芒果GGPPS基因的克隆与表达分析

Cloning and Expression Analysis of GGPPS Gene from Mango (Mangifera indica)

在植物中,异戊烯焦磷酸(GGPP)是赤霉素、胡萝卜素、叶绿素等异戊二烯类化合物的公共前体物质。从芒果栽培品种贵妃芒的新鲜叶片中提取DNA和RNA,根据转录组测序拼接结果设计引物扩增得到香叶基香叶基焦磷酸合成酶基因(Min GGPPS1)。通过测序、多重比对和聚类分析,证实该片段属于GGPPS的全长基因并且为最大的ORF,全长为1 100 bp。与芒果Min GPS2(AFJ52722.1)氨基酸序列同源性为92%。蛋白质序列分析表明该序列含有两个DDx_(2-4)D保守基元(FARM和SARM)基元和一个Cxxx C基元。我们选取了拟南芥中12个GGPPS基因、2个FPPS基因、2个SPPS基因、一个GPS基因及其他物种的相关基因,通过聚类分析表明,Min GGPPS1属于香叶基香叶基焦磷酸合成酶大亚基。表达分析表明:Min GGPPS1在叶片中表达量最高,茎部表达量最低,青果果皮和成熟果果皮中表达量相似,在成熟果肉中的表达量大约是青果果肉表达量的5倍,暗示Min GGPPS1具有调控果肉后熟的功能。

Geranylgeranyl diphosphate（GGPP） is the precursor for the biosynthesis of gibberellins, carotenoids,chlorophylls, isoprenoid quinones and geranylgeranylated proteins in plants. A Min GGPPS1 gene was cloned from mature mango fruit by using c DNA as template through transcriptome RNA-Seq. Through sequencing, multiple alignment and phylogenetics analyses, we found the sequence was likely a GGPPS gene and predicted coding region is the largest ORF, with the full-length c DNA as 1 100 bp. Protein sequence comparison indicated that Min GGPPS1 is closely related（92%） to the Min GPS2 of Mangifera indica. We selected 12 GGPPS genes, 2 FPPS genes, 2 SPPS genes, and 1 GPS gene in Arabidopsis thaliana as well as other related genes. BLAST comparisons of available GGPPS, FPPS, and GPS sequences revealed the presence of two DDx2-4D motives and a conserved Cxxx C motif in each GGPPS sequences. It was found that the protein encoded by the gene had close relationship with GGPPS through Mrbayes analysis. Expression of Min GGPPS1 gene in different tissues and organs are as follows： the high expression of green leaves, and the lower expression in stems, similar amount between unripe and ripe peels. The expression in the ripe flesh was almost five times as much as it was in the unripe flesh, which indicates that GGPPS has the function of regulating flesh ripening.