牦牛GDF9和BMP15基因的克隆、表达分析及miRNA研究

Cloning,Expression Analysis and mi RNA Study of Gene GDF9 and BMP15 in Yak

以牦牛GDF9和BMP15基因为研究对象,采用PCR方法分别克隆出GDF9和BMP15的两个外显子片段,RT-PCR方法分析了GDF9和BMP15及靶定mi RNA在各组织中的表达情况。实验结果显示,克隆扩增获得外显子区进行生物信息学分析。牦牛GDF9基因外显子片段长分别411 bp和1 082 bp,编码453个氨基酸;BMP15基因外显子片段长分别为323 bp和802 bp,编码372个氨基酸。GDF9编码蛋白是亲水蛋白,含有一个信号肽和15个潜在磷酸化位点;BMP15编码蛋白是亲水蛋白,不含信号肽,有6个潜在磷酸化位点。通过实时荧光定量PCR方法分析牦牛GDF9和BMP15基因在不同组织中的表达量,结果表明GDF9在卵巢和子宫中表达相对较高,且卵巢表达显著高于其他组织,心、肾、胰和脾中无表达。BMP15仅在卵巢中表达。Target Scan预测靶定牦牛GDF9和BMP15基因的靶定mi RNA,分析GDF9靶基因bta-mi R-193a-3p和bta-mi R-193b在心、脾、肺、肾和子宫中的表达量,结果显示bta-mi R-193a-3p脾中表达量显著高于其他组织,但在心、肺和子宫中均不表达。bta-mi R-193b在心和肾中没检测到表达,在肺中的表达量显著低于脾和子宫中。

Gene GDF9（growth differentiation factor 9）and BMP15（bone morphogenetic protein 15）of yak were used as research subject;then 2 exon fragments of GDF9 and BMP15 were cloned by PCR, and the expressions of GDF9 and BMP15 as well as targeted mi RNA were analyzed by RT-PCR. The results were as follows. The cloned and amplified exon fragments were analyzed by bioinformatics;the exons of yak gene GDF9 were 411 bp and 1 082 bp, encoding 453 amino acids;the lengths of exons in yak gene BMP15 were 323 bp and 802 bp separately, encoding 372 amino acids. The protein encoded by GDF9 was hydrophilic, containing one signal peptide and 15 potential phosphorylation sites;the protein encoded by BMP15 was also hydrophilic, and owing no signal peptide and 6 potential phosphorylation sites. The expressions of GDF9 and BMP15 in different tissues were examined by RT-PCR. The results showed that GDF9 expressed relatively high in ovary and uterus, and higher in ovary than others tissues, and no expression in heart, kidney, pancreas, and spleen;BMP15 was only detected in ovary. With Target Scan, the target mi RNA of yak gene GDF9 and BMP15 were predicted and the expressions of target gene bta-mi R-193a-3pand bta-mi R-193 b of GDF9 in heart, spleen, lung, kidney and uterus were analyzed. mi RNAs genes were examined with RT-PCR;the results indicated that bta-mi R-193a-3p in the spleen expressed significantly higher than in others tissues, and no expression in heart, lung and uterus;bta-mi R-193 b expressed lower in lung than spleen and uterus, and no expression was detected in heart and kidney.