大豆类受体蛋白激酶基因(rlpk2)RNAi双元表达载体的构建及其转基因

植物类受体蛋白激酶(plant receptor-like kinases RLKs)以其特有的结构在植物的生长、发育和防御等多种生理生化过程中发挥着重要的作用。利用RNA干扰技术(RNA interference RNAi)来研究RLKs的功能已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理,以大豆类受体蛋白激酶基因rlpk2为靶基因,在rlpk2-cDNA序列3'端选择312bp作为构建RNAi的序列,借助中间克隆载体,经过三次亚克隆,最后形成含rlpk2-RNAi表达盒的双元表达载体pART27-R2,并转入农杆菌LBA4404。采用农杆菌介导大豆子叶节转化方法,共获得了三株转基因植株。转基因植株 RT-PCR分析表明rlpk2基因已被成功敲减(knock-down),并且发现敲减大豆叶片中的rlpk2基因表达明显改善大豆叶片的光合能力,结合前期研究结果,表明rlpk2基因可能在维持叶绿体的结构及保护叶绿体膜系统的完整性方面起负调节作用。

CONSTRUCTION OF RNAi BINARY VECTOR OF SOYBEAN RECEPTOR-LIKE KINASE GENE (rlpk2) AND ITS SOYBEAN TRANSFORMATION

Plant receptor-like kinases (RLKs) play important roles in plant growth, development and responses to environmental stress. RNA interference (RNAi) is a powerful tool to study functions of RLKs. In this study, a 312bp 3'end cDNA fragment of the soybean receptor-like ki-nase gene rlpk2 was chosen to construct the rlpk2-RNAi expression cassette. The binary vector pART27-R2 harboring rlpk2 -RNAi expression cassette was constructed through three times sub-cloning via mid-clone vector and then transformed into Agrobacterium LBA4404. Three independent transgenic plants were obtained by Agrobactium -mediated soybean cotyledon transformation method. RT-PCR analysis showed that rlpk2 gene was successfully knocked down in all transgenic plants. It was found that photosynthesis activities of rlpk2-RNAi transgenic leaves were much improved, suggesting rlpk2 may function as a negative regulator of soybean leaf functions and/or chloroplast structure.