大豆BCAT基因家族鉴定及GmBCAT3在干旱胁迫中的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0462

生物技术通报 ›› 2025, Vol. 41 ›› Issue (10): 196-209.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0462

大豆BCAT基因家族鉴定及GmBCAT3在干旱胁迫中的功能分析

王碧成¹^,²(), 景海青¹, 万坤¹^,², 张莹莹¹^,², 丁家豪¹, 李润植¹, 薛金爱¹(), 张海平²()

^1.山西农业大学农学院山西省特用作物遗传与代谢工程研究中心，太谷 030801
^2.山西农业大学农业基因资源研究中心农业农村部黄土高原作物基因资源与种质创制重点实验室，太原 030031

收稿日期:2025-05-06 出版日期:2025-10-26 发布日期:2025-10-28
通讯作者: 张海平，女，博士，研究员，研究方向：大豆种质资源；E-mail: nkyzhp@126.com；
薛金爱，女，博士，教授，研究方向：分子遗传与基因工程；E-mail: 306214803@qq.com
作者简介:王碧成，男，硕士研究生，研究方向：种质创新与遗传工程；E-mail: 18656762826@163.com
基金资助:
山西省重大专项计划“揭榜挂帅”项目(202201140601025);国家重点研发计划(2021YFD1600601-03);科技创新2030—重大项目(2023ZD0403703);大豆种业创新良种联合攻关项目(NYGG27-01-01);山西省现代农业产业技术体系建设专项资金(2025CYJSTX05)

Identification of Soybean BCAT Gene Family and Functional Analysis of GmBCAT3 in Soybean Responses to Drought Stress

WANG Bi-cheng¹^,²(), JING Hai-qing¹, WAN Kun¹^,², ZHANG Ying-ying¹^,², DING Jia-hao¹, LI Run-zhi¹, XUE Jin-ai¹(), ZHANG Hai-ping²()

^1.College of Agronomy, Shanxi Agricultural University, Shanxi Engineering Research Center for Genetics and Metabolism of Specific Crops, Taigu 030801
^2.Center for Agricultural Genetic Resources Research, Shanxi Agricultural University, Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture and Rural Affairs, Taiyuan 030031

Received:2025-05-06 Published:2025-10-26 Online:2025-10-28

摘要/Abstract

摘要：

目的支链氨基酸（branched-chain amino acids, BCAAs）是植物在干旱胁迫下积累的主要代谢物，支链氨基酸转氨酶（branched-chain amino acid transaminase, BCAT）是支链氨基酸合成最后一步反应的关键酶。研究大豆GmBCAT的生物学功能，为大豆及其他作物抗旱遗传改良奠定理论基础，亦为培育优异抗逆性能的大豆新种质提供科学参考。方法从大豆全基因组鉴定GmBCAT基因家族成员，并预测GmBCAT蛋白亚细胞定位，通过烟草遗传转化研究GmBCAT3在BCAA合成积累及干旱胁迫应答中的功能。结果从大豆基因组中共鉴定到10个GmBCATs基因，同一类群GmBCAT成员具有相似的保守结构域及基因结构；GmBCATs启动子含有多种与植物生长发育及非生物胁迫相关的顺式元件；RT-qPCR分析发现，GmBCATs在大豆不同组织及种子不同发育时期差异表达，GmBCAT2和GmBCAT3在根和叶中表达水平显著高于其他组织；干旱胁迫处理后，大豆幼苗GmBCAT3表达量最高。亚细胞定位结果表明，GmBCAT3蛋白定位在叶绿体。烟草遗传转化显示，正常条件下，GmBCAT3转基因烟草缬氨酸、亮氨酸和异亮氨酸含量分别增加37.48%、63.46%和72.62%。干旱胁迫下，转基因烟草亮氨酸含量增加57.42%，而缬氨酸和异亮氨酸含量分别增加2.00和1.90倍；同时，转基因烟草脯氨酸含量增加3.40倍，丙二醛含量降低23.01%，SOD和POD活性分别升高72.33%和74.91%；过表达GmBCAT3烟草影响了BCAAs代谢相关基因的表达。结论大豆GmBCAT3异源过表达显著提高转基因烟草BCAAs含量，提高植株耐旱性。

