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

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

Abstract

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

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.

Figures/Tables 13

References 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