大豆Kti基因ihpRNA种子特异性表达载体的构建及转化

Construction and transformation of seed-specific expression RNAi vector of Kunitz tripsin inhibitor gene from soybean

大豆Kunitz型胰蛋白酶抑制剂(trypsin inhibitor,Kti)是大豆品质的主要营养抑制因子之一,培育低Kti含量的大豆品种是提升大豆品质的关键。本研究采用RT-PCR技术克隆得到大豆Kti基因的核心保守序列,并构建成种子特异性表达的、具有Kti基因反向重复结构的RNAi载体,利用农杆菌介导法侵染大豆品种吉林30子叶节,通过潮霉素筛选和PCR检测,获得4株转基因阳性植株。RT-PCR检测表明,构建的种子特异性RNAi载体可以在转基因植株的种子中特异表达,且Kti基因表达量均有降低;通过胰蛋白酶抑制剂活性检测发现,转基因大豆籽粒中胰蛋白酶抑制剂活性平均降低了77.5%。为进一步评价RNAi技术对大豆Kti基因的表达调控效果奠定良好基础。

Kunitz trypsin inhibitor（Kti） from soybean is one of the main inhibitive factors of nutrition. Soybean breeding with low content of trypsin inhibitor was the key to promote the soybean quality. In this study,we cloned the consensus sequence of Kti gene in soybean seeds by RT-PCR. Then,the RNAi vector for silenced Kti gene was reconstructed and transformed to soybean（Jilin 30） by Agrobacterium tumefaciens-mediated method.Four positive transgenic plants were obtained by hygromycin screening and PCR identification. RT-PCR analyses revealed that Kti mRNA was specifically expressed at a low level in transgenic soybean seeds,and the activity of Kunitz trypsin inhibitor was decreased about 77. 5%. It provided a foundation for further evaluation of RNAi technology for soybean Kti gene regulation.