甜瓜β‍-淀粉酶基因家族的鉴定及对非生物胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2024-0926

摘要/Abstract

摘要：

目的 β‍-淀粉酶（β‍-amylase, BAM）能水解淀粉，在植物生长和响应非生物胁迫中起重要作用。从全基因组水平对甜瓜BAM基因家族成员进行鉴定，为甜瓜BAM基因功能研究及抗性育种提供基因资源。方法分析BAM家族蛋白理化性质、系统进化、基因结构、保守基序、启动子顺式作用元件以及通过RT-qPCR对其在干旱、ABA、低温、盐胁迫下的表达模式进行分析。结果在甜瓜全基因组中鉴定出9个CmBAM基因家族成员，分别位于第1、4、5、6、7和11号染色体上，蛋白平均氨基酸个数为543，平均分子量为58.7 kD，等电点为5.47‒8.89。系统进化分析表明，甜瓜BAM基因家族成员与黄瓜的亲缘关系最近，同源性在94.5%以上，且存在一一对应的关系。甜瓜CmBAM成员启动子中主要顺式作用元件为激素响应元件、光响应元件、非生物胁迫元件和蛋白质结合元件。CmBAMs在干旱、ABA、低温和盐处理下的表达模式分析发现，CmBAMs不同程度地响应4种非生物胁迫，其中CmBAM1、CmBAM3和CmBAM9受干旱强烈诱导，CmBAM3、CmBAM5、CmBAM9受ABA强烈诱导，CmBAM1、CmBAM5、CmBAM9受低温胁迫强烈诱导；CmBAM3的表达受盐胁迫强烈诱导。结论从甜瓜全基因组中鉴定出9个CmBAMs，CmBAM1、CmBAM3、CmBAM5和CmBAM9对4种非生物胁迫最为敏感，表明其可能在非生物胁迫中发挥关键作用，可作为后续研究的候选基因。

关键词: 甜瓜, β?-淀粉酶, 基因家族, 生物信息学, 非生物胁迫

Abstract:

Objective β‍-amylase (BAM) catalyzes the hydrolysis of starch and plays a crucial role in plant growth and stress response. Identifying the BAM gene family members from the whole melon genome may provide genetic resources for function study of BAM gene and resistance breeding in melon. Method This work analyzed the physicochemical properties, phylogenetic evolution, gene structure, conserved motifs, promoter cis-acting regulatory elements of the BAM family's protein. RT-qPCR was used to analyze their expression patterns under drought, ABA, low temperature, and salt stress. Result Nine CmBAMs were identified from the whole-genome of melon (Cucumis melo), and they were located on chromosome 1, 4, 5, 6, 7, and 11, with an average length of 543 amino acids. The average molecular weight was approximately 58.7 kD, and the isoelectric point ranged from 5.47 to 8.89. Phylogenetic analysis demonstrated that BAMs in melon were highly homologous with cucumbers (Cucumis sativus) with over 94.5% homology and one-to-one correspondence. CmBAMs promoter in melon mainly harbored hormone-responsive elements, photo-responsive elements, abiotic stress elements, and protein binding elements. Expression pattern analysis of CmBAMs under drought, ABA, low temperature, and salt stresses revealed that CmBAMs responded to these four abiotic stresses at varied level. Notably, CmBAM1, CmBAM3, and CmBAM9 were dramatically induced by drought, while CmBAM3, CmBAM5, and CmBAM9 showed strong induction by ABA. CmBAM1, CmBAM5, and CmBAM9 were obviously induced by cold stress whereas CmBAM3 showed the highest induction under salt stress. Conclusion Nine CmBAMs are identified from the whole genome of melon, and CmBAM1, CmBAM3, CmBAM5, and CmBAM9 were the most sensitive to the four abiotic stresses, suggesting that they might play a key role in abiotic stress and thus can be selected as candidate genes for further research.

Key words: melon, β?-amylase, gene family, bioinformatics, abiotic stress

韩江涛, 张帅博, 秦雅蕊, 韩硕洋, 张雅康, 王吉庆, 杜清洁, 肖怀娟, 李猛. 甜瓜β‍-淀粉酶基因家族的鉴定及对非生物胁迫的响应[J]. 生物技术通报, 2025, 41(3): 171-180.

HAN Jiang-tao, ZHANG Shuai-bo, QIN Ya-rui, HAN Shuo-yang, ZHANG Ya-kang, WANG Ji-qing, DU Qing-jie, XIAO Huai-juan, LI Meng. Identification of β‍-amylase Gene Family in Melon and Their Response to Abiotic Stresses[J]. Biotechnology Bulletin, 2025, 41(3): 171-180.

