玉米甲硫氨酸合酶基因METS的克隆及表达特性

doi:10.13560/j.cnki.biotech.bull.1985.2021-1226

摘要/Abstract

摘要：

在植物中,5-甲基四氢叶酸和同型半胱氨酸在甲硫氨酸合酶（methionine synthase,METS）的作用下生成甲硫氨酸,甲硫氨酸合酶是连接甲基代谢和一碳代谢的枢纽,克隆玉米甲硫氨酸合酶基因METS并分析其表达特性可为解析玉米籽粒的甲硫氨酸和叶酸的积累机制奠定基础。通过逆转录聚合酶链反应克隆得到玉米METS的编码序列,通过生物信息学方法分析其蛋白的特点,同时运用实时荧光定量技术分析了基因的组织表达特性及在萌发过程中的表达模式。结果发现玉米包含3个编码甲硫氨酸合酶的同源基因ZmMETS1、ZmMETS2、ZmMETS3,基因长度分别为2 301、2 298和2 298 bp,编码蛋白的氨基酸数量分别为766、765和765个,蛋白质分子质量约为84.5 kD,3个蛋白均含有N端和C端2个结构域,每个结构域由α螺旋环绕平行的β片层构成立体结构。系统发育树分析3个蛋白与高粱SbMETS的相似程度最高。表达分析发现它们在玉米各组织中均表达,各组织中转录水平最高的是ZmMETS1;相对来说雌穗中3个基因的转录水平较其他组织高;另外,在萌发过程中,ZmMETS1和ZmMETS3的转录水平随着萌发的时间迅速增加,之后迅速下降。玉米中存在3个具有组成型表达特征的玉米甲硫氨酸合酶基因,ZmMETS1可能在雌穗发育和萌发过程中起主要作用。

关键词: 玉米, 甲硫氨酸合酶, METS, 萌发

Abstract:

In plants,methionine is produced by methionine synthase（METS）using 5-methyltetrahydrofolate and homocysteine as substrates. Methionine synthase is the hub connecting methyl metabolism and one carbon metabolism. Cloning and expression analysis of METS coding sequence can provide basis information for the regulation of methionine- and folate- accumulation in maize（Zea mays）grains. The coding sequences of maize METS were cloned by RT-PCR,and its characteristics were analyzed by bioinformatics methods. The expression patterns in different tissues and during germination were detected by real-time quantitative PCR. Maize contained three homologous genes of ZmMETS1,ZmMETS2,and ZmMETS3 encoding methionine synthase,the gene length was 2 301 bp,2 298 bp and 2 298 bp,encoding 766,765 and 765 amino acids,respectively,and the protein molecular weights were all about 84.5 kD. All these three proteins contained two domains at N- and C-terminals. Each domain consisted of α helices surrounded by parallel β lamellae to form a stereoscopic structure. Phylogenetic tree analysis showed that 3 proteins were most similar to SbMETS in Sorghum bicolor. Expression analysis indicated that three ZmMETSs were expressed in all tissues of maize,and the highest transcription level was ZmMETS1. The transcription levels of three genes in ear were higher than those in other tissues. In addition,the transcription levels of ZmMETS1 and ZmMETS3 increased rapidly during germination and then decreased rapidly after germination. In conclusion,there are three constitutive expressed methionine synthase genes in maize. ZmMETS1 may play a role in ear development and germination.

Key words: Zea mays, methionine synthase, METS, germination

任莹, 连通, 张春义, 姜凌. 玉米甲硫氨酸合酶基因METS的克隆及表达特性[J]. 生物技术通报, 2022, 38(4): 79-85.

REN Ying LIAN Tong ZHANG Chun-yi JIANG Ling. Gene Cloning and Expression Characteristics of Methionine Synthase METS in Maize[J]. Biotechnology Bulletin, 2022, 38(4): 79-85.

图/表 6

表1 试验所用引物

Table 1 Primers used in the experiment

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Function
ZmMETS1-F1	CAGAAAGATGGCGTCCCACATTG	基因克隆 Gene cloning
ZmMETS1-R1	GACGACGCCCTCCTCGGAAACG
ZmMETS2-F1	GGGAGCAACACATGCAGTATGA- ATTC
ZmMETS2-R1	CACATACCCCCAGAAACATAGCCC
ZmMETS3-F2	CCTCAGACAGTCAGACGTCCCGTG
ZmMETS3-R2	CTGTCCTGTACATACGTTGAGC- CTGG
METS1-QRT-F1	GATCCAGGACACCACCCAGATC	qRT-PCR
METS1-QRT-R1	CTCCTCGGAAACGAAAAAGGC
METS2-QRT-F1	TAAGATTTTACTGTTATATAGAA- AGATGGC
METS2-QRT-R1	GGGACAATAAAGTGGTAGTTTG- TATC
METS3-QRT-F1	CTCTACAAATGAAACCCCTCAGAC
METS3-QRT-R1	GTATCCAACAATATGGGACGCC
ZmGAPDH-F	CCCTTCATCACCACGGACTAC	Internal primers for qRT-PCR
ZmGAPDH-R	AACCTTCTTGGCACCACCCT	Internal primers for qRT-PCR

图1 ZmMETS的亲疏水性和结构域预测

Fig. 1 Prediction of hydrophilicity,hydrophobicity,and protein domain of ZmMETS

图2 ZmMETS的三维结构预测,甲硫氨酸位于C端结构域

Fig. 2 Three-dimensional structure prediction of ZmME-TS,and methionine locates in C-terminal domain

图3 METS同源基因的进化分析 SbMETS1：高粱Sorghum bicolor,A0A1B6PEC9;SbMETS2：高粱Sorghum bicolor,Q8W0Q7;ZmMETS1：玉米Zea mays,C0P5Y3;ZmMETS2：玉米Zea mays,B8A1R8;ZmMETS3：玉米Zea mays,A0A1D6KZ65;OsMETS1：水稻Oryza sativa L.,Q2QLY5;OsMETS2：水稻Oryza sativa L.,Q2QLY4;AtMS1：拟南芥Arabidopsis thaliana,O50008;AtMS2：拟南芥Arabidopsis thaliana,Q9SRV5;AtMS3：拟南芥Arabidopsis thaliana,Q0WNZ5;ScMS：酵母Saccharomyces cerevisiae,P05694;HsMTR：人Homo sapiens,Q99707;EcMS：大肠杆菌Escherichia coli,P13009。所有蛋白序列来自Uniprot数据库,All information of proteins is from www.uniprot.org

Fig.3 Evolutionary analysis of METS homologous proteins

图4 ZmMETS1/ZmMETS2/ZmMETS3在不同组织中的相对表达量误差线表示标准偏差,n=3;星号代表与根中表达水平相比显著差异（*P<0.05;**P<0.01;***P<0.001;****P<0.0001）。下同

Fig. 4 Relative expressions of ZmMETS1/ZmMETS2/ZmMETS3 in different tissues The error line refers to the standard deviation,n=3;significant difference compared with root（*P<0.05;**P<0.01;***P<0.001;****P<0.0001）. The same below

图5 ZmMETS1/ZmMETS2/ZmMETS13在萌发过程中的相对表达量

Fig. 5 Relative expressions of ZmMETS1/ZmMETS2/ZmMETS3 during germination

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