药学学报

Gene symbol	Sequencing ID	Protein length (aa)	Molecular weight (Da)	pI	GRAVY	Instability index	Fatty coefficient	Alpha helix	Extended strand	Beta turn	Random coil
RpMYB1	Isoform0001686	720	78 633.76	6.07	-0.781	52.08	60.39	24.03%	5.42%	2.22%	68.33%
RpMYB2	Isoform0002152	720	78 556.62	6.12	-0.79	52.35	60.39	25.97%	6.25%	2.64%	65.14%
RpMYB3	Isoform0002173	658	74 424.35	8.34	-0.974	55.08	62.07	56.84%	5.47%	4.26%	33.43%
RpMYB4	Isoform0002641	659	74 547.59	8.34	-0.955	54.14	63.31	57.21%	5.46%	4.25%	33.08%
RpMYB5	Isoform0002840	720	78 575.66	6.12	-0.782	52.08	60.39	24.44%	7.22%	2.22%	66.11%
RpMYB6	Isoform0004774	635	69 756.57	5.17	-0.664	48.11	71.67	24.57%	11.34%	3.15%	60.94%
RpMYB7	Isoform0005245	614	68 171.04	8.92	-0.643	58.12	72.07	21.99%	12.38%	3.75%	61.89%
RpMYB8	Isoform0005378	690	75 187.64	8.34	-0.592	50.34	75.42	17.68%	12.61%	3.48%	66.23%
RpMYB9	Isoform0006986	640	72 243.68	6.29	-0.957	55.89	63.05	54.37%	5.62%	3.91%	36.09%
RpMYB10	Isoform0010223	670	75 137.03	8.69	-1.165	72.4	62.15	39.85%	8.21%	4.63%	47.31%
RpMYB11	Isoform0010351	514	55 678.65	6.07	-0.625	46.78	62.72	21.98%	6.23%	2.14%	69.65%
RpMYB12	Isoform0010794	608	68 280.64	9.51	-0.926	60.17	67.38	36.68%	4.11%	2.30%	56.91%
RpMYB13	Isoform0012565	470	53 946.03	6.23	-0.785	49.73	80.26	52.98%	2.34%	1.49%	43.19%
RpMYB14	Isoform0014170	432	46 499.83	5.58	-0.506	51.11	66.67	34.95%	5.09%	0.93%	59.03%
RpMYB15	Isoform0014609	514	55 610.57	6.13	-0.613	45.69	62.92	22.76%	6.23%	2.53%	68.48%
RpMYB16	Isoform0015432	405	44 703.66	8.45	-0.577	54.01	76.25	18.27%	11.60%	1.98%	68.15%
RpMYB17	Isoform0015860	374	41 792.19	9.44	-0.693	59.47	65.45	20.32%	12.83%	2.41%	64.44%
RpMYB18	Isoform0019461	402	44 436.33	8.45	-0.586	54.34	75.85	22.14%	10.20%	1.49%	66.17%
RpMYB19	Isoform0021823	402	44 463.36	8.45	-0.593	54.34	75.85	18.66%	11.69%	2.24%	67.41%
RpMYB20	Isoform0022646	293	31 216.49	6.41	-0.687	47.52	59.32	20.14%	12.29%	4.10%	63.48%
RpMYB21	Isoform0023114	399	44 513.38	9.53	-0.972	51.18	67.09	60.90%	1.75%	3.26%	34.09%
RpMYB22	Isoform0023982	370	40 697.27	5.9	-0.823	60.57	68.57	30.00%	5.68%	1.62%	62.70%
RpMYB23	Isoform0024607	427	46 837.45	9.44	-0.928	44.17	55.76	34.19%	4.22%	2.11%	59.48%
RpMYB24	Isoform0024646	375	40 075.85	6.7	-0.592	61.63	56.99	13.07%	9.87%	2.13%	74.93%
RpMYB25	Isoform0025237	370	40 628.16	5.77	-0.813	59.9	68.57	28.92%	6.76%	2.43%	61.89%
RpMYB26	Isoform0025261	353	38 441.88	7.14	-0.71	65.39	61.73	31.16%	7.37%	1.70%	59.77%
RpMYB27	Isoform0025338	307	33 080.65	6.01	-0.386	52.66	72.77	44.95%	7.49%	3.91%	43.65%
RpMYB28	Isoform0027902	306	33 609.99	8.3	-0.593	42.34	64.41	28.10%	6.86%	4.25%	60.78%
RpMYB29	Isoform0027961	333	34 781.7	8.71	-0.413	59.52	64.23	7.21%	17.12%	3.00%	72.67%
RpMYB30	Isoform0029831	348	39 259.05	7.36	-0.818	41.14	74.28	32.18%	9.48%	2.87%	55.46%
RpMYB31	Isoform0031429	309	34 015.51	8.9	-0.638	34.23	76.31	47.25%	8.41%	3.56%	40.78%
RpMYB32	Isoform0032110	303	32 954.93	8.78	-0.656	47.02	61.88	24.09%	7.92%	3.63%	64.36%
RpMYB33	Isoform0033061	290	31 756.23	6.16	-0.932	53.13	65.97	36.21%	5.52%	4.83%	53.45%
RpMYB34	Isoform0033819	274	30 662.11	8.79	-0.498	45.09	66.2	39.85%	8.21%	4.63%	47.31%
RpMYB35	Isoform0034193	355	38 644.09	7.14	-0.704	65.31	61.66	30.42%	5.07%	1.13%	63.38%
RpMYB36	Isoform0036039	305	33 516.15	9.09	-0.505	43.44	72.52	43.28%	8.85%	3.61%	44.26%
RpMYB37	Isoform0036743	310	345 36.43	8.61	-0.464	48.9	71.42	22.58%	8.06%	3.87%	65.48%
RpMYB38	Isoform0037363	301	33 028.59	9.09	-0.502	45.84	73.49	46.18%	7.64%	3.65%	42.52%
RpMYB39	Isoform0037744	289	31 634.73	9.42	-0.741	38.65	66.99	47.06%	7.61%	2.08%	43.25%
RpMYB40	Isoform0038308	293	31 243.52	6.41	-0.696	46.86	59.32	19.45%	12.29%	3.75%	64.51%
RpMYB41	Isoform0039688	303	32 998.98	8.78	-0.656	47.02	62.18	24.09%	7.92%	3.63%	64.36%
RpMYB42	Isoform0039877	283	31 021.6	8.5	-0.576	53.12	67.84	20.49%	12.01%	1.77%	65.72%
RpMYB43	Isoform0039969	282	30 351.76	9.04	-0.411	63.8	74.65	19.86%	14.54%	4.26%	61.35%
RpMYB44	Isoform0041222	266	28 533.8	7.94	-0.486	56.9	68.98	15.41%	15.41%	2.63%	66.54%
RpMYB45	Isoform0042536	331	34 629.55	8.71	-0.4	58.08	65.5	10.88%	12.99%	2.42%	73.72%
RpMYB46	Isoform0043169	275	30 598.69	7.55	-0.699	51.93	74.62	46.18%	5.45%	3.64%	44.73%
RpMYB47	Isoform0043290	260	28 148.4	9.39	-0.492	74.89	70.12	16.92%	21.54%	3.85%	57.69%
RpMYB48	Isoform0044981	269	28 623.89	6.4	-0.384	43.88	68.25	32.71%	8.18%	5.20%	53.90%
RpMYB49	Isoform0048867	260	28 134.38	9.39	-0.492	74.89	70.12	16.54%	16.15%	4.23%	63.08%

