Molecular Cloning And Expression Analysis Of Glutathione S-transferase From Small Brown Planthopper(Laodelphax Striatellus)

The small brown planthopper(SBPH),Laodelphax striatellus(Fallen)is one of the important rice pests in China.SBPH can not only sucking on rice gramineous plants,but also can transfer important plant viruses.It caused a threat to rice production.Glutathione S-transferases(GSTs)are multifunctional enzymes.GSTs were thought to be involved in insecticide detoxification in pests and the overexpression of GST genes may lead to insect resistance.In this paper,the gene fragments of GSTs were selected from the transcriptome data of SBPH,and GSTs fragments were cloned to full-length.The expression levels of GSTs in different resistant strains of SBPH were analyzed by fluorescence quantitative real time PCR to identify the GSTs which were possibly involved in insecticide detoxification.The results of this study are summarized as follows:1.Molecular cloning and sequence analysis of Glutathione S-transferases of SBPHNineteen GSTs gene fragments were identified from the SBPH transcriptome data by local BLAST search.After removal of two redundant sequences,seventeen sequences were remained.Among the remaining seventeen sequences,nine sequences were published previously,and the other eight sequences were newly discovered SBPH GSTs.The newly discovered eight genes were cloned and verified by PCR,and named as LsGSTd2,LsGSTo2,LsGSTo3,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4,respectively.The sequences of LsGSTd2,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4 have the complete open reading frames,while the 3' ends of LsGSTo2 and LsGSTo3 are deletion.The 3' terminal sequences of LsGSTo2 and LsGSTo3 genes were cloned by 3' RACE PCR,and their open reading frames were verified.LsGSTo2 is 1158bp with an 1119bp open reading frame,encoding 372 amino acids.LsGSTo3 is 648bp with an 636bp open reading frame,encoding 211 amino acids.Seventeen SBPH GSTs sequences(nine previously published GSTs and eight newly discovered GSTs)were aligned and submitted to phylogenetic analysis,the result show that there are two GSTs in delta subfamily,one GST in epsilon subfamily,three GSTs in omega subfamily,three GSTs in sigma subfamily,one GST in theta subfamily,two GSTs in zeta subfamily and five GSTs in microsomal subfamily.2.Expression analysis of Glutathione S-transferases in three different resistant strains of SBPHIn order to screen the glutathione S-transferases might involved in pesticide detoxification,the expression levels of seventeen GSTs genes in three different resistant stains(imidacloprid resistant strains,chlorpyrifos resistant strains and deltamethrin resistant strain)of SBPH were analyzed.The results showed that the expression levels of LsGSTd2,LsGSTm1,LsGSTo2,LsGSTel and LsGSTz1 are significantly up-regulated in imidacloprid resistant strain than that in the susceptible stain,while the expression of LsGSTm4 in imidacloprid resistant strain was decreased to thirty percent than the susceptible stain.The expression level of LsGSTo2 in chlorpyrifos resistant strain is significantly higher than that in the susceptible strain,while the expression of LsGSTm4 in chlorpyrifos resistant strains was decreased to fifty percent than the susceptible stain.In the deltamethrin resistant strain of SBPH,the expression levels of LsGSTd2,LsGSTo2,LsGSTsl,LsGSTe1 and LsGSTz1 are increased significantly than that in the susceptible stain.From the above results,it can be found that in addition to the delta and epsilon subfamily GSTs,omega,sigma zeta and microsomal subfamily GSTs are also up-regulated in the resistant strains of SBPH.In summary,this study identified GSTs and found some GSTs might involved in the insecticide detoxification by GST molecular cloning and expression analysis,which providing a basis for further study of GST in insecticide resistance.