高温干旱复合胁迫下铁皮石斛MYB基因家族鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0151

摘要/Abstract

摘要：

【目的】研究铁皮石斛Dendrobium catenatum MYB（DcMYB）基因转录因子家族成员的结构和功能，鉴定参与高温干旱复合胁迫的主要成员及其表达模式，为今后研究铁皮石斛适应逆境胁迫的生理机制提供参考。【方法】基于铁皮石斛的全基因组与高温干旱复合胁迫处理后的转录组数据，筛选出响应高温干旱复合逆境的关键MYB转录因子，利用生物信息学方法进行分析和鉴定，实时荧光定量PCR分析在不同胁迫处理下的表达模式。【结果】鉴定出38个DcMYBs基因，可分为1R-MYB、R2R3-MYB和3R-MYB三种类型，其中R2R3-MYB占比89%；DcMYBs蛋白长为203-581aa，热稳定性佳，多具亲水性；亚细胞定位预测显示均存在于细胞核中，部分并存于其他细胞器；MEME分析揭示同亚组的MYB基因具有相似的结构特征；DcMYBs进化发育后将其分为19个亚组（D1-D19）；顺式作用元件预测结果表明DcMYBs参与生物、非生物胁迫与激素诱导，DcMYB6、DcMYB15、DcMYB16、DcMYB21、DcMYB23、DcMYB35均含有干旱响应元件MBS，DcMYB6、DcMYB35还具有4个ABA响应元件ABRE；在高温干旱复合胁迫下，R2R3-MYB亚家族基因被显著诱导，关键基因的RT-qPCR验证与转录组数据一致，DcMYB6、DcMYB35在胁迫下上调，DcMYB15、DcMYB16、DcMYB21、DcMYB23下调；外源脱落酸和20% PEG6000处理具有干旱响应元件MBS基因结果显示在处理后都呈现先上升后下降的趋势，这6个基因可能参与ABA信号途径与干旱胁迫的响应。【结论】逆境胁迫下从铁皮石斛中鉴定出38个DcMYBs基因，R2R3-MYB为主要类型，编码蛋白均具有良好的热稳定性和亲水性且预测主要定位于细胞核；DcMYBs基因与拟南芥MYB具有较高的同源性；特定基因被干旱和外源脱落酸显著诱导，其可能参与生物、非生物胁迫与激素诱导，在铁皮石斛逆境生长发育中发挥重要作用。

关键词: 铁皮石斛, 高温干旱, MYB转录因子, 基因家族鉴定, RT-qPCR验证

Abstract:

【Objective】To study the structure and function of the transcription factor（TF）family in the Dendrobium catenatum MYB（DcMYB）gene, And identify the prime members and their expression under high-drought stress, which may provide aiding in elucidating D. catenatum's stress adaptation mechanisms.【Method】Utilizing whole-genome transcriptomic data from the combined stress of high temperature & drought, key MYB TFs responsive to this stress were screened, then analyzed, identified via informatics, and their expression patterns under various stresses were validated via real-time PCR.【Result】The 38 DcMYBs genes were identified, they were divided into three types: 1R-MYB, R2R3-MYB, and 3R-MYB, and R2R3-MYB accounted for 89%. The DcMYBs proteins’ lengths were 203-581 aa, they were of good thermal stability, and most of them were of hydrophilic properties. Prediction of subcellular localization revealed that all were present in the nucleus, and some of them coexisted in other organelles. The MEME analysis uncovered that the MYB genes in the same subgroup had similar structural characteristics. DcMYBs was divided into 19 subgroups（D1-D19）after evolution. The prediction for cis-acting element showed that DcMYBs were involved in bio/abiotic stress & hormonal induction, DcMYB6, DcMYB15, DcMYB16, DcMYB21, DcMYB23, and DcMYB35 contained drought-responsive MBS. DcMYB6 and DcMYB35 also harbored 4 ABA-responsive ABREs. Under the combined stress of high temperature and drought, the R2R3-MYB subfamily's genes were significantly induced.The the key genes were consistent with the transcriptome data by RT-qPCR validation. DcMYB6 and DcMYB35 were upregulated under stress, DcMYB15, DcMYB16, DcMYB21, and DcMYB23 were downregulated. Treatment with exogenous abscisic acid, 20% PEG6000, and drought-responsive MBS gene showed an initial rising followed by a declining, suggesting the involvement of six genes in ABA signaling during drought drought. 【Conclusion】Under stress, 38 DcMYBs genes were identified in D. catenatum, dominated by R2R3-MYB type. The encoded proteins are of thermal stability, hydrophilicity, and are predicted to be in nucleus. DcMYBs genes have high homology with Arabidopsis MYB. The specific genes are significantly induced by drought and ABA, and they might be likely involved in biological, abiotic stress, hormone induction, and play important roles in the growth development of D. catenatum under stress environment.

