Functional Study Of Chicken Mismatch Repair Related To Gene MLH1

Mismatch repair systems play an important role in maintaining genome integrity.The system buys time for the body to repair by participating in the regulation of the cell cycle and generating different periods of blockage,and mediates apoptosis when the repair task is not completed during the blocked time.Tumor formation is closely related to defects in the mismatch repair system,and many mismatch repair gene mutations and microsatellite abnormalities are currently found in a variety of tumors,but little research has been reported on chicken mismatch repair genes and their role in tumorigenesis.In this study,we cloned the chicken mismatch repair-related gene MLH1 and performed prokaryotic expression and antibody preparation,and then silenced the expression of MLH1 gene and detected the response of MLH1 gene to DNA damage after silencing.In this study,the MLH1 gene was cloned from the conserved region of the MLH1 gene and the tissue RNA obtained from SPF chicken embryos,reconstructed it into prokaryotic expression vector pET 32a.Inducting by IPTG and purifying by nickel column method,the MLH1 recombinant protein was required.The MLH1 protein was used to immunize the New Zealand Great White Rabbit.The results showed that the full length of the MLH1 gene was 2382 bp(KX602287),encoding 757 amino acids,and the full length of the chicken MLH1 gene amino acid sequence was 79.5%homologous to the human MLH1 gene amino acid sequence.The ATP-binding site in the structural domain of histidine kinase-like ATPases is 80%homologous to human and may have a function similar to that of the human MLH1 gene.Polyclonal rabbit antiserum was obtained by immunization after purification of recombinant proteins,and the ELISA method was used to determine the polyvalence of the antiserum above 1:20 000,and the Western blot technique was used to make the serum and MLH1 protein-specific binding bands clear.To provide a molecular and antibody basis for later studies of its role in mismatch repair function.By designing silencing plasmids psi-U6-MLH1 shRNAl,psi-U6-MLH1 shRNA2,psi-U6-MLH1 shRNA3,psi-U6-MLH1 shRNA4 and control plasmid NC shRNA for transfection,the MLH1 gene silent cell model was successfully established,the expression of the indicated transfected green fluorescent protein was observed using fluorescence inverted microscopy,the proliferative capacity of DF-1 cells was detected by CCK8 method,the MLH1 mRNA transcript level was detected by qRT-PCR technique,and the cell cycle and apoptosis changes were analyzed by flow cytometry.The results showed that the MLH1 mRNA transcript level detected by qRT-PCR was the best time point of silencing effect 48h after transfection,mRNA transcript was effectively suppressed,psi-U6-MLH1 shRNA 4 and psi-U6-MLH1 shRNA 3 had significant suppressive effect on MLH1 gene(2-ΔΔCT:0.25±0.01 and 2-ΔΔCT:0.53 ± 0.05):fluorescence inverted microscopy observation indicated high expression of transfected green fluorescent protein,proliferation ability of DF-1 cells was slightly suppressed after silencing MLH1,cell cycle was blocked at G0/G1 stage,and apoptosis occurred.A cell damage model was established by UV radiation or cisplatin drug induction,and the effects of DNA damage on DF-1 cells were analyzed by applying CCK8 method,qRT-PCR technique,flow cytometry,etc.The results showed that cisplatin induction had little effect on the mRNA transcript level of MLH1 gene and cell proliferation ability was not significant;UV radiation-induced DNA damage increased the mRNA transcript level of MLH1 gene in DF-1 cells significantly(P<0.05)and cell proliferation ability was inhibited significantly(P<0.05),and cell cycle G2 blockade appeared and apoptosis was not significant;silencing the MLH1 gene and then UV radiation induction increased the mRNA transcript level of MLH1 gene in DF-1 cells and at the same time increased cell cycle G1 blockade and cell apoptosis.In summary,we have a preliminary understanding of the function of the chicken mismatch repair-related gene MLH1,which causes cell blockade at G0/G1 phase and apoptosis when MLH1 expression is reduced;after DNA damage,the MLH1 gene participates in the DF-1 cell cycle regulatory process by generating the repair link of G1 phase blockade effect.