Differential Genes Screening And Analysis Of Bacillus Thuringiensis Sip Protein Treated Colaphellus Bowringi Baly

Cruciferous vegetables are widely grown in China and have always been the main edible vegetables of Chinese people.As a representative insect of coleoptera,Colaphellus bowringi Baly beetle is an important pest that damages cruciferous crops.Bacillus thuringiensis is a kind of gram-positive rod-shaped bacteria,Bt preparations have already has extensive application in the agricultural pests prevention and control,found in the study of Bt resources,secretory protein Sip of coleoptera insects(such as Colaphellus bowringi Baly,CPB)showed high insecticidal activity,the pest control has a good application prospect.However,the present study found that some pests also showed resistance to Bt preparations.Therefore,researchers have begun to pay attention to the genomic information and transcriptome information of insects,in order to screen the target genes of the toxin responsive protein of pests and fundamentally prevent and control pests.However,the complete genome information of Colaphellus bowringi Baly has not been published,and various related gene information cannot be known,which leads to the slow progress of the research on the gene function of Colaphellus bowringi Baly.Laboratory preliminary obtained the full of Colaphellus bowringi Baly transcriptome database,the SSR label information as well as their interactions with Sip toxin proteins in the process of information,find differences between gene expression related to metabolic detoxification,among the common cytochrome P450 genes,ribosomal protein S8 and L14 and muscle protein LIM have taken place in difference expression.This study analyzed the PCR amplification differences of SSR tags before and after Sip toxin protein treatment by Colaphellus bowringi Baly.Four differentially expressed genes(P450,S8,L14 and LIM)were selected for PCR amplification,and prokaryotic expression vectors were constructed,respectively expressing target proteins with high efficiency of about 56 k Da,30 k Da,25 k Da and 89 k Da.Amplification P450 respectively,the S8 L14 and LIM four core difference gene fragments,extraction of sequencing the right forward insertion fragment and reverse insertion fragment plasmid,single enzyme after linearization for template synthesis of double-stranded RNA,RNA concentration measurement and diluted to 5.0 μg/μL,injection to the age of 3/4 of age at the end of Colaphellus bowringi Baly larvae(about 2 μL),respectively,after the success of the injection of 1 d,3 d,5 d and 7 d in intestinal rt-pcr test,the results show that injection,a day afterthe S8,L14 and LIM expression were not significantly changed,while P450 expression was significantly decreased.On the third day,S8,L14 and P450 gene expressions were rather weak,indicating that the interference was successful.However,the expression level of LIM gene was only slightly decreased on the third day,and slightly decreased on the fifth day compared with the first day,but the difference was not obvious between the two days.Until the seventh day,the expression level of the gene was significantly reduced,indicating that the interference was successful.The untreated Colaphellus bowringi Baly and RNA interfered Colaphellus bowringi Baly were fed to Sip protein respectively.The results showed that no deaths were observed in each group after 12 hours of protein feeding.After 24 hours,the mortality of S8 was almost no different from that of the control group.After 48 hours,the mortality of L14 was slightly higher than that of the control group.From the third day,the death rate of the four interference groups was significantly higher than that of the control group until the seventh day of death.The results were consistent with those of RT-PCR.The results of this study provide good materials for the screening of Colaphellus bowringi Baly target genes and further control of coleoptera such as Colaphellus bowringi Baly,as well as new ideas for the further screening of corresponding Bt toxin protein receptors,and provide preliminary theoretical basis for the future modification and practical application of Sip protein.