Screening And Optimizing Of DsRNA Fragments For RNAi-based Pest Management In Apolygus Lucorum

Apolygus lucorum is an important agricultural pest with a wide range of hosts including many economic crops such as cotton,apple,pear,date and grape,causes huge economic losses.The method of controlling A.lucorum mainly relys on chemical pesticides.However,long-term and unreasonable use of pesticides not only pollutes the environment,but also easily makes pests resistant to pesticides.Therefore,it is urgent to develop new environmentally friendly pest control methods.The RNAi-based pest management is a new type of green method for pest control.The main component of RNAi-based pest management is dsRNA,which specifically designed for target pests.It has the characteristics of strong specificity,easy degradation,and non-toxic side effects,displayed great potential for application.The core issue of RNAi-based pest management is to screen target genes that have high negative effects(such as lethality)on pests and are safe for humans and other beneficial organisms.In this study,three target genes were screened and obtained by injection-based RNAi in A.lucorum.After homology and off-target analysis,dsRNA fragments were optimized by removing same sequence fragments compared to non-pest organisms,and the lethal effect of each dsRNA fragment was verified.In addition,effects of dsRNA on different lengths(300-360 bp,200-300 bp,100-150 bp,30-90 bp)and different target sites were confirmed.dsRNA with short length and high lethal effect were identified in three target genes,and finally,these short dsRNA fragments were assembled into a new artificial recombinant dsRNA and was proved to have a higher lethal effect for A.lucorum.This study verifies the possibility of using dsRNA of multi-target genes to control pests,provides a theoretical basis for the development of RNAi-based pest management using single dsRNA contained multi-target genes in controlling one kind of pests,or even using single dsRNA in controlling multi-target pests.The main results are as follows:1.Three high lethal effect target genes were screened and obtained in A.lucorum.The lethal effect of four RNAi-based target genes were verified by injection-based RNAi.Among them,interference with Ubx(Ultrabithorax),wupA(wings up A)and Dpp(Decapentaplegic7)significantly increased the mortality rate of the 3rd instar nymph of A.lucorum compared to the controls.The mortality rate(7 d)of A.lucorum after injected dsUbx was 56.67±3.33%.The mortality rate(7 d)of A.lucorum after injected dswupA was 94.44±1.11%.The mortality rate(7 d)of A.lucorum after injected dsDpp was 92.22±1.11%.2.The dsRNA fragments of the three target genes were optimized by off-target analysis,and their lethal effects were verified.The results showed that the dsRNA fragments of all the three target genes had off-target sites.Among them,sequences of more than 19 bp consecutively same with non-pest organisms were considered to be risk sites and needed to be deleted.It is worth noting that sequences of some off-target sites were only found in pests,indicating that these sites have the potential to simultaneously control different pests other than A.lucorum.These sites were defined as super sites and should be remained.After sequence optimization,the longest fragments of Ubx,wupA and Dpp were 75 bp,241 bp and 282 bp in length,respectively.Furthermore,lethal effects caused by optimized dswupA and dsDpp(length>200 bp)were verified,with the mortality rates of 77.78±2.94%and 70.00±1.93%,respectively.3.Shortened the dsRNA length of the above genes,compare the different lethal effect caused by different target sites,and screened highly effective dsRNA fragments.The Dpp(282 bp)was divided into 4 s-level fragments with different target sites in length of 100-150 bp.The results show that injected dsRNA of Dpp-s-1(135 bp)and Dpp-s-2(140 bp)significantly increased the mortality rate of the 3rd instar nymphs of A.lucorum.The 7-day mortality rate of A.lucorum after injecting dsDpp-s-1(135 bp)was 58.89±12.94%.The 7-day mortality rate of A.lucorum after injecting dsDpp-s-2(140 bp)was 62.22±2.94%.The above efficient s-level fragments were further refined into ss-level fragments(30-90 bp),and the Dpp-ss-2(65 bp)was obtained,with the mortality rate of 55.56±2.94%for 7 d.Similarly,The 7-day mortality rate of A.lucorum after injecting dswupA-s-1(135bp)and dswupA-s-2(132bp)were 67.78±2.94%and 70.00±0.00%,respectively.Further refinement was carried out to obtain ss-level small fragments wupA-ss-1(66 bp)and wupA-ss-2(66 bp),The 7-day mortality rate of A.lucorum after injecting dswupA-ss-1 and wupA-ss-2 were 56.67±3.85%and 52.22±1.11%,respectively.In addition,the 7-day mortality rate of A.lucorum after injecting dsUbx-ss-1(75 bp)was 47.78±1.11%.4.The high-lethal-efficiency small dsRNA fragments from different genes were artificially recombined and its lethal effects were verified.dsRNA fragments of Dpp-ss-2,wupA-ss-1,wupA-ss-2 and bx-ss-1 were artificially recombined into Udw(262 bp).Off-target sites of Udw were re-excluded.The 7-day mortality rate of A.lucorum after injecting dsUdw-4 was 72.22±2.94%.Besides,injection of ds Udw obtained significant lethal effects on Day 2 and Day 3,and caused the highest relative mortality rate on Day 3,displaying a faster effect of mortality compared to other single-target dsRNAs.Dose response of dsUdw indicated that injecting ds Udw at lower concentrations could still induce highly lethal effects.The 7-day mortality rate of A.lucorum after injecting 10,5 and 1.67 times dilution of dsUdw were 64.44±2.94%,66.67±1.93%and 67.78±2.94%,respectively.