Gene symbol	Sequencing ID	Protein length (aa)	Molecular weight (Da)	pI	GRAVY	Instability index	Fatty coefficient	Alpha helix	Extended strand	Beta turn	Random coil
RpMYB1	Isoform0001686	720	78 633.76	6.07	-0.781	52.08	60.39	24.03%	5.42%	2.22%	68.33%
RpMYB2	Isoform0002152	720	78 556.62	6.12	-0.79	52.35	60.39	25.97%	6.25%	2.64%	65.14%
RpMYB3	Isoform0002173	658	74 424.35	8.34	-0.974	55.08	62.07	56.84%	5.47%	4.26%	33.43%
RpMYB4	Isoform0002641	659	74 547.59	8.34	-0.955	54.14	63.31	57.21%	5.46%	4.25%	33.08%
RpMYB5	Isoform0002840	720	78 575.66	6.12	-0.782	52.08	60.39	24.44%	7.22%	2.22%	66.11%
RpMYB6	Isoform0004774	635	69 756.57	5.17	-0.664	48.11	71.67	24.57%	11.34%	3.15%	60.94%
RpMYB7	Isoform0005245	614	68 171.04	8.92	-0.643	58.12	72.07	21.99%	12.38%	3.75%	61.89%
RpMYB8	Isoform0005378	690	75 187.64	8.34	-0.592	50.34	75.42	17.68%	12.61%	3.48%	66.23%
RpMYB9	Isoform0006986	640	72 243.68	6.29	-0.957	55.89	63.05	54.37%	5.62%	3.91%	36.09%
RpMYB10	Isoform0010223	670	75 137.03	8.69	-1.165	72.4	62.15	39.85%	8.21%	4.63%	47.31%
RpMYB11	Isoform0010351	514	55 678.65	6.07	-0.625	46.78	62.72	21.98%	6.23%	2.14%	69.65%
RpMYB12	Isoform0010794	608	68 280.64	9.51	-0.926	60.17	67.38	36.68%	4.11%	2.30%	56.91%
RpMYB13	Isoform0012565	470	53 946.03	6.23	-0.785	49.73	80.26	52.98%	2.34%	1.49%	43.19%
RpMYB14	Isoform0014170	432	46 499.83	5.58	-0.506	51.11	66.67	34.95%	5.09%	0.93%	59.03%
RpMYB15	Isoform0014609	514	55 610.57	6.13	-0.613	45.69	62.92	22.76%	6.23%	2.53%	68.48%
RpMYB16	Isoform0015432	405	44 703.66	8.45	-0.577	54.01	76.25	18.27%	11.60%	1.98%	68.15%
RpMYB17	Isoform0015860	374	41 792.19	9.44	-0.693	59.47	65.45	20.32%	12.83%	2.41%	64.44%
RpMYB18	Isoform0019461	402	44 436.33	8.45	-0.586	54.34	75.85	22.14%	10.20%	1.49%	66.17%
RpMYB19	Isoform0021823	402	44 463.36	8.45	-0.593	54.34	75.85	18.66%	11.69%	2.24%	67.41%
RpMYB20	Isoform0022646	293	31 216.49	6.41	-0.687	47.52	59.32	20.14%	12.29%	4.10%	63.48%
RpMYB21	Isoform0023114	399	44 513.38	9.53	-0.972	51.18	67.09	60.90%	1.75%	3.26%	34.09%
RpMYB22	Isoform0023982	370	40 697.27	5.9	-0.823	60.57	68.57	30.00%	5.68%	1.62%	62.70%
RpMYB23	Isoform0024607	427	46 837.45	9.44	-0.928	44.17	55.76	34.19%	4.22%	2.11%	59.48%
RpMYB24	Isoform0024646	375	40 075.85	6.7	-0.592	61.63	56.99	13.07%	9.87%	2.13%	74.93%
RpMYB25	Isoform0025237	370	40 628.16	5.77	-0.813	59.9	68.57	28.92%	6.76%	2.43%	61.89%
RpMYB26	Isoform0025261	353	38 441.88	7.14	-0.71	65.39	61.73	31.16%	7.37%	1.70%	59.77%
RpMYB27	Isoform0025338	307	33 080.65	6.01	-0.386	52.66	72.77	44.95%	7.49%	3.91%	43.65%
RpMYB28	Isoform0027902	306	33 609.99	8.3	-0.593	42.34	64.41	28.10%	6.86%	4.25%	60.78%
RpMYB29	Isoform0027961	333	34 781.7	8.71	-0.413	59.52	64.23	7.21%	17.12%	3.00%	72.67%
RpMYB30	Isoform0029831	348	39 259.05	7.36	-0.818	41.14	74.28	32.18%	9.48%	2.87%	55.46%
RpMYB31	Isoform0031429	309	34 015.51	8.9	-0.638	34.23	76.31	47.25%	8.41%	3.56%	40.78%
RpMYB32	Isoform0032110	303	32 954.93	8.78	-0.656	47.02	61.88	24.09%	7.92%	3.63%	64.36%
RpMYB33	Isoform0033061	290	31 756.23	6.16	-0.932	53.13	65.97	36.21%	5.52%	4.83%	53.45%
RpMYB34	Isoform0033819	274	30 662.11	8.79	-0.498	45.09	66.2	39.85%	8.21%	4.63%	47.31%
RpMYB35	Isoform0034193	355	38 644.09	7.14	-0.704	65.31	61.66	30.42%	5.07%	1.13%	63.38%
RpMYB36	Isoform0036039	305	33 516.15	9.09	-0.505	43.44	72.52	43.28%	8.85%	3.61%	44.26%
RpMYB37	Isoform0036743	310	345 36.43	8.61	-0.464	48.9	71.42	22.58%	8.06%	3.87%	65.48%
RpMYB38	Isoform0037363	301	33 028.59	9.09	-0.502	45.84	73.49	46.18%	7.64%	3.65%	42.52%
RpMYB39	Isoform0037744	289	31 634.73	9.42	-0.741	38.65	66.99	47.06%	7.61%	2.08%	43.25%
RpMYB40	Isoform0038308	293	31 243.52	6.41	-0.696	46.86	59.32	19.45%	12.29%	3.75%	64.51%
RpMYB41	Isoform0039688	303	32 998.98	8.78	-0.656	47.02	62.18	24.09%	7.92%	3.63%	64.36%
RpMYB42	Isoform0039877	283	31 021.6	8.5	-0.576	53.12	67.84	20.49%	12.01%	1.77%	65.72%
RpMYB43	Isoform0039969	282	30 351.76	9.04	-0.411	63.8	74.65	19.86%	14.54%	4.26%	61.35%
RpMYB44	Isoform0041222	266	28 533.8	7.94	-0.486	56.9	68.98	15.41%	15.41%	2.63%	66.54%
RpMYB45	Isoform0042536	331	34 629.55	8.71	-0.4	58.08	65.5	10.88%	12.99%	2.42%	73.72%
RpMYB46	Isoform0043169	275	30 598.69	7.55	-0.699	51.93	74.62	46.18%	5.45%	3.64%	44.73%
RpMYB47	Isoform0043290	260	28 148.4	9.39	-0.492	74.89	70.12	16.92%	21.54%	3.85%	57.69%
RpMYB48	Isoform0044981	269	28 623.89	6.4	-0.384	43.88	68.25	32.71%	8.18%	5.20%	53.90%
RpMYB49	Isoform0048867	260	28 134.38	9.39	-0.492	74.89	70.12	16.54%	16.15%	4.23%	63.08%