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参考文献 39

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引物名称 Primer name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	PCR产物 PCR product/bp
CmBAM1	ATCAACAAGCAGCAGTCC	CCACCAAACATCAATCATTACC	184
CmBAM2	AAATGAACCCTGTGAGTAGC	CCCTCGACATCTTTACAACC	144
CmBAM3	AGGAGGAGGAGAAAGAAGG	CCACACATCCACCATAACC	164
CmBAM4	CCAGTGTATGTGATGATGCC	AGTCTCTCTCCACCAATCC	151
CmBAM5	TCTTGCAGCATTCCTTTACC	ACTTGGATTGGTGTTCTTCC	155
CmBAM6	GGTGCTGCTTTACAATTTCC	TAACCCATCCTTGTCAAACC	168
CmBAM7	GTGAAAGCCCTATCTCATCC	TCATTCTTTGCATCTCTCTCC	148
CmBAM8	GTGATCGGGTACTTTATCTGG	GCCATAATTGCAGCATAACC	163
CmBAM9	GTGAGCTTCGATATCCTTCC	AGGGCATTTCGTCATAGC	171
CmActin7	GTGATGGTGTGAGTCACACTGTTC	ACGACCAGCAAGGTCCAAAC	83

引物名称 Primer name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	PCR产物 PCR product/bp
CmBAM1	ATCAACAAGCAGCAGTCC	CCACCAAACATCAATCATTACC	184
CmBAM2	AAATGAACCCTGTGAGTAGC	CCCTCGACATCTTTACAACC	144
CmBAM3	AGGAGGAGGAGAAAGAAGG	CCACACATCCACCATAACC	164
CmBAM4	CCAGTGTATGTGATGATGCC	AGTCTCTCTCCACCAATCC	151
CmBAM5	TCTTGCAGCATTCCTTTACC	ACTTGGATTGGTGTTCTTCC	155
CmBAM6	GGTGCTGCTTTACAATTTCC	TAACCCATCCTTGTCAAACC	168
CmBAM7	GTGAAAGCCCTATCTCATCC	TCATTCTTTGCATCTCTCTCC	148
CmBAM8	GTGATCGGGTACTTTATCTGG	GCCATAATTGCAGCATAACC	163
CmBAM9	GTGAGCTTCGATATCCTTCC	AGGGCATTTCGTCATAGC	171
CmActin7	GTGATGGTGTGAGTCACACTGTTC	ACGACCAGCAAGGTCCAAAC	83

名称 Name	登录号 Accession ID	染色体位置 Chromosomal location	长度 Length/aa	分子量 Molecular weight /kD	理论等电点 pI	稳定性 Instability index	亲水性 GRAVY	脂肪系数 Aliphatic index
CmBAM1	MELO3C018794	chr01：2 866 533-2 869 946（-）	229	25.85	6.83	46.74	-0.27	86.77
CmBAM2	MELO3C022739	chr04：20 844 211-20 850 106（+）	546	61.51	8.70	42.96	-0.33	70.20
CmBAM3	MELO3C023067	chr05：6 925 334-6 929 085（+）	576	65.07	8.47	42.86	-0.53	66.74
CmBAM4	MELO3C006362	chr06：2 807 369-2 810 480（-）	577	64.02	5.83	35.07	-0.40	68.35
CmBAM5	MELO3C013887	chr06：34 421 144-34 424 228（+）	537	60.03	8.64	37.52	-0.44	74.12
CmBAM6	MELO3C014105	chr06：36 954 661-36 958 280（+）	524	59.95	8.89	42.93	-0.30	77.04
CmBAM7	MELO3C016213	chr07：22 268 773-22 275 513（+）	668	75.11	5.90	43.75	-0.40	73.91
CmBAM8	MELO3C021214	chr11：31 280 641-31 285 843（-）	698	78.20	5.74	37.39	-0.43	73.60
CmBAM9	MELO3C021362	chr11：30 050 890-30 053 716（-）	533	59.21	6.07	36.11	-0.42	73.36

名称 Name	登录号 Accession ID	染色体位置 Chromosomal location	长度 Length/aa	分子量 Molecular weight /kD	理论等电点 pI	稳定性 Instability index	亲水性 GRAVY	脂肪系数 Aliphatic index
CmBAM1	MELO3C018794	chr01：2 866 533-2 869 946（-）	229	25.85	6.83	46.74	-0.27	86.77
CmBAM2	MELO3C022739	chr04：20 844 211-20 850 106（+）	546	61.51	8.70	42.96	-0.33	70.20
CmBAM3	MELO3C023067	chr05：6 925 334-6 929 085（+）	576	65.07	8.47	42.86	-0.53	66.74
CmBAM4	MELO3C006362	chr06：2 807 369-2 810 480（-）	577	64.02	5.83	35.07	-0.40	68.35
CmBAM5	MELO3C013887	chr06：34 421 144-34 424 228（+）	537	60.03	8.64	37.52	-0.44	74.12
CmBAM6	MELO3C014105	chr06：36 954 661-36 958 280（+）	524	59.95	8.89	42.93	-0.30	77.04
CmBAM7	MELO3C016213	chr07：22 268 773-22 275 513（+）	668	75.11	5.90	43.75	-0.40	73.91
CmBAM8	MELO3C021214	chr11：31 280 641-31 285 843（-）	698	78.20	5.74	37.39	-0.43	73.60
CmBAM9	MELO3C021362	chr11：30 050 890-30 053 716（-）	533	59.21	6.07	36.11	-0.42	73.36

名称 Name	α-螺旋 α-helix/%	延伸链 Extended strand/%	β‍-转角 β‍-sheet/%	不规则卷曲 Random coil/%
CmBAM1	26.20	22.71	7.86	43.23
CmBAM2	32.60	14.10	7.14	46.15
CmBAM3	41.67	12.33	5.56	40.45
CmBAM4	38.13	11.79	5.55	44.54
CmBAM5	32.03	16.20	6.52	45.25
CmBAM6	35.50	13.93	6.68	43.89
CmBAM7	38.17	11.68	5.99	44.16
CmBAM8	34.38	13.18	6.73	45.70
CmBAM9	33.40	15.20	5.63	45.78