technique, its open reading frame was816bp,and encoded a polypeptide containing 271 amino acids. Blastx analysis indicatedthis gene had 70% homologies with a peroxidase of Arabidopsis thaliana, so this gene wasdesignated as GhPOD1; the insert fragment of the other cDNA clone was 1355bp,its openreading frame was 999bp,and encoded a polypeptide containing 332 amino acids. Blastxanalysis indicated this gene had a 99% identity with peroxidase of Gossypium hirsutum Lreported (pod8, L08199) and their protein sequences were almost similar. We designatedthis gene as GhPOD2;(4)pectate lyase (PL), the insert fragment of the eDNA clone was1539bp,its open reading frame was 1236bp,and encoded a polypeptide containing 411 amino acids. Blastx analysis indicated this gene had an 85% identity with pectate lyase ofMalus×doraestica.So this gene was designated as GhPL (GenBank Accession number:DQ073046).
We predicted their signal peptide via signalP process.They all had typical signalpeptide except GhHGD.We also analyzed their conserved domain and phylogenetic tree.
Judging from their expression characters,GhCFE was preferential expressed in fibercells and did not express in root or stem but only had a weak expression in leaf, while itcould be detected in ovules and fiber cells of different developing period, especially inelongating fibers;GhHGD could be detected in every tissue,but in leaf and ovules itsexpression was weak and it was preferential expressed in fiber cells from 5DPA to 14 DPA;GhPOD2 just did not express in root and its expression was decreased after 14DPA in fiberdevelopmental stages;GhPL was specifically expressed in fiber cells, while it could not bedetected in root, stem or leaf;GhPOD1 was constitutive expression and detected in everytissue.
Southern blotting analysis shows that there are two copies of these five genes in thegenome of upland cotton; sub-genome A and sub-genome D contained each.
Sense expression vector containing 35S promoter and both sense and antisenseexpression vectors containing E6 promoter using pBI121 plasmid were constructed forGhCFE and GhPL; both sense and antisense expression vectors containing 35S promoterusing pBI121 plasmid were constructed for GhPOD2 and RNAi vector was constructed forGhPL.The work of transferring these recombined vectors into Gossypium hirsutum L. byAgrobactedum tumefaciens is ongoing.
GhCFE, GhHGD and GhPL were integrated into yeast expression vector (pREP5N),and electro-transformed into fission yeast S. pombe Q-01. The results showed that GhHGDleaded a significant change of length and length/width ratio; GhPL leaded a significantchange of width and length/width ratio; GhCFE had no significant effect on elongatingcells or thickening cell wall in the transformed yeast.
We have used the BC_1 mapping population derived from the hybridization between theupland cultivar TM-1 and the island cultivar Hai7124, further TM-1 as recurrent parent.GhCFE and GhPL were localized on the chromosome 18 and chromosome 3 respectively.
