Effects Of Three Microbial Pesticides On Subunits Of The V-ATPase V₁ Domain Of Helicoverpa Armigera

Vacuolar type proton ATPase(V-ATPase)is widely distributed in the lysosomes,Golgi bodies and vacuoles of eukaryotes and prokaryotes.Its main function is to transport Na+ or K+ out of cells by coupling K+/2H+ reverse transport carrier,and promote the absorption of nutrient substances,thus providing energy for various life activities of organisms.Helicoverpa armigera,as an important agricultural pest,is widely distributed in China and all over the world.It is one of the important model organisms of Lepidoptera and Noctuidae.At present,the use of insecticides and planting insect resistant cotton are still the main means to control the cotton bollworm.However,with the aggravation of the problem of insecticide resistance and pesticide safety,the control of cotton bollworm urgently needs new solutions.To find and find the genes necessary for the growth of Helicoverpa armigera,and to study and explore the function and mechanism of these genes in Helicoverpa armigera can provide clear targets and directions for the development of new insecticidal drugs and biological pesticides.CRISPR/cas9 gene editing technology is a new method to study gene function,which provides a convenient and quick tool for the study of insect genome function.In recent years,CRISPR/cas9 mediated target gene mutations have been applied in Lepidoptera,Diptera,Coleoptera and Orthoptera.However,due to the lack of genomic information and immature embryo micro injection technology,CRISPR/Cas9 system has not been widely used in Helicoverpa armigera.The open reading frame(ORF)sequences of C,D and G subunits of Helicoverpa armigera V-ATPase were cloned in this experiment.The effects of three kinds of microbial pesticides,including Bacillus brevis,Metarhizium anisopliae and Beauveria bassiana,on the expression of each subunit of V-ATPase Ⅵ domain of Helicoverpa armigera were determined and analyzed by fluorescence quantitative PCR.The V-ATPase of Helicoverpa armigera was successfully knocked out by CRISPR/Cas9.The expression of V-ATPase V1 domain in G0 mutant was detected by fluorescence quantitative PCR.The main results are as follows(1)The open reading frame(ORF)of V-ATPase C subunit of Helicoverpa armigera was amplified by PCR with specific primers.The full length of the open reading frame was 1155bp,encoding 384 amino acids.The predicted isoelectric point and molecular weight were 8.46 and 44.03Kd,respectively,and the molecular formula was C1980H3100N532O581S12,The stability index was 30.09,belonging to the stable protein,the fat index was 87.55,the average hydrophilic coefficient was-0.395,suggesting that it was a hydrophilic protein;the open reading frame sequence of D subunit was 741 bp,encoding 246 amino acids,and its isoelectric point and molecular weight were predicted to be 9.44 and 27.49kD,respectively,and the molecular formula was C1227H2035N339O360S6,The stability index was 22.69,belonging to the stable protein,the fat index was 103.58,and the average hydrophilic coefficient was-0.229,suggesting that it was a hydrophilic protein;the full length of G subunit open reading frame was 354bp,encoding 246 amino acids,and its isoelectric point and molecular weight were predicted to be 9.40 and 13.70kD,respectively,and the molecular weight was C595H996N178O183S4,The instability index was 40.74,belonging to the unstable protein,the fat index was 75.98,and the average hydrophilic coefficient was-1.009,suggesting that it was a hydrophilic protein.(2)After the third instar larvae of Helicoverpa armigera treated with LC50,the expression of V-ATPase V1 domain was changed as follows:a subunit expression increased,B,C,D,E,G,H subunit expression decreased,and F subunit expression tended to remain unchanged;after LC50 Metarhizium anisopliae treatment,the expression of V-ATPase in the third instar larvae of Helicoverpa armigera was changed The results showed that the expression level of each subunit in V1 domain was as follows:A,G subunit expression increased,B,C,D,E,F,H subunit expression decreased;LC50 Beauveria bassiana treatment of cotton bollworm third instar larvae,the expression of each subunit of VATPase V1 domain was as follows:a subunit expression increased,B,C,D,E,F,G,H decreased.(3)The CRISPR/cas9 system was used to knock out C,D and G subunits,and the mutants of C,D and G subunits of Helicoverpa armigera V-ATPase were successfully obtained.The results of qPCR analysis of each subunit of V-ATPase V1 domain in G0 generation mutant showed that in the mutant after C subunit knockout,V-ATPase in the mutant body was significantly higher than that in the mutant without C subunit.The changes of the expression levels of each subunit in V1 domain were as follows:A subunit expression increased,B,D,E,F,G,H subunit expression decreased;after D subunit knockout,the expression of V-ATPase V1 domain subunits decreased,while the expression of H subunit increased;the expression of V-ATPase in the mutant after G subunit knockout was decreased The results showed that the expression of A and C subunits increased,while that of B,D,E,F and H decreased.The mutation of the three subunits not only led to the change of the expression of each subunit in the V-ATPase V1 domain,but also in the phenotype,we found that most of the G0 mutant eggs could not hatch normally,most of them died 1-2 days before hatching,and the hatching rate was significantly lower than that of the control group.Therefore,we speculate that the C,D,G subunits of V-ATPase or the interaction of C,D,G subunits and other subunits of V-ATPase played an important role in the embryonic development of Helicoverpa armigera.These results indicate that V-ATPase plays an important role in the growth and development of Helicoverpa armigera.Next,further study on the specific role and mechanism of each subunit in the growth and development of Helicoverpa armigera can provide new targets and directions for the control of Helicoverpa armigera.Gene editing technology will become one of the important methods to study gene function in the future.