基于全长转录组测序的掌叶大黄R1-MYB基因家族鉴定分析

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赵霞 ¹^,²^,^# , 李元敏 ¹^,³^,^# , 李依民 ¹^,²^,^* , 肖光辉 ⁴ , 张明英 ¹ , 程文萍 ¹ , 高静 ¹^,² , 彭亮 ¹ , 张岗 ¹^,²^,^*

药学学报 | 研究论文 2023,58(5): 1354-1363

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药学学报 | 研究论文 2023, 58(5): 1354-1363

基于全长转录组测序的掌叶大黄R1-MYB基因家族鉴定分析

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赵霞¹^,²^,^#, 李元敏¹^,³^,^#, 李依民¹^,²^,^*, 肖光辉⁴, 张明英¹, 程文萍¹, 高静¹^,², 彭亮¹, 张岗¹^,²^,^*

作者信息

1.陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心, 陕西西安 712046

2.陕西中医药大学陕西省中医药管理局"秦药"研发重点实验室, 陕西西安 712046

3.陕西中医药大学第二附属医院, 陕西西安 712046

4.陕西师范大学生命科学学院, 陕西西安 710119

通讯作者:

*李依民, E-mail: 2051058@sntcm.edu.cn;

张岗, E-mail: jay_gumling2003@aliyun.com

Identification and analysis of R1-MYB gene family in Rheum palmatum L. based on full-length transcriptome sequencing

Xia ZHAO¹^,², Yuan-min LI¹^,³, Yi-min LI¹^,²^,^*, Guang-hui XIAO⁴, Ming-ying ZHANG¹, Wen-ping CHENG¹, Jing GAO¹^,², Liang PENG¹, Gang ZHANG¹^,²^,^*

Affiliations

1. College of Pharmacy and Shaanxi Qinling Application Development and Engineering Center of Chinese Herbal Medicine, Shaanxi University of Chinese Medicine, Xi'an 712046, China

2. Key Laboratory for Research and Development of "Qin Medicine" of Shaanxi Administration of Traditional Chinese Medicine, Shaanxi University of Chinese Medicine, Xi'an 712046, China

3. The Second Affiliated Hospital of Shaanxi University of Chinese Medicine, Xi'an 712046, China

4. College of Life Science, Shaanxi Normal University, Xi'an 710119, China

出版时间: 2023-05-12 doi: 10.16438/j.0513-4870.2022-1009

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摘要

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作为MYB (v-myb avian myeloblastosis viral oncogene homolog) 转录因子家族的一大类, R1-MYB (MYB-related) 家族在植物生长发育、环境胁迫及激素信号转导等方面发挥重要的调节作用。本研究基于全长转录组测序分析, 系统筛选掌叶大黄R1-MYB家族基因, 分析其编码蛋白的理化、结构域及分子进化等特性, 利用RNA-seq进行基因组织表达分析, 并检测RpMYB24响应激素、非生物胁迫的表达模式。结果表明, 共鉴定到掌叶大黄49个R1-MYB家族基因, 主要编码热稳定的亲水蛋白, 大部分定位于细胞核。各蛋白无规卷曲和α螺旋所占比例较大, 存在MYB家族标志性的W型保守氨基酸。掌叶大黄R1-MYB家族成员分布于CCA1 (circadian clock associated 1)-like、I-box (GATAAG)-like、CPC (CAPRICE)-like、TRF (telomere repeat binding factor)-like和TBP (TATA binding protein)-like 5个亚组, 且CCA1-like数量居多。RNA-seq揭示49个R1-MYBs基因在掌叶大黄根、根茎及叶中差异表达, 根及根茎中分别有15、23个基因表达量高于叶中。发现RpMYB24基因受脱落酸(abscisic acid, ABA)、水杨酸(salicylic acid, SA) 和茉莉酸甲酯(methyl jasmonate, MeJA) 的诱导, 且能显著应答损伤、低温或高温胁迫, 但对干旱胁迫响应不显著。本研究系统鉴定了掌叶大黄R1-MYB家族基因及其分子特征, 为下一步开展基因功能验证提供基础, 进而为大黄药材品质形成的转录调控机制研究提供科学依据。

