锦绣黄桃漆酶基因PpLAC7的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of PpLAC7 Gene from Jinxiu Yellow Peach

为克隆锦绣黄桃漆酶基因PpLAC7序列,分析PpLAC7基因序列信息及其在果肉褐变中的作用。以锦绣黄桃为试材,采用同源克隆方法从锦绣黄桃中获得漆酶基因PpLAC7的cDNA序列。采用生物信息学软件对PpLAC7基因的启动子元件、蛋白质理化性质、蛋白质二级、三级结构、系统发育进化树和同源氨基酸序列比对进行分析。此外还对PpLAC7基因进行亚细胞定位以及在果肉褐变过程中的表达规律进行了分析。生物信息学分析表明,PpLAC7的启动子区域含有光响应元件、茉莉酸响应元件、干旱响应元件、种子特异性调控等元件。PpLAC7基因全长为1692 bp,其蛋白质共编码563个氨基酸,分子质量为61.867 ku,总原子数为8621个,亲水系数为-0.028,理论等电点为5.95,不稳定指数为36.48,脂溶性指数为87.26。PpLAC7蛋白的α-螺旋、延长链、β-转角、无规则卷曲分别占氨基酸总数的14.74%,28.77%,6.22%,50.27%。进化树结果显示,PpLAC7基因与苹果MdLAC7基因具有较高的同源性,PpLAC7基因的氨基酸序列同样含有3个典型的铜离子结构域。亚细胞定位结果表明,PpLAC7定位于内质网。PpLAC7基因在锦绣黄桃果肉褐变过程中呈大量上调表达。结合锦绣黄桃果肉褐变指数与PpLAC7表达量的关系,推测PpLAC7可能在锦绣黄桃果肉褐变过程中起重要的作用。

To clone the PpLAC7 gene sequence of Jinxiu yellow peach,investigating its gene sequence information and its role in flesh browning.The cDNA sequence of PpLAC7 was cloned from Jinxiu yellow peach by homologous cloning.The promoter region elements,physicochemical properties,secondary and tertiary structure,phylogenetic tree and amino acid sequence alignment of PpLAC7 gene were performed bioinformatics by using bioinformatics software.In addition,it also investigated the subcellular localization and the expression pattern of PpLAC7 during the process of fresh browning in Jinxiu yellow peach.The results of bioinformatics analysis indicated that the promoter region included light responsiveness,MeJA-responsiveness,drought-inducibility,seed-specific regulation elements,etc.The full length of PpLAC7 gene was 1692 bp,encoding 563 amino acids,molecular weight was 61.867 ku,total number of atoms was 8621,GRAVY was-0.028,theoretical pI was 5.95,instability index was 36.48,aliphatic index was 87.26.The secondary structure of PpLAC7 protein mainly containedα-helix of 14.74%,extended strand of 28.77%,β-turn of 6.22%and random coil of 50.27%.Phylogenetic analysis showed that PpLAC7 had high similarity to MdLAC7.Deduced amino acid sequence indicated that PpLAC7 protein,the same as other species,also contained three typical copper ion domains.Subcellular localization analysis revealed that PpLAC7 was located in endoplasmic reticulum.The expression of PpLAC7 gene was up-regulated during the storage of Jinxiu yellow peach fruit.Combining the relationship of flesh browning index and PpLAC7 expression,it indicated that PpLAC7 may play an important role in the browning process of Jinxiu yellow peach fruit.