| Laodelphax striatellus is one of the most important agricultural pests,and was usually controlled by using insecticides.However long-term and extensive use of insecticides led resistance developed in this pest.Aquaporin(AQP),a member of the major intrinsic protein superfamily,functions mainly for transporting water and small molecule compounds across cell membrane.In the study of protozoan and helminth parasites,it is found that AQPs are directly or indirectly related to the absorption of anti parasitic drugs.In order to investigate the function of the AQPs and their possible contribution to insecticide resistance in L.striatellus,we carried out the following studies.Based on the transcriptome data,AQP like genes were cloned with PCR and RACE cloning techniques.Then,the cloned genes were subjected to sequence homogeny analysis and phylogenetic relationship analysis by constructing phylogenetic tree with insect AQP genes previously reported,detection of the polymorphic sites and possible resistance related mutations,tests of the expression level in the susceptible and resistant strains by real-time quantitative PCR,and functional verification for their transporting capability by combinant expression in vito.The main results are summarized as follows:1.Cloning and sequence analysis of AQP genes in L.striatellusThe AQP like gene fragments were first searched from the transcriptome data of L.striatellus and valided by PCR.Then,corresponding special primers were designed for RACE to clone full length genes.After end-to-end test,six full-length AQP sequences were obtained from L.striatellus and named as LsAQP1,LsAQP2,LsAQP3,LsAQP4,LsAQP5,LsAQP6,respectively.They encode 272 amino acids,248 amino acids,346 amino acids,247 amino acids,274 amino acids,273 amino acids,respectively.BLAST analysis showed that all the cloned genes showed their highest similarity to the AQP gene amino acid sequences of other insects,and had six transmembrane regions,NPA triplet sites and almost all Ar/R(aromatic/arginine)residues,indicating that they are the homogenous AQP genes of L.striatellus.The AQP genes of L.striatellus were phylogenetic analyzed with 78 AQP genes from 22 other insects which had been previously verified,and as expected the result showed that there were at least four subfamilies of insect AQPs,including Drosophila integral protein(DRIPs),Pyrocoelia rufa integral protein(PRIPs),D.melanogaster big brain protein(BIBs)and the fourth unclassified subfamily.Among the cloned genes,LsAQP1 belongs to the PRIPs subfamily,LsAQP2 belongs to the DRIPs subfamily,LsAQP3 belongs to BIBs subfamily,LsAQP4,LsAQP5 and LsAQP6 belong to unclassified subfamily.2.Expression analysis of the AQP genes in L.striatellusThe quantitative expression of 6 AQP genes in the four differentstrains of L.striatellus(susceptible and resistant to imidacloprid,chlorpyrifos and ethiprole)was compared by using q-RT PCR.The results showed that all the resistant strains showed no significant increase in the expression of LsAQP1,LsAQP2,LsAQP3,LsAQP4 and LsAQP5 as compared with the susceptible strain.However,LsAQP6 was significantly high expressed in all the three resistant strains.The detection was performed in successing 3 generations.In the first generation,the relative expression ratio of LsAQP6 gene in the 3 strains resistant to imidacloprid,chlorpyrifos and ethiprole was 5.64,2.75 and 3.19 times over the susceptible strain,respectively,in the second generation it was 3.93,4.29 and 1.95 times,and the third generation was 1.03,3.50 and 8.99 times.These results imply that LsAQP6 might play a role in the resistance development of L.striatellus.3.Mutation analysis of AQP gene in imidacloprid resistant strain of L.striatellusBased on the 6 cloned full-length AQP sequences in L.striatellus,special primers were designed to clone the full-length AQPs from both imidacloprid resistant and susceptible strains for checking resistance related mutations.For each gene,10 sequences were cloned from a 10-insect sample prepared for both resistant and susceptible strains.The alignment analysis revealed that all the 6 genes have many different polymorphic loci,of which the most up to 38 were found in LsAQP4,while the least only 18 in LsAQP5.However,there was no significant difference between the sequences of the resistant and susceptible strains.All the sequences have their NPA and Ar/R sites intact.Thus,these results indicate that none of these genes had resistance-related mutations.4.Expression in vitro and functional analysis of AQP1 and AQP6 in Xenopus oocytesFor functional analysis of the gene highly expressed in the resistant strains,LsAQP6 recombinant gene was injected into Xenopus laevis oocytes.The controls used the oocytes intact,injected with water,and LsAQP1 presenting the genes without expression increase in resistant strain.Then,the treated oocytes were checked for their capability to transport water,glycerol and insecticides.Because of the large molecular weight and poor water solubility of major insecticides,this study selected only methomyl and bisultap as representatives.The results indicated that LsAQP6 had high transport ability for water,and significant transport ability to methomyl,but not for glycerol and bisultap.The LsAQP1 that was not highly expressed in the resistant strain has no significant transport ability for all the four substances.It is thought that LsAQP6 can transport water and some small molecule insecticides in L.striatellus.In summary,this study cloned 6 full-length AQP genes in L.striatellus based on transcriptome data,and found that LsAQP6 not only highly expressed in different resistant strains,but also capable of transporting water and some insecticides.Thus,its over-expression might play roles in resistance development of L.striatellus.These results laid the foundation for further study of aquaporins in L.striatellus,revealing the functions of aquaporins in insecticide resistance of agricultural pests,and were also significant for insecticide resistance monitorring,management and new insecticide development. |
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