Identification Of Novel Isoforms Of The Boule Gene And Their Function Study In Goat

As a conserved gene during meiosis, Boule encodes RNA-binding protein being requiring for sperm meiosis and the deletion of Boule gene is responsible for meiotic arrest of sperms and male sterility. Boule gene alternative splicing（AS） have been found in human,cattle and bats, and its methylation has also been shown to link with male sterility of cattle-yak. However, the studies on alternative splicing and their functions, single nucleotide polymorphism（SNP） and methylation of the Boule gene have not been reported in goat.Therefore, this study focused on Xinong Saneng dairy goat and other goat breeds, using DNA sequencing, quantitative real-time polymerase chain reaction（q RT-PCR）, bisulfite sequencing PCR（BSP） and other technologies, combining bioinformatics and identified the Boule gene transcripts and their m RNA expression patterns, and analyzed the relationship between Boule gene novel transcripts with genetic variation and methylation in promoter region. In addition,over expression vector of different transcripts was constructed and transfected to model animal of the mouse germ cells to study the effects of different transcripts on meiosis related-genes, which could provide a scientific information reference for further study on regulation mechanism on Boule gene as well as benefit for the goat genetics, breeding and reproduction.1. Identification and m RNA expression analysis of the novel transcripts of the Boule gene in goatSince the Boule gene was specific expressed in testis, the testis cDNA pool of Xinong Saneng dairy goat was used as template to amplify CDS region of the Boule gene. A total of three novel transcripts were identified, named Boule-a, Boule-b and Boule-c. Compared to the reference sequence（NC₀22294）, Boule-a lacked exon 7 and exon 8; Boule-b lacked exon 8;and Boule-c retained only the exon 1 and exon 2. The q RT-PCR showed that different transcripts differently expressed in the testis, and the expression of Boule-a had a significant difference with Boule-b and Boule-ab and Boule-b might be the main transcript form of the Boule gene.2. The relationship between mRNA expression of novel transcripts and genetic variation with the Boule gene in GoatIn this experiment, the individuals from Xinong Saanen（XNSN） dairy goat, Guanzhong（GZ） dairy goat, Hainan（HN） black goat, and Inner Mongolia white Cashmere（IMWC）goat breeds were chosen as experimental subjects. Herein, two new SNPs loci were found in goat Boule gene, which located in intron 2（NC₀22294:g.7243） and intron 5（NC₀22294:g.12350） respectively. These SNPs had significant impacts on production traits（P<0.05）. They could be required as the molecular markers for the early selection breeding of goats. At the same time, DNA sequencing was used for genotyping Boule SNPs loci of testicular samples and the relationship between m RNA expression of different transcripts and genetic variation was analyzed. The results showed that, SNP3-P5（NC₀22294:g.12350）locus signifivantly affected the total m RNA expression of Boule-ab（P<0.05）.3. The relationship between m RNA expression of Boule novel transcripts and promoter methylation in goatThe promoter of Boule gene was in high methylation status in testis（above 90%）.Correlation analysis showed the promoter overall methylation level and the total m RNA expression of all transcripts was negatively correlated. Cp G3 and Cp G7 loci were significantly correlated with the total m RNA expression level Boule-ab of Boule-a and Boule-b（P<0.01）.4. The effect of goat Boule gene novel transcripts on expression patterns of meiosis related genes in male mice GC-1 cellThe over-expression vectors of CD513B-Boule-a, CD513B-Boule-b and CD513B-Boule-c were constructed and transfected to GC-1 germ cells of model mouse to analyze the expression level of Boule gene different isoforms and meiosis-related genes. Due to the low transfection efficiency of the over-expression vector of Boule-b transcript, only over-expression of Boule-a and Boule-c transcripts were analyzed. The results showed that after transfection of CD513B-Boule-a and CD513B-Boule-c, the expression abundance of CDC2 gene was significantly down-regulated（P<0.01）, implying that these two transcripts may inhibit meiosis.