| Water buffalo is an important local species in South China, which have strong characteristics of resistance and resistance to crude feed. In addition, buffalo milk has a higher protein and fat percentage rate as compared to Holstein cow milk which marks that buffalo has a great potential in meat and dairy, etc. However, the deficiencies of lower reproductive performance in buffalo have restricted to be further developed. Inhibin(INH) as one of the important reproductive hormones in body, and its main function is to inhibit Follicle-stimulating hormone(FSH) synthesis and secretion through negative feedback to affect the ovarian development, follicular maturation and spermatogenesis and maturity. From now on, many studies have been carried out to study individual INH gene immunity to enhance animal fertility of pigs and mice.In order to improve and enhance the reproductive performance of water buffalo by interfering INH α gene, this study utilized transgenic mice as research materials which buffalo INH α gene RNAi(fragment B and C) and mouse INH α gene RNAi(fragments of M and C, where fragment C was focused on the homologous CDs sequences of buffalo and mice) were integrated into the genome by micro-surgical fertilization and transgenic technology to analyze the silencing effect and influence on reproductive performance after interfering INH α gene of three different RNAi fragments, the results will be used for the functional study of INH α gene and improve reproductive performance. The main results are as follows: 1. Comparative analysis of interference efficiency of three different INH α gene RNAi fragments(1) Interference efficiency analysis of INH α gene RNAi fragments: three specific INH α gene RNAi fragments(designated as B, C, and M) were injected into female fertilized egg by using micro-injection technique(with company assistance) to obtain transgenic mice, it was detected by PCR method to confirm positive mice. The efficiency of three interference fragments showed to be the efficiency of C(96.1%)> M(75.1%)>B(27.5%) fragment in three-week-old transgenic positive female mice in F3 generation, this may due to C fragment was based on the common CDs of buffalo and mice INH α gene sequences and has a strong interference of transcription target sites;(2) The positive rate analysis of INH α gene RNAi fragments: extracted individual genomic DNA of F3 generation, using PCR to analyze the positive rate of transgenic offspring mice, the positive rates of B, C and M fragments were 81.3%, 51.1% and 77.6% in F3 generation of transgenic mice, graded as B> M> C;(3) The litter size analysis of INH α gene RNAi fragments: statistical analysis three successive generations of the first litter size of different interference segments transgenic mice, compared with wild-type, the litter size in transgenic mice has increased and the performance of B and M fragments in F1, F2 generation increased, but the significant was not(P>0.05); Besides, compared with the litter size of different interference fragments in multi-parity, the litter size was found to decline in transgenic groups with the parity increased but lower than wild-type mice;(4) The reproductive hormones, gonadal growth and development analysis of INH α gene RNAi fragments: ELISA method to detect the FSH, estrogen and androgen levels in transgenic mice blood, and these three hormone levels have risen both; besides, compared to wild-type mice, the testes, ovaries and birth weight of three-week-old transgenic mice were significantly gained in F3 generation transgenic mice(P<0.05); 2. Comparative analysis of different mating groups carrying B interference fragment:Based on the above results, choose a high genetic stability, good reproductive performance and interference fragment designed according to buffalo INH α gene sequences and mated in different groups(B×B, B×WT and WT×WT as control).(5) The interference efficiency analysis of INH α gene RNAi fragments in different mating groups: the interference efficiency of B×WT mice was lower than homozygous offspring mice carrying B×B fragments, but the interference efficiency all reached 70%, in addition, the positive rate was higher than its homozygous offspring mice;(6) The mice survival rate and immune-related blood indicators analysis of INH α gene RNAi fragments in different mating groups: Survival rates in transgenic mice(B×B, B×WT) were higher than wild-type; blood analysis showed that monocytes MO reduced and the number of lymphocytes LY increased, while the number of red blood cells RBC increased significantly(P<0.05);(7) The apoptosis analysis of testicular sertoli cells of INH α gene RNAi fragments in different mating groups: Using flow cytometry to detect the apoptosis of Sertoli cell and found that the apoptosis rate of Sertoli cells(5.4%) in transgenic mice(B×B) was significantly higher than that of wild-type mice(1.46%), q PCR detected the factor-related apoptosis P53 and Bcl2 and showed that both m RNA levels were significantly down-regulated, but anti-apoptotic factor Bcl2 decreased more than pro-apoptotic factor P53;(8) The gene expression analysis of INH α gene RNAi fragments in different mating groups: the expression of osteopontin(OPN) in Sertoli cells was significantly increased in transgenic mice(B×B, B×WT)(P<0.05), while FSHR, receptor gene of FSH decreased; Cell assay showed that both genes located on Sertoli cell membrane and the existence of the interaction and its function requires further study;(9) Interfere with efficiency between 20 to 70% may improve the reproductive traits; between 70 to 80% may have little effect on the reproductive performance; interfere with efficiency of more than 80% may cause cancer and not conducive to improve reproductive performance. |
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