本研究从优质材料7235不同发育时期的棉纤维混合cDNA文库中分离出五个cDNA克隆：(1)陆地棉纤维表达蛋白(cotton fiber expressed protein，GhCFE)(GenBank登录号：DQ073045)，该cDNA克隆插入片段长度为1274bp，开放读码框长度为996bp，编码331个氨基酸，Blastx结果表明，该cDNA序列与已报道的3个陆地棉纤维表达蛋白cDNA克隆相似性均很高，与CFE1，CFE2和CFE3分别达到了95％，98％和96％；(2)2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase，HGD)，该cDNA克隆插入片段长度为1697bp，开放读码框长度为1422bp，编码473个氨基酸，Blastx结果表明，该cDNA序列与已报道的番茄(Lycopersicon esculentum)的2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase)的相似性为84％，与拟南芥(Arabidopsis thaliana)的2，5-二羟苯乙酸1，2-加双氧酶(homogentisate 1，2-dioxygenase)(At5g54080)相似性为81％；(3)两个过氧化物酶(peroxidase，POD)，其中一个是利用5’RACE技术得到的长度为1489bp的完整cDNA序列，开放读码框长度为816bp，编码271个氨基酸，Blastx结果表明，该基因最大的ORF编码的氨基酸和拟南芥中一个过氧化物酶基因的氨基酸相似性为70％，我们将其命名为GhPOD1；另一个cDNA克隆插入片段长度为1355bp，开放读码框长度为999bp，编码332个氨基酸，Blastx结果表明，该cDNA序列与已报道的陆地棉纤维过氧化物酶基因(pod8，L08199)相似性很高，达到了99％，并且氨基酸比较也基本相似，将其命名为GhPOD2；(4)果胶裂解酶(pectate lyase，PL)，该cDNA克隆插入片段长度为1539bp，开放读码框长度为1236bp，编码411个氨基酸，Blastx比较结果显示，我们克隆的这个cDNA序列与苹果(Malus x domestica)的一个果胶裂解酶相似性最高，达到85％，与辣椒(Capsicum annuum)、草莓(Fragaria x ananassa)、百日菊(Zinnia elegans)、葡萄(Vitis vinifera)等物种的果胶裂解酶也都有一定的相似性。因此，我们将这个基因命名为GhPL，它在棉花中是首次报道(GenBank登录号：DQ073046)。
我们采用SignalP程序对这五个基因进行了N端信号肽预测，除了GhHGD没有典型的信号肽，其余四个基因都有典型的信号肽，说明这四个基因可能是分泌蛋白。并且对这五个基因进行了保守区域及进化树分析等。
从表达特征来看，GhCFE在根、茎中都不表达，在叶中微弱表达，在胚珠和纤维细胞中表达，并在开花后5～20天的纤维中持续高效表达，20天后减弱，是一个棉纤维优势表达基因：GhHGD在根、茎、叶、胚珠和纤维细胞中都表达，但是在叶、开花后1天和3天的胚珠中表达较弱，在开花后5天的纤维中表达增强，在17天的纤维中表达又减弱，所以该基因是一个在开花后5天到14天的纤维中优势表达的基因；GhPOD2在根中不表达，在茎、叶、胚珠和纤维细胞中表达，并在开花后14天的纤维中表达开始逐渐减弱；GhPL在根、茎、叶中都不表达，在纤维和胚珠中表达，是一个棉纤维特异表达基因；GhPOD1是组成性表达的基因。
Southern杂交结果表明这五个基因在陆地棉基因组中都存在两个拷贝，其中A，D亚组中各有一个拷贝。
我们对GhCFE，GhPL构建了pBI121-35s启动子正义载体及E6启动子正义和反义载体，对GhPOD2构建了pBI121-35s启动子正义和反义载体，并对GhPL构建了RNAi载体，现在正在进行棉花转基因功能验证。
将GhCFE，GhHGD和GhPL转入酵母表达载体pREP5N，通过电击转化法转化酵母细胞，结果表明GhHGD的导入使酵母细胞在长度及长／宽上有显著差异，GhPL的导入使酵母细胞在宽度及长／宽上有显著差异，而GhCFE的导入对酵母细胞的长度、宽度及长／宽都没有显著变化，说明该基因的表达与细胞的极性生长无关。
利用本实验室陆地棉遗传标准系TM-1和海岛棉海7124培育的140个BC_1作图群体，将GhCFE和GhPL分别定位在第18号染色体和第3号染色体上。
Cotton fibers are important textile materials that play important role in nationaleconomy and people's lives. Each cotton fiber is a single cell of ovule epidermis thatelongates to 2.5-3.0 cm within approximately 16 days post anthesis (DPA). There are agreat deal of cellulosic and noncellulosic polysacchafide been synthesized during cottonfiber development to participate in the formation of fiber configuration, and thisdevelopmental process consists of myriads of saccharide progressing complicatedbiochemical reactions. So cloning the developmentally regulated genes from cotton fibersmay play an important role not only in improving the quality of cotton fiber, but also instudying plant cell development and cellulose biosynthesis.