关键词

掌叶大黄 / R1-MYB / 转录因子 / 基因表达 / 胁迫

Abstract

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As one kind of v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors, R1-MYB (MYB-related) family plays an important role in plant growth and development, as well as environmental stress and hormone signal transduction. In this study, R1-MYB family genes in Rheum palmatum L. were systematically screened based on full-length transcriptome sequencing analysis. Firstly, the physicochemical, protein domain and molecular evolution characteristics of the coding proteins were analyzed. Furthermore, the tissue expression levels of R1-MYB genes were analyzed by RNA-seq. We also investigated the expression pattern of RpMYB24 in response to various hormones and abiotic stresses. The results showed that a total of 49 R1-MYB genes were identified, which mainly encoded thermally stable hydrophilic proteins. Most of the deduced proteins were predicted to locate in nucleus. Each protein had a large proportion of random curl and α helix, and also had the W-type conserved amino acids which were the signature of MYB. R1-MYB family members were distributed in five subgroups, including circadian clock associated 1 (CCA1)-like, I-box (GATAAG)-like, CAPRICE (CPC)-like, telomere repeat binding factor (TRF)-like and TATA binding protein (TBP)-like, and the number of CCA1-like was the majority. RNA-seq revealed that 49 R1-MYB genes were differentially expressed in roots, rhizomes and leaves of R. palmatum, and the expression levels of 15 and 23 genes in roots and rhizomes were higher than those in leaves, respectively. RpMYB24 transcript was induced by abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) treatment, and could also significantly respond to injury, low temperature and high temperature stresses except drought stress. This study systematically identified the R1-MYB family genes and their molecular characteristics, better for further gene functional validation, and then provide a scientific basis for the transcriptional regulation mechanism research into rhubarb quality formation.

Key words

Rheum palmatum L. / R1-MYB / transcription factor / gene expression / stress

引用本文

赵霞, 李元敏, 李依民, 肖光辉, 张明英, 程文萍, 高静, 彭亮, 张岗. 基于全长转录组测序的掌叶大黄R1-MYB基因家族鉴定分析. 药学学报, 2023 , 58 (5) : 1354 -1363 . DOI: 10.16438/j.0513-4870.2022-1009

Xia ZHAO, Yuan-min LI, Yi-min LI, Guang-hui XIAO, Ming-ying ZHANG, Wen-ping CHENG, Jing GAO, Liang PENG, Gang ZHANG. Identification and analysis of R1-MYB gene family in Rheum palmatum L. based on full-length transcriptome sequencing[J]. Acta Pharmaceutica Sinica, 2023 , 58 (5) : 1354 -1363 . DOI: 10.16438/j.0513-4870.2022-1009

正文

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掌叶大黄Rheum palmatum L.系《中华人民共和国药典》2020年版正品大黄三基源植物之一, 其药材具有泻下攻积、清热泻火、凉血解毒、逐瘀通经、利湿退黄等功效^[1]。掌叶大黄主要分布于甘肃、青海、陕西等高海拔地区, 野生资源有限, 临床需求量大, 目前主要为栽培品^[2]。大黄的主要活性成分为蒽醌类成分, 其生物合成途径主要由莽草酸、甲羟戊酸、磷酸甲基赤藓醇及聚酮途径复合而成^[3]。苯丙烷代谢途径起始于上游糖酵解, 与莽草酸途径共同产生苯丙氨酸, 故大黄蒽醌类成分与苯丙氨酸同享莽草酸途径^[4]。研究发现, 通过抑制苯丙氨酸解氨酶活性阻断苯丙酸途径, 使代谢流偏向合成蒽醌途径, 可提高蒽醌类物质的积累量^[5]。MYB (v-myb avian myeloblastosis viral oncogene homolog) 转录因子被证实广泛参与调控植物细胞苯丙烷类代谢^[6]。因此, 挖掘掌叶大黄MYB并阐明作用机制对解析蒽醌类成分生物合成的调控研究具有重要意义。

药用植物在生长发育过程中常经受各种胁迫(也称逆境)^[7]。基于“顺境出产量, 逆境促品质”, 中药材“拟境栽培”生态种植便是模拟药用植物野生环境, 以实现药用植物品质的“天地人药合一”^[8]。植物在面对不良环境时其细胞迅速从“舒适”状态转变进入“胁迫响应”状态, 这依赖于植物对环境胁迫的感知、激素信号的激发、转录因子的活化等一系列复杂的生理生化反应^[9]。MYB是应答外界环境指导细胞转录调控的重要参与者, 也是植物中成员众多且功能多样的一大类转录因子。MYB蛋白N端含有约50个高度保守的氨基酸残基和间隔序列组成的R结构域, 保守氨基酸残基以螺旋-转角-螺旋(helix-turn-helix, HTH) 形式参与结合DNA。根据R结构域的重复数量, MYB家族分为R2R3-MYB、3R-MYB、4R-MYB和R1-MYB/MYB-related四类, 分别含有2、3、4个和单一R结构域^{[10, 11]}。R2R3-MYB在植物中最常见, 普遍参与植物细胞分化、激素应答、次生代谢及环境胁迫生理适应等多种生物学过程^[12]。但关于R1-MYB的相关报道较少, 限制了对植物MYB功能的深入研究。

植物中的第一个R1-MYB基因分离自马铃薯MybSt1^[13]。随着基因组测序技术的发展, 逐渐在拟南芥^[14]、三七^[15]、山楂^[16]等多种植物中鉴定了R1-MYB类转录因子。该家族蛋白主要由CCA1 (circadian clock associated 1)-like、I-box (GATAAG)-like、CPC (CAPRICE)-like、TRF (telomere repeat binding factor)-like和TBP (TATA binding protein)-like 5个亚组构成; 其中CCA1-like和TBP-like亚组所含成员最多, 具有典型的保守基序, CCAI-like亚组在MYB重复序列的第3个螺旋中含有高度保守的SHAQK (Y/F) F基序, TBP-like亚组含有高度保守的LKDKW (R/K) (N/T) 基序^[17]。大量研究揭示, R1-MYB参与植物生长发育、环境胁迫及激素信号转导等生理过程。如拟南芥韧皮部特异FE蛋白通过调控韧皮部细胞中与开花相关蛋白的合成和运输来调节开花过程^[18]。干旱胁迫条件下, 过表达OsMYB48的转基因水稻在萌发和萌发后阶段对脱落酸(abscisic acid, ABA) 高度敏感, 促进体内更多内源ABA的积累^[19]。羊草LcMYB2在转基因拟南芥中直接结合渗透胁迫标记基因AtDREB2A、AtLEA14和AtP5CS1的启动子并激活其表达, 以抵御干旱胁迫^[20]。

