Clonging The Subunit A And Subunit B Of Agrotis Ypsilon And The Construction Of The Baculovirus Silence Vector Of Subunit B

It is a new pest control strategy that using RNAi technology to control pest. V-ATPase is not only a H+ proton pump related to transport energy, but a key protein to maintain the vital sign of insects. In this research, we cloned full-length cDNA of subunit A and subunit B from Agrotis ypsilon, and construct a baculovirus silence vector of subunit B based on the sequence of subunit B. And we got several results:(1)The full-length gene of subunit A and subunit B of V-ATPase were cloned using RACE technology, and analysis the sequence of the two genes. Full-length of subunit A gene was 2693 bp, with a CDS region of 1851 bp, coding amino acid 616, the relative molecular mass of the protein is 68.06 KD. Full-length of subunit B gene was 2583 bp, with a CDS region of 1491 bp, coding amino acid 496, the relative molecular mass of the protein is 50.01 KD. Currently, these two genes have been submitted to the GenBank, and got the accession numbers(subunit A: KM434187; subunit B: KM434187).(2)We had designed three siRNA based on the sequence of subunit B, injected them into the third instar larvae of Agrotis ypsilon, and the effect of interference was detected by q-PCR. The si1 that has the best interfere effect(with interfere effect of 44.90%) was used to construct the silence vector.(3)The U6 promoter of mice and shRNA of subunit B were built into baculovirus transfer vector pBacPAK9, and transfect cell sf21 to screen a recombinational baculovirus that can silence the V-ATPase specially.(4)The recombinational baculovirus was enlargement cultured, and continuous passage cultured to 15 th generation. Every generation of the recombinant fragment was detected by PCR, and the result show that the 15 th generation still can expression the silence fragment, indicated that the recombinational baculovirus has a good genetic stability.(5)Fed the third instar Agrotis ypsilon with recombinational baculovirus, and the interfere effect was detected by q-PCR, the result show that the expression of subunit B was dramatically declined from the third day, indicated that recombinational baculovirus could propagate and express silence fragment in insect.This study successfully constructed baculovirus vector capable of expressing the siRNA, and silencing the B subunit gene.we also analyzed the genetic stability of Baculovirus. We expect to provide a new way for pest control.