茶树‘黄金芽’叶绿体铁氧还蛋白-NADP+氧化还原酶基因的克隆与表达分析

Cloning and Expression Analysis of Chloroplast Ferredoxin-NADP+Oxidoreductase Gene of Tea Plants'Huangjinya'

高等植物叶绿体定位的铁氧还蛋白-NADP+氧化还原酶(LFNR)是一种亲水性黄素蛋白,位于光合电子传递链末端,是将光合作用电子运输与叶绿体氧化还原代谢联系起来的枢纽。为探究LFNR基因与茶树品种‘黄金芽’受光调控黄化之间的关系,本研究分别以‘黄金芽’、‘舒茶早’(CK)1芽2叶为材料克隆CsLFNR基因,获得了相似度为100%的序列,将其命名为CsLFNR1.1(GenBank登录号:MT311318)。经生物信息学分析,其cDNA基因全长1095 bp,编码364个氨基酸,蛋白分子量为40.623 kD,理论等电点为8.86,为碱性蛋白。CsLFNR1.1无跨膜结构和信号肽,含一条叶绿体转运肽,二级结构含23.35%的α-螺旋,5.49%的β-转角,28.85%延伸链和42.31%的无规则卷曲,亚细胞定位于叶绿体。通过blastn、blastp及DNAMAN软件比对分析发现CsLFNR1.1及其翻译氨基酸序列与NCBI‘舒茶早’茶树基因CsLFNR1(XM_028233617.1)和蛋白(XP_028089418.1)序列分别有4个碱基和3个氨基酸的差异,相似性分别达到了99.63%和99.18%,预测的蛋白在理化性质及二级结构上有微小差异,三级结构预测所用模板一致,且拥有相同的保守结构域及活性结构位点。以不同遮荫度‘黄金芽’叶片为材料进行了RT-qPCR实验,结果显示CsLFNR1.1基因表达响应光照强度变化,随着光强增加表达量提高。本研究结果为进一步探究CsLFNR1.1基因在‘黄金芽’新梢叶片光系统受光调控过程中所发挥的作用提供了理论基础和科学依据。

The chloroplast-targeted ferredoxin-NADP+oxidoreductase(LFNR)is a hydrophilic flavin protein located at the end of the photosynthesis electron delivery chain in higher plants.It's a hub for linking photosynthesis electronic transportation with chloroplast redox metabolism.In order to explore the relationship between LFNR gene and yellowing of'Huangjinya'controlled by light,we took separately one bud two leaves of'Huangjinya''Shuchazao'(CK)as the material to clone CsLFNR gene and then we obtained the sequence with a similarity of 100%,named CsLFNR1.1(GenBank accession No.MT311318).Through bioinformatics analysis,we know its cD-NA gene length is 1095 bp,coding 364 amino acids.The protein molecular weight is 40.623 kD,the theoretical isoelectric point is 8.86.CsLFNR1.1 is an alkaline protein without transmembrane structure and signal peptide.It has a chloroplast transport peptide(cTP).The secondary structure contains 23.35%alpha helix,5.49%beta turn,28.85%extended strand and 42.31%random coil.It is located in the chloroplast.Through the nucleotide BLAST,protein BLAST and DNAMAN software,we found that CsLFNR1.1 and its translated amino acid sequences had 4 bases and 3 amino acid differences with NCBI'Shuchazao'gene CsLFNR1(XM_028233617.1)and protein(XP_028089418.1),the similarity reached 99.63%and 99.18%.CsLFNR1.1 and CsLFNR1 have small differences in the physical and chemical properties and secondary structure.The tertiary structure prediction template of them is consistent.In addition,they have the same conserved domain and active site.We took different shade'Huangjinya'blades as the material for RT-qPCR.The result showed that the expression of CsLFNR1.1 gene response to the light intensity,its expression increased with the increase of light intensity.This study provides a theoretical basis and scientific basis for further exploring the role of CsLFNR1.1 gene in the light regulation process of the new shoots and leaves of'Huangjinya'.