水牛SND1基因克隆及生物信息学分析

试验旨在利用电子克隆法对水牛SND1（staphylococcal nuclease and tudor domain containing 1）基因进行克隆和序列分析，为探究该基因对水牛泌乳性能的作用机制奠定基础。以奶牛SND1基因（GenBank登录号：NM_205784.1）作为种子序列，利用Primer Premier 5.0设计3对引物，以水牛基因组DNA为模板，PCR扩增水牛SND1基因mRNA序列，扩增获得序列连接pMD18-T载体，通过测序拼接获得水牛SND1基因mRNA全序列，并对其进行生物信息学分析。结果显示，水牛SND1基因完整编码序列长为3 503 bp，包含长为2 733 bp CDS序列，编码910个氨基酸，蛋白分子式为C₄₅₂₃H₇₁₈₃N₁₂₈₁O₁₃₅₄S₂₆，分子质量为102.01 ku，理论等电点（pI）为6.74，不稳定系数为42.07，平均亲水性为-0.419，属可溶酸性蛋白。二级结构以α-螺旋和无规则卷曲为主，其中α-螺旋占37.80%，无规则卷曲占34.18%。结合Protfun 2.2在线软件对SND1的功能进行预测分析表明，该蛋白在嘌呤和嘧啶、中央中间代谢、能量代谢、氨基酸生物合成发挥功能的可能性分别为0.449、0.401、0.303和0.262。SND1基因编码区序列与黄牛、绵羊、山羊、猪、马、人的同源性分别为98.7%、97.8%、97.8%、93.8%、93.1%和91.7%，物种之间同源性较高，系统进化情况与其亲缘关系远近一致。利用SMART在线软件预测蛋白结构域，结果显示，水牛SND1蛋白包含有4个SN区域，说明SND1基因编码区在进化过程中较为保守。

Cloning and Bioinformatics Analysis of SND1 Gene in Buffalo

This study was aimed to clone and sequence the buffalo staphylococcal nuclease and tudor domain containing 1 (SND1) gene using the electronic cloning method,and analyze its genetic struction with bioinformatics,which laid a foundation for investigating the effect of SND1 gene on the milk performance in buffaloes.In this experiment,three pairs of specific primers were designed by Primer Premier 5.0 according the sequence of Bos taurus SND1 gene in GenBank (accession No.NM_205784.1),the buffalo DNA was used as the template,the sequence of buffalo SND1 gene mRNA was ampified by PCR,the target fragment linked into the cloning vector pMD18-T was sequenced and systemically analyzed by bioinformatics.The results showed that the length of SND1 gene mRNA was 3 503 bp,containing a CDS of 2 733 bp,which encoded 910 amino acids.The protein molecular formula was C₄₅₂₃H₇₁₈₃N₁₂₈₁O₁₃₅₄S₂₆, the molecular weight was 102.01 ku,the theory isoelectric point was 6.74,the instability coefficient was 42.07,grand average of hydropathy was -0.419,SND1 was the soluble acid protein.The secondary structures prediction of buffalo SND1 protein showed that alpha helices and random coils accounted for 37.80% and 34.18%,which were the main structure of the protein.Gene function prediction showed that the possibility of the gene function in the purine and pyrimidine,central intermediary metabolism,energy metabolism,biosynthesis of amino acids were 0.449,0.401,0.303 and 0.262,respectively.Sequence homology analysis indicated that the buffalo SND1 showed 98.7%,97.8%,97.8%,93.8%,93.1% and 91.7% identity with those of Bos taurus,Ovis aries,Capra hircus,Sus scrofa,Equus caballus and Homo sapiens,respectively.Sequence analysis revealed that buffalo SND1 gene was conserved,and showed high sequence similarity with other species.Online predicting tool SMART was used to predict protein structure domain,it showed that buffalo SND1 protein contains four SN area.This results suggested that the SND1 gene CDS was more conservative in the evolution process.