桃蚜MpAChE基因RNAi表达载体构建及转化

通过害虫取食植物表达害虫发育关键基因dsRNA的转基因植株,分析能否通过抑制害虫特定基因的表达来防控害虫。本研究利用RT-PCR技术从桃蚜中克隆乙酰胆碱酯酶基因383 bp cDNA片段,命名为MpAChE。进一步利用该MpAChE基因片段构建植物RNAi表达载体RNAi-MpAChE,并通过浸花法转化野生型拟南芥,通过卡那霉素抗性筛选转化植株,PCR及Southern杂交进一步鉴定转基因植株。结果表明:克隆的cDNA片段与桃蚜中已克隆的乙酰胆碱酯酶(GenBank登录号AY147797)cDNA序列核苷酸一致性为99%。卡那霉素抗性初步筛选和PCR进一步鉴定,获得25株阳性转基因植株。从25株中随机选择的5株阳性植株,Southern杂交均为阳性。经接种桃蚜初步鉴定,转基因植株对蚜虫的抗性效果不显著。

Transformation and Construction of RNAi Plant Expression Vector Carrying MpAChE Gene from Myzus persicae

To analyze whether the plant-mediated RNAi may be a way for gene-silencing in herbivorous insect by suppressing a critical insect gene by feeding insects with genetically modified plant tissues to produce a specific dsRNA.We used RT-PCR method to clone a 383 bp cDNA fragment of acetylcholine esterase(AChE) from Myzus persicae,and the fragment was termed MpAChE.The RNAi plant expression vector was constructed harboring both the positive-sense strand and antisense strand of MpAChE,which named RNAi-MpAChE and then was introduced into wild-type Columbia Arabidopsis thaliana plants by the floral dip method.Transformed Arabidopsis plants were identified by growth on solid 0.5×MS medium containing 50 mg/L kanamycin,PCR and Southern blotting.The results showed that the 383 bp fragment exhibits 99% identity at nucleotide acids level with corresponding part of the cDNA sequences of AChE2 from Myzus persicae(Genbank accession number AY147797).Twenty-five transgenic A.thaliana plants were obtained by identification using kanamycin and PCR.Among these plants,five independent transformants selected at random were further identified as positive transgenic plants by Southern blotting.Transgenic plants show non-resistance to Myzus persicae by feeding insects with genetically modified plants.