不同品种忍冬ANR基因克隆、表达模式及原核表达分析  

Cloning and expression analysis of ANR genes from different species of Lonicera japonica Thunb.

作　　者：余永亮[1] 鲁丹丹 谭政委 杨红旗[1] 李磊 许兰杰[1] 杨青 董薇[1] 安素妨 郭水柱 高松 梁慧珍[1] YU Yong-liang;LU Dan-dan;TAN Zheng-wei;YANG Hong-qi;LI Lei;XU Lan-jie;YANG Qing;DONG Wei;An Su-fang;GUO Shui-zhu;GAO Song;LIANG Hui-zhen(Henan Sesame Research Center,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Zhongjing Wanxi Pharmaceutical Co.,Ltd.,Nanyang 474550,China)

机构地区：[1]河南省农业科学院芝麻研究中心,河南郑州450002 [2]仲景宛西制药股份有限公司,河南南阳474550

出　　处：《药学学报》2023年第11期3449-3460,共12页Acta Pharmaceutica Sinica

基　　金：河南省重大科技专项(221100310400);河南省科技攻关项目(222102110379,232102110198,222102110466,232102110243,232102110262);中央本级重大增减支项目(2060302);河南省农科院自主创新专项基金(2023ZC083);财政部和农业农村部:国家现代农业产业技术体系资助(CARS-21);河南省农科院新兴学科发展专项(2023XK03)。

摘　　要：花青素还原酶(anthocyanidin reductase,ANR)是黄酮合成途径中的关键酶,该酶的催化活性对花青素含量有重要的调控作用。本研究根据忍冬转录组数据,设计特异性引物,克隆忍冬和红白忍冬ANR基因的CDS、gDNA及启动子序列。结果表明,从忍冬和红白忍冬克隆得LjANR和rLjANR的CDS序列均为1002 bp,gDNA序列分别为2017和2026 bp,启动子序列分别为1170和1164 bp。LjANR和rLjANR均含有6个外显子和5个内含子,外显子长度一致,内含子差异较大,二者启动子序列中均含有大量光响应、激素应答、非生物胁迫应答元件。生物信息学分析发现,LjANR和rLjANR均编码333个氨基酸,均为稳定的疏水性蛋白,无跨膜结构,无信号肽,二级结构主要由α-螺旋和无规卷曲组成。序列比对及系统进化分析表明,LjANR和rLjANR蛋白与猕猴桃、茶树、油茶聚为一类,亲缘关系较近。qRT-PCR分析发现,LjANR和rLjANR均在花蕾中表达量最高,根中表达量最低,不同花期的表达量变化模式相似,S1和S2期表达量较高,然后表达量逐渐下降,至S4期达到最低,之后在S5期有一个缓慢的上升后表达量再次下降,两个品种中ANR基因的表达量在根、S2、S5期差异显著,茎、花蕾、S1、S3、S6期差异极显著。将LjANR基因构建到原核表达载体pET-32a上进行诱导表达,在59 kD处成功表达出目的蛋白。该研究为进一步研究ANR基因的功能奠定了基础,同时也为忍冬新品种的选育提供理论指导。Anthocyanidin reductase(ANR)is one of the key enzyme in the flavonoid biosynthetic pathway,and its catalytic activity is important for the synthesis of plant anthocyanin.In this study,specific primers were designed according to the transcriptome data of Lonicera japonica Thunb.,and the CDS,gDNA and promoter sequences of ANR genes from Lonicera japonica Thunb.and Lonicera japonica Thunb.var.chinensis(Wats.)Bak.were cloned.The results showed that the CDS sequences of LjANR and rLjANR were 1002 bp,the gDNA sequences were 2017 and 2026 bp respectively,and the promoter sequences were 1170 and 1164 bp respectively.LjANR and rLjANR both contain 6 exons and 5 introns,which have the same length of exons and large differences in introns.The promoter sequences both contain a large number of light response,hormone response and abiotic stress response elements.Bioinformatics analysis showed that both LjANR and rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides.The secondary structures of LjANR and rLjANR were predicted to be mainly consisted ofα-helix and random coil.Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with Actinidia chinensis var.chinensis,Camellia sinensis and Camellia oleifera,and were closely related to them.The expression levels of LjANR and rLjANR were the highest in flower buds and the lowest in roots.The expression patterns at different flowering stages were similar,with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage,after a slow increase in S5 stage,the expression levels decreased again.The expression levels of ANR genes in the two varieties showed significant differences in roots,S2 and S5 stages,while the differences in stems,flower buds,S1,S3 and S6 stages were extremely significant.The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression.The target protein was successfully ex

关 键 词：忍冬 花青素还原酶 基因克隆 表达模式 原核表达 

分 类 号：R931[医药卫生—生药学]