小飞蓬捕光叶绿素结合蛋白基因CcLhca-J9克隆及表达分析

doi:10.3864/j.issn.0578-1752.2021.01.007

摘要/Abstract

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。

关键词: 小飞蓬, 光捕获蛋白, 基因克隆, 分子对接, 羊脂酸

Abstract:

【Background】Conyza canadensis, a pernicious weed, is harmful and lack of the effective management strategy. The caprylic acid can efficiently inhibit the photosynthesis of C. canadensis, which is a potential bio-herbicidal compound. Light harvesting chlorophyll a/b-binding protein (LHC) is the key complex protein in PSI, and plays an important role in photosynthesis.【Objective】The objective of this study is to explore the LHC gene in response to caprylic acid, and to provide a theoretical basis for the development of bio-herbicide.【Method】The open reading frame (ORF) of CcLhca-J9 was cloned from C. canadensis leaf by homologous PCR and RACE. The nucleotide sequence was analyzed by DNAMAN. The phylogenetic tree was constructed with the high similarity nucleotide from NCBI by neighbor-joining method and MEGA software. The molecular weight, isoelectric point and protein structure were analyzed using SWISS-MODEL and ExPaSy websites. Using homologous modeling results as a model, the affinity of caprylic acid and CcLhca-J9 protein was analyzed using AutoDock 4.2 software. RT-qPCR was used to analyze the difference of CcLhca-J9 expression level of caprylic acid, water and ferulic acid treatments.【Result】The CcLhca-J9 ORF is 744 bp, encoding 247 amino acid residues with a molecular weight of 26.766 kD and theoretical pI of 6.43, which belongs to Chloroa b-bind family protein. The phylogenetic analysis showed that the CcLhca-J9 had the most closed relationship with the Lhca protein of Tanacetum cinerariifolium (GEW73959.1) and Artemisia annua (PWA35049.1), and was in the same branch of Asteraceae group, with consistency of more than 85%, indicating that the gene family was conserved. The second structure of CcLhca-J9 protein had alpha helix, random coil, extended strand, beta turn. The homologous three-dimensional model of CcLhca-J9 protein was compared by 4y28.1.O (2.80Á), the tertiary structure was a monomolecular object with six chlorophyll a ligands, which was a typical light harvesting chlorophyll a/b-binding protein. Molecular docking result showed that hydrogen and p-π bond interaction were produced between caprylic acid and CcLhca-J9 amino acid residues Gly68, Phe67, Phe69 and Arg197. The result of RT-qPCR showed that the expression level of CcLhca-J9 was significantly decreased after 0-8 h caprylic acid treatment. Compared with ferulic acid control and water treatment, caprylic acid treatment inhibited the expression of CcLhca-J9.【Conclusion】CcLhca-J9 has the typical function of light harvesting chlorophyll a/b-binding protein, which may be involved in the process of inhibiting the growth of C. canadensis leaves and is a herbicidal target with the potential to develop herbicides.

Key words: Conyza canadensis, light harvesting protein, gene clone, molecular docking, caprylic acid

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.

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https://www.chinaagrisci.com/CN/Y2021/V54/I1/86

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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