The Mechanism Of Gossypol Metabolism In Helicoverpa Armigera

Cotton bollworm is one of the most important agricultural pests in the world.Due to its high fecundity,strong adaptability and wide host range,more and more difficult to control this pest.Using the metabolic enzyme system to degrade the heterologous compounds such as pesticides and plant secondary metabolite is an important strategy for insect to respond to heterogenous substances.By interfering with the key detoxifying enzyme gene and blocking the detoxification and metabolic process of the insect,it can achieve the goal of controlling insect pests.In this dissertation,determined that CYP4L11,CYP6AB9 and CCE001 a gene were related to the detoxification and metabolism of gossypol using RNA-seq and RNAi;RNA-seq and genomic data were used to analyze the expression patterns of ABC transporeters in different developmental stages,different tissues and the expression patterns under different host plants treatment;the ABCB6 gene was knockout using CRISPR/Cas9 and screened out a homozygous line;the bioassay showed that the sensitivity of cotton bollworm to gossypol was changed after the knockout of ABCB6 gene;achieved the deletion of genomic fragment of Helicoverpa armigera mediated by CRISPR/Cas9 system;verified the universal applicability of the system through the editing of HaABCC2.The main results of this thesis are as follows: 1 CYP6AB9,CYP4L11,and CCE001 a participate in the metabolism of gossypol in cotton bollwormRAN-seq was used to analyze the expression patterns of metabolic enzymes after feeding on cotton,soybean,pepper,corn and artificial feed.The difference gene cluster analysis showed that corn and soybean treatment's gene expression patterns were similar,while gene expression pattern in cotton treatment was different from corn,soybean and pepper treatment.The metabolic enzyme gene with significant difference was screened by q-value < 0.005 & |log2FoldChange>1.5|,among them,9 genes belong to P450,15 belong to UGTs,3 belong to GSTs,6 belong to CarEs.By further screening,6 detoxification genes were screened out of q-value <0.001 and log2(fold-change)>1.5 in all three comparisons(cotton/chili,cotton/soybean,and cotton/corn).To confirm the expression profile data,we used RT-qPCR to further quantify the relative expression levels of six genes(CYP6AB9,CYP4L11,CYP18B3,CCE001 a,CCE001d,GSTD5)between cotton and other treatments.We also measured the relative expression of the candidate genes fed on artificial diet or the gossypol diet.The relationship between 6 candidate genes and gossypol metabolism was studied using RNAi.The results indicated reasonably good silencing of all the candidate genes by 24 h after dsRNA injection.After the 24 h recovery,the injected bollworms were transferred to gossypol-containing food for 2 days.Larval mass gain was significantly inhibited only in larvae injected with dsCYP4L11,dsCYP6AB9,or dsCCE001 a,not with the dsRNA for the three genes.Furthermore,for insects feeding on the normal artificial diet,injected larvae and the controls did not differ significantly in mass gain.2 HaABCB6 participate in the metabolism of gossypol in cotton bollwormBased on the genome sequence and transcriptome data,the ABC transporter family of cotton bollworm was identified.A total of 54 ABC transporters were identified,belonging to 8 subfamilies(A-H).According to the structural domain,the transtransport proteins are divided into full-transport proteins,half-transporters,and a quarter protein,the numbers are 21,30 and 3,respectively.ABCD,E,F and H subfamilies contain fewer genes,respectively 2,1,3 and 3,and ABCG is the largest subfamiliy in cotton bollworm,with 17 genes.There are 49 genes in the ABC transporter that contain the characteristic amino acid fragment LSGGQ,accounting for 90% of the total,which also indicates that ABC transporter has a high structural conservatism.The spatial and temporal expression of ABC transporter protein in cotton bollworm was analyzed by RNA-seq.The expression profiles in different developmental stages showed a variety of expression levels in different developmental stages.The results of differential cluster analysis showed that the five instar larvae were clustered in one branch,and the eggs were clustered to one branch with the pupa and the female moth,and the male