植物介导大豆食心虫RNA干扰的机制研究

oybean pod borer (Leguminivora glycinivorella (Mats.) obraztsov) is one of the most serious soybean pests in Northeastern China. The hatched larvae enters the pod and uses the young beans as a food source until the formation of mature larvae. The larvae of soybean pod borer feed actively on the immature beans can cause a considerable yield and seed quality loss. The control of this pest is difficult due to the inability to achieve the contact between the pesticide and the larvae inside the soybean pods. Therefore, the breeding of pest-resistant variety of soybean is the most avaluable methodology to restrict the attack of soybean pod borer. However, the development of resistant variety is time cost and the effect of resistance is limited. Our previous results showed that the transformation of 18rDNA and P0dsRNA of soybean pod borer into soybean cultivar distinctly enhanced the plant resistance to pod borer. But how the fragments of 18rDNA and P0 dsRNA worked on against soybean pod borer remained unknown. The objectives of the present proposal are 1) to perform the transcriptom sequencing of the larvae RNA collected from transgenic soybean pods (integreded with the insect dsRNA) and non-transgenic pods, and to find the differential expressed genes. 2) to determine the protein interactions between 18rDNA/P0 proteins and other related proteins by yeast hybrid. 3) investigate the expression and reaction parterns of SID and SR genes when the dsRNA is intaken into larvae. The results will illustrate the mechanism by which dsRNA enter the midgut epithelium and suppressed the endogenous genes and further kill larvaes. The present finding showed a great potential for the control of soybean pod borer through RNAi technique.

大豆食心虫是危害东北大豆生产最严重的害虫，培育抗虫大豆品种是防治食心虫最经济有效的方法。但是，传统育种周期长，效率低。通过前期研究，我们首次利用RNAi技术将食心虫18SrDNA和P0dsRNA片段分别导入转入大豆品种，创制了高度抗食心虫的转基因大豆株系。然而，目前尚不清楚dsRNA介导食心虫靶基因沉默，提高大豆抗食心虫的机制。本项目在前期工作基础上，首先利用转录组测序，分析取食转基因和非转基因豆荚食心虫幼虫的基因表达谱变化，确定差异表达基因；进而：(1)通过酵母双杂交技术筛选与18SrDNA或P0互作的蛋白；(2)克隆食心虫SID和SR基因，通过RNA干扰技术，研究SID和SR基因在食心虫吸收转基因大豆表达的dsRNA途径中的功能。通过系统研究，阐明植物介导食心虫RNA干扰的机制，为培育新一代抗虫转基因大豆品种提供科学依据和实践基础。

大豆食心虫是危害东北大豆生产最严重的害虫，培育抗虫大豆品种是防治食心虫最经济有效的方法。本研究对已获得表达食心虫18SrDNA 或P0 dsRNA的转基因大豆株系进行抗虫性鉴定，筛选出3个表达P0dsRNA T5代抗虫品系（ds-18、ds-22和ds-25），1个表达18SrDNA dsRNA的T6代抗虫品系（18SrDNA-26）；并利用农杆菌介导将18SrDNA dsRNA导入垦丰16中，经田间抗虫鉴定，筛选出5个T3代抗虫品系（KFr-5、KFr-1、KFr-3、KFr-4和KFr-6）。利用T4 代表达P0 dsRNA转基因豆荚和东农50豆荚喂食食心虫1龄幼虫，取食转基因豆荚幼虫P0基因表达受到干扰且死亡率明显高于对照，存活的幼虫呈现蜕皮不完全或蜕皮后不合成新表皮的畸形。. 分析取食表达P0dsRNA转基因和非转基因豆荚幼虫的转录组，共获得获得36.04Gb和60,685条Unigenes。差异表达基因大部分属于GO库的Biological Processs分类； 56个COG差异表达基因中38基因参与核糖体组成，氨基酸运输，蛋白质翻译及翻译后修饰等过程；主要涉及KEGG核糖体通路。. 食心虫核心RNA干扰基因Dicer，dsRBM和Argonaute基因家族与果蝇和赤拟谷盗同源性最高；系统RNA干扰基因SID1与秀丽线虫同源最高，SID2和SID3与家蚕同源性最高，SR基因与家蚕和赤拟谷盗同源性较高；色素基因Lac-2沉默后显著抑制食心虫幼虫头部色素形成且不影响生长发育，因此选择Lac-2作为Marker基因。幼虫取食Spbsil-2和SpbSRB-3, SpbSRB-4，SpbSRB-7 dsRNA后，抑制maker基因lac基因沉默，表明Spbsil-2和SpbSRB-3, SpbSRB-4，SpbSRB-7基因参与食心虫的系统RNA干扰。. 对大豆食心虫转录组数据进行挖掘，获得35个与昆虫免疫相关基因，幼虫喂食Toll13-2 和Toll-E dsRNA后，生长发育受到抑制，从而增加死亡率；而喂食itype dsRNA幼虫可发育到老熟，但易受细菌的感染，不能羽化成成虫；因此，Toll13-2 ，Toll-E和itype 可以作为靶基因用于大豆食心虫的防治研究。