双角山羊草紫色酸性磷酸酶PAP1基因的克隆及生物信息学分析

摘要

通过对双角山羊草紫色酸性磷酸酶 PAP1基因的核酸和氨基酸序列进行相关生物信息学分析，旨在进一步利用基因工程手段将 AeBiPAP1基因转入到小麦中，为获得耐低磷胁迫能力增强的小麦品种提供种质基因。以双角山羊草叶片为材料，根据小麦中的PAP1基因和二穗短柄草的PAP1基因设计兼并引物，采用同源克隆的方法，获得了双角山羊草紫色酸性磷酸酶 PAP1基因的 cDNA序列，并将其命名为 AeBiPAP1，该双角山羊草紫色酸性磷酸酶 PAP1基因长1.124 kb，共编码335个氨基酸，相应的氨基酸分子量为38.131 kDa，等电点为5.77。与小麦中的 PAP1基因相比，双角山羊草 PAP1的 cDNA编码区发生碱基替代的地方共有35处，包含13处颠换与22处转换，有15处编码的氨基酸残基不同，其序列一致性达95.52%。对 AeBiPAP1基因编码的蛋白质进行生物信息学分析发现，该蛋白具有一个信号肽但却没有跨膜结构，对 AeBiPAP1编码蛋白进行三级结构预测发现，该编码蛋白包含金属离子结合中心，预测它属于金属蛋白。因此，将其定位于质膜比分泌到胞外的概率要高，通过对同源蛋白质氨基酸序列进行系统进化树分析，结果表明，双角山羊草紫色酸性磷酸酶 PAP1基因与普通小麦的亲缘关系最近，与粳稻、短柄草、谷子亲缘关系较近，与大麦、乌拉尔图小麦、水稻以及节节麦等植物的亲缘关系较远。%This work would help for wheat breeding that we could get some low phosphorus tolerance germplasm resources via the methods of the genetic transformation of AeBiPAP1 into wheat cultivals. In this study,PAP1 gene was cloned from Aegilop bicornis with designed primers according the sequence of PAP1 genes from wheat and slender falsebrome based on homologous cloning method and named AeBiPAP1. The cloned AeBiPAP1 gene was 1. 124 kb in size and encoded 335 amino acids,with molecular weight of 38. 131 kDa and isoelectric point 5. 77. Sequence alignment of AeBiPAP1 with PAP1 of wheat showed that there were 35 single nucleotide polymorphisms,22 locus transitions and 13 locus transversion and AeBiPAP1 had high homology to PAP1 of wheat with the identities of 95. 52%. Amino acid sequence alignment showed that 15 deduced amino acids of AeBiPAP1 were different from PAP1 of wheat. Bioinformatics analysis of AeBiPAP1 gene encoding protein found that the protein has a signal pep-tide but have not the across-membrane structure and the AeBiPAP1 coding protein with tertiary structure prediction found that the encoded protein contains the metal ions center predicted it belongs to the metal protein. Besides,its possibility of localization in the plasma membrane is higher than being secreted to outside of the cell,the phylogenic tree results showed that PAP1 gene from Aegilop bicornis shared high similarities to the previously reported PAP1 genes from other plants,such as Triticun aestivun,Brachypodiun distachyon,and Setaria italica,among them,AeBi-PAP1 gene shared the highest similarities to PAP1 gene from Triticun aestivun.