摘要
【目的】棉蚜(Aphis gossypii)是棉花主要害虫之一,严重危害棉花的生产。利用靶标昆虫重要基因的双链RNA进行干涉从而防治农业害虫是当今发展的1种新技术。通过转基因的方法,在棉花中表达棉蚜靶标基因的双链RNA(Double stranded RNA,dsRNA),特异性地抑制棉蚜内源基因的表达,从而控制蚜虫对棉花的危害。【方法】本研究利用棉蚜腺苷三磷酸酶E亚基(Adenosine triphosphatase subunit E,ATPase E)基因,构建其RNA干扰载体,并通过农杆菌介导转化棉花。PCR技术初步筛查45个独立转化植株,对PCR阳性植株进行DNA印迹(Southern blot)分析,确认外源基因插入的拷贝数。同时利用RT-PCR (Reverse transcription polymerase chain reaction)方法检测dsRNA的表达情况。最后结合网棚和大田抗蚜性鉴定分析转基因株系的抗蚜效果。【结果】从45个转基因再生植株中获得22株阳性转基因株,其中12株为单拷贝插入。根据农艺性状和dsRNA表达量分析结果,筛选出4个转基因株系,分别为CZ1、CZ2、CZ3和CZ4。2年的网棚和大田的蚜虫抗性鉴定结果表明:转基因棉花CZ1、CZ2和CZ3具有较高的抗蚜性,2015年和2016年的蚜害减退率分别达70%和60%以上。【结论】利用RNAi技术将蚜虫腺苷三磷酸酶E亚基基因片段转化棉花,可以提高棉花的抗蚜性,为棉花抗蚜育种提供了新种质和新方法。
[Objective]The cotton aphid(Aphis gossypii)seriously affects cotton growth and development.Recently,RNA interference(RNAi)technology has been employed as a new technique to control damage caused by agricultural pests.There is an urgent demand to develop transgenic cotton expressing double stranded RNA(dsRNA)targeting insect essential genes to control damage by aphids.[Method]In this study,we isolated a gene that encodes adenosine triphosphatase(ATPase)subunit E from the cotton aphid,and constructed a plant RNAi expression vector with the fragment of this gene.The transgenic cotton plants were developed through Agrobacterium-mediated transformation.The putative transgenic plants were screened by polymerase chain reaction(PCR)analysis,and the PCR-positive transgenic plants were subjected to Southern blot assay to detect the copy number of T-DNA insertions.Then,reverse transcription PCR(RT-PCR)was employed to analyze the expression level of dsRNA.[Result]Twenty-two transgenic plants were confirmed from 45 putative transgenic plants.The dsRNA had been introduced into the cotton genome with a single-or multi-copy insertion.Among the 22 transgenic plants,12 plants contained only one copy of T-DNA,which had been stably expressed in cotton cells.The four transgenic lines,CZ1,CZ2,CZ3,and CZ4,with one-or two-copy dsRNA insertions and obviously normal agricultural traits were selected for analysis of aphid resistance.According to identification of aphid resistance in both net room and field experiments,the results showed that the CZ1,CZ2 and CZ3 transgenic lines possessed high toxicity against cotton aphids,with over 70%and 60%decline rates of aphid damage in 2015 and 2016,respectively,while the control exhibited susceptibility to aphids,leading to leaf curl and stunted growth.[Conclusion]The cotton aphid resistance in transgenic cotton plants with the fragment of aphid ATPase subunit E gene by RNAi provides a new idea,method and germplasm for aphid-resistant breeding.
作者
罗晓丽
张安红
肖娟丽
王志安
陈晓英
吴家和
Luo Xiaoli;Zhang Anhong;Xiao Juanli;Wang Zhi’an;Chen Xiaoying;Wu Jiahe(Institute of Cotton Research,Shanxi Academy of Agricultural Science,Yuncheng,Shanxi 044000,China;Institute of Microbiology,Chinese Academy of Sciences/State Key Laboratory of Plant Genomics,Beijing 100101,China)
出处
《棉花学报》
CSCD
北大核心
2018年第5期353-362,共10页
Cotton Science
基金
山西省农业科学院重点项目(YZD1501)
山西省农业科学院农业创新研究课题(ZDSYS1501)
国家科技重大专项--转基因生物新品种培育科技重大专项(2014ZX08010-004)
