Characterization Of The Molecular Elements And Construction Of Plasmid For The Development Of Genetically Modified Cydia Pomonella(Lepidoptera:Olethreutidae)

The codling moth Cydia pomonella(L.)is an important quarantine pest of of fruits and nuts.Efforts to control the codling moth in the past mostly relied on the chemical pesticides with broad spectrum,although alternative non-chemical control methods such as mating disruption and granulovirus of C.pomonella(CpGV)are increasingly being adopted,the population could not be eradicated or suppressed effectively.As a result,the demand for new control tactics that are not only effective but also friendly to the environment has drastically increased.Genetically modified insects technique which is a genetics-based control system based on the traditional sterile insect technique(SIT)is a novel,environment-friendly,promising method.Promoter as one of the key components in constructing genetic transformed strains was used in insect population genetic control by regulating the expression of the lethal gene purposefully.And the control effect mainly depended on the function of the lethal gene.In this study,we take Cydia pomonella as the target insect and tried to construct genetic transformation vector of this pest.Given all that,we conduct the following experiments:(1)In order to isolate the highly expressed female-specific gene,homologous cloning,genomic walking technique combined with RACE PCR strategy were performed to isolate the Vitellogenin from Cydia pomonella.Quantitative real time PCR was used to analyze the relative expression pattern of CpVg at different developmental stages and in different tissues.The expression profile revealed that the CpVg was highly expressed in female adult.And its transcript was more abundant in the hemolymph than in other tissues.Based on the genome database of Cydia pomonella,the promoter region from the CpVg 5'-flanking region was isolated,and its transcription initiation site and the transcriptional regulators were predicted by BDGP and JASPAR software,respectively.Subsequently,we conducted 6 luciferase(LUC)report vectors drived by different partly deleting pattens of CpVg promoter for investigating the correlative regulation elements.The results of transient expression of these vectors in Sf9 cellline indicate that the-1306 nt?+51 nt region of CpVg promoter enhanced the transcription level of lueiferase gene.(2)In order to isolate the highly expressed embryonic stage-specific gene,routine PCR combined with RACE PCR strategy were performed to isolate the serendipity-a(Sry-a)from Cydia pomonella.The expression profiles of CpSry-a at different developmental stages and in different tissues indicated that the high expression of CpSry-a occurred during early to mid-embryogenesis.Its transcript was respective more abundant in female hemolymph and male fatbody.Based on the genome database of Cydia pomonella,the promoter region(1542 bp)from the CpSry-a 5'-flanking region was isolated.Based on the prediction result of the transcription initiation site and the transcriptional regulators,we conduct 5 LUC report vectors drived by different partly deleting pattens of CpSry-a promoter to investigate the correlative regulation elements.The results revealed that the-309 nt?+105 nt region of CpSry-a promoter enhanced the transcription level of lueiferase gene.(3)In order to isolate a strong promoter which can drive the lethal gene expression in all tissues regulated by temperature,the promoter region(1707 bp)was cloned from the Cphsp70-1 5'-flanking region through Inverse-PCR.According to the prediction result of the transcription initiation site and the transcriptional regulators,we conduct 4 LUC report vectors drived by different partly deleting pattens of its promoter to investigate the correlative regulation elements.The results revealed that both positive and negative transcription elements existed in this region.And the-994 nt?-+49 nt region of Cphsp70-1 promoter enhanced the transcription level of lueiferase gene.(4)In order to isolate the candidate dominant lethal gene,routine PCR combined with RACE PCR strategy was performed to identify cell death protease effector caspase CpCasp 1 and two initiator caspases CpCasp 4-1 and CpCasp 4-2.All the novel CpCasps contain the typical domain of the Caspase gene family,CASc and Peptidase_C14.Their expression profiles at different developmental stages revealed that an increase in transcripts of CpCasp 1 coincideds with variable developmental stages changes in C.pomonella,suggesting that CpCasp 1 could play a role in developmental apoptosis.CpCasp 4-1 and CpCasp 4-2 were highly related to the developmental stages only in male insect.The tissue-specific expression profiles indicated that the three CpCasp genes were highly expressed in the hemolymph of female insect.The expression levels of two CpCasp 4 were increased under moderate UV stimuli,but the expression level of CpCasp 1 was down-regulated.Besides,the effect of the RNAi for each gene was not significant.So,Based on the analysis results of amino acid sequences,the apoptosis-related expression pattern for each gene during the developmental stages,and the reference which elucidated the Caspase 1 could induced apoptosis in Lepidoptern,we concluded that the effector caspase CpCasp 1 could be the optimal candidate dominant lethal gene for constructing the effector vector.(5)The promoter of the female-specific gene and the highly expressed embryonic stage-specific gene were used to construct the female-specific and the embryonic-specific driver plasmids for genetically modified Cydia pomonella.The Mediterranean fruit fly driver vector KNE006 as basic carrier,were recombinated with the promoter of CpVg,CpSry and Cphsp70-1 to drive tTA expression,and the corresponding plasmid were named Cp-Vg-driver,Cp-Sry-driver and Cp-hsp-driver,respectively.The effect vector AH426 as the carrier,was recombinated with CpCasp 1 gene to obtain a new effector plasmid and named as CpCas-Effector.All of the new vectors were the essential material for the development of the genetically modified Cydia pomonella.