甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析

doi:10.3724/SP.J.1006.2020.94171

作物学报 ›› 2020, Vol. 46 ›› Issue (6): 844-857.doi: 10.3724/SP.J.1006.2020.94171

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析

郑清雷¹,余陈静¹,姚坤存¹,黄宁¹,阙友雄^1,⁴,凌辉^2,^3,^*(),许莉萍^1,^4,^*()

¹福建农林大学 / 农业农村部福建甘蔗生物学与遗传育种重点实验室, 福建福州 350002
²福建农林大学生命科学学院, 福建福州 350002
³玉林师范学院农学院, 广西玉林 537000
⁴福建农林大学 / 教育部作物遗传育种与综合利用重点实验室, 福建福州 350002

收稿日期:2019-11-10 接受日期:2020-01-15 出版日期:2020-06-12 网络出版日期:2020-02-17
通讯作者: 凌辉,许莉萍
作者简介:E-mail: zhengqinglei1666@163.com.
基金资助:
国家自然科学基金项目(31801424);国家现代农业产业技术体系建设专项(CARS-17)

Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC

ZHENG Qing-Lei¹,YU Chen-Jing¹,YAO Kun-Cun¹,HUANG Ning¹,QUE You-Xiong^1,⁴,LING Hui^2,^3,^*(),XU Li-Ping^1,^4,^*()

¹Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Area / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
²College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
³College of Crop Science, Yulin Normal University, Yulin 537000, Guangxi, China
⁴Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

Received:2019-11-10 Accepted:2020-01-15 Published:2020-06-12 Published online:2020-02-17
Contact: Hui LING,Li-Ping XU
Supported by:
National Natural Science Foundation of China(31801424);China Agriculture Research System(CARS-17)

摘要/Abstract

摘要：

细胞色素b₆f复合体还原型铁硫蛋白前体(Cytochrome b₆f complex Rieske Fe/S precursor protein, PetC)是由细胞核PetC基因编码的蛋白, 其成熟蛋白参与构成细胞色素b₆f复合体, 是电子传递的重要元件。基于前期构建的受高粱花叶病毒(Sorghum mosaic virus, SrMV)侵染的甘蔗转录组数据库, 从主栽甘蔗品种‘新台糖22号’叶片中成功克隆到该基因, 并命名为ScPetC (GenBank登录号为MH333037.1)。生物信息学分析发现, ScPetC基因cDNA全长824 bp, 包含了一个678 bp的开放阅读框, 编码226个氨基酸。ScPetC属于PRK13473超家族, 其C末端具有典型的Rieske保守结构域; 蛋白理论等电点为8.19, 属于稳定的、亲水性蛋白; 二级结构多为无规则卷曲, 三级结构预测其比其他植物PetC多出一段α螺旋结构。在本氏烟(Nicotiana benthamiana)叶片瞬时表达中, ScPetC定位于叶绿体、细胞质和细胞膜。尽管前人研究表明, ScPetC的表达量会受SrMV侵染的影响, 不同于半夏(Pinellia ternata) PetC与大豆花叶病毒(Soybean mosaic virus, SMV) P1蛋白之间的互作, ScPetC不能与SrMV-P1蛋白互作, 但能与甘蔗黄叶病毒(Sugarcane yellow leaf virus, SCYLV) P0蛋白互作。实时荧光定量PCR分析表明, ScPetC基因在甘蔗不同组织中均有表达, 但在叶片中的表达量最高。甘蔗受脱落酸胁迫3 h时, ScPetC表达量显著上调, 但是随着处理时间的延长, 表达受到抑制; 在茉莉酸甲酯、水杨酸、氯化铜、氯化镉和氯化钠胁迫下, ScPetC表达量均显著下调。本研究通过对ScPetC生物学功能、环境外源激素与重金属等胁迫下的表达模式及其与甘蔗病原病毒蛋白互作的初步探究, 增加了对甘蔗PetC的了解, 也为深入研究其在甘蔗受黄叶病毒侵染中的作用奠定基础。

关键词: 甘蔗, 细胞色素b₆f复合体还原型铁硫蛋白前体, 生物信息学, 亚细胞定位, 蛋白互作, 实时荧光定量PCR

Abstract:

Cytochrome b₆f complex Rieske Fe/S precursor protein (PetC) is encoded by the nuclear PetC gene, and its mature protein involved in the formation of the cytochrome b₆f complex, which is important for electron transfer. Based on our previous transcriptome data of sugarcane (Saccharum spp. hybrids) infected by Sorghum mosaic virus (SrMV), a cytochrome b₆f complex reduced iron-sulfur precursor protein gene was cloned from leaves of sugarcane superior elite cultivar ‘ROC22’, and named as ScPetC (GenBank accession number: MH333037.1). Bioinformatics analysis showed that the ScPetC gene was 824 bp in length, containing a 678 bp open reading frame (ORF), and encoding a peptide of 226 amino acids. ScPetC belongs to the PRK13473 superfamily and has a typical Rieske domain at its C-terminus of the amino acid chain. ScPetC is a stable and hydrophilic protein with pI 8.19. Most of the secondary structural elements in ScPetC were random coil. Compared to the PetC from the other plants, ScPetC contained one more fragment of helix in its third dimensional structure. The transient expression of YFP-fused protein in Nicotiana benthamiana leaves showed that ScPetC was located in the chloroplast, cytoplasm and cell membrane. Although the previous study indicated that the expression of ScPetC was affected by SrMV infection in sugarcane, different to the interaction between Pinellia ternata PetC and SMV-P1, without interaction between ScPetC and the SrMV-P1, it did interact with SCYLV-P0, i.e. the P0 protein of Sugarcane yellow leaf virus (SCYLV). Real-time quantitative PCR analysis showed that ScPetC gene was expressed constitutively in different tissues of sugarcane, and the highest level of its expression was found in leaves. When sugarcane plant was exposed to abscisic acid stress for 3 h, the expression of ScPetC was significantly up-regulated, but the expression level was then inhibited with longer treatment time. Under the stress of methyl jasmine, salicylic acid, copper chloride, cadmium chloride and sodium chloride, the expression of ScPetC was significantly down-regulated. The study on biological function, expression pattern and interaction of ScPetC with the proteins of sugarcane pathogenic virus will improve the understanding of the happening of yellow leaf symptom, which caused by SCYLV.