本研究以掌叶大黄为对象, 利用全长转录组测序技术结合RNA-seq挖掘R1-MYB转录因子家族基因并分析其差异表达特征, 同时进行RpMYB24基因响应激素、非生物胁迫的表达模式检测, 为后续深入研究其在大黄生长发育及蒽醌类成分代谢调控中的作用机制提供支撑。

材料与方法

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主要试剂和仪器 茉莉酸甲酯(methyl jasmonate, MeJA)、水杨酸(salicylic acid, SA)、脱落酸(abscisic acid, ABA) (上海源叶生物科技有限公司); Trizol试剂盒(Life technologies公司); SMARTer PCR cDNA Synthesis Kit (美国Clontech公司); RN38-EASYspin Plus植物RNA提取试剂盒(北京艾德莱公司); PrimeScript^TM RT Master Mix反转录试剂盒、TB Green^® Premix ExTaq^TM Ⅱ (TliRNaseH Plus) (TaKaRa公司)。NanoDrop^TM 2000分光光度计(美国Thermo-fisher公司); PacBio Seque Ⅲ测序仪(美国PacBio公司); StepOnePlus^TM Real-Time PCR (qPCR) 仪(美国Applied Biosystems公司); K5800自动检测超微量分光光度计(凯奥公司)。

材料 2021年8月在甘肃省陇南市甘肃中医药大学和政药用植物园分别采集掌叶大黄R. palmatum L.一年生植株和成熟种子, 经陕西中医药大学胡本祥教授鉴定。

选择大小一致、饱满的种子点播于装有200 g泥炭土(klasmann) 的黑色塑料花盆中(直径9 cm、高12.5 cm), 每盆点播4颗种子, 23 ± 2 ℃, 光周期16/8 h, 光照强度9 000 Lx条件下培养。初次浇水至盆底有水排出即可, 每隔3天补充水50 mL。生长至1月龄时选取长势均匀、生命旺盛的掌叶大黄幼苗3株, 将根、叶等量混合, 液氮中速冻后置于干冰中送广州基迪奥生物科技有限公司进行全长转录组测序分析。取一年生植株3株, 根、根茎、叶3个部位各等量混合后进行RNA-Seq分析。

同时, 选取生命旺盛、长势均匀的一月龄幼苗, 激素处理组分别喷施200 μmol·L^-1 MeJA、SA、ABA, 喷施溶剂模拟对照组; 非生物胁迫处理组分别进行干旱(10% PEG 6000)、盐(100 mmol·L^-1 NaCl)、高温(40 ℃)、低温(4 ℃)、机械损伤(针刺叶片), 喷施无菌水模拟对照组; 两组材料均以0 h为空白对照, 所有样品重复3次, 分别于处理后1、3、6、12和24 h取样处理, 液氮速冻后置-80 ℃冰箱保存备用。

全长转录组测序及R1-MYB鉴定 利用Trizol试剂盒提取总RNA, NanoDrop^TM 2000分光光度计检测其完整性, 检测合格后的总RNA使用SMARTer PCR cDNA Synthesis Kit及PCR扩增合成全长cDNA并进行SMRT bell文库构建。采用PacBio Seque Ⅲ进行上机测序, 利用SMRT Link V8.0.0分析原始数据。先提取高质量的环形一致性序列(circular consensus sequencing, CCS), 聚类FLNC reads, 得到完整的isoform。通过BlastX (http://www.ncbi.nlm.nih.gov/BLAST/) 将isoform比对到蛋白数据库Nr (http://www.ncbi.nlm.nih.gov)、SwissProt (http://www.expasy.ch/sprot) 和KEGG (http://www.genome.jp/keggs) 进行功能注释。

使用转录因子数据库plant TFdb (http://planttfdb.gao-lab.org/index.php) 在蛋白序列中找到有已知转录因子的motif, 进行转录因子家族归类。挑选R1-MYB转录因子家族基因, 再次进行BlastX比对, 结合ORF Finder分析筛选出具有完整开放阅读框(ORF) 的全长基因。用ExPASy (https://prosite.expasy.org/prosite.html) 分析基因编码蛋白质的结构域, 同时用CDD (https://www.ncbi.nlm.nih.gov/cdd) 进行验证。

蛋白序列分析 利用在线工具ProtParam (https://web.expasy.org/protparam/)和SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) 对掌叶大黄R1-MYB转录因子的一级、二级结构进行理化性质分析。SignalP-5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0) 和TMHMM预测信号肽和跨膜区域。WoLF PSORT (https://www.genscript.com/wolf-psort.html) 预测亚细胞定位。MEME (https://meme-suite.org/meme/tools/streme) 分析保守氨基酸基序, 并用TBtools将其可视化。用Weblogo^[21]分析蛋白序列位点。借助MEGA 7邻接法(Neighbor-joining, NJ) 进行掌叶大黄R1-MYB系统进化树构建分析, 自举值为1 000次。

基因组织表达分析 基于RNA-Seq转录组测序数据, 筛选出R1-MYB转录因子在掌叶大黄根、根茎和叶中的表达数据, 以FPKM值表示丰度, 经log2标准化后运用TBtools绘制R1-MYB基因在不同组织中的相对表达量热图, 分析其在掌叶大黄不同组织部位的表达模式。

RpMYB24的表达模式分析参照RN38-EASYspin Plus植物RNA提取试剂盒提取掌叶大黄各样品总RNA, 1.0%琼脂糖凝胶电泳检测完整性, 用K5800自动检测超微量分光光度计检测RNA浓度和纯度。用PrimeScript^TM RT Master Mix反转录试剂盒合成cDNA第一链, -20 ℃保存备用。

基于RNA-Seq转录组测序数据选择CCA1-like亚组中于根中高表达的RpMYB24基因进行表达模式分析, 利用qPCR检测其在一年生掌叶大黄根、根茎、叶中的表达水平及其响应激素或非生物胁迫的相对表达量, 以β-actin为内参基因^[22]。RpMYB24基因qPCR引物: RpMYB24-qPCR-F 5'-CCTGGACATCTGAGGAGCATAG-3', RpMYB24-qPCR-R 5'-CATTTGTTCTTCGGGCACTG-3', 扩增产物长239 bp。使用TB Green^® Premix ExTaq^TM Ⅱ (TliRNaseH Plus) 进行qPCR。20 μL反应体系: 2× TB Green^® Premix ExTaq^TM Ⅱ (TliRNaseH Plus) 10 μL、forward/reverse primer各0.8 μL、cDNA模板2 μL、50× ROX Reference Dye 0.4 μL、ddH₂O 7 μL。反应程序: 预变性95 ℃ 30 s, 变性95 ℃ 5 s, 退火60 ℃ 30 s, 延伸60 ℃ 34 s, 40个循环, 反应结束后于95 ℃ 15 s, 60 ℃ 1 min, 95 ℃ 15 s条件下绘制熔解曲线。包括不加模板的对照在内, 所有qPCR反应技术重复和实验重复各3次, 应用2^-∆∆Ct法^[23]计算相对表达量。

