水牛DDR1基因生物信息学及细胞学功能分析

Bioinformatics and cytological functional analysis of buffalo DDR1 gene

为了了解水牛DDR1蛋白的生物信息学和细胞学功能,试验采用ProtParam、ProtScale、SOPMA、SWISS-MODEL、PSORTⅡPredictio、NetPhos 3.1在线软件对水牛DDR1蛋白进行生物信息学分析,采用基因干扰的方法研究水牛DDR1基因对水牛乳腺上皮细胞增殖、凋亡、基因表达和迁移的影响。结果表明:水牛DDR1蛋白由915个氨基酸组成,分子式为C_(4538)H_(7029)N_(1261)O_(1287)S_(43),分子量为101222.99 u,半衰期为30 h,理论等电点为6.22,属于酸性、不稳定、可溶性蛋白质。DDR1蛋白的二级结构由256个α-螺旋(27.98%)、175个延伸链(19.13%)、55个β-折叠(6.01%)、429个无规则卷曲(46.89%)构成。亚细胞定位于内质网(44.4%)、高尔基体(33.3%)和质膜中(22.2%)中。DDR1蛋白共含有65个磷酸化位点,其中丝氨酸磷酸化位点35个,苏氨酸磷酸化位点18个,酪氨酸磷酸化位点12个。DDR1基因的最佳干扰片段为siDDR1-1,最佳干扰时间为48 h。DDR1基因干扰72小时时可极显著抑制水牛乳腺上皮细胞的增殖(P<0.01)。DDR1基因干扰显著或极显著上调凋亡相关基因BCL-2(P<0.01)、XIAP(P<0.05)、TP53(P<0.01)和增殖相关基因CCND1(P<0.01)的表达,下调凋亡相关基因CASPASE3(P<0.05)和CCDB1(P>0.05)的表达,但对细胞凋亡率的影响不显著(P>0.05)。此外,DDR1基因干扰可极显著降低水牛乳腺上皮细胞的迁移率(P<0.01)。说明DDR1基因干扰可极显著影响水牛乳腺上皮细胞的增殖和迁移。

In order to further understand the biological and cytological functions of buffalo DDR1 protein, for in this study, ProtParam, ProtScale, SOPMA, SWISS-MODEL, PSORT Ⅱ Predictio and NetPhos 3.1 online analysis softwares were used to bioinformatics analysis. Meanwhile, the effects of DDR1 gene on proliferation, apoptosis, gene expression and migration in buffalo breast epithelial cells were studied by gene interference method. Results showed that buffalo DDR1 protein was composed of 915 amino acids with a formula of C_(4538)H_(7029)N_(1261)O_(1287)S_(43), molecular weight of 101 222.99 u, half-life of 30 h, and theoretical isoelectric point of 6.22, which was an acidic, unstable and soluble protein. The secondary structure of DDR1 protein consisted of 256 alpha helixes(27.98%), 175 extended strands(19.13%), 55 beta turns(6.01%), and 429 random coils(46.89%). Subcells were localized in the endoplasmic reticulum(44.4%), golgi apparatus(33.3%), and plasma membrane(22.2%). The DDR1 protein contained a total of 65 phosphorylation sites, including 35 serine phosphorylation sites, 18 threonine phosphorylation sites, and 12 tyrosine phosphorylation sites. The optimal interference fragment of the DDR1 gene was siDDR1-1, and the optimal interference time was 48 hours. At 72 hours of DDR1 gene interference, the proliferation of buffalo mammary epithelial cells was significantly inhibited(P<0.01). DDR1 gene interference significantly up-regulated the expression of apoptosis-related genes(BCL-2[P<0.01], XIAP[P<0.05], and TP53[P<0.01]) and proliferation-related gene CCND1,and down-regulated the expression of apoptotic-related genes(CASPASE3[P<0.05] and CCDB1[P<0.05]), but had no significant effects on apoptosis(P>0.05). In addition, cell migration in the interfering group was significantly reduced(P<0.01). The results indicated that DDR1 gene interference could affect the proliferation and migration of buffalo mammary epithelial cells.