Functional Analyses Of Wheat Protease Inhibitors For Different Biological Types Of Sitobion Avenae

Sitobion avenae,as the most important agricultural pests in the world,is widespresd among wheat producing area in China,and is one of the dominant species of pests in the wheat areas.The species has evolved a high genetic diversity,due to oligophagy,adaptability,complex life history,wing shape differentiation,migratory ability,that facilitate them to lead to genetic diversity and pest differentiation.In the history of co-evolution,plants have formed a set of defense mechanism to cope withThe stress from external organisms.Protease inhibitors are plant defense related proteins,which interfere with the metabolic system of insects,and inhibit their normal growth and development.Correspondingly,Aphids differentiate into various biotype according to plant resistance,which is an important adaptive mechanism.Therefore,based on plant biochemistry,molecular biology and ecology,to investigate the relationships between plant protease inhibitors and biotypes,and identify the roles of protease inhibitors in biotype differentiation,and thus to reveal the mechanisms of biotype differentiation.with respect to Sitobion avenae will provide insight into the role of protease inhibitor in biological differentiation.Furthermore,the study is conducive to the development of aphid prevention and control strategies,and the formation of sustainable aphid defense methods,which will lay the foundation for the control of wheat aphids,and the improvement of the and the quality of wheat in China.In this study,the published wheat protease inhibitor gene sequences were downloaded from NCBI database,and PCR amplification was used to verify the presence of the gene in wheat strain mingxian 169.Xinong 979;186.the TM12-34;zhong mang in 4;ai kang 58,The results showed that the cysteine protease inhibitor genes WC1,WT005＿K06 and WC4 had a bright band.The gene cloning vector was successfully constructed and expressed in escherichia coli competent cells(DH5α).The correct gene sequence was obtained by sequencing and comparing with the published wheat protease inhibitor gene sequence in NCBI database.The result of Fluorescence real-time quantitative showed as followed.The expression levels of WC1,WT005＿K06 and WC4 genes were not significantly different among wheat strains,but the expression levels of WT005＿K06 were hundreds of times higher than those of WC1 and WC4 genes.It is speculated that whether WT005＿K06 has insect resistance remains to be verified,and its biological function remains to be further studied.To study the effects of WC1,WT005＿K06 and WC4 genes on different biotypes of wheat aphid,we successfully constructed the expression vectors of PET-28a(+)-WC1,PET-28a(+)-WT005＿K06 and PET-28a(+)-WC4.The optimal concentration of IPTG and temperature condition were identified to ensure efficient gene expression through pre-experiments.Further,the recombinant vector was induced to express in E.coli competent cells BL21.After a small amount of expression,the results indicated that all three recombinant proteins were expressed in the supernatant and the precipitate,and the expression level of the supernatant was very high.After a large amount of expression to improve protein purification efficiency,the optimal concentration of imidazole for WC1,WT005＿K06,and WC4 proteins was screened.The cathepsin B and cathepsin L were found in the digestive tract of wheat aphid,and the specificity of cathepsin L was consistent with that of cysteine protease inhibitor.The design idea of this experiment is to synthesize the fluorescent substrate of Cathepsin L,and infer the activity of protease inhibitor by analyzing the consumption of fluorescent substrate.The results from different biotypes of Aphis gossypii indicated that the activities of total protease and cysteine protease activities for different biotypes were significantly different,while the activity of recombinant protein was not significantly different among the biotypes of Sitobion avenae.Implying that the proteases may be involved in biological differentiation.However,due to the differences in protease content and the harmfulness of biological types,it is difficult to identify the effects of recombinant protein on the biological types of wheat aphid.The inhibition effect of recombinant protein on the biological types of wheat aphid was observed by artificial feeding experiment.The results show that: after feeding for 24 h,the recombinant protein had a low inhibitory effect on the survival amount of the biological type,and the survival amount of the treatment group was consistent with that of the control group.After feeding for 72 hours,the recombinant protein had a significant effect on the system and the survival amount was generally reduced.In summary,there are four aspects to this study,(1)gene screens and sequence analysis of wheat cysteine protease inhibitor,(2)the analysis of the activity of wheat aphid protease and cysteine protease for different biotype,(3)the measurement of the activity of recombinant protein and inhibition of protease,and(4)the investigation for the effects of recombinant proteins on the survival and reproductive rate of biotype.The result from these experiments confirmed the insect resistance of cysteine protease inhibitor,which provides new evidences for studying other effects of protease inhibitors,and a theoretical basis for the application of protease inhibitors in genetic engineering.