中国农业科学 ›› 2021, Vol. 54 ›› Issue (1): 86-94.doi: 10.3864/j.issn.0578-1752.2021.01.007
李祖任1(),罗丁峰1,柏浩东1,徐晶晶1,韩进财1,徐强2,王若仲2,柏连阳1,2(
)
收稿日期:
2020-04-12
接受日期:
2020-06-28
出版日期:
2021-01-01
发布日期:
2021-01-13
通讯作者:
柏连阳
作者简介:
李祖任,E-mail: 基金资助:
LI ZuRen1(),LUO DingFeng1,BAI HaoDong1,XU JingJing1,HAN JinCai1,XU Qiang2,WANG RuoZhong2,BAI LianYang1,2(
)
Received:
2020-04-12
Accepted:
2020-06-28
Online:
2021-01-01
Published:
2021-01-13
Contact:
LianYang BAI
摘要:
【背景】恶性杂草小飞蓬(Conyza canadensis)危害严重,有效治理手段匮乏。植物源羊脂酸可高效抑制小飞蓬光合作用,是一种具有开发潜力的灭生型植物源除草化合物。捕光叶绿素a/b结合蛋白(LHC)是光合系统I(PSI)中重要复合体蛋白,在植物进行光合作用中发挥关键作用。【目的】挖掘羊脂酸抑制小飞蓬的潜在靶标基因,为植物源羊脂酸除草剂的开发提供理论依据。【方法】采用同源克隆和RACE技术从小飞蓬叶片中克隆CcLhca-J9的全长序列,并利用DNAMAN分析其核酸序列特征。在NCBI中搜索LHC的高相似度氨基酸序列,采用邻接法构建系统进化树。利用SWISS-MODEL和ExPaSy在线预测分子量、等电点和蛋白结构。以同源建模结果作为模型,采用AutoDock 4.2软件分析羊脂酸与CcLhca-J9蛋白之间的亲和力。采用实时荧光定量PCR(RT-qPCR)分析羊脂酸和对照药剂阿魏酸及清水处理小飞蓬叶片后CcLhca-J9表达差异。【结果】成功获得小飞蓬CcLhca-J9,基因编码区全长为744 bp,编码247个氨基酸,分子量为26.766 kD, 理论等电点为6.43,属于Chloroa_b-bind家族蛋白。系统进化分析表明,CcLhca-J9与除虫菊(GEW73959.1,Tanacetum cinerariifolium)和黄花蒿(PWA35049.1,Artemisia annua)Lhca蛋白进化程度最为接近,同处于菊科这一分支,一致性超过85%,表明该基因家族保守性较强。CcLhca-J9蛋白二级结构具有螺旋、β转角、延伸链、无规则卷曲;以4y28.1.O (2.80Á)为模板进行同源建模,三级结构是单分子物体,具有6个叶绿体a配体,是一个典型的捕光复合物I叶绿素a/b结合蛋白。分子对接显示,羊脂酸与CcLhca-J9蛋白的氨基酸残基Gly68、Phe67、Phe69和Arg197在结合过程中产生了氢键和p-π的作用力。RT-qPCR结果显示,羊脂酸胁迫处理小飞蓬叶片条件下,CcLhca-J9的表达量存在明显差异,药后0—8 h内随时间延长表达量表现出下降的趋势。与对照阿魏酸和清水处理相比,羊脂酸处理抑制了CcLhca-J9的表达。【结论】CcLhca-J9具有典型的捕光叶绿素a/b结合蛋白功能,可能参与了羊脂酸抑制小飞蓬叶片生长过程,是具有开发除草剂潜力的抑草靶标。
李祖任,罗丁峰,柏浩东,徐晶晶,韩进财,徐强,王若仲,柏连阳. 小飞蓬捕光叶绿素结合蛋白基因CcLhca-J9克隆及表达分析[J]. 中国农业科学, 2021, 54(1): 86-94.
LI ZuRen,LUO DingFeng,BAI HaoDong,XU JingJing,HAN JinCai,XU Qiang,WANG RuoZhong,BAI LianYang. Cloning and Expression Analysis of Light Harvesting Chlorophyll a/b Protein Gene CcLhca-J9 in Conyza canadensis[J]. Scientia Agricultura Sinica, 2021, 54(1): 86-94.
表1
CcLhca-J9克隆及实时荧光定量PCR表达引物"
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 引物用途 Use of primers |
---|---|---|
CcJ9-1 | Forward: GAGYTGCGGYATCBCCGCCGT Reverse: GTTGTGCCATGGRTCAGCCAA | 核心片段扩增 Fragment cloning |
CcJ9-F2 | Forward: CGGATTTGACCCACTTCGTCT | 5′ RACE扩增 5′ cDNA end amplification |
CcJ9-R2 | Reverse: TATTGCCAGTATGGTGGGTAGGGT | 5′ RACE扩增 5′ cDNA end amplification |
CcJ9-R3 | Reverse: GGCAGCCCATTCTTGTGCTTTTAC | 5′ RACE扩增 5′ cDNA end amplification |
CcJ9-F3 | Forward: GGCAACTGGGTAAAAGCACAAGAAT | 3′ RACE扩增 3′ cDNA end amplification |
CcJ9-F4 | Forward: AAGACCCAAAAACATTCGCAGAGTA | 3′ RACE扩增 3′ cDNA end amplification |
qR-J9 | Forward: CATTCTTGTGCTTTTACCCAG Reverse: GAGGTTTAAAGAGTCCGAGCT | 实时荧光定量PCR RT-qPCR |
ACTIN | Forward: CCGATCCAGACGCTGTATTT Reverse: TGCTGATCGTATGAGCAAGG | 实时荧光定量PCR RT-qPCR |
图3
CcLhca-J9与同源基因编码区氨基酸序列比对 同源氨基酸及其登录号The source and accession number of the amino acids are as follows: TcLhca (Tanacetum cinerariifolium, GEW73959.1), AaLhca (Artemisia annua, PWA35049.1), HaLhca (Helianthus annuus, XP021976973.1), LsLhca (Lactuca sativa, XP023729531.1), CcLhca (Conyza canadensis)"
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