Key words: sugarcane, cytochrome b₆f complex Rieske Fe/S precursor protein, bioinformatics, subcellular location, protein interaction, real-time flourescent quantitative PCR

郑清雷,余陈静,姚坤存,黄宁,阙友雄,凌辉,许莉萍. 甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析[J]. 作物学报, 2020, 46(6): 844-857.

ZHENG Qing-Lei,YU Chen-Jing,YAO Kun-Cun,HUANG Ning,QUE You-Xiong,LING Hui,XU Li-Ping. Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC[J]. Acta Agronomica Sinica, 2020, 46(6): 844-857.

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)	用途 Usage
ScPetC-F	TGGACGCCAGGGAGTGAAC	基因克隆 Gene cloning
ScPetC-R	TGTAGCAACAACGCCTTCGG	基因克隆 Gene cloning
ScPetC-QF	GTACAACAACCAGGGCAAGG	实时荧光定量PCR qRT-PCR
ScPetC-QR	CAGGGACAGGAGGCATACAT	实时荧光定量PCR qRT-PCR
CAC-F	ACAACGTCAGGCAAAGCAAA	内参基因 Reference gene
CAC-R	AGATCAACTCCACCTCTGCG	内参基因 Reference gene
CUL-F	TGCTGAATGTGTTGAGCAGC	内参基因 Reference gene
CUL-R	TTGTCGCGCTCCAAGTAGTC	内参基因 Reference gene
ScPetC-Subloc-F	CGAACGATACTCGAGGTCGACATGGCGACCTCCGCG	亚细胞定位载体 Vector for subcellular localization
ScPetC-Subloc-R	GCTCACCATACTAGTGGATCCTGCCTTCCACCACGG	亚细胞定位载体 Vector for subcellular localization
ScPetC-AD-F	CCATGGAGGCCAGTGAATTCATGGCGACCTCCGCGGCGCT	酵母双杂交载体 Vector for Y2H
ScPetC-AD-R	AGCTCGAGCTCGATGGATCCTGCCTTCCACCACGGGTCCT	酵母双杂交载体 Vector for Y2H
SrMV-P1-BK-F	TGGCCATGGAGGCCGAATTCATGGCAGGAGCATGGAGCAC	酵母双杂交载体 Vector for Y2H
SrMV-P1-BK-R	CGCTGCAGGTCGACGGATCC AAAATAAGTAATTTCATTGA	酵母双杂交载体 Vector for Y2H
SCYLV-P0-BK-F	TGGCCATGGAGGCCGAATTCATGCTTTTCAACGAATTCTC	酵母双杂交载体 Vector for Y2H
SCYLV-P0-BK-R	CGCTGCAGGTCGACGGATCCTATATCATGAGAATAGGTGC	酵母双杂交载体 Vector for Y2H
ScPetC-YN-F	TCTGAGGAGGATCTTCCCGGGATGGCGACCTCCGCGGCGCT	双分子荧光互补实验载体 Vector for BiFC
ScPetC-YN-R	AGGGCATGCCTGCAGGTCGACGTGCCTTCCACCACGGGTCCT	双分子荧光互补实验载体 Vector for BiFC
SrMV-P1-YC-F	TCTAGGAGCTCGGTACCCGGGATGGCAGGAGCATGGAGCAC	双分子荧光互补试验载体 Vector for BiFC
SrMV-P1-YC-R	ATCGTATGGGTACATACTAGTAAAATAAGTAATTTCATTGA	双分子荧光互补试验载体 Vector for BiFC
SCYLV-P0-YC-F	CTAGGAGCTCGGTACCCGGGATGCTTTTCAACGAATTCTC	双分子荧光互补实验载体 Vector for BiFC
SCYLV-P0-YC-R	TCGTATGGGTACATACTAGTTATATCATGAGAATAGGTGC	双分子荧光互补实验载体 Vector for BiFC

工具Tool	网址Website
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/
ExPaSy	http://web.expasy.org/protparam/
Prabi	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html
SWISSMODEL	http://swissmodel.expasy.org/interactive
SignalP 4.1 Server	http://www.cbs.dtu.dk/servers/SignalP/
NCBI conserved domain	http: //blast.ncbi.nlm.nih.gov/
WoLFPSORT	https://wolfpsort.hgc.jp/
TMHMM 2.0 Server	http://www.cbs.dtu.dk/servers/TMHMM/
ProtScale	https://web.expasy.org/protscale/
MEME	http://meme-suite.org/

一级结构特性Primary structure characteristic	预测结果Predicted result
编码的氨基酸个数Number of coding amino acids	225
等电点 Theoretical pI	8.19
分子量 Molecular weight (×10³)	23.85
分子式 Formula	C₁₀₅₄H₁₆₆₈N₂₉₄O₃₁₅S₁₁
不稳定系数 Instability index (II)	21.61
平均疏水性Grand average of hydropathicity (GRAVY)	-0.146

甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析

Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC

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