苎麻UDPGDH基因cDNA的克隆及功能分析
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摘要
苎麻(Boehmeria nivea)在我国有着悠久的栽培历史，是一种重要的纤维作物。为达到纺织要求，必须经过脱胶过程将苎麻韧皮中以果胶、半纤维素、木质素等为代表的胶质成分去除，不仅提高了生产成本，也容易对环境造成显著污染。因此，研究苎麻半纤维素和果胶合成的分子机理，从分子水平改善纤维作物的胶质成分，具有非常重要的理论意义和应用价值。
前人的研究表明，尿苷二磷酸葡萄糖脱氢酶(UDPGDH)是半纤维素、果胶合成代谢途径中的关键酶之一。在本研究中，采用现代分子生物学技术，克隆了苎麻UDPGDH全长cDNA；运用植物转基因技术，实现了苎麻UDPGDH基因在异源植株中的表达，并分析了其功能；初步建立了苎麻离体培养体系和遗传转化技术，为苎麻分子育种的深入研究奠定了基础。
主要研究结果：
根据对已知的UDPGDH基因序列同源性分析，设计简并PCR引物，以由湘苎三号中提取的总RNA反转录所得的cDNA为材料，扩增得到苎麻UDPGDH基因片段，运用RACE技术，获得总长度为1837 bp的UDPGDH全长cDNA序列(Gene Bank No．EF178294)，其读码框编码的蛋白质含有480个氨基酸残基。
采用生物信息学方法，对所获得的苎麻UDPGDH全长cDNA以及其所编码的蛋白质进行分析，结果表明，该基因及其所编码的蛋白质与已知的多种植物UDPGDH具有高度的同源性，其蛋白质结构中含有UDPGDH所特有的功能性结构域、酶活性部位关键残基及修饰位点，初步说明该基因为苎麻的UDPGDH全长cDNA。
将所获得的基因克隆到质粒pET32和pMAL中，在大肠杆菌中表达获得该基因所编码的蛋白质，生物学活性研究表明，该蛋白质具有UDPGDH酶活性，从而证实所获得的基因序列为苎麻UDPGDH全长cDNA序列。
为了研究UDPGDH基因的表达和纤维形成的关系，运用RT-PCR技术对苎麻UDPGDH基因在苎麻不同组织中的表达进行了半定量分析。结果表明，在苎麻根、茎、叶、皮中均可以检测到UDPGDH基因的表达，其表达水平按茎、皮、叶、根的顺序呈下降趋势，与上述组织中纤维成分的相对含量呈正相关。
运用分子克隆的手段构建了苎麻UDPGDH基因的干扰、反义、正义表达载体，以农杆菌介导将其转化模式植物烟草(WS38)，PCR检测结果表明转化阳性率分别达到了100％、90％、70％，证实外源UDPGDH基因片段已经整合到烟草基因组中，酶活性分析同样证实了苎麻UDPGDH基因在转基因植物中的表达。初步的组织学分析表明，反义和干扰表达的转基因植株中，茎段与叶横切面细胞结构较为松散，基本组织细胞间隙增大，而在UDPGDH基因过量表达的转基因植株中则观察到相反的现象，进一步证实UDPGDH基因在苎麻半纤维素和果胶合成中所具有的重要作用。
以苎麻叶片为外植体，初步建立了苎麻的离体培养体系。湘苎三号形成优良愈伤组织的较优激素组合为：TDZ 0．3mg／L+2,4-D 0．01-0．03 mg/L，城步青麻为：TDZ 0．45 mg/L+2,4-D 0．03 mg/L、湘苎二号为：TDZ 0．15 mg/L+2,4-D 0．03 mg/L。湘苎三号诱导芽分化较优激素组合为TDZ 1 mg/L+2,4-D 0．1 mg/L，城步青麻品种为TDZ 0．5 mg/L+2,4-D 0．3 mg/L，湘苎二号为TDZ 0．75 mg/L+2,4-D0．3 mg/L。同时采用叶盘法并对苎麻进行了遗传转化，获得了较多的PCR检测阳性苎麻植株。这些工作将为下一步苎麻分子育种研究打下了基础。
Ramie (Boehmeria nivea) is one of the most important fiber crops in China. In textile industry, a pretreatment procedure of degumming is always required to get rid of the non-cellulose materials such as pectin, hemicellulose, lignin, et al. This pretreatment procedure, not only increases the cost, but also polutes the environment. Therefore, it is of great importance to investigate the molecular mechanism(s) of pectin, hemicellulose biosynthesis and improve the molecular composition of the fiber crops.