Key words: Dendrobium catenatum, heat and drought, MYB transcription factor, genetic family identification, RT-qPCR validation

田姗姗, 黄诗宇, 杨天为, 高曼熔, 张尚文, 何龙飞, 张向军, 李婷, 石前. 高温干旱复合胁迫下铁皮石斛MYB基因家族鉴定及表达分析[J]. 生物技术通报, 2024, 40(12): 145-159.

TIAN Shan-shan, HUANG Shi-yu, YANG Tian-wei, GAO Man-rong, ZHANG Shang-wen, HE Long-fei, ZHANG Xiang-jun, LI Ting, SHI Qian. Identification and Expression Analysis of MYB Gene Family in Dendrobium catenatum Under the Combined Stress of High Temperature and Drought[J]. Biotechnology Bulletin, 2024, 40(12): 145-159.

图/表 13

参考文献 34

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引物名字Primer name	引物序列F Primer sequence F（5'-3'）	引物序列R Primer sequence R（5'-3'）
DcMYB6	GCTCCTCAACCCAATGCTCT	TGCAGGAGATTTGAGGCGAA
DcMYB15	AAGCAGAAGCTAAGGAAAGGCT	TGCTGTGAAAATGTGCCTCT
DcMYB16	GCCGAACACAGGCACATCTA	GCACTCGGACTCGAACATGA
DcMYB23	ATGGTCTTTGATAGCGGCGA	CTTGTTGCTCGTGGAGTGGA
DcMYB24	AGCCATGAGATGTGGGAGGA	TGAAGGTAGGGGAAGAAGGCT
DcMYB35	CCGAGCTGGCAAGAAGAAGA	GCCAAACGACTGACTGAGGA
18S rRNA	CAAGCCCACGCTCTGGATAATTAG	ATGCTTTCGCAGTGGTTCTCTTTC

引物名字Primer name	引物序列F Primer sequence F（5'-3'）	引物序列R Primer sequence R（5'-3'）
DcMYB6	GCTCCTCAACCCAATGCTCT	TGCAGGAGATTTGAGGCGAA
DcMYB15	AAGCAGAAGCTAAGGAAAGGCT	TGCTGTGAAAATGTGCCTCT
DcMYB16	GCCGAACACAGGCACATCTA	GCACTCGGACTCGAACATGA
DcMYB23	ATGGTCTTTGATAGCGGCGA	CTTGTTGCTCGTGGAGTGGA
DcMYB24	AGCCATGAGATGTGGGAGGA	TGAAGGTAGGGGAAGAAGGCT
DcMYB35	CCGAGCTGGCAAGAAGAAGA	GCCAAACGACTGACTGAGGA
18S rRNA	CAAGCCCACGCTCTGGATAATTAG	ATGCTTTCGCAGTGGTTCTCTTTC