moths were relatively special,and were clustered into one branch alone.The patterns of expression between different tissues are also diverse.There are many high expression of ABC genes in the head,including members of the A/C/H subfamilies.Compared to the midgut and hindgut,expression of ABC gene in the foregut is low.The expression of ABC transporter was analyzed by RNA-seq in response to different host plants.The results of cluster analysis showed that corn and pepper were gathered in one,and then clustered with cotton and soybean.Cotton,one of the most suitable host plant,the expression pattern was low,and only a few genes belong to B/C subfamily were up-regulated,such as HaOG200336 and HaOG200302.The effect of gossypol on the expression of HaABCB6 was analyzed using qPCR.The results showed that the expression of HaABCB6 was down-regulated after feeding on 0.1% concentration of gossypol diet.With the increase of the concentration of gossypol,the expression of HaABCB6 gene also increased.In odrde to further analyze the HaABCB6 gene,combined with the transcriptome data,the whole sequence of HaABCB6 gene was obtained by using RACE technique.The ORF length 2541 bp and encoding 847 amino acids.Transmembrane protein online prediction software shows HaABCB6 contains 11 hydrophobic peptides,and have five unique hydrophobic peptides in the n-terminal.Homologous comparison with ABCB6 gene of other species showed high similarity.In addition,subcellular localization results showed that ABCB6 gene and lysosomal co-localization.The HaABCB6 gene was knocked out using CRISPR/Cas9.Two different sgRNA were designed,and the results showed that sgRNA1 mediated editing efficiency was 56.2%,while sgRNA mediated editing efficiency was only 16.7%.The results showed that the choice of sgRNA directly affect editing efficiency.A mutation of 7bp was selected for purification,and a homozygous HaABCB6 knockout strain was obtained after two generations of screening,named 96s-b6-/-.The function of HaABCB6 gene in the metabolism of gossypol was studied by using the knockout strain.Gossypol stress bioassay results showed that the weight growth of knockout strain was slightly lower than that of control strain at low concentration,but no significant difference.When gossypol content reached 0.4%,the weight gain of knockout strain was severely inhibited,showing a significant difference compared with the control strain.Above all,HaABCB6 gene was involved in the metabolism of pesticides and gossypol,and may provides new targets for the control of cotton bollworm.3 Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigeraExtened the application of CRISPR/Cas9 system on Helicoverpa armigera by double sgRNAs mediated chromosomal deletion.By designing the sgRNA targeted two different locations and adjusting the concentration ratio of sgRNAs and Cas9 protein,the expected chromosomal deletion mutation were obtained and the mutation efficiency was 34%.A mutant with 338 bp fragment deletion was selected for purification.The homozygous mutant strain was finally obtained,named 96-cad.It is proved that CRISPR/Cas9 mediated chromosomal deletion can be inherited.qPCR was used to detect the expression of HaCad in the 96-cad.The results showed that the expression of HaCad was not detected in 96-cad,however,the expression of HaCad gene in the 96S strain was normal.The changes in sensitivity of the knockout strain 96-cad and the sensitive strain 96S to Cry1Ac were detected by bioassay.The results showed that the sensitivity of the knockout strain 96-cad to Cry1Ac was significantly declined,and the resistance level reached 794 times.In addition,the HaABCC2 gene was selected as the target gene to verify that the chromosomal deletion mediated by CRISPR/Cas9 system was widely applicable in cotton bollworm.The results showed that 26%of the individuals had a chromosomal deletion.This section of the thesis was obtained and verified that CRISPR/Cas9 system could be used as an efficient tool to engineer genomes with chromosomal deletion in H.armigera.In conclusion,the results not only promoted the understanding of metabolic mechanism of gossypol,and enrich the information of ABC transporters,and provided new targets for the control of cotton bollworm,and also proved that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H.armigera.