中国桔梗PgF3’5’H及其突变型基因在毕赤酵母中的表达差异分析

Differential Expression Analysis of Platycodon grandiflorus PgF3’5’H and Its Mutant Gene in Pichia pasteris

F3’5’H是催化合成蓝色花色素的关键酶,F3’5’H蛋白序列中一段编码9个氨基酸的特殊序列区段可能与F3’5’H催化合成的蓝色花色素有关,但该特殊序列区段的催化功能和作用机制尚不清楚。为了探究PgF3’5’H中,该特殊序列结构的催化功能及作用机制,本研究通过重叠延伸PCR技术去除PgF3’5’H编码的9个氨基酸特殊序列区段,获得缺失突变型基因;将PgF3’5’H野生型及其突变型基因同时导入毕赤酵母;通过qRT-PCR检测野生基因及其突变基因在酵母中转录水平基因表达量的差异;通过SDS-PAGE检测PgF3’5’H野生型基因及其突变型基因在酵母中的合成蛋白量的差异。qRT-PCR分析结果显示PgF3’5’H野生型基因较其突变型在酵母中呈现更高的表达量;SDS-PAGE电泳结果显示重组子分泌生成了分子量大小约为52.0 k D的目标蛋白,且野生型蛋白量相比其突变型蛋白量高。因此,推测Pg F3’5’H蛋白序列中9个氨基酸的特殊序列与调控该基因在RNA和蛋白水平的表达量有关,进而可能影响该基因的催化功能。研究结果将为观赏植物蓝色花色形成分子机制及蓝色花色分子育种提供一定研究基础。

Favonoid 3’,5’-hydroxylases(F3’5’H) is a key enzyme that catalyzes the synthesis of blue anthocyanin. A special sequence segment encoding 9 amino acids in the F3’5’H protein sequence may be related to the blue anthocyanin catalyzed by F3’5’H, but its true function and mechanism remains unclear. To investigate the function and mechanism of the PgF3’5’H special sequence structure, in this study, the special sequence segment encoding 9 amino acids of PgF3’5’H was removed by overlapping extension PCR, and its mutant gene was obtained. The PgF-3’5’H wild type and its mutated genes were simultaneously introduced into Pichia pasteris. qRT-PCR was used to detect the transcription level of wild genes and their mutated genes in yeast. Differences in the amount of protein synthesized by PgF3’5’H wild-type gene and its mutant gene in yeast were detected by SDS-PAGE. The qRT-PCR analysis showed that the PgF3’5’H wild type gene displayed a higher transcription level than the PgF3’5’H mutant gene in yeast. The molecular weight of the recombinant protein was about 52.0 kD analyzed by SDS-PAGE, and the expression of PgF3’5’H wild type gene in the recombinant strain was more than its Pg F3’5’H mutant gene. It was speculated that the Pg F3’5’H special sequence structure was related to regulate the gene expression at RNA and protein levels, which might affect the PgF3’5 ’H catalytic function. The results will provide a basis for the molecular mechanism of blue color formation and molecular breeding of ornamental plants.