大豆咖啡酰辅酶A-O-甲基转移酶（CCoAOMT）基因克隆及结构分析

郭子雯; 司修洋; 焦莉苹; 刘大伟

doi:10.19303/j.issn.1008-0384.2023.05.013

大豆咖啡酰辅酶A-O-甲基转移酶（CCoAOMT）基因克隆及结构分析

doi: 10.19303/j.issn.1008-0384.2023.05.013

1.
东北农业大学植物保护学院，黑龙江　哈尔滨　150030
2.
浙江大学农业与生物技术学院，浙江　杭州　310058

基金项目: 黑龙江省自然科学基金（LH2019C034）

详细信息

作者简介:
郭子雯（1999−），女，硕士研究生，研究方向：植物病原线虫学（E-mail：1057138000@qq.com）

通讯作者:
刘大伟（1983−），男，博士，副教授，研究方向：植物病原线虫学（E-mail：liudawei353@163.com）

中图分类号: S435
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出版历程
- 收稿日期: 2022-11-10
- 修回日期: 2023-03-13
- 网络出版日期: 2023-05-24
- 刊出日期: 2023-05-28

Cloning and Bioinformatics of CCoAOMT Relating to Resistance of Soybean to Cyst Nematodes

1.
College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang　150030, China
2.
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang　310058, China

摘要

摘要: 目的对大豆中咖啡酰辅酶A-O-甲基转移酶（CCoAOMT）基因进行克隆和生物信息学分析，为研究木质素代谢途径关键基因CCoAOMT在大豆抗胞囊线虫的作用机制奠定基础。方法以高抗大豆胞囊线虫（Heterodera glycines Ichinohe）3号生理小种品种灰皮支黑豆根系为材料，利用RT-PCR技术进行克隆。结果克隆得到1条大豆 CCoAOMT 基因的cDNA全长序列，将其命名为 GmCCoAOMT ，GenBank登录号为MW480860。该基因全长为848 bp，含有1个741 bp的开放阅读框，编码246个氨基酸组成的蛋白，分子量为27.6 kDa，等电点为5.67；亚细胞定位预测显示，CCoAOMT蛋白可能位于细胞质，该蛋白含有1个保守的AdoMet_MTases结构域，属于AdoMet_MTases超级家族；在与其他豆科植物CCoAOMT编码的蛋白序列比对及系统进化树分析中，GmCCoAOMT编码的蛋白序列与其他豆科植物具有较高的相似性。结论获得大豆CCoAOMT基因的cDNA全长序列，通过构建进化树确定大豆与豇豆、木豆、菜豆和赤豆的CCoAOMT蛋白进化关系较近，而与白羽扇豆和鹰嘴豆进化关系较远。
- 大豆 /
- 大豆胞囊线虫 /
- 咖啡酰辅酶A-O-甲基转移酶 /
- 基因克隆 /
- 生物信息学分析
Abstract: Objective Full-length cDNA of Caffeoyl-CoA-O-methyltransferase gene (CCoAOMT) in the lignin metabolism pathway of soybean was cloned, and bioinformatics analyzed to study the resistance mechanism to cyst nematode (Heterodera glycines Ichinohe) of the plant. Method CCoAOMT sequence from roots of a cyst nematode-resistant Huipizhiheidou soybean plant was cloned by RT-PCR. Results The full length cDNA of GmCCoAOMT was cloned and then submitted to GenBank with the accession number of MW480860. The length of GmCCoAOMT was 848 bp, containing an ORF of 741bp and encoding 246 amino acids with a predicted molecular weight of 27.6 kDa and a theoretical isoelectric point of 5.67. It was located in the cytoplasm with a conserved AdoMet_MTases domain and belonged to that superfamily. The multiple alignment on amino acid sequence and phylogenetic tree indicated a high similarity between the gene and those of other leguminous plants. Conclusion The cDNA of GmCCoAOMT was successfully cloned and shown with a close evolutionary relationship with Vigna unguiculata (Linn.) Walp., Cajanus cajan (Linn.) Millsp., Phaseolus vulgaris Linn. and Vigna angularis (Willd.) Ohwi et Ohashi but far from Lupinus albus and Cicer arietinum Linn.
- Soybean /
- Heterodera glycines Ichinohe /
- Caffeoyl-CoA-O-methyltransferase /
- gene cloning /
- bioinformatics analysis

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图 1 根系总RNA的电泳检测

Figure 1. Electrophoresis of total RNA in roots

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图 2 CCoAOMT基因的RT-PCR结果

M: DNA marker；1、2: RT-PCR 产物

Figure 2. RT-PCR on CCoAOMT

M: DNA marker；1 and 2: RT-PCR products.