基因ID Gene ID	基因名 Gene name	MYB类型 MYB symbol	氨基酸数 Number of amino acids	理论分子量Molecular weight/Da	等电点 Point isoelectric	不稳定指数Instability index	脂肪族氨基酸指数Aliphatic index	亲水度 Average of hydropathicity	亚细胞定位预测Prediction of subcellularlocalization
LOC110097606	DcMYB1	3R-MYB	581	64501.81	8.88	63.81	71.86	-0.605	nucl: 14
LOC110098032	DcMYB2	2R3R-MYB	203	23543.65	7.68	57.57	68.28	-0.865	nucl: 14
LOC110101288	DcMYB3	2R3R-MYB	247	28192.25	4.93	56.44	60.49	-0.809	nucl: 14
LOC110114505	DcMYB4	2R3R-MYB	526	57624.91	5	53.08	66.2	-0.621	Nucler: 13, pero: 1
LOC110115927	DcMYB5	2R3R-MYB	340	38337.7	7.64	54.12	84.62	-0.481	nucl: 14
LOC110098979	DcMYB6	2R3R-MYB	336	38188.52	5.38	45.77	75.15	-0.652	nucl: 14
LOC110114551	DcMYB7	2R3R-MYB	314	35140.56	6.47	53.83	70.19	-0.665	nucl: 13, pero: 1
LOC110103484	DcMYB8	1R-MYB, ZnF	221	24557.44	9.94	49.5	61.76	-0.714	nucl: 5, chlo: 4, mito: 3, plas: 1, cysk: 1
LOC110105838	DcMYB9	2R3R-MYB	287	32412.67	5.99	56.18	74.84	-0.589	nucl: 14
LOC110094774	DcMYB10	2R3R-MYB	275	30978.88	6.37	55.97	65.27	-0.672	nucl: 14
LOC110111576	DcMYB11	2R3R-MYB	243	27921.06	8.94	58.35	56.58	-0.875	nucl: 14
LOC110112067	DcMYB12	2R3R-MYB	231	26786.37	9.67	60.98	63.77	-0.805	nucl: 14
LOC110109430	DcMYB13	2R3R-MYB	238	26549.02	8.28	55.86	71.39	-0.513	nucl: 13, cyto: 1
LOC110111434	DcMYB14	2R3R-MYB	213	24546.74	9.86	49.62	63.19	-0.8	nucl: 14
LOC110115321	DcMYB15	2R3R-MYB	409	45603.97	7.21	68.2	68.66	-0.633	nucl: 14
LOC110113344	DcMYB16	2R3R-MYB	316	35293.45	5.46	45.84	67.25	-0.591	nucl: 14
LOC110104025	DcMYB17	2R3R-MYB	327	37236.83	8.92	56.94	67.13	-0.654	nucl: 14
LOC110102853	DcMYB18	1R-MYB, ZnF	316	34892.05	6.81	63	63.96	-0.652	chlo: 6, mito: 5, nucl: 2, cysk: 1
