棉花咖啡酸-O-甲基转移酶基因（GhCOMT）的克隆、表达及功能分析
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摘要
棉花纤维是从胚珠外表皮细胞分化而来的单细胞,是纺织工业的重要原料,具有重要的经济价值。棉花体内通过苯丙氨酸途径进入木质素特异途径合成木质素,这条代谢途径涉及许多酶的参与,其中咖啡酸-O-甲基转移酶(caffeic acid-O-methyltransferase, COMT)是木质素特异途径中的一个关键酶,可催化咖啡酸,5-羟基松柏醛和5-羟基松柏醇甲基化分别生成阿魏酸、芥子醛和芥子醇,参与S-木质素的合成。本研究从以下几个方面对咖啡酸-O-甲基转移酶进行分析：
(1)本研究以陆地棉徐州142为材料,克隆了3个木质素合成关键酶基因GhCOMT1(GenBank登录号为FJ479708)、GhCOMT2(GenBank登录号为FJ479709)和GhCOMT3(GenBank登录号为FJ848869)并将GhCOMT1/2/3基因构建到原核表达载体pET-28a中,转化至大肠杆菌BL21(DE3)中。用0.2 mmol/L IPTG诱导融合蛋白表达,再经过蛋白标记亲和层析柱(His TrapTM HP)纯化分别得到GhCOMT1/2/3融合蛋白,用SDS-PAGE和Western blotting检测纯化效果,鉴定表达产物,目的蛋白相对分子量分别为39.748 kD,40.062 kD,39.119 kD,检测结果与预期一致。
(2)本研究构建了由棉纤维组织特异性启动子E6驱动的GhCOMT1基因正义、反义表达载体；同时克隆了GhCOMT1基因的一段417 bp高度保守区序列,构建了GhCOMT1基因的RNAi干扰载体,并将上述载体转入农杆菌菌株LBA4404中。经测序表明,目的基因成功整合至表达质粒中。
(3)构建GhCOMT1基因的瞬时表达载体,以pMD-T-GhCOMT1质粒为模板,获得GhCOMTl基因目的片段,然后将其连接到瞬时表达载体pRTL2-GUS/NIa中,获得35S启动子驱动的pGUS-GhCOMT1融合表达载体,将pGUS-GhCOMT1转化到棉花胚珠中进行培养,经检测表明构建的瞬时表达载体可在棉纤维细胞中高效表达。并利用透射电镜观察了培养棉纤维细胞横截面,结果显示pGUS-GhCOMT1基因对棉纤维细胞壁的增厚有显著的影响。
通过对棉花咖啡酸-O-甲基转移酶基因(GhCOMT)初步研究的结果显示,GhCOMT基因在棉纤维发育过程中起着十分重要的作用,从而对从分子水平改良纤维品质提供新的候选目的基因,并为研究棉花纤维发育调控的分子机理提供理论基础。
Cotton fiber is a single and highly elongated epidermal cell of the outer integument of the ovule. Lignin in cotton is synthesized via the lignin specific biosynthesis pathway which is just downstream of the common phenylpropanoid pathway, and many enzymes are involved in this process. Caffeic acid-3-O-methyltransferase (COMT) is a key enzyme of lignin specific pathway and catalyzes caffeic acid,5-Hydrocy-coniferyl aldehyde and 5-Hydrocy-coniferyl-coniferyl alcohol to produce ferulaic acid sinapyl aldehyde and sinapyl alcohol. It participates into the pathway of synthesizing S-lignin. Here we examined the role of GhCOMT in cotton development.
1. In this study, full-length cDNAs of a key enzyme genes GhCOMT1/2/3(GenBank accession no.FJ479708, FJ479709, FJ848869), related to lignin metabolism in cotton (Gossypium hirsutum, cv. Xuzhou 142.) were isolated. And the GhCOMT1/2/3 genes were inserted into the pET-28a vector to construct recombinant vector pET-28a-GhCOMT1/2/3, which were then transformed into E.coli BL21(DE3) strain. After induction and expression, the target proteins were purified by using His TrapTM HP to get degenerated protein, SDS-PAGE and Western blotting detection were then employed to identify target proteins which had the molecular mass about 39.748kD,40.062 kD, and 39.119 kD respectively, the results were consistent with expectation.
2. To investigate the function of GhCOMT1 gene, in the present study, we constructed the sense and anti-sense expression vectors of GhCOMT1 gene, and the coding region of the gene was placed under the E6 promoter in either sense or anti-sense orientation. In addition, a 450 bp fragment of GhCOMT1 was also cloned, and this fragment was inserted into plant vector to construct RNAi expression vector of GhCOMT1. These constructs were then transformed into Agrobacterium LBA4404. The sequencing result of different recombinant expression vectors showed that the GhCOMT1 gene has been correctly constructed.
3. In this study transient expression vector of cotton GhCOMT1 gene was constructed. Firstly, the GhCOMT1 gene cDNA was obtained from pGEM-T-GhCOMT1 by PCR, then inserted into transient expression vector pRTL2-GUS/NIa. The transient expression vector pGUS-GhCOMTl is driven by 35S promoter with GUS reporter gene and the target gene, which expressed simultaneously and formed a fusion protein. Secondly, the vector pGUS-GhCOMT1 was transformed into cotton ovule by using PDS-1000/He biolistic particle delivery system. The results indicated that the transient expression vector pGUS-GhCOMT1 could be expressed efficiently in the epidermal cells of cotton fibre.