Five cDNA clones were separated from developmentally different cotton fiber pool ofpremium material 7235 library:(1)cotton fiber expressed protein (GhCFE, GenBankAccession numbe:DQ073045), the insert fragment of the cDNA clone was 1274bp,its openreading frame was 996bp, and encoded a polypeptide containing 331 amino acids. Blastxanalysis indicated GhCFE had 95%,98% and 96% homologies with CFE1,CFE2 and CFE3respectively;(2)homogentisate 1,2-dioxygenase (HGD), the insert fragment of the cDNAclone was 1697bp,its open reading frame was 1422bp,and encoded a polypeptidecontaining 473 amino acids. Blastx analysis indicated this gene had a 84% identity withhomogentisate 1,2-dioxygenase of Lycopersicon esculentum and 81% with homogentisate1,2-dioxygenase of Arabidopsis thaliana(At5g54080);(3)two peroxidases (POD), one wasa 1489bp cDNA sequence obtained via 5'RACE technique, its open reading frame was816bp,and encoded a polypeptide containing 271 amino acids. Blastx analysis indicatedthis gene had 70% homologies with a peroxidase of Arabidopsis thaliana, so this gene wasdesignated as GhPOD1; the insert fragment of the other cDNA clone was 1355bp,its openreading frame was 999bp,and encoded a polypeptide containing 332 amino acids. Blastxanalysis indicated this gene had a 99% identity with peroxidase of Gossypium hirsutum Lreported (pod8, L08199) and their protein sequences were almost similar. We designatedthis gene as GhPOD2;(4)pectate lyase (PL), the insert fragment of the eDNA clone was1539bp,its open reading frame was 1236bp,and encoded a polypeptide containing 411 amino acids. Blastx analysis indicated this gene had an 85% identity with pectate lyase ofMalus×doraestica.So this gene was designated as GhPL (GenBank Accession number:DQ073046).
We predicted their signal peptide via signalP process.They all had typical signalpeptide except GhHGD.We also analyzed their conserved domain and phylogenetic tree.
Judging from their expression characters,GhCFE was preferential expressed in fibercells and did not express in root or stem but only had a weak expression in leaf, while itcould be detected in ovules and fiber cells of different developing period, especially inelongating fibers;GhHGD could be detected in every tissue,but in leaf and ovules itsexpression was weak and it was preferential expressed in fiber cells from 5DPA to 14 DPA;GhPOD2 just did not express in root and its expression was decreased after 14DPA in fiberdevelopmental stages;GhPL was specifically expressed in fiber cells, while it could not bedetected in root, stem or leaf;GhPOD1 was constitutive expression and detected in everytissue.
Southern blotting analysis shows that there are two copies of these five genes in thegenome of upland cotton; sub-genome A and sub-genome D contained each.
Sense expression vector containing 35S promoter and both sense and antisenseexpression vectors containing E6 promoter using pBI121 plasmid were constructed forGhCFE and GhPL; both sense and antisense expression vectors containing 35S promoterusing pBI121 plasmid were constructed for GhPOD2 and RNAi vector was constructed forGhPL.The work of transferring these recombined vectors into Gossypium hirsutum L. byAgrobactedum tumefaciens is ongoing.
GhCFE, GhHGD and GhPL were integrated into yeast expression vector (pREP5N),and electro-transformed into fission yeast S. pombe Q-01. The results showed that GhHGDleaded a significant change of length and length/width ratio; GhPL leaded a significantchange of width and length/width ratio; GhCFE had no significant effect on elongatingcells or thickening cell wall in the transformed yeast.
We have used the BC_1 mapping population derived from the hybridization between theupland cultivar TM-1 and the island cultivar Hai7124, further TM-1 as recurrent parent.GhCFE and GhPL were localized on the chromosome 18 and chromosome 3 respectively.
引文
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