LOC110099916	DcMYB19	2R3R-MYB	291	33548.13	9.07	62.25	75.05	-0.69	nucl: 14
LOC110108743	DcMYB20	2R3R-MYB	216	24044.51	9.29	54.61	73.7	-0.667	nucl: 14
LOC110113232	DcMYB21	2R3R-MYB	409	45585.94	7.21	68.2	69.61	-0.627	nucl: 14
LOC110109300	DcMYB22	2R3R-MYB	361	39400.05	6.76	62.16	63.88	-0.67	nucl: 14
LOC110112867	DcMYB23	2R3R-MYB	251	28687.7	8.68	72.15	69.96	-0.795	nucl: 14
LOC110111024	DcMYB24	2R3R-MYB	326	37055.28	5.23	56.37	70.31	-0.664	nucl: 14
LOC110107869	DcMYB25	2R3R-MYB	347	38031.44	6.1	54.8	62.8	-0.663	nucl: 14
LOC110115620	DcMYB26	2R3R-MYB	370	40224.06	6.34	68.96	65.19	-0.525	nucl: 14
LOC110094240	DcMYB27	2R3R-MYB	292	32526.71	6.16	48.86	74.55	-0.451	nucl:13，pero: 1
LOC110094080	DcMYB28	2R3R-MYB	312	35182.39	7.01	58.72	70.67	-0.746	nucl: 14
LOC110105411	DcMYB29	2R3R-MYB	286	32205.26	5.38	57.37	79.51	-0.657	nucl: 13, cyto: 1
LOC110095044	DcMYB30	2R3R-MYB	242	28182.63	6.34	52.67	66.12	-0.776	nucl: 14
LOC110113544	DcMYB31	2R3R-MYB	272	31107.96	6.11	59.23	72.76	-0.626	nucl: 14
LOC110113885	DcMYB32	1R-MYB，H15	275	30415.83	9.42	51.22	69.31	-0.687	nucl: 14
LOC110112113	DcMYB33	2R3R-MYB	298	33583.52	6.61	46.48	66.44	-0.737	nucl: 14
LOC110107882	DcMYB34	2R3R-MYB	348	38967.19	7.37	60.89	56.72	-0.735	nucl:13.5, cyto_nucl: 7.5
LOC110099272	DcMYB35	2R3R-MYB	280	31255.11	6.09	58.79	72.89	-0.59	nucl: 13, pero: 1
LOC110103760	DcMYB36	2R3R-MYB	257	29168.99	6.75	52.42	83.07	-0.535	nucl: 9.5, cyto_nucl: 5.5, mito: 2,plas:1.5, golg_plas: 1.5
LOC110107293	DcMYB37	2R3R-MYB	235	27563.73	6.38	74.32	61.4	-0.989	nucl: 14
LOC110093341	DcMYB38	2R3R-MYB	373	41852.49	5.87	57.95	67.24	-0.8	nucl: 14