RpMYB24编码蛋白的分子进化分析通过NCBI和PlantTFDB数据库检索RpMYB24同源序列, 使用MEGA 7邻接法进行分子进化分析。

统计学分析 应用SPSS 24软件对RpMYB24基因响应外源激素及非生物胁迫的表达量进行t检验分析, P < 0.05代表有显著性差异。

结果与分析

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1 R1-MYB转录因子序列分析

掌叶大黄全长转录组包含55个TF家族, R1-MYB家族有92个isoforms, 包含完整ORF的isoform有58个。整理ORF差异位点并合并重复, 最终获得49个全长R1-MYB转录因子, 编号RpMYB1~RpMYB49 (表 1)。蛋白氨基酸序列最长的是RpMYB1/2/5, 有720个残基, 最短的是RpMYB49, 编码260个残基。

对49个R1-MYB蛋白序列的一级结构特征分析发现, 蛋白的分子质量在28.134 (RpMYB49)~78.633 (RpMYB1) kD; 理论等电点介于5.17~9.53, 包括32个碱性蛋白, 17个酸性蛋白, 表明在不同的微环境中R1-MYB转录因子发挥着不同生物学功能。所有R1-MYB蛋白的平均亲水性数值是负值, 说明其均为亲水性蛋白; RpMYB31/39的稳定系数 < 40, 推测其为稳定蛋白, 其余47个为不稳定蛋白; 脂肪系数为55.76~80.26, 说明R1-MYB转录因子基因编码蛋白的热稳定性较好。二级结构分析发现, 掌叶大黄R1-MYB家族蛋白均具有α螺旋、延伸链、β转角和无规卷曲, 主要由α螺旋和无规卷曲构成(所占比平均值分别为30.33%、57.78%), 延伸链和β-折叠所占比例较小(所占比平均值分别为8.83%、3.06%), 散布于整个蛋白中。SignalP-5.0和TMHMM在线分析一致表明掌叶大黄R1-MYB蛋白均无信号肽和跨膜结构域。亚细胞定位结果显示, 掌叶大黄R1-MYB蛋白仅RpMYB48定位于细胞质, 其余成员均定位于细胞核。

2 进化树分析

利用MEGA7.0构建掌叶大黄与拟南芥R1-MYB转录因子家族系统进化树。图 1结果表明, 49个R1-MYB蛋白分为CCA1-like、TBP-like、CPC-like、TRF-like和I-box-like 5个亚组, 其中CCA1-like是最大的1个亚组, 含有RpMYB家族成员24个, 占总数的48.98%, 又分成多个小的亚支; 其次是TBP-like、TRF-like、CPC-like亚组, 分别含有9、9、4个RpMYB基因家族成员, 依次占总数的18.37%、18.37%、8.16%; 与拟南芥I-box-like相近的转录因子在掌叶大黄中较少, 只有3个, 仅占6.12%。其中, RpMYB28与拟南芥AT3G09600.2/RVE8亲缘关系较近, 可能具有相似的生物学功能。

3 保守基序分析

利用在线软件MEME对掌叶大黄49个R1-MYB蛋白序列进行保守基序分析(图 2)。结果表明, 不同R1-MYB转录因子基因包含的保守元件数量及种类存在差异, RpMYB10/12/14基因包含的保守元件数量最少(1个), RpMYB1/2/5/11/15基因包含的保守元件数量最多(13个), 说明RpMYBs成员具有功能冗余现象, 也具有功能差异性。Motif2存在于所有MYB序列中, 且大多分布于近N端, 可能是掌叶大黄R1-MYB转录因子的特征基序。Motif15是CCA1-like亚组的特征基序, 且是最短的基序, 含有8个氨基酸残基, 最长含有50个氨基酸残基。此外, Motif3、Motif7和Motif17也存在于CCA1-like亚组, 其中, Motif7位于该亚组的近N端。CPC-like亚组的RpMYB3/4和TBP-like亚组成员均拥有Motif18, 但所处位置有差异。

MYB蛋白结合位点序列分析结果表明(图 3), R1-MYB转录因子的每个重复结构域约为51 aa, 且存在MYB标志性的W型保守氨基酸, R1-MYB的第3个色氨酸被甲硫氨酸(k) 或半胱氨酸(D) 取代, 第一个色氨酸残基高度保守, 说明其对于形成疏水核和维持三维结构非常重要。

4 不同组织表达量热图

将49个RpMYBs基因在掌叶大黄3个部位的差异表达数据进行层级聚类。图 4结果表明, 绝大部分基因的表达不恒定, 在不同组织具有相对较高的表达量, 叶、根和根茎表达量较高的基因数分别有23、15和25个。其中RpMYB6/17/22/25/29/41/45/46在根和根茎中表达量均较高, 呈现类似的表达模式。CCA1-like、TRF-like、CPC-like及I-box-like基因在掌叶大黄根、茎、叶中均表达; 而TBP-like基因在根茎中高表达。

5 RpMYB24的表达模式

以叶为参考样本, qPCR分析检测RpMYB24在掌叶大黄一年生植株根、根茎、叶中表达水平, 结果显示, RpMYB24在根、根茎中的表达量分别为叶中的7.81、4.36倍, 根及根茎中表达量显著高于叶中。

RpMYB24响应外源激素处理不同时间点的表达见图 5A。以0 h (control check, CK) 为空白对照, Mock在处理3 h内迅速下调后缓慢恢复; 与之相反的是, MeJA和ABA处理分别持续上调至峰值为CK的1.74 (3 h)、2.63 (1 h) 倍后逐渐下调; SA处理在24 h内缓慢促进表达, 在6 h上调明显。