Previous research results suggested that UDP-glucose dehydrogenase (UDPGDH) plays a critical role in the regulation of the biosynthesis of pectin and hemicellulose. In this study, the full length ramie UDPGDH cDNA was cloned. The cloned ramie cDNA was expressed in exogenous plants using transgenic technologies and the functions were analyzed. A in vitro ramie culture system was established whereas a genetic transformation technique was developed. These results will greatly facilitate the further study of the molecular breeding of ramie.
The major research results presented in this study are briefly summarized as following:
Based on the homology comparision of the known UDPGDH genes, a pair of degenerate PCR primers were designed. Using the cDNA synthesis from the total RNA isolated from ramie strain Xiangzhu No. 3, a fragment of ramie UDPGDH gene was amplified. The RACE procedure was carried out to obtain the full length ramie cDNA, the resulted cDNA contains 1837 bp (Gene Bank No. EF178294), which contains an open reading frame that encodes a protein of 480 amino acid residues.
Bioinformatic analysis was performed on the obtained ramie full length UDPGDH cDNA and the deduced protein. The results demonstrated that the gene is highly homologous to the known plant UDPGDH genes.. The deduced protein contains all the functional domains of UDPGDH, the critical amino acid residues of the enzyme activity core and important modification sites are also presented in the deduced protein.
The obtained ramie UDPGDH gene was cloned inton vectors pET32 and pMAL and expressed in E. coli. The expressed protein clearly demonstrated the UDPGDH enzyme activity in vitro. In combination with the bioinformatic analysis, it was concluded that the full length ramie UDPGDH cDNA was successfully cloned in this study.
To investigate the relationship of the expression level of UDPGDH gene and the formation of fiber, the expression levels of UDPGDH in different ramie tissues were examined using semi-quantitative RT-PCR. The results suggested that in ramie, UDPGDH gene is expressed in stem, phloem, leaf, and root. The expression level of UDPGDH gene in ramie decreases in different tissues in the sequence decribed above.
The core region of UDPGDH was cloned into the binary expression vectors (pFGC5941, pWM101) and transformed into tobacco via leaf disc infection with Agrobacterium tumefaciens system. The result demonstrated that UDPGDH gene was successfully integrated into tobacco genome. Tissue analysis indicated that three expression vectors in the tobacco may effect hemicelluloses and pectin synthesis. The cells in cross-section of leaf and stem become swollen and intercellular space of the ground tissue increass when the expression of UDPGDH gene was inhibited in tobacco. When UDPGDH gene was over expresed, opposite physiological changes were observed. These results further suggested that UDPGDH gene may play an important role in the synthesis of pectin and hemicelluloses in ramie.
Using ramie leaves as explant, a in vitro culture system of ramie was established. Xiang Zhu No. 3 in MS supplemented with TDZ 0.3 mg/L + 2,4-D 0.01-0.03 mg/L, Cheng Bu Qing Ma in MS with TDZ 0.45 mg/L + 2,4-D 0.03 mg/L, Xiang Zhu No. 2 in MS with TDZ 0.15 mg/L + 2,4-D 0.03 mg/L, they formed the fine callus on the hormone combinations. Induced high frequency shoots of Xiang Zhu No. 3 in MS with TDZ 1 mg/L + 2,4-D 0.1 mg/L, and the Cheng Bu Qing Ma in MS with TDZ 0.5 mg/L + 2,4-D 0.3 mg/L, Zhu Xiang No. 2 in MS with TDZ 0.75 mg/L + 2,4-D0.3 mg/L. PCR positive plants were obtained with leaves disk transformation. The further molecular breeding research of ramie might be carried out based on the results presented in this work.
