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木薯MeGWD3基因克隆及植物表达载体构建
作者: 付莉莉 韩冰莹 谭德冠 孙雪飘 张家明* 
单位: (中国热带农业科学院 热带生物技术研究所 海口 571101) 
关键词: 木薯 葡聚糖水合双激酶 克隆 植物表达载体 
分类号:S533
出版年,卷(期):页码:2016,47(7):1057-1063
摘要:

【目的】克隆木薯葡聚糖水合双激酶(MeGWD3)基因并构建其植物表达载体,为开展木薯MeGWD3基因调控及功能研究提供参考。【方法】采用RT-PCR从木薯华南124(SC124)中克隆MeGWD3基因,对其进行序列分析,并将其连接至改造的pCAMBIA2300载体,构建植物表达载体pCAMBIA2300-MeGWD3。【结果】MeGWD3基因全长3522 bp,编码1173个氨基酸。经序列比对,发现MeGWD3基因序列与木薯Phytozome数据库中公布的GWD基因(cassava4.1_000497m)只存在2个碱基差异,同源性高达99.94%;与NCBI数据库中收录的木薯GWD基因(JN618460)同源性高达99.00%,与蓖麻GWD基因(XM_002518566)同源性达86.00%;成功构建植物表达载体pCAMBIA2300-MeGWD3。【结论】成功构建的植物表达载体pCAMBIA2300可用于MeGWD3基因功能及木薯淀粉代谢研究。

【Objective】The present experiment was conducted to clone cassava(Manihot esculenta) glucan water dikinase(MeGWD3) and construct its plant expression vector, in order to provide references for further studying function and regulation of MeGWD3 gene in cassava. 【Method】MeGWD3 gene was cloned from cassava variety South China 124(SC124) by RT-PCR, and its sequence was analyzed. Then MeGWD3 gene was ligated into pCAMBIA2300 vector, so as to construct plant expression vector pCAMBIA2300-MeGWD3. 【Result】The full-length cDNA of MeGWD3 was 3522 bp, encoding 1173 amino acids. The sequence alignment results showed that, MeGWD3 gene shared homology of 99.94% with its reference gene(cassava4.1_000497m) from cassava Phytozome database, only with two base differences, and shared homologies of 99.00% and 86.00% with GWD genes of Manihot esculenta Crantz and Ricinus communis, respectively. Furthermore, the plant expression vector pCAMBIA2300-MeGWD3 was constructed successfully. 【Conclusion】The constructed plant expression vector pCAMBIA2300-MeGWD3 can be used to research MeGWD3 gene function and cassava starch metabolism.

基金项目:
海南省自然科学基金项目(314123)
作者简介:
*为通讯作者,张家明(1966-),研究员,主要从事植物分子生物学研究工作,E-mail:zhangjiaming@itbb.org.cn。付莉莉(1982-),主要从事植物分子生物学研究工作,E-mail:fulili@itbb.org.cn
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