Interfering Effect On V-ATPase Of Spodoptera Frugiperda Research On Recombinant Insect Baculovirus

Spodoptera frugiperda is an omnivorous agricultural pest that feeds on more than 350species of plants.It has a widespread distribution and strong adaptability,and causes serious damage to agricultural production.Meanwhile,under the selective pressure of pesticides,Spodoptera frugiperda has developed resistance to a variety of insecticides.A series of negative influence on food security,ecological environment and biodiversity emerged because of the blind use and abuse of pesticides.Therefore,it is urgent to explore new technologies to prevent and control Spodoptera frugiperda.As a new pest control strategy,RNA interference(RNAi)technique,has been paid more and more attention and been gradually used in agricultural production owing to its characteristics of strong specificity,high efficiency and green safety in recent years.Insect baculovirus,as a traditional and unique resource for biological control,has been widely utilized in the control of agricultural pests.V-ATPase plays an important role in the growth and development of insects.Its main function is to hydrolyze ATP and use the generated energy to maintain the co-transport of protons(H~+).It is a ubiquitous proton pump in living organisms and one of the potential targets of new pesticides.For all these reasons,we propose to construct a recombinant baculovirus with the short hairpin RNA(sh RNA)of the V-ATPase A and B subunits of Spodoptera frugiperda,utilizing the proliferative properties of the virus to generate sustainably expressed si RNA to achieve lasting interference with Spodoptera frugiperda V-ATPase and ultimately kill the pest.The main results are as follows:1.Bioinformatics analysis showed that the full length of V-ATPase A subunit of Spodoptera frugiperda was 2522 bp,encoding 616 amino acids;and the full length of B subunit was 2218 bp,encoding 494 amino acids.The double chain RNAs(ds RNAs)with lengths of 321,378 and 372 bp were synthesized based on the sequence of the A subunit,and ds RNAs with the lengths of 432,409 and 378 bp were synthesized in accordance with the sequence of the B subunit,respectively.The synthesized ds RNA was injected into the third instar larvae by microinjector,and the interference efficiency of different fragments on the target gene was detected by RT-q PCR.The results illustrated that the expression levels of the target gene were significantly suppressed by corresponding ds RNA fragments.There was a tendancy to fall and then rise,and the interference efficiency of various fragments was slightly different.Among them,the 378 bp ds RNA of the A subunit had the best inhibitory effect on the expression level,and the expression level of the A subunit was most significantly down-regulated at 24 h,which reached 80.67%.The 409 bp ds RNA in the B subunit had the best inhibitory influence on the expression level,and the expression level of the B subunit was most significantly down-regulated at 48 h,which reached 77.67%.Using the best fragment as a template,sh RNAs targeting the A and B subunits of Spodoptera frugiperda V-ATPAse were designed.2.Four U6 snRNAs(located on chromosomes 6,15 and 28,respectively)were cloned from Spodoptera frugiperda genome,and 300-2000 bp before U6 sn RNAs were used as candidate U6 promoters.The results indicated that the sequence similarity of U6sn RNA between Spodoptera frugiperda and Bombyx mori,Plutella xylostella,Drosophila melanogaster,Melitaea cinxia were up to 96%.Through the analysis of the software Promoter-2.0,it was concluded that the sequence located on chromosome 6 has a transcription start site at 1000 bp,and the sequence on chromosome 15 might have a transcription start site at 600 bp or 900 bp.Two sequences on chromosome 28 did not predict the presence of a transcription start site.The obtained sequences were functionally verified by cell transfection,and the results showed that the candidate promoter sequence U61,which located on chromosome 6 with a full length of 1695 bp had the best sh RNA expression effect for driving the fluorescent protein gene.3.The above screened U61 promoter and designed sh RNA were constructed into the nuclear polyhedrosis baculovirus genome of Spodoptera frugiperda to obtain a recombinant insect baculovirus.The growth and development of the third instar larvae of Spodoptera frugiperda were significantly inhibited after fed with the recombinant baculovirus sh A and sh B.The survival rates of larval treatments were 18.77%and 41.05%,pupation rates were18.75%and 35.4%,and eclosin rates were 18.75%and 22.95%,respectively.After microinjection of 100 n L recombinant baculovirus sh A into the third instar larvae of Spodoptera frugiperda,the expression levels of V-ATPAse A subunit decreased to 56%,38%,and 26%in the 2 d,4 d,and 5 d,respectively;body weight decreased by 6.3 mg and body length decreased by 2 mm.After microinjection of recombinant baculovirus sh B,the expression levels of V-ATPAse B subunit of Spodoptera frugiperda decreased to 78%,42%,and 30%in the 2 d,4 d,and 5 d,respectively,Body weight decreased by 6.31 mg and body length decreased by 2.43 mm.Five days after administration,the tested insects showed typical symptoms of decay and disintegration.4.After 10 generations continuous passage,the insect recombinant baculovirus still showed high efficiency expression of the interference fragment detected by fluorescence analysis and PCR amplification,indicating that the recombinant baculovirus had good genetic stability.In conclusion,in this study,the recombinant insect baculovirus was obtained by using gene cloning,RNAi,cell transfection and other technologies.Subsequently,the continuous silencing of Spodoptera frugiperda V-ATPAse A and B subunits was successfully achieved.The establishment of this interference system provides a theoretical and mental basis for the future use of RNAi technique to control Spodoptera frugiperda and other lepidopteran pests.