生物技术通报 ›› 2015, Vol. 31 ›› Issue (8): 82-87.doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.012

• 研究报告 • 上一篇    下一篇

大豆基因GmGW2的克隆与转化

熊鹤雯1, 吴志慧1, 段思宇2, 谢皓1, 秦玉涛2, 郭蓓2, 3   

  1. 1.北京农学院植物科学技术学院,北京 102206; 2.北京农学院生物科学与工程学院,北京 102206; 3. 农业部都市农业 (北方) 重点开放实验室,北京 102206
  • 收稿日期:2014-11-05 出版日期:2015-08-21 发布日期:2015-08-22
  • 作者简介:熊鹤雯,女,硕士研究生,研究方向:大豆分子遗传育种;E-mail:344356549@qq.com

Cloning and Transformation of Gene GmGW2 in Soybean

Xiong Hewen1, Wu Zhihui1, Duan Siyu2, Xie Hao1, Qin Yutao2, Guo Bei2, 3   

  1. 1. College of Plant Science and Technology,Beijing 102206; 2. College of Biological Science and Engineering,Beijing University of Agriculture,Beijing 102206; 3. Key Laboratory of Urban Agriculture(North)of Ministry of Agriculture China,Beijing 102206
  • Received:2014-11-05 Published:2015-08-21 Online:2015-08-22

摘要: GW2 编码一个环型E3 泛素连接酶,位于细胞质中,通过将其底物锚定到蛋白酶体进行降解,从而负调节细胞的分裂。之前有文献报道GW2位点突变可导致水稻籽粒大小的改变。根据水稻中GW2基因序列,在GenBank中找到与之同源的大豆基因序列GmGW2,克隆目的片段并进行了生物信息学分析。构建转化载体pCAMBIA1300∷GmGW2∷EYFP,并对大豆品种中黄10进行转化。利用含有重组载体的农杆菌EHA105侵染萌发24 h的中黄10幼胚。当外植体长出3片复叶后,提取基因组DNA,对报告基因EYFP进行PCR检测。得到的阳性株在激光共聚焦显微镜下观察,EYFP蛋白含量显著增加,实验结果显示目的基因已转化成功。序列分析表明GW2基因与苜蓿,鹰嘴豆中的GW2基因具有较高的同源性。

关键词: GmGW2, 大豆, 系统进化树, 遗传转化, EYFP

Abstract: GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is located in cytoplasm. It is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers. It was reported that the mutation of GW2 lead to change the size of rice seeds. According to rice GW2 gene sequence, blast in the NCBI, we found a soybean GW2 gene which is homologous. We cloned the target gene and carried on bioinformatics analysis, then we constructed a over-expression vector pCAMBIA1300∷GmGW2∷EYFP, and transformed into soybean named zhonghuang10 by Agrobacterium-mediated method. Seeds were germinated on sterile moistened paper towels in Petri dishes for 24 h then infected the immature embryo by Agrobacterium EHA105 which obtained recombining vector. When the explants have 3 compound leaves, we extract the genome DNA and PCR identified the report gene EYFP. The content of EYFP is enhanced in PCR positive plant was observed by LSCM. It is demonstrated the transformation of target gene is successful. The sequence analysis showed that the target gene have higher homology with those of Alfalfa and Gram.

Key words: GmGW2, soybean, phylogenetic tree, transformation, EYFP