Molecular Mechanism Of GSS Expression In Response To The Glucosinolate Of Host Plant

Glucosinolates are a type of defensive secondary metabolite common in cruciferous plants that can be degraded to toxic chemicals by plant myrosinase.However,the crucifer specialist diamondback moth(DBM),plutella xylostella,can use glucosinolate sulfatase(GSS)to prevent the formation of toxic compounds by the plant myrosinase and convert glucosinolates into nontoxic desulfoglucosinolates.A previous study showed that significantly higher expression of GSS was coupled with higher expression of sulfatase-modifying factor gene SUMF in the midgut of DBM larva,the development stage,when it abundantly feeds on cruciferous plants and contacts with glucosinolates.In this study we aimed to confirm whether the expression of GSS and SUMF can respond to glucosinolates and identify the cis-acting elements involved.The main results are outlined below.1.We used artificial diet and radish leaves treated with different concentrations of sinigrin(SIN)or allyl isothicyanate(AITC)to feed the 3rd instar larvae.The qPCR method was used to detect the expression levels of GSS and SUMF in the midgut and whole body 48 hours after treatment.The results suggested that the expression of target genes did not show obvious changes when larvae were fed with artificial diet and radish leaves treated either with AITC or SIN.However,the expressions of GSS was upregulated at 48 h after feeding with artificial diet containing three different concentrations of SIN.The expression of GSS1 in the midgut was similar to that in the whole body after feeding on artificial diet containing 25 ?g/?L SIN.2.Four types of GSS1 promoters were cloned from the FZ,HN and G88 strains of DBM.They showed large fragment deletions or base mutations in the region from-540 to-1200 bp upstream of transcription start site(TSS).Four other types of SUMF1? promoters were also cloned from the FZ strain and showed large fragment insertions or deletions between-1700 and-2200 bp upstream of the TSS.There were genetic polymorphisms and several AT-rich sequences in both promoters of two genes,which might affect the transcription.3.The prediction of regulatory elements using a bioinformatics approach showed that transcription factor binding sites,such as those for Bcd,En,Dfd,Eve,Hb and SGF-3,existed in the 5' upstream sequence of GSS1 and SUMF1a.And one ER binding site conservatively existed near TSS in different genotypes of GSS1-pro and SUMFla-pro.We constructed the expression vectors,which consisted of GSS1 promoters,IE1 promoter and EGFP reporter,and transfected the vectors into Sf9 cells to identify the SIN-responsive elements.The results of semiquantitative analysis,based on the images processed by ImageJ,located a potential element responding to SIN between-1702 and-874 upstream from TSS.However,the results of EGFP expression,as detected by qPCR,revealed that SIN reduced IE1 promoter activity.But there might existed a SIN-responsive element between-1702 and-874,compensating the reduction of IE1 promoter activity.Also a potential active promoter element was predicted in the region from-412 to +36.4.The transcriptome study of the larval midgut showed that SIN had no significant effect on other classical detoxification enzymes(such as P450s and GSTs)and,instead repressed the expression of growth-and development-related genes and induced digestive enzyme genes.Furthermore,an upregulated transcription factor(TF)gene,Px003655,was identified.We suggested that this TF may be involved in the regulatory pathway of detoxifying glucosinolates by GSS1.Our study confirmed that the expression of GSS1 could respond to SIN.By isolating the promoters of GSS1 and localizing the SIN-responsive elements,we identified an upregulated transcription factor through transcriptome sequencing,thus,providing basis for the further study of molecular regulatory mechanism of SIN responsiveness in DBM.This study was significant for illuminating the molecular details of the coevolution between DBM and its host plants and provided the basis for host plant resistance-based ecological pest management.