基因ID Gene ID	基因名 Gene name	MYB类型 MYB symbol	氨基酸数 Number of amino acids	理论分子量Molecular weight/Da	等电点 Point isoelectric	不稳定指数Instability index	脂肪族氨基酸指数Aliphatic index	亲水度 Average of hydropathicity	亚细胞定位预测Prediction of subcellularlocalization
LOC110097606	DcMYB1	3R-MYB	581	64501.81	8.88	63.81	71.86	-0.605	nucl: 14
LOC110098032	DcMYB2	2R3R-MYB	203	23543.65	7.68	57.57	68.28	-0.865	nucl: 14
LOC110101288	DcMYB3	2R3R-MYB	247	28192.25	4.93	56.44	60.49	-0.809	nucl: 14
LOC110114505	DcMYB4	2R3R-MYB	526	57624.91	5	53.08	66.2	-0.621	Nucler: 13, pero: 1
LOC110115927	DcMYB5	2R3R-MYB	340	38337.7	7.64	54.12	84.62	-0.481	nucl: 14
LOC110098979	DcMYB6	2R3R-MYB	336	38188.52	5.38	45.77	75.15	-0.652	nucl: 14
LOC110114551	DcMYB7	2R3R-MYB	314	35140.56	6.47	53.83	70.19	-0.665	nucl: 13, pero: 1
LOC110103484	DcMYB8	1R-MYB, ZnF	221	24557.44	9.94	49.5	61.76	-0.714	nucl: 5, chlo: 4, mito: 3, plas: 1, cysk: 1
LOC110105838	DcMYB9	2R3R-MYB	287	32412.67	5.99	56.18	74.84	-0.589	nucl: 14
LOC110094774	DcMYB10	2R3R-MYB	275	30978.88	6.37	55.97	65.27	-0.672	nucl: 14
LOC110111576	DcMYB11	2R3R-MYB	243	27921.06	8.94	58.35	56.58	-0.875	nucl: 14
LOC110112067	DcMYB12	2R3R-MYB	231	26786.37	9.67	60.98	63.77	-0.805	nucl: 14
LOC110109430	DcMYB13	2R3R-MYB	238	26549.02	8.28	55.86	71.39	-0.513	nucl: 13, cyto: 1
LOC110111434	DcMYB14	2R3R-MYB	213	24546.74	9.86	49.62	63.19	-0.8	nucl: 14
LOC110115321	DcMYB15	2R3R-MYB	409	45603.97	7.21	68.2	68.66	-0.633	nucl: 14
LOC110113344	DcMYB16	2R3R-MYB	316	35293.45	5.46	45.84	67.25	-0.591	nucl: 14
LOC110104025	DcMYB17	2R3R-MYB	327	37236.83	8.92	56.94	67.13	-0.654	nucl: 14
LOC110102853	DcMYB18	1R-MYB, ZnF	316	34892.05	6.81	63	63.96	-0.652	chlo: 6, mito: 5, nucl: 2, cysk: 1
LOC110099916	DcMYB19	2R3R-MYB	291	33548.13	9.07	62.25	75.05	-0.69	nucl: 14
LOC110108743	DcMYB20	2R3R-MYB	216	24044.51	9.29	54.61	73.7	-0.667	nucl: 14
LOC110113232	DcMYB21	2R3R-MYB	409	45585.94	7.21	68.2	69.61	-0.627	nucl: 14
LOC110109300	DcMYB22	2R3R-MYB	361	39400.05	6.76	62.16	63.88	-0.67	nucl: 14
LOC110112867	DcMYB23	2R3R-MYB	251	28687.7	8.68	72.15	69.96	-0.795	nucl: 14
LOC110111024	DcMYB24	2R3R-MYB	326	37055.28	5.23	56.37	70.31	-0.664	nucl: 14
LOC110107869	DcMYB25	2R3R-MYB	347	38031.44	6.1	54.8	62.8	-0.663	nucl: 14
LOC110115620	DcMYB26	2R3R-MYB	370	40224.06	6.34	68.96	65.19	-0.525	nucl: 14
LOC110094240	DcMYB27	2R3R-MYB	292	32526.71	6.16	48.86	74.55	-0.451	nucl:13，pero: 1
LOC110094080	DcMYB28	2R3R-MYB	312	35182.39	7.01	58.72	70.67	-0.746	nucl: 14
LOC110105411	DcMYB29	2R3R-MYB	286	32205.26	5.38	57.37	79.51	-0.657	nucl: 13, cyto: 1
LOC110095044	DcMYB30	2R3R-MYB	242	28182.63	6.34	52.67	66.12	-0.776	nucl: 14
LOC110113544	DcMYB31	2R3R-MYB	272	31107.96	6.11	59.23	72.76	-0.626	nucl: 14
LOC110113885	DcMYB32	1R-MYB，H15	275	30415.83	9.42	51.22	69.31	-0.687	nucl: 14
LOC110112113	DcMYB33	2R3R-MYB	298	33583.52	6.61	46.48	66.44	-0.737	nucl: 14
LOC110107882	DcMYB34	2R3R-MYB	348	38967.19	7.37	60.89	56.72	-0.735	nucl:13.5, cyto_nucl: 7.5
LOC110099272	DcMYB35	2R3R-MYB	280	31255.11	6.09	58.79	72.89	-0.59	nucl: 13, pero: 1
LOC110103760	DcMYB36	2R3R-MYB	257	29168.99	6.75	52.42	83.07	-0.535	nucl: 9.5, cyto_nucl: 5.5, mito: 2,plas:1.5, golg_plas: 1.5
LOC110107293	DcMYB37	2R3R-MYB	235	27563.73	6.38	74.32	61.4	-0.989	nucl: 14
LOC110093341	DcMYB38	2R3R-MYB	373	41852.49	5.87	57.95	67.24	-0.8	nucl: 14