前人的研究表明，尿苷二磷酸葡萄糖脱氢酶(UDPGDH)是半纤维素、果胶合成代谢途径中的关键酶之一。在本研究中，采用现代分子生物学技术，克隆了苎麻UDPGDH全长cDNA；运用植物转基因技术，实现了苎麻UDPGDH基因在异源植株中的表达，并分析了其功能；初步建立了苎麻离体培养体系和遗传转化技术，为苎麻分子育种的深入研究奠定了基础。
主要研究结果：
根据对已知的UDPGDH基因序列同源性分析，设计简并PCR引物，以由湘苎三号中提取的总RNA反转录所得的cDNA为材料，扩增得到苎麻UDPGDH基因片段，运用RACE技术，获得总长度为1837 bp的UDPGDH全长cDNA序列(Gene Bank No．EF178294)，其读码框编码的蛋白质含有480个氨基酸残基。
采用生物信息学方法，对所获得的苎麻UDPGDH全长cDNA以及其所编码的蛋白质进行分析，结果表明，该基因及其所编码的蛋白质与已知的多种植物UDPGDH具有高度的同源性，其蛋白质结构中含有UDPGDH所特有的功能性结构域、酶活性部位关键残基及修饰位点，初步说明该基因为苎麻的UDPGDH全长cDNA。
将所获得的基因克隆到质粒pET32和pMAL中，在大肠杆菌中表达获得该基因所编码的蛋白质，生物学活性研究表明，该蛋白质具有UDPGDH酶活性，从而证实所获得的基因序列为苎麻UDPGDH全长cDNA序列。
为了研究UDPGDH基因的表达和纤维形成的关系，运用RT-PCR技术对苎麻UDPGDH基因在苎麻不同组织中的表达进行了半定量分析。结果表明，在苎麻根、茎、叶、皮中均可以检测到UDPGDH基因的表达，其表达水平按茎、皮、叶、根的顺序呈下降趋势，与上述组织中纤维成分的相对含量呈正相关。
运用分子克隆的手段构建了苎麻UDPGDH基因的干扰、反义、正义表达载体，以农杆菌介导将其转化模式植物烟草(WS38)，PCR检测结果表明转化阳性率分别达到了100％、90％、70％，证实外源UDPGDH基因片段已经整合到烟草基因组中，酶活性分析同样证实了苎麻UDPGDH基因在转基因植物中的表达。初步的组织学分析表明，反义和干扰表达的转基因植株中，茎段与叶横切面细胞结构较为松散，基本组织细胞间隙增大，而在UDPGDH基因过量表达的转基因植株中则观察到相反的现象，进一步证实UDPGDH基因在苎麻半纤维素和果胶合成中所具有的重要作用。
以苎麻叶片为外植体，初步建立了苎麻的离体培养体系。湘苎三号形成优良愈伤组织的较优激素组合为：TDZ 0．3mg／L+2,4-D 0．01-0．03 mg/L，城步青麻为：TDZ 0．45 mg/L+2,4-D 0．03 mg/L、湘苎二号为：TDZ 0．15 mg/L+2,4-D 0．03 mg/L。湘苎三号诱导芽分化较优激素组合为TDZ 1 mg/L+2,4-D 0．1 mg/L，城步青麻品种为TDZ 0．5 mg/L+2,4-D 0．3 mg/L，湘苎二号为TDZ 0．75 mg/L+2,4-D0．3 mg/L。同时采用叶盘法并对苎麻进行了遗传转化，获得了较多的PCR检测阳性苎麻植株。这些工作将为下一步苎麻分子育种研究打下了基础。
Ramie (Boehmeria nivea) is one of the most important fiber crops in China. In textile industry, a pretreatment procedure of degumming is always required to get rid of the non-cellulose materials such as pectin, hemicellulose, lignin, et al. This pretreatment procedure, not only increases the cost, but also polutes the environment. Therefore, it is of great importance to investigate the molecular mechanism(s) of pectin, hemicellulose biosynthesis and improve the molecular composition of the fiber crops.
