Preliminary Study On Cloning,analysis And Functional Verification Of RAC2,KRAS,MEK2 And CD3Z Genes Of Bufo Gargarizans

According to Pharmacopoeia of the People’s Republic of China(2020 edition),Bufo gargarizans Cantor is one of the primitive animals of precious medicinal materials,such as Bufonis Venenum,Bufonis Cutis and Bufo Siccus.It belongs to the Bufo genus of the Bufo family of Amphibian Anura,and was officially listed as a protected animal with important ecological,scientific and social values in 2014.At present,there are few reports of gene related research on Bufo gargarizans,which mainly focus on genome analysis,the effects of environmental stress on gene expression,and biosynthesis of active components and so on.In the early stage,we carried out transcriptome analysis on the parotoid gland,dorsal skin,and abdomen skin of Bufo gargarizans,and then excavated the Natural Killer Cell-Mediated Cytotoxicity signaling pathway.This pathway can identify and kill abnormal or infected cells without prior stimulation,which plays a key role in immune monitoring of cancer and viral infectious diseases.In previous studies,37 differentially expressed genes were obtained from this pathway.In this study,RAC2,KRAS,MEK2 and CD3Z genes were sreened out by constructing Protein Protein Interaction(PPI)and consulting relevant literature.CD3Z gene,playing a key role in T cell signal transduction,is located upstream of RAC2,KRAS and MEK2 genes in the pathway.As well as,RAC2,KRAS and MEK2 genes can participate in cell proliferation,migration,differentiation and other cellular physiological activities.In this study,the interaction relationship between RAC2,KRAS,MEK2 and CD3Z genes and the effect on proliferation and migration of HeLa cells were preliminarily explored by overexpressing RAC2,KRAS,MEK2 and CD3Z genes in HeLa cells.First of all,we screened the RAC2,KRAS,MEK2 and CD3Z genes from the NK cell-mediated cytotoxic pathway.Then the full-length cDNA sequences of four genes were cloned and analyzed by bioinformatics and tissue expression profile.The qPCR results showed that the relative expression of BgsRAC2 and BgsCD3Z genes was the highest in kidney,while the relative expressions of BgsKRAS and BgsMEK2 genes were the highest in liver,tongue and heart.Secondly,BgsRAC2,BgsKRAS,BgsMEK2 and BgsCD3Z genes were constructed in pcDNA3.1(+)/myc-His A and pEGFP-C1 vectors by molecular cloning,and sequenced for verification.And then the eukaryotic expression recombinant plasmids were transfected on HeLa cells,all of which were highly expressed.The results of qPCR showed that the mRNA exprssion levels of the RAC2 gene of pcDNA3.1(+)/myc-His A-BgsRAC2 and pEGFP-C1-BgsRAC2 recombinant plasmid transfection group were about 120,000 and 80,000 times higher than those in the empty vector groups,and caused upregulation of the MEK2 gene expression in the downstream.The mRNA exprssion level of the CD3Z gene of pcDNA3.1(+)/myc-His A-BgsCD3Z recombinant plasmid transfection group was about 88,000 times higher than that in the empty vector group,and the expression level of the downstream RAC2 gene(P<0.01)was significantly upregulated,as well as the expression of the downstream KRAS gene was upregulated.The mRNA exprssion level of the KRAS gene of pEGFP-C1-BgsKRAS recombinant plasmid transfection group was about 90,000 times higher than that in the empty vector group,and caused the downstream MEK2 gene expression level to be upregulated.At the same time,the mRNA exprssion levels of the MEK2 gene of pcDNA3.1(+)/myc-His A-BgsMEK2 and pEGFP-C1-BgsMEK2 recombinant plasmid transfection group were about 2,000 and 6,000 times higher than those in the empty vector groups.Finally,the effects of BgsRAC2,BgsKRAS,BgsMEK2 and BgsCD3Z genes on the migration and proliferation capacity of HeLa cells were detected by cell scratching experiment and cell counting kit-8.The results showed that the overexpression of BgsKRAS,BgsMEK2 and BgsCD3Z genes could promote HeLa cells migration,meanwhile,the overexpression of BgsRAC2 gene had no significant effect on HeLa cells migration.In addition,the overexpression of BgsRAC2,BgsKRAS,BgsMEK2 and BgsCD3Z genes can significantly promote HeLa cells proliferation(P<0.01).