Improvement Of Rice Resistance By Using RNAi To Knockdown An Important Gene Of Nilaparvata Lugens

The brown planthopper Nilaparvata lugens(BPH),is one of the major pests in rice field in China and Asia.At present,the control of BPH mainly depends on the spraying of pesticides.However,overuse of pesticides usually results in environmental pollution and leads to development of BPH resistance to pesticides.Thus,breeding resistant rice has become the preferred method for control of BPH forthe safety,economy,and effectiveness reason.This study aims to use RNAi to increase rice resistance level and to eliminate the food risk of genetically modified rice.As we all know,the middle intestine is an important organ for insects to digest food and absorb nutrients.Food is preferentially exposed to midgut epithelial cells after being ingested by an insect.Therefore,midgut is an important interface between BPH and rice.By employing RNAi technology to knockdown the important functional genes in midgut cells,the development and reproduction of BPH will be inhibited for repression of normal digestion of food and absorption of nutrients.The works carried out in this study are as follows:1.The rice endosperm-specific promoter Glub-4 and the phloem-specific promoter RSsl were cloned;the RNAi expression cassette containing the NQO gene from the biotype I of BPH was constructed and subcloned into a Ti plasmid vector,in which also harboring the deletion enzyme gene with its recognition sites flanking the two genes.Rice Minghui 63(MH63)was used to create the transgenic rice line.The expression levels of siRNA in the recombinant plants were confirmed by Northern blot hybridization.The results showed that siRNA molecules were expressed at a high level.We also revealed siRNA molecules preferentially expressed in phloem tissue by using mRNA in situ hybridization.There were no significant hybridization signals in phloem of transgenic rice when hybridized with the sense NQO RNAs as probes,while the hybridization signals presented clearly when using the antisense NQO RNAs as probes.The signals were mainly distributed in the parenchyma of the phloem,as well as partially in the vascular parenchyma.It is possible that the expressed small RNAs may permeate therein,or the promoter used has a basal activity in the vascular parenchyma cells.2.Diet effects of transgenic rice on NQO gene of BPH.To determine the effect of the RNAi molecules,northern blot hybridization was used to detect the expression NQO gene in BPH feeding on transgenic rice for 24 hours.The results showed that the RNAi molecules had a significant inhibitory effect on NQO gene.To determine the time-elapse knockdown of NQO gene,BPH treated on transgenic rice for 6,12,24,48,and 72 h were collected respectively for RNA isolation and hybridization detection.We found that the transcript levels of NQO in the treated BPH decreased greatly compared with the control.3,Diet effects of transgenic rice on the relative growth rate(RGR),the honeydew amount and the mortality rate of BPH.The results demonstrated that the RGR dropped from 0.15 mg.mg-1d-1 in control insects to 0 mg.mg-1d-1 in the insects kept on transgenic rice for 24 h,the honeydew amount decreased from 21.00 mg.d-1 in control to 8.50 mg.d’1 in treated BPH,and the mortality rate increased significantly from 47.00%in control up to 80.20%in treated insects.Subsequently,the insect-resistant index and the antibiotic index of the transgenic rice to BPH were measured to evaluate the insect-resistant effect of transgenic rice.The results displayed that after challenged by the 3-4th instar nymphs,the insect-resistant index decreased from 0.746 of control seedlins to 0.4518 of transgenic seedlings;but the insect-resistant index decreased slightly from 0.881 of adult plants to 0.7349 of adult transgenic plants correspondingly.However,after challenged by the 3-4th instar nymphs,the antibiotic index increased significantly from 0.0384 of control seedlings to 0.6833 of transgenic seedlings;the antibiotic index increased dramatically from 0.0225 of adult plants to 0.8052 of adult transgenic rice correspondingly.The data demonstrated that the NQO-RNAi molecules in the transgenic rice provided a strong and persistent resistance to BPH.4.The average starch content of the transgenic rice line was detected as 15.4%.Further study showed there were no heterogeneous genes in rice endosperm due to the function of deletion enzyme.These data demonstrated that the safety of the transgenic rice food was satisfactory.This study focused on the expression profiles of NQO-RNAi molecules in transgenic rice,impact of the RNAi molecules on the growth and development of BPH,evaluation of the insect-resistant effects of the transgenic rice and the deletion of heterogeneous genes in the rice endosperm.This work explored a novel way to improve rice resistance to BPH and to address the food safety of transgenic rice.In the following work,we will employ the molecular marker-assisted selection techniqueas well as backcross breeding to create new rice lines in conventional rice breeding and hybrid rice breeding.We hope to explore bran-new rice varieties for application in production.