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图 3 大豆CCoAOMT基因全长序列及其编码的氨基酸序列

Figure 3. Full-length cDNA and amino acid sequence of CCoAOMT in soybean

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图 4 大豆CCoAOMT蛋白保守结构域的预测

Figure 4. Predicted conservative domain of GmCCoAOMT protein

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图 5 大豆CCoAOMT蛋白亲疏水性分析

Figure 5. Hydrophobicity of GmCCoAOMT protein

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图 6 大豆CCoAOMT蛋白拓扑结构分析

Figure 6. Topological structure of GmCCoAOMT protein

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图 7 大豆与其他豆科植物CCoAOMT蛋白氨基酸多重序列比对

Figure 7. Multiple alignment of amino acid sequences between CCoAOMTs in soybean and other legumes

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图 8 大豆与其他植物CCoAOMT蛋白的进化树

Figure 8. Phylogenetic tree of CCoAOMT proteins in soybean and other plants

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图 9 大豆CCoAOMT蛋白的二级结构预测

Figure 9. Predicted secondary structure of soybean CCoAOMT protein

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图 10 大豆CCoAOMT蛋白的三维建模

Figure 10. Predicted tertiary structure of soybean CCoAOMT protein

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表 1 大豆和其他豆科植物CCoAOMT蛋白的氨基酸组成和理化性质对比

Table 1. Amino acid composition and physicochemical properties of CCoAOMTs in soybean and other plants

序号 Serial number	物种 Species	登录号 Accession No.	氨基酸数量 Number of amino acids	相对分子量 Relative molecular mass/kDa	理论等电点 Theoretical isoelectric point	比例 Percentage/%
序号 Serial number	物种 Species	登录号 Accession No.	氨基酸数量 Number of amino acids	相对分子量 Relative molecular mass/kDa	理论等电点 Theoretical isoelectric point	BA	AC	AR	AL
1	大豆 Glycine max	MW480860	246	27.6	5.67	14.2	13.8	7.3	84.6
2	菜豆 Phaseolus vulgaris	AGV54319.1	246	27.8	5.32	13.8	14.6	8.5	83.4
3	虎爪豆 Mucuna pruriens	RDX78299.1	248	28.0	5.51	14.1	14.2	7.6	84.8
4	木豆 Cajanus cajan	XP_020208653.1	246	27.8	5.72	15.1	14.2	6.9	84.1
5	密花豆 Spatholobus suberectus	TKY55699.1	246	27.9	5.47	13.8	14.2	8.1	84.6
6	相思子 Abrus precatorius	XP_027347195.1	246	27.9	5.50	14.7	14.6	7.3	83.7
7	白羽扇豆 Lupinus albus	KAE9597637.1	247	28.0	5.40	14.1	14.6	8.1	84.2
8	豇豆 Vigna unguiculata	QCD81613.1	246	27.8	5.48	15.1	15.0	7.3	83.7
9	赤豆 Vigna angularis	XP_017427550.1	246	27.8	5.90	14.2	13.4	8.5	83.0
10	红车轴草 Trifolium pratense	PNY11893.1	248	27.9	5.43	12.4	12.9	7.2	86.0
11	鹰嘴豆 Cicer arietinum	NP_001351681.1	247	28.0	5.69	15.0	14.6	8.1	84.6
BA：碱性氨基酸；AC：酸性氨基酸；AR：芳香族氨基酸；AL：脂肪族氨基酸。 BA: Basic amino acids; AC: acidic amino acids; AR: aromatic amino acids; AL: aliphatic amino acids.

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