关键词: 大豆, 支链氨基酸转氨酶, 基因组鉴定, 表达分析, 干旱胁迫应答, 烟草遗传转化

Abstract:

Objective Branched-chain amino acids (BCAAs) are the main metabolites accumulated by plants under drought stress, and branched-chain amino acid transaminase (BCAT) is the key enzyme in the final step reaction of BCAAs biosynthesis. The study of the biological functions of GmBCAT in soybean (Glycinemax) aims to lay a theoretical foundation for the genetic improvement of drought resistance in soybeans and other crops, and offers scientific references for the breeding of new soybean germplasms with excellent stress resistance. Method GmBCAT gene family members were identified in the soybean genome database, and the subcellular localization of GmBCAT protein was conducted. The functions of GmBCAT3 in the biosynthesis and accumulation of BCAA and in response to drought stress were studied through tobacco genetic transformation. Result A total of 10 GmBCAT genes were identified from the soybean genome, and GmBCAT members in the same group had similar conserved domains and gene structures. The promoters of the GmBCAT contained a various cis-acting elements related to plant growth and development as well as abiotic stress. RT-qPCR analysis revealed that the GmBCAT were differentially expressed in various tissues of the soybean and at different developmental stages of seeds. The expressions of GmBCAT2 and GmBCAT3 were significantly higher in the roots and leaves than those in other tissues. After drought stress treatment, the expressions of GmBCAT3 in soybean seedlings was the highest. The results of subcellular localization indicated that the GmBCAT3 protein was localized in chloroplasts. The genetic transformation of tobacco showed that the contents of valine, leucine and isoleucine in transgenic tobacco increased by 37.48%, 63.46% and 72.62%, respectively under normal conditions. Under drought stress, the leucine content of transgenic tobacco increased by 57.42%, while the levels of valine and isoleucine increased by 2.00 and 1.90 times respectively. Meanwhile, the proline content of transgenic tobacco increased by 3.40 times, the malondialdehyde content decreased by 23.01%, the activities of SOD and POD increased by 72.33% and 74.91% respectively. Moreover, the overexpression of GmBCAT3 in tobacco affected the expressions of genes related to BCAAs metabolism. Conclusion Heterologous overexpression of soybean GmBCAT3 significantly increases the content of BCAAs in transgenic tobacco and improves the drought tolerance of the plants.

Key words: soybean, branched-chain amino acid transaminase, genomic identification, expression analysis, drought stress response, tobacco genetic transformation

王碧成, 景海青, 万坤, 张莹莹, 丁家豪, 李润植, 薛金爱, 张海平. 大豆BCAT基因家族鉴定及GmBCAT3在干旱胁迫中的功能分析[J]. 生物技术通报, 2025, 41(10): 196-209.

WANG Bi-cheng, JING Hai-qing, WAN Kun, ZHANG Ying-ying, DING Jia-hao, LI Run-zhi, XUE Jin-ai, ZHANG Hai-ping. Identification of Soybean BCAT Gene Family and Functional Analysis of GmBCAT3 in Soybean Responses to Drought Stress[J]. Biotechnology Bulletin, 2025, 41(10): 196-209.

