Cloning And Expression Study Of ZAR1 And GDF9 Gene In New Zealand White Rabbits

Maternal effect genes(MEG) were generally considered to express specifically in ovarian tissue and oocyte, and now has been confirmed to play important roles in the process of maternal zygotic transition(MZT),the early development of mammal follicles and embryos.ZAR1 and GDF9 genes,as the minority members of MEG, has been reported in the species like mice, rats, frogs, zebrafish, cattle, sheep and pigs.ZAR1 not only affect the growth of oocytes and the early development of embryos,but also has been proved that some locus mutations on this gene was significantly associated with the reproduction traits such as litter size. And GDF9 gene,as a member of the super-family of TGF-β, has been confirmed to be a endogenous cell factor of the mammal ovarian, and be important in regulating the development of follicle and the process of oocyte maturation.What’s more, because its key role in the reproductive process of mammals,it nowadays has been the hot spot of the animal reproduction biology research. But at present there were still only a few researches of these two genes about rabbit at home and abroad,and the mechanism of these two genes was still not understood thoroughly.Basing on the above situation and the previous work of the laboratory,in this research,we cloned the sequences of the ZAR1 and GDF9 genes in the New Zealand white rabbits,and bioinformatics software were used to predict and analyze the molecular structure and characteristics of the two genes.As well as, in order to clarify the action mechanism and the tissue expression laws in the gene transcription levels,the method of relative real-time PCR was adopted to measure the m RNA levels of the two genes in the heart, liver, spleen, lung, kidney, ovary and uterus tissues respectively of New Zealand white rabbits with high and low reproductive traits.Through the above methods,the test results were as follows:1、According to the published rabbit ZAR1 and GDF9 m RNA sequences on NCBI, specific primers were designed. And finally three sequences of the ZAR1 exon2-exon4、exon4 and 3 ’flanking region、exon1-exon2 were got from New Zealand white rabbit kidney:357,338,141 bp respectively.In fact,a total number of 709 bp were gotten after splicing and integration.As for GDF9,two sequences were got from the New Zealand white rabbit liver:the 5 ’flanking region and exon 1、part sequences of the exon 2:796 bp and 614 bp,respectively.2、Bioinformatics and related softwares were used to analyze the ZAR1 nucleotide sequences genehomology of rabbit with the other species.The BLAST analysis for cloned ZAR1 gene indicated that the identities to the released rabbit ZAR1 gene were100%; the identities to the people,cattle, sheep and pig were91%, 88%, 88%and 87%, respectively;the identities to the mice and rat were 86%,87%;and the identities to the zebrafish and xenopus laevis were 76% and 82%,respectively.As for the translated amino acids sequences of the cloned ZAR1 gene,its identities to the released rabbit, people,mice and rats amino acid sequences were 100%, 84%, 93%, 94%, respectively;its identities to the cattle and pig were both 97%;and its identities to the zebrafish and xenopus laevis were 89% and 96%,respectively.3、Bioinformatics and related softwares were used to analyze the GDF9 nucleotide sequences genehomology of rabbit with the other species.The BLAST analysis for cloned GDF9 gene indicated that the identities to the released rabbit GDF9 gene were99%; the identities to the people,cattle, monkey, sheep and pig were79%, 79%, 77%, 76% and 75%, respectively;the identities to the mice and rat were both 72%;and the identities to the zebrafish and frogs were 68% and 73%,respectively.As for the translated amino acids sequences of the cloned GDF9 gene,its identities to the released rabbit, people, monkeys, mice, rats and pigs amino acid sequences were 97%, 71%, 72%, 66%, 65%, 69%, respectively;its identities to the cattle and sheep were both 68%;however,it showed lower identities to the zebrafish(43%) and frog(49%).4、The method of semi-quantitative RT-PCR was used to detect the different tissue expression levels of the ZAR1 and GDF9 gene in the heart, liver, spleen, lung, kidney, ovary and uterus.Results showed that,there were m RNA expression of the two genes in all of the above organizations,which indicated that the ZAR1 and GDF9 has no expression specificity in New Zealand white rabbits.5、 In the New Zealand white rabbits with high and low reproductive traits,ZAR1 genes were expressed the highest in the lung tissue, the expression in the spleen and kidney took the second place, and the expression in the heart tissue was lowest; GDF9 gene were expressed the highest in ovarian and liver tissue, and lowest in the heart and spleen.6、The relative m RNA expression in the same tissue but in different yield New Zealand white rabbit showed that:the gene expression of ZAR1 was highly significant in the liver, kidney and ovary(P < 0.01), and significant in the heart and uterus(P < 0.05),however,there were no significant differences in expression of the spleen and lung(P > 0.05);When it comes to GDF9,the gene expression was highly significant in liver and uterus(P < 0.01),and significant in the heart, spleen and ovarian tissue(P < 0.05), however,there were no significant differences in expression of the lung and kidney(P > 0.05).7、The expression differences between ZAR1 and GDF9 gene in New Zealand white rabbit showed that: GDF9 expression levels were always significantly higher than that of ZAR1 respectively in the heart, liver, kidney, ovary and uterus tissues.