杧果脱镁叶绿素酶基因MiPPH的克隆及植物表达载体构建

Cloning of MiPPH Gene and Construction of Plant Expression Vector from Mango(Mangifera indica)

叶绿素降解是果实色泽形成过程中一个主要的影响因素。脱镁叶绿素酶(pheophytinase, PPH)是叶绿素降解代谢的关键酶。本研究提取杧果果皮总RNA,采用RACE方法,克隆得到一个脱镁叶绿素酶基因,并对其进行了初步的生物信息学分析。序列分析表明MiPPH基因cDNA序列全长1 267 bp,开放阅读框为1 113 bp,可编码370个氨基酸,分子量为41.73 kD,等电点为5.22。使用NCBI上的Conserved domains分析MiPPH蛋白结构域,结果显示MiPPH含有PLN02578、Abhydrolase_1共2个结构域,其中PLN02578为保守域,其他物种PPH也都含有这个水解酶(Hydrolase)保守域,说明PPH蛋白有高度的保守性。通过BLAST上其他植物的氨基酸序列进行相似性比对,利用DNAMAN生成的系统进化树发现杧果MiPPH蛋白序列和与同为漆树科的开心果的PPH蛋白序列相似度较高,亲缘关系较近。利用定量PCR技术对不同品种中的MiPPH基因表达量进行分析,发现绿色‘桂七’品种表达量最高,而黄色‘金煌’品种表达量最低,黄色‘金煌’品种和红色‘贵妃’品种表达量相差不大。本试验成功构建pCAMBIA2300-GFP-MiPPH植物表达载体,通过农杆菌介导的烟草瞬时表达,亚细胞定位MiPPH主要定位于细胞核。本试验为后续进行PPH基因的功能验证提供试验基础。

Chlorophyll degradation is a major factor in fruit color formation.Pheophytinase(PPH)is a key enzyme in chlorophyll degradation.In this study,the total RNA of mango pericarp was extracted,and a pheophytinase(PPH)gene was cloned by RACE method,and the preliminary bioinformatics analysis was carried out.Sequence analysis showed that the full length of MiPPH gene cDNA sequence was 1267 bp,with an open reading frame(ORF)of 1113 bp and an open reading frame(ORF)of 1113 bp,which could encode 370 amino acids with molecular weight of 41.73 kD and isoelectric point of 5.22.Conserved domains on NCBI were used to analyze the domain of MiPPH protein.The results showed that MiPPH contained two domains,PLN02578 and Abhydrolase_1,among which PLN02578 was a conserved domain.PPH in other species also contained this hydrolase conserved domain,indicating that PPH protein was highly conserved.The amino acid sequences of other plants were compared by BLAST,and the phylogenetic tree generated by DNAMAN showed that the MiPPH protein sequence of mango was highly similar to that of PPH protein sequence of pistachio,and the phylogenetic relationship was close.Quantitative PCR was used to analyze the expression of MiPPH gene in different varieties.It was found that the expression level of green'Guiqi'was the highest,while that of yellow'Jinhuang'was the lowest,and that of yellow'Jinhuang'and red'Guifei'were similar.The plant expression vector pCAMBIA2300-GFP-MiPPH was successfully constructed.MiPPH was mainly localized in the nucleus by Agrobacterium-mediated transient expression in tobacco.This experiment provided experimental basis for the subsequent functional verification of PPH gene.