图/表 13

参考文献 37

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引物 Primer	正向引物 Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）	用途 Purpose
GmBCAT1-q	TCCGCCACTCTGTCTTCTGATCC	GTTCCACCTCGTGTGCATTTCATG	RT-qPCR
GmBCAT2-q	TTTGGCTCCTGCACCTCAATACAC	GCACGGTCAAAGTTCTCCTCCAC
GmBCAT3-q	TTGCTTCTTCGGGCTGGTTGTTC	CTAGGGTTGTGGCTCGGTAGAGG
GmBCAT4-q	GGAACAGGAGCATCGTTAGGTGTG	GTGGTAGCTGCCAACAGGAGAAC
GmBCAT5-q	CCAGTTGGCAGCTACCAGAAGG	GCACATCAGAGAATCCGTTGGC
GmBCAT6-q	TGGCACCTTCTGTTGAGCAGTA	TGTCGTCCTTTGTAAGCGTTGG
GmBCAT7-q	ACCAGTTTGTTGCTGCTGTGAA	CGGTTCCACCAGTGCCAGATAT
GmBCAT8-q	AGCGAAGAATCAGCCTGTCAGAAG	ATTATGCGGCCATCGACCTTCAC
GmBCAT9-q	GCCATCGCCTACTGTTGAGCAG	CGGTCCACTTCCCATTAGCAAAGG
GmBCAT10-q	ATGCTTCTTGGCAGCGGAGTTC	CGAACAGTGGAGGGACCTGATTTC
GmActin-q	AAGCTGTTCTCTCCTTGTACGCC	GCACAGTGTGAGACACACCATCA	内参基因
GmBCAT3-1302	acgggggactcttgaccatggGAGATGCAATGTTCCAAAAAGGA	aagttcttctcctttactagtTTAATCAACTTCGACAATCCATCC	过表达载体构建
GmBCAT3-1300	atacaccaaatcgactctagaGAGATGCAATGTTCCAAAAAGGA	gcccttgctcaccatggtaccATCAACTTCGACAATCCATCCC	亚细胞定位载体构建
NtActin-q	CAGTGGCCGTACAACAGGTA	AACCGAAGAATTGCATGAGG	内参基因
NtDHAD-q	CTATGGTGATGGTCATGGCACTTGG	ACCGCATCGCTAACCTTCTGGAA	RT-qPCR
NtBCKDH-q	CATTCTACATCCGATGACTCCACCAA	TCTTTGTCGTTCCACCAGCCATTT
NtIVD-q	GCAGGCACAAGTGAGATCAGAAGAA	GAGAGGTGGGAGGTCGTCAATTTG

引物 Primer	正向引物 Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）	用途 Purpose
GmBCAT1-q	TCCGCCACTCTGTCTTCTGATCC	GTTCCACCTCGTGTGCATTTCATG	RT-qPCR
GmBCAT2-q	TTTGGCTCCTGCACCTCAATACAC	GCACGGTCAAAGTTCTCCTCCAC
GmBCAT3-q	TTGCTTCTTCGGGCTGGTTGTTC	CTAGGGTTGTGGCTCGGTAGAGG
GmBCAT4-q	GGAACAGGAGCATCGTTAGGTGTG	GTGGTAGCTGCCAACAGGAGAAC
GmBCAT5-q	CCAGTTGGCAGCTACCAGAAGG	GCACATCAGAGAATCCGTTGGC
GmBCAT6-q	TGGCACCTTCTGTTGAGCAGTA	TGTCGTCCTTTGTAAGCGTTGG
GmBCAT7-q	ACCAGTTTGTTGCTGCTGTGAA	CGGTTCCACCAGTGCCAGATAT
GmBCAT8-q	AGCGAAGAATCAGCCTGTCAGAAG	ATTATGCGGCCATCGACCTTCAC
GmBCAT9-q	GCCATCGCCTACTGTTGAGCAG	CGGTCCACTTCCCATTAGCAAAGG
GmBCAT10-q	ATGCTTCTTGGCAGCGGAGTTC	CGAACAGTGGAGGGACCTGATTTC
GmActin-q	AAGCTGTTCTCTCCTTGTACGCC	GCACAGTGTGAGACACACCATCA	内参基因
GmBCAT3-1302	acgggggactcttgaccatggGAGATGCAATGTTCCAAAAAGGA	aagttcttctcctttactagtTTAATCAACTTCGACAATCCATCC	过表达载体构建
GmBCAT3-1300	atacaccaaatcgactctagaGAGATGCAATGTTCCAAAAAGGA	gcccttgctcaccatggtaccATCAACTTCGACAATCCATCCC	亚细胞定位载体构建
NtActin-q	CAGTGGCCGTACAACAGGTA	AACCGAAGAATTGCATGAGG	内参基因
NtDHAD-q	CTATGGTGATGGTCATGGCACTTGG	ACCGCATCGCTAACCTTCTGGAA	RT-qPCR
NtBCKDH-q	CATTCTACATCCGATGACTCCACCAA	TCTTTGTCGTTCCACCAGCCATTT
NtIVD-q	GCAGGCACAAGTGAGATCAGAAGAA	GAGAGGTGGGAGGTCGTCAATTTG

