茶儿茶素合成关键酶基因CsANS和CsLAR的功能鉴定
Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis
张芷苓 1张媛媛 1林晓蓉 1李斌 2陈忠正2
作者信息
- 1. 华南农业大学食品学院,广州 510642
- 2. 华南农业大学食品学院,广州 510642;华南农业大学,广东省功能食品活性物重点实验室,广州 510642
- 折叠
摘要
以'英红9号'茶树[Camellia sinensis(L.)O.Kuntze]为材料,克隆了儿茶素合成途径的两个关键酶基因CsANS和CsLAR,并利用原核表达、亚细胞定位以及转基因表达分析对其功能进行鉴定.结果表明,CsANS和CsLAR开放阅读框(open reading frame,ORF)分别长1 068和1 029 bp,与TPIA数据库中来源于'舒茶早'的对应基因同源性均高达99％.CsANS和CsLAR可溶性蛋白均能在原核表达系统诱导获得,并经GST标签成功纯化.本氏烟草叶片的瞬时表达结果显示,CsANS和CsLAR编码的蛋白定位于细胞质和细胞核.利用农杆菌介导的遗传转化方法在茶愈伤组织中超表达CsANS和CsLAR,结果显示其均能显著促进总儿茶素的合成,其中CsANS主要促进顺式儿茶素(EC和EGCG)含量的增加,而CsLAR主要促进反式儿茶素(C、GC、CG和GCG)的积累.推测在'英红9号'茶树中CsANS的功能主要是促进顺式儿茶素的合成,而CsLAR更利于反式儿茶素的合成.
Abstract
Two key enzyme genes CsANS and CsLAR involved in the catechin synthetic pathway from'Yinghong 9'were cloned,and successfully identified their functions by prokaryotic expression,subcellular localization and transgenic overexpression analyses.The results showed that the ORFs(open reading frame)of CsANS and CsLAR were 1 068 and 1 029 bp,respectively,which were 99％homologous to the corresponding genes from'Shuchazao'in TPIA database.Soluble proteins of CsANS and CsLAR can be obtained in prokaryotic induced expression system and can be successfully purified by GST tag.Transient expression of CsANS and CsLAR in Nicotiana benthamiana leaves showed that proteins they encoded were localized in the cytoplasm and nucleus.Furthermore,CsANS and CsLAR were overexpressed in tea calli by Agrobacterium-mediated genetic transformation.The results showed that both of them could significantly promote the biosynthesis of total catechins in tea calli,in which CsANS mainly increased content of EC and EGCG,while CsLAR mainly promoted the accumulation of trans-catechins C,GC,CG and GCG.It is speculated that the function of CsANS is mainly to promote the synthesis of cis-catechins,while CsLAR is more conducive to the synthesize trans-catechins in'Yinghong9'.
关键词
茶树/儿茶素/ANS/LAR/过表达Key words
Camellia sinensis/catechins/ANS/LAR/overexpression引用本文复制引用
基金项目
广东省科技厅公益研究与能力建设项目(2015A030302065)
出版年
2024
NETL
NSTL
万方数据