白木香查尔酮异构酶基因的克隆鉴定与表达分析

Chalcone isomerase (CHI) is one of the key enzymes in the biosynthetic pathway of flavonoids and plays an important role in plant defense responses. In this study, based on the transcriptome sequencing results of Aquilaria sinensis and combined with RT - PCR technology, a CHI gene was cloned from the callus of Aquilaria sinensis for the first time and named AsCHI1. The open reading frame (ORF) of the AsCHI1 gene in Aquilaria sinensis is 654 bp long, encoding a protein containing 217 amino acids, and its protein molecular mass is 23.11 kDa. The AsCHI1 protein has a conserved active site of chalcone isomerase, and the phylogenetic tree shows that the AsCHI1 protein is a type I CHI protein and has a relatively close genetic relationship with the CHI protein of cotton (Gossypium hirsutum). The prokaryotic expression vector pET28a - AsCHI1 was constructed and AsCHI1 was successfully expressed in the E. coli BL21 (DE3) strain. The soluble AsCHI1 recombinant protein was purified by Ni~(2+) affinity chromatography. In vitro enzyme activity analysis proved that the recombinant protein AsCHI1 can catalyze the conversion of naringenin chalcone to naringenin. The results of real - time fluorescence quantitative PCR detection showed that after the callus of Aquilaria sinensis was induced by salt, mannitol, low temperature and heavy metal stress, the expression level of the AsCHI1 gene increased significantly; plant hormones abscisic acid, gibberellin and salicylic acid could all induce the expression of the AsCHI1 gene in the callus, indicating that AsCHI1 plays a role in the self - defense response of Aquilaria sinensis. The results of this study provide a reference for further exploring the biosynthesis of flavonoids in Aquilaria sinensis and their role in the defense response of Aquilaria sinensis.

查尔酮异构酶(chalcone isomerase, CHI)是黄酮类成分生物合成途径中的关键酶之一,在植物防御反应中发挥重要作用。本研究根据白木香转录组测序结果并结合RT-PCR技术首次从白木香愈伤组织中克隆得到1个CHI基因,命名为AsCHI1。白木香AsCHI1基因的开放阅读框(ORF)长654 bp,编码蛋白含217个氨基酸,其蛋白分子质量为23.11 kDa。AsCHI1蛋白具有查尔酮异构酶保守的活性位点,系统进化树显示AsCHI1蛋白为I型CHI蛋白,与棉花(Gossypium hirsutum) CHI蛋白亲缘关系较近。构建原核表达载体pET28a-AsCHI1并在E. coli BL21 (DE3)菌株中成功表达AsCHI1,利用Ni~(2+)亲和色谱纯化得到可溶性AsCHI1重组蛋白。体外酶活性分析证明重组蛋白AsCHI1可以催化柚皮素查尔酮转化为柚皮素。实时荧光定量PCR检测结果表明白木香愈伤组织经盐、甘露醇、低温以及重金属胁迫诱导后, AsCHI1基因的表达量明显上升;植物激素脱落酸、赤霉素和水杨酸均能够诱导愈伤组织中AsCHI1基因表达,说明AsCHI1在白木香自我防御反应中发挥作用。本研究结果为进一步探讨白木香中黄酮类成分的生物合成及其在白木香防御反应中的作用提供参考。