蛋白Protein	基因IDGene ID	氨基酸数Number of amino acids	分子量Molecular weight (kD)	理论等电点Theoretical pI	不稳定系数Instability index	脂溶指数Aliphatic index	亲水性GRAVY	亚细胞定位Subcellular location
GmBCAT1	Glyma.01G196300	406	44.58	5.77	54.72	94.09	-0.036	叶绿体 Chloroplast
GmBCAT2	Glyma.04G049200	384	41.99	6.76	43.72	84.82	-0.209	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT3	Glyma.06G050100	462	50.80	8.51	45.85	80.43	-0.223	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT4	Glyma.07G186000	358	38.79	5.23	38.97	81.70	-0.121	叶绿体 Chloroplast
GmBCAT5	Glyma.07G186100	359	38.57	6.83	42.88	88.27	-0.043	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT6	Glyma.08G063000	354	38.96	5.44	33.76	80.14	-0.172	细胞质 Cytoplasm
GmBCAT7	Glyma.08G063300	357	38.47	5.52	46.83	87.93	-0.025	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT8	Glyma.10G156701	244	27.07	8.78	33.47	97.09	-0.211	细胞质 Cytoplasm
GmBCAT9	Glyma.11G045300	412	45.18	6.08	56.27	92.96	-0.072	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT10	Glyma.17G190800	382	42.24	9.05	40.49	88.51	-0.15	叶绿体 Chloroplast

蛋白Protein	基因IDGene ID	氨基酸数Number of amino acids	分子量Molecular weight (kD)	理论等电点Theoretical pI	不稳定系数Instability index	脂溶指数Aliphatic index	亲水性GRAVY	亚细胞定位Subcellular location
GmBCAT1	Glyma.01G196300	406	44.58	5.77	54.72	94.09	-0.036	叶绿体 Chloroplast
GmBCAT2	Glyma.04G049200	384	41.99	6.76	43.72	84.82	-0.209	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT3	Glyma.06G050100	462	50.80	8.51	45.85	80.43	-0.223	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT4	Glyma.07G186000	358	38.79	5.23	38.97	81.70	-0.121	叶绿体 Chloroplast
GmBCAT5	Glyma.07G186100	359	38.57	6.83	42.88	88.27	-0.043	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT6	Glyma.08G063000	354	38.96	5.44	33.76	80.14	-0.172	细胞质 Cytoplasm
GmBCAT7	Glyma.08G063300	357	38.47	5.52	46.83	87.93	-0.025	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT8	Glyma.10G156701	244	27.07	8.78	33.47	97.09	-0.211	细胞质 Cytoplasm
GmBCAT9	Glyma.11G045300	412	45.18	6.08	56.27	92.96	-0.072	叶绿体、线粒体 Chloroplast， mitochondrion
GmBCAT10	Glyma.17G190800	382	42.24	9.05	40.49	88.51	-0.15	叶绿体 Chloroplast

大豆BCAT基因家族鉴定及GmBCAT3在干旱胁迫中的功能分析

Identification of Soybean BCAT Gene Family and Functional Analysis of GmBCAT3 in Soybean Responses to Drought Stress

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