摘要
PA1b是一种来源于豌豆种子的生物活性多肽,具有非常重要的生物杀虫活性,本研究以豌豆幼苗为材料,利用RT-PCR技术克隆抗虫基因PA1b,通过酶切连接技术构建了适用于豌豆植物的瞬时表达载体pCAPE2-PA1b,转化农杆菌GV3101后,利用真空侵染技术将含有PA1b及报告基因GFP的农杆菌分别接种发芽的豌豆种子,在0.08 MPa真空条件下侵染3~5 min后播种豌豆种子,预期在豌豆幼苗中瞬时表达抗虫蛋白PA1b,幼苗出土后利用手提式紫外灯跟踪观察,在报告基因GFP表达高峰期(幼苗出土后5~7 d)提取豌豆幼苗可溶性总蛋白,利用His标签抗体检测豌豆抗虫基因PA1b的表达,实验结果表明抗虫基因PA1b在豌豆幼苗中成功表达,为进一步分析PA1b抗虫机制提供基础。
PA1 b is a biological active polypeptide derived from pea seeds and has important biological insecticidal activity. In this study, peaseedlings were used as materials to clone the insect-resistant gene PA1 b by RT-PCR. The transient expression vector pCAPE2-PA1 b was constructed by enzyme digestion and ligation method for pea plants. After transformation of Agrobacterium GV3101, Agrobacterium containing PA1 b and reporter GFP were inoculated into the pea seeds respectively with vacuum infection technique. Pea seeds were sowed after infiltration for 3-5 minutes under vacuum conditions of 0.08 MPa. The insect-resistant protein PA1 b was transiently expressed in the seedlings, the seedlings were observed with hand-held UV lamps. And the total soluble protein of pea seedlings was extracted at the peak of the reporter gene GFP expression. The expression of the insect-resistant gene was detected by His-tag antibody. The experimental results showed that the insect-resistant gene PA1 b was successfully expressed in pea seedlings, this may help to analysis the mechanism about insect resistance of PA1 b protein.
作者
范亚军
赵轩
刘琼馨
盛晓珏
Fan Yajun;Zhao Xuan;Liu Qiongxin;Sheng Xiaoyu(College of Life Science,Changchun Normal University,Changchun,130024)
出处
《分子植物育种》
CAS
CSCD
北大核心
2019年第20期6680-6684,共5页
Molecular Plant Breeding
基金
国家自然科学基金项目(No.31350003)资助
关键词
抗虫蛋白PA1b
豌豆
瞬时真空侵染
蛋白杂交
Insect resistance protein PA1b
Pea
Transient vacuum infection
Protein hybridization