Based on this data, we suggest that GhCOMT may play an important role in the morphogenesis and secondary wall thickening of cotton by positively/negatively regulating the structure of cell wall in growing course. Our study presents important experimental evidence for the function of lignin and provides gene candidates for genetic improvement of cotton fiber quality.
(1)本研究以陆地棉徐州142为材料,克隆了3个木质素合成关键酶基因GhCOMT1(GenBank登录号为FJ479708)、GhCOMT2(GenBank登录号为FJ479709)和GhCOMT3(GenBank登录号为FJ848869)并将GhCOMT1/2/3基因构建到原核表达载体pET-28a中,转化至大肠杆菌BL21(DE3)中。用0.2 mmol/L IPTG诱导融合蛋白表达,再经过蛋白标记亲和层析柱(His TrapTM HP)纯化分别得到GhCOMT1/2/3融合蛋白,用SDS-PAGE和Western blotting检测纯化效果,鉴定表达产物,目的蛋白相对分子量分别为39.748 kD,40.062 kD,39.119 kD,检测结果与预期一致。
(2)本研究构建了由棉纤维组织特异性启动子E6驱动的GhCOMT1基因正义、反义表达载体；同时克隆了GhCOMT1基因的一段417 bp高度保守区序列,构建了GhCOMT1基因的RNAi干扰载体,并将上述载体转入农杆菌菌株LBA4404中。经测序表明,目的基因成功整合至表达质粒中。
(3)构建GhCOMT1基因的瞬时表达载体,以pMD-T-GhCOMT1质粒为模板,获得GhCOMTl基因目的片段,然后将其连接到瞬时表达载体pRTL2-GUS/NIa中,获得35S启动子驱动的pGUS-GhCOMT1融合表达载体,将pGUS-GhCOMT1转化到棉花胚珠中进行培养,经检测表明构建的瞬时表达载体可在棉纤维细胞中高效表达。并利用透射电镜观察了培养棉纤维细胞横截面,结果显示pGUS-GhCOMT1基因对棉纤维细胞壁的增厚有显著的影响。
通过对棉花咖啡酸-O-甲基转移酶基因(GhCOMT)初步研究的结果显示,GhCOMT基因在棉纤维发育过程中起着十分重要的作用,从而对从分子水平改良纤维品质提供新的候选目的基因,并为研究棉花纤维发育调控的分子机理提供理论基础。
Cotton fiber is a single and highly elongated epidermal cell of the outer integument of the ovule. Lignin in cotton is synthesized via the lignin specific biosynthesis pathway which is just downstream of the common phenylpropanoid pathway, and many enzymes are involved in this process. Caffeic acid-3-O-methyltransferase (COMT) is a key enzyme of lignin specific pathway and catalyzes caffeic acid,5-Hydrocy-coniferyl aldehyde and 5-Hydrocy-coniferyl-coniferyl alcohol to produce ferulaic acid sinapyl aldehyde and sinapyl alcohol. It participates into the pathway of synthesizing S-lignin. Here we examined the role of GhCOMT in cotton development.
1. In this study, full-length cDNAs of a key enzyme genes GhCOMT1/2/3(GenBank accession no.FJ479708, FJ479709, FJ848869), related to lignin metabolism in cotton (Gossypium hirsutum, cv. Xuzhou 142.) were isolated. And the GhCOMT1/2/3 genes were inserted into the pET-28a vector to construct recombinant vector pET-28a-GhCOMT1/2/3, which were then transformed into E.coli BL21(DE3) strain. After induction and expression, the target proteins were purified by using His TrapTM HP to get degenerated protein, SDS-PAGE and Western blotting detection were then employed to identify target proteins which had the molecular mass about 39.748kD,40.062 kD, and 39.119 kD respectively, the results were consistent with expectation.
2. To investigate the function of GhCOMT1 gene, in the present study, we constructed the sense and anti-sense expression vectors of GhCOMT1 gene, and the coding region of the gene was placed under the E6 promoter in either sense or anti-sense orientation. In addition, a 450 bp fragment of GhCOMT1 was also cloned, and this fragment was inserted into plant vector to construct RNAi expression vector of GhCOMT1. These constructs were then transformed into Agrobacterium LBA4404. The sequencing result of different recombinant expression vectors showed that the GhCOMT1 gene has been correctly constructed.
3. In this study transient expression vector of cotton GhCOMT1 gene was constructed. Firstly, the GhCOMT1 gene cDNA was obtained from pGEM-T-GhCOMT1 by PCR, then inserted into transient expression vector pRTL2-GUS/NIa. The transient expression vector pGUS-GhCOMTl is driven by 35S promoter with GUS reporter gene and the target gene, which expressed simultaneously and formed a fusion protein. Secondly, the vector pGUS-GhCOMT1 was transformed into cotton ovule by using PDS-1000/He biolistic particle delivery system. The results indicated that the transient expression vector pGUS-GhCOMT1 could be expressed efficiently in the epidermal cells of cotton fibre.
Based on this data, we suggest that GhCOMT may play an important role in the morphogenesis and secondary wall thickening of cotton by positively/negatively regulating the structure of cell wall in growing course. Our study presents important experimental evidence for the function of lignin and provides gene candidates for genetic improvement of cotton fiber quality.
引文
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