图 5B为RpMYB24应答非生物胁迫处理不同时间点的表达分析, 以0 h (CK) 为对照, 发现对比Mock, 干旱组中的表达变化不显著; 受损伤、低温、高温胁迫表达变化显著, 分别在3、6、24 h达峰值, 为CK的10.01、5.46、16.88倍; 盐胁迫12 h显著上调, 后下调。

6 RpMYB24蛋白的分子进化分析

从NCBI和PlantTFDB数据库下载拟南芥、丹参、水稻、玉米、大豆、青蒿、花生等7种植物R1-MYB蛋白家族成员, 采用MEGA 7邻接法分析RpMYB24基因编码蛋白的分子进化关系。图 6结果显示, RpMYB24与大豆GmMYB62、花生Ahy011271关系最近。

讨论

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R1-MYB基因家族在大多数物种中已有鉴定, 不仅在维持染色体结构的完整性方面发挥作用, 还参与生长发育、激素应答、环境胁迫等众多生命活动过程^[9]。被子植物中存在大量R1-MYB基因, 藻类等低等植物中相对较少^[11]。本研究基于全长转录组测序技术挖掘到92个R1-MYB isoforms, 通过ORF Finder结合BlastX分析筛选出具有完整ORF的全长基因, 人工手动去除重复的isoforms, 最终确定49个R1-MYB基因。相较于水稻^[24]、红花^[25]、川白芷^[26]基因组的70、128、122个基因, 掌叶大黄的R1-MYB基因数量较少, 这是由于物种在进化过程中, 基因不断复制与分化, 外加环境适应, 进而导致不同物种间基因数量的差异。随着后续大黄基因组测序研究的推进, 可能会补充本研究所鉴定的R1-MYB, 结合基因共线性、染色体定位等分析, 系统阐明掌叶大黄R1-MYB基因分子特征。

系统进化分析显示, 掌叶大黄R1-MYB家族成员在5个亚组中均有不均匀分布, 且CCA1-like亚组中数量最多。研究表明, CCA1-like蛋白参与植物的昼夜节律调节^[27], 如CCA1/LHY参与拟南芥早期的光形态建成、光周期节律的维持、调控和光周期开花^[28]。RpMYB1/2/5/11/15与拟南芥AT1G01060.1/LHY高度同源, 且包含的保守元件数量最多, 说明RpMYB1/2/5/11/15可能在大黄光周期节律的诱导和保持机制中起重要作用。该亚家族成员的二级结构也有明显的特点, 主要有α-螺旋、无规卷曲。相对于TBP-like亚家族庞大的数量, 目前只有少数基因进行了功能验证。其中, Smh1^[29]可参与编码末端结合转录因子, 调控端粒酶活性; PcMYB1^[30]在体内与光调节启动子单元(LRU1) 相互作用, 调控植物生长发育。本研究揭示了24个CCA1-like基因, 9个TBP-like基因, 亚家族成员之间蛋白二级结构预测结果十分相似, 但序列间同源性相对较低, 说明这些亚组成员可能在掌叶大黄生长发育方面发挥广泛的生物功能。

掌叶大黄R1-MYB家族成员蛋白二级结构具有HTH结构, 符合MYB蛋白的结构特点^[31], 从结构上证实预测结果属于MYB转录因子蛋白。川白芷R1-MYB家族基因中存在多个保守结构域, 其中Motif6和Motif9保守性较高, Motif2出现频率最高^[27]。马铃薯R1-MYB中含有Motif1~Motif20, Motif1和Motif2出现频次最高^[32]。掌叶大黄R1-MYB转录因子蛋白序列含有Motif1~Motif20, Motif2出现频次最高。三者有一定区别, Motif2均出现频率高且稳定, 同一亚组基因含有相似基序, 但所含Motif数量与分布不同的特点, 表明R1-MYB类转录因子的空间位置保守性相对较低。保守序列中3个均匀分布的氨基酸(W) 残基存在被替换的情况, 但第1个W高度保守, 且存在其他保守的氨基酸残基, 推测可能对维持其HTH结构具有重要意义。

R1-MYB基因家族成员通常具有组织表达特异性, 这与其所调控的生命活动有关。本研究组织表达热图揭示CCA1-like、TRF-like及I-box-like基因在掌叶大黄根、根茎、叶中均表达; 有趣的是, I-box-like类基因在叶片组织中有较高的表达量, 说明该类基因虽然缺少C端转录激活区, 但具有广泛的表达谱^[11], 推测其参与调控掌叶大黄叶的发育和相关生理代谢过程, 如番茄I-box-like基因Lefsml, 可特异调控早期果实发育^[33]。选择一个在根中高表达的R1-MYB基因, RpMYB24组织表达量热图与qPCR结果一致, 其表达量次序为根 > 根茎 > 叶, 说明其主要在掌叶大黄地下部分发挥作用。

内源激素在调节植物体生理生化反应中起重要信号转导的作用, 而植物MYB基因表达也与激素密切相关^[9]。如ABA和干旱处理下大豆GmMYB174基因的表达量下调, 盐胁迫下表达量上调^[34]。黑果枸杞LrMYB1R1在不同时间NaCl (250 mmol·L^-1) 胁迫条件下差异表达, 可能参与黑果枸杞盐胁迫的响应^[35]。本研究发现RpMYB24基因受ABA、SA和MeJA处理显著诱导, 损伤、低温、高温胁迫均显著提高RpMYB24表达水平, 说明RpMYB24可能在掌叶大黄应对干旱、盐胁迫、微生物胁迫等生理过程起作用, 但其具体作用机制有待深入研究。RpMYB24系统进化分析表明RpMYB24与GmMYB62亲缘关系最近, 研究发现, GmMYB62与GmMYB176相互作用可调节大豆CHS8基因的表达和异黄酮的生物合成^[36], 猜测RpMYB24可能作用于苯丙烷类生物代谢途径。同时RpMYB24与OsMYBS3也聚在一大支, 而OsMYBS3参与低温应激反应, 结合本研究中发现该基因受低温调控的结果, 推测其功能可能与低温应答有关^[37]。

目前对MYB转录因子的研究主要集中于R2R3-MYB, 对R1-MYB的研究还相对较少, 本研究主要鉴定了49个掌叶大黄R1-MYB转录因子家族成员, 通过生物信息学对其蛋白理化特性与结构特征进行初步分析, 实时荧光定量表达分析的方法对RpMYB24进行鉴定与表达分析, 为探究掌叶大黄生长发育、代谢过程及非生物胁迫的调控机制提供参考依据, 同时丰富了R1-MYB类转录因子的研究, 为后期的基因功能研究奠定了基础。

