Preliminary Study On The Function Of α-S1-Casein Gene In Mammary Gland Of Dairy Goats

αS1-casein（Alpha S1 Casein,CSN1S1）is an important protein in milk protein.It can provide active peptides,essential amino acids and other nutrients for the growth and development of animal.It can also regulate the physiological state and function of many kinds of cells,such as mammary epithelial cells.Therefore,it is of great significance to explore the regulation of α S1-casein gene on milk protein synthesis in dairy goats.In this study,the CDS region of CSN1S1 gene of dairy goat was cloned,and the recombinant adenovirus overexpression vector of CSN1S1 gene was constructed.At the same time,the specific siRNA.targeting CSN1S1 gene was designed and synthesized on-line.The expression of CSN1S1 gene was overexpressed and interfered in goat mammary epithelial cells,and the expression of milk protein related genes and some milk proteins were detected by RT-qPCR and Western Blot,respectively.It lays a theoretical foundation for further clarifying the function of α S1 casein gene in milk protein synthesis.The following are the main findings of this study:1.The mammary gland tissue of Xinong Saanen dairy goat was collected and total RNA,was extracted from the breast tissue.The primers were designed according to the CSN1S1 gene sequence（KT253650.1）of Capra hircus in GenBank.The CDS sequence of CSN1S1 gene was cloned by RT-PCR technique.The length of the CDS sequence was 642 BP,encoding 213 amino acids.The homology of CSN1S1 CDS region between dairy goat and Ovis aries（FJ440845.1）,Bos taurus（EU908730.1）,Mus musculus（AF275367.1）,Sus scrofa（NM₀01004029.2）,Homo sapiens(NM₀01890.2 was 98.91%,97.83%,respectively.84.87%,92.52% and 85.69%.2.The homologous recombinant plasmid pAdEasy-CSN1S1,containing the target gene was constructed and transfected into 293 A cells.After four generations of infection and amplification,the high titre adenovirus was obtained.The titer of the recombinant plasmid was 108 U / ml ·L-1.When goat mammary epithelial cells were infected with different volume of virological solution,the optimal complex number of virus infection（MOI）was 200..The total RNA or total protein of goat mammary epithelial cells were collected 48 hours after virus infection.The results of RT-qPCR showed that the expression of CSN1S1 gene was up-regulated significantly（P<0.01）,the expression of CSN2 gene was significantly down-regulated（P<0.05）,and the expression level of CSN1S2,CSN3,LALBA,BLG was not significantly changed.Western blot results showed that α S1-casein expression was inhibited（P < 0.05）,β-casein expression was significantly up-regulated（P < 0.05）,and the expression of α s2-casein,κ-casein and β-lactoglobulin did not change significantly.3.According to the CDS region sequence of the CSN1S1 gene,the specific siRNA targeting the CSN1S1 gene is designed,and the goat mammary epithelial cell is transfected,and the siR-507 with the best interference efficiency is screened to obtain the siR-507（P <0.01）.After interfering with the CSN1S1 gene,the expression level of CSN2 gene was significantly up-regulated（P <0.05）,and the mRNA level of CSN1S2,CSN3,LBA and BLG gene was not significantly changed.The results of Western blot showed that the expression of α S1-casein was inhibited（P <0.05）,and the expression level of β-casein was up-regulated（P <0.05）.There was no significant change in the expression of s2-casein,κ-casein and β-lactoglobulin.In conclusion,we successfully cloned the CDS region of α S1-casein gene of dairy goats,constructed adenovirus overexpression vector,and infected goat mammary epithelial cells to over-express CSN1S1 gene.The mRNA expression level and protein expression of CSN2 gene were significantly down-regulated,and α S1-casein gene interference could up-regulate the expression of CSN2 gene and β-casein gene.This study provides a theoretical basis for the regulation of CSN1S1 gene in the synthesis of milk protein in the mammary gland cells of Xinong Saanen dairy goat.