抗虫基因Cry8-like在大豆中的遗传转化及抗虫性初步鉴定

Transformation of Cry8-like gene and analysis of insect resistance in soybean(Glycine max)

利用无缝克隆技术把抗虫基因Cry8-like重组到pCAMBIA3301载体上,成功构建了含有抗草铵膦筛选标记的植物表达载体pCAMBIA3301-RSP-Cry8-like-nos。将该载体通过农杆菌介导法转入两个受体大豆品种吉农28和吉农42中,经PCR检测,吉农28和吉农42分别获得了17株和13株T_1阳性植株,36株和25株T_2阳性植株。T_2转基因植株Southern杂交结果显示,目的基因以单拷贝的形式整合到大豆基因组中。荧光定量PCR检测结果表明,Cry8-like在转基因植株幼根得到了表达,而非转化受体对照未检测到荧光信号。室内抗暗黑鳃金龟幼虫鉴定结果表明,转基因植株提高了抗暗黑鳃金龟幼虫的能力。

Cry8-like gene was recombined into pCAMBIA3301 vector and a root-specific plant expression vector, and pCAMBIA3301-RSP-Cry8-like-nos with glufosinate resistant marker was constructed successfully by Seamless Assembly Cloning. Two different soybean varieties ？Jinong 28？ and ？Jinong 42？ were transformed by Agrobacterium-mediated genetic transformation. Detection of PCR showed that 17 transformed plants in T1 generation of Jinong 28 and 13 of Jinong 42 were obtained, and 36 transformed plants in T2 generation of Jinoing 28 and 25 of Jinong 42 were then produced. Southern blotting of T2 generation indicated that target gene was integrated into the genome of oybean with single copy. Detection of the transcription of Cry8-like by using quantitative real-time PCR （qRT-PCR） showed that Cry8-like expressed in root of transformed plants, while fluorescence signal was not detected in non-transformed control. The results of insect resistance to Holotrichia parallela larvae in laboratory showed that the ability of insect resistance was improved in transgenic plants.