Previous research results suggested that UDP-glucose dehydrogenase (UDPGDH) plays a critical role in the regulation of the biosynthesis of pectin and hemicellulose. In this study, the full length ramie UDPGDH cDNA was cloned. The cloned ramie cDNA was expressed in exogenous plants using transgenic technologies and the functions were analyzed. A in vitro ramie culture system was established whereas a genetic transformation technique was developed. These results will greatly facilitate the further study of the molecular breeding of ramie.
The major research results presented in this study are briefly summarized as following:
Based on the homology comparision of the known UDPGDH genes, a pair of degenerate PCR primers were designed. Using the cDNA synthesis from the total RNA isolated from ramie strain Xiangzhu No. 3, a fragment of ramie UDPGDH gene was amplified. The RACE procedure was carried out to obtain the full length ramie cDNA, the resulted cDNA contains 1837 bp (Gene Bank No. EF178294), which contains an open reading frame that encodes a protein of 480 amino acid residues.
Bioinformatic analysis was performed on the obtained ramie full length UDPGDH cDNA and the deduced protein. The results demonstrated that the gene is highly homologous to the known plant UDPGDH genes.. The deduced protein contains all the functional domains of UDPGDH, the critical amino acid residues of the enzyme activity core and important modification sites are also presented in the deduced protein.
The obtained ramie UDPGDH gene was cloned inton vectors pET32 and pMAL and expressed in E. coli. The expressed protein clearly demonstrated the UDPGDH enzyme activity in vitro. In combination with the bioinformatic analysis, it was concluded that the full length ramie UDPGDH cDNA was successfully cloned in this study.
To investigate the relationship of the expression level of UDPGDH gene and the formation of fiber, the expression levels of UDPGDH in different ramie tissues were examined using semi-quantitative RT-PCR. The results suggested that in ramie, UDPGDH gene is expressed in stem, phloem, leaf, and root. The expression level of UDPGDH gene in ramie decreases in different tissues in the sequence decribed above.
The core region of UDPGDH was cloned into the binary expression vectors (pFGC5941, pWM101) and transformed into tobacco via leaf disc infection with Agrobacterium tumefaciens system. The result demonstrated that UDPGDH gene was successfully integrated into tobacco genome. Tissue analysis indicated that three expression vectors in the tobacco may effect hemicelluloses and pectin synthesis. The cells in cross-section of leaf and stem become swollen and intercellular space of the ground tissue increass when the expression of UDPGDH gene was inhibited in tobacco. When UDPGDH gene was over expresed, opposite physiological changes were observed. These results further suggested that UDPGDH gene may play an important role in the synthesis of pectin and hemicelluloses in ramie.
Using ramie leaves as explant, a in vitro culture system of ramie was established. Xiang Zhu No. 3 in MS supplemented with TDZ 0.3 mg/L + 2,4-D 0.01-0.03 mg/L, Cheng Bu Qing Ma in MS with TDZ 0.45 mg/L + 2,4-D 0.03 mg/L, Xiang Zhu No. 2 in MS with TDZ 0.15 mg/L + 2,4-D 0.03 mg/L, they formed the fine callus on the hormone combinations. Induced high frequency shoots of Xiang Zhu No. 3 in MS with TDZ 1 mg/L + 2,4-D 0.1 mg/L, and the Cheng Bu Qing Ma in MS with TDZ 0.5 mg/L + 2,4-D 0.3 mg/L, Zhu Xiang No. 2 in MS with TDZ 0.75 mg/L + 2,4-D0.3 mg/L. PCR positive plants were obtained with leaves disk transformation. The further molecular breeding research of ramie might be carried out based on the results presented in this work.
引文
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