异砧嫁接转基因杨树中外源Bt-Cry1Ac蛋白的运输及抗虫性检测  

Transportation of the Foreign Bt-Cry1Ac Protein and Insect Resistance Detection in Transgenic Poplar(Populus spp.) with Different Anvil Grafting

作　　者：陈兴浩 张军 李政 于晓跃 张德建[2] 杨敏生 CHEN Xing-Hao;ZHANG Jun;LI Zheng;YU Xiao-Yue;ZHANG De-Jian;YANG Min-Sheng(Hebei Key Laboratory for Tree Genetic Resources and Forest Protection / College of Forestry Science, Hebei Agricultural University, Baoding 071000, China;College of Life Science, Inner Mongolia University, Hohhot 010021, China)

机构地区：[1]河北农业大学林学院/河北省林木种质资源与森林保护重点实验室,保定071000 [2]内蒙古大学生命科学学院,呼和浩特010021

出　　处：《农业生物技术学报》2019年第5期836-843,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31660211);河北省基础研究计划项目(No.18966801D)

摘　　要：随着我国杨树(Populus spp.)的广泛种植,杨树的虫害问题也随之而来。如今,化学杀虫剂的应用不但不能有效地降低害虫对杨树的危害,同时还严重地破坏了生态环境。因此,通过转基因杨树与非转基因杨树嫁接繁殖的方式来提高杨树自身抗虫能力具有现实可行性。为了解嫁接转基因杨树生态安全性,本研究以杂种毛白杨和转Bt-Cry1Ac基因欧美杨107杨(Populus×euramericana cv.'Neva')抗虫株系Hb1互为接穗和砧木进行嫁接,对Cry1Ac基因的mRNA和蛋白是否在接穗和砧木之间运输以及嫁接苗叶片对美国白蛾(Hyphantria cunea)的抗虫性进行了探究。逆转录PCR (reverse transcription-PCR, RT-PCR)检测和第3代数字PCR结果表明:以杂种毛白杨为接穗或砧木的嫩枝和嫩叶中均没有检测到Cry1Ac基因的mRNA,且Cry1Ac基因的拷贝数远远低于阳性对照,表明外源Cry1Ac基因的mRNA不能在砧木与接穗间进行运输。通过酶联免疫吸附测定(enzyme-linked immuno sorbent assay, ELISA),在两个嫁接组合接穗和砧木的叶片、韧皮部以及木质部中均检测到了Cry1Ac蛋白的存在,证明Cry1Ac蛋白可以通过嫁接的方式在砧木和接穗间进行运输。用各嫁接处理的接穗叶片饲养美国白蛾幼虫,发现嫁接在转基因杨树上的非转基因杨树可以抑制美国白蛾幼虫的生长发育,使其发育进度明显减慢,表现出一定程度的抗虫性。本研究明确了Cry1Ac基因表达产物在转基因和非转基因组织之间的表达情况、传递规律以及抗虫性,为嫁接转基因杨树在抗虫实践中的合理利用提供理论依据和科学指导。With the extensive planting of poplar (Populus spp.) in China, the pest problem of poplar comes along.Nowadays, the application of chemical insecticides can not only fail to effectively reduce the harm of pests to poplar trees, but also seriously damage the ecological environment.Therefore, it is feasible to improve the insect resistance of poplar through grafting and propagation of transgenic poplar and non-transgenic poplar.In this study, in order to understand the ecological safety of grafted transgenic poplar, hybrid Populus tomentosa and Hb1 transgenic Populus × euramericana cv.'Neva' with Bt-Cry1Ac gene were used as scions and rootstocks to study whether the mRNA and protein of Cry1Ac gene were transported between scions and rootstocks, and whether the leaves of grafted seedlings were resistant to the insect pest.The results of reverse transcription-PCR (RT-PCR) and third-generation digital PCR showed that no Cry1Ac gene mRNA was detected in the shoots and leaves of hybrid P.tomentosa as scions or rootstocks, and the copy number of Cry1Ac gene was much lower than that of positive control, indicating that the exogenous Cry1Ac gene mRNA could not be transported between rootstocks and scions.Cry1Ac protein was detected in leaves, phloem and xylem of scions and rootstocks in 2 grafting combination by enzyme-linked immuno sorbent assay (ELISA), which proved that Cry1Ac protein could be transported between rootstocks and scions by grafting.The non-transgenic poplar grafted on the transgenic poplar could inhibit the growth and development of the larvae of the Hyphantria cunea, and slow down the development of the larvae, showing a certain degree of insect resistance.This study clarified the expression, transmission and insect resistance of Cry1Ac gene expression products between transgenic and non-transgenic tissues, and could provide theoretical basis and scientific guidance for the rational use of grafted poplar in insect resistance practice.

关 键 词：嫁接 mRNA移动 Cry1Ac蛋白 抗虫性 

分 类 号：S722.36[农业科学—林木遗传育种]