作者贡献: 赵霞和李元敏是本研究的执行人; 程文萍参与数据整理及论文初稿的写作; 肖光辉、张明英、高静和彭亮参与实验设计和实验结果分析; 张岗和李依民是项目的构思者及负责人, 指导实验设计、数据分析、论文写作与修改。

利益冲突: 所有作者均声明不存在利益冲突。

基金

收起

国家自然科学基金资助项目(82104334)
国家自然科学基金资助项目(81973430)
咸阳市中青年科技创新领军人才项目(L2022CXNLRC009)
陕西省重点产业创新链资助项目(2021ZDLSF04-01)
陕西中医药大学“秦药”品质评价与资源开发学科创新团队项目(2019-QN01)

参考文献

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文献

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2023年第58卷第5期

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文章信息

doi: 10.16438/j.0513-4870.2022-1009

接收时间：2022-08-23
首发时间：2025-11-21
出版时间：2023-05-12

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出版历史

收稿日期：2022-08-23
修回日期：2022-09-23

基金

国家自然科学基金资助项目(82104334)

国家自然科学基金资助项目(81973430)

咸阳市中青年科技创新领军人才项目(L2022CXNLRC009)

陕西省重点产业创新链资助项目(2021ZDLSF04-01)

陕西中医药大学“秦药”品质评价与资源开发学科创新团队项目(2019-QN01)

作者信息

1.陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心, 陕西西安 712046

2.陕西中医药大学陕西省中医药管理局"秦药"研发重点实验室, 陕西西安 712046

3.陕西中医药大学第二附属医院, 陕西西安 712046

4.陕西师范大学生命科学学院, 陕西西安 710119

通讯作者:

*李依民, E-mail: 2051058@sntcm.edu.cn;

张岗, E-mail: jay_gumling2003@aliyun.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Gene symbol	Sequencing ID	Protein length (aa)	Molecular weight (Da)	pI	GRAVY	Instability index	Fatty coefficient	Alpha helix	Extended strand	Beta turn	Random coil
RpMYB1	Isoform0001686	720	78 633.76	6.07	-0.781	52.08	60.39	24.03%	5.42%	2.22%	68.33%
RpMYB2	Isoform0002152	720	78 556.62	6.12	-0.79	52.35	60.39	25.97%	6.25%	2.64%	65.14%
RpMYB3	Isoform0002173	658	74 424.35	8.34	-0.974	55.08	62.07	56.84%	5.47%	4.26%	33.43%
RpMYB4	Isoform0002641	659	74 547.59	8.34	-0.955	54.14	63.31	57.21%	5.46%	4.25%	33.08%
RpMYB5	Isoform0002840	720	78 575.66	6.12	-0.782	52.08	60.39	24.44%	7.22%	2.22%	66.11%
RpMYB6	Isoform0004774	635	69 756.57	5.17	-0.664	48.11	71.67	24.57%	11.34%	3.15%	60.94%
RpMYB7	Isoform0005245	614	68 171.04	8.92	-0.643	58.12	72.07	21.99%	12.38%	3.75%	61.89%
RpMYB8	Isoform0005378	690	75 187.64	8.34	-0.592	50.34	75.42	17.68%	12.61%	3.48%	66.23%
RpMYB9	Isoform0006986	640	72 243.68	6.29	-0.957	55.89	63.05	54.37%	5.62%	3.91%	36.09%
RpMYB10	Isoform0010223	670	75 137.03	8.69	-1.165	72.4	62.15	39.85%	8.21%	4.63%	47.31%
RpMYB11	Isoform0010351	514	55 678.65	6.07	-0.625	46.78	62.72	21.98%	6.23%	2.14%	69.65%
RpMYB12	Isoform0010794	608	68 280.64	9.51	-0.926	60.17	67.38	36.68%	4.11%	2.30%	56.91%
RpMYB13	Isoform0012565	470	53 946.03	6.23	-0.785	49.73	80.26	52.98%	2.34%	1.49%	43.19%
RpMYB14	Isoform0014170	432	46 499.83	5.58	-0.506	51.11	66.67	34.95%	5.09%	0.93%	59.03%
RpMYB15	Isoform0014609	514	55 610.57	6.13	-0.613	45.69	62.92	22.76%	6.23%	2.53%	68.48%
RpMYB16	Isoform0015432	405	44 703.66	8.45	-0.577	54.01	76.25	18.27%	11.60%	1.98%	68.15%
RpMYB17	Isoform0015860	374	41 792.19	9.44	-0.693	59.47	65.45	20.32%	12.83%	2.41%	64.44%
RpMYB18	Isoform0019461	402	44 436.33	8.45	-0.586	54.34	75.85	22.14%	10.20%	1.49%	66.17%
RpMYB19	Isoform0021823	402	44 463.36	8.45	-0.593	54.34	75.85	18.66%	11.69%	2.24%	67.41%
RpMYB20	Isoform0022646	293	31 216.49	6.41	-0.687	47.52	59.32	20.14%	12.29%	4.10%	63.48%
RpMYB21	Isoform0023114	399	44 513.38	9.53	-0.972	51.18	67.09	60.90%	1.75%	3.26%	34.09%
RpMYB22	Isoform0023982	370	40 697.27	5.9	-0.823	60.57	68.57	30.00%	5.68%	1.62%	62.70%
RpMYB23	Isoform0024607	427	46 837.45	9.44	-0.928	44.17	55.76	34.19%	4.22%	2.11%	59.48%
RpMYB24	Isoform0024646	375	40 075.85	6.7	-0.592	61.63	56.99	13.07%	9.87%	2.13%	74.93%
RpMYB25	Isoform0025237	370	40 628.16	5.77	-0.813	59.9	68.57	28.92%	6.76%	2.43%	61.89%
RpMYB26	Isoform0025261	353	38 441.88	7.14	-0.71	65.39	61.73	31.16%	7.37%	1.70%	59.77%
RpMYB27	Isoform0025338	307	33 080.65	6.01	-0.386	52.66	72.77	44.95%	7.49%	3.91%	43.65%
RpMYB28	Isoform0027902	306	33 609.99	8.3	-0.593	42.34	64.41	28.10%	6.86%	4.25%	60.78%
RpMYB29	Isoform0027961	333	34 781.7	8.71	-0.413	59.52	64.23	7.21%	17.12%	3.00%	72.67%
RpMYB30	Isoform0029831	348	39 259.05	7.36	-0.818	41.14	74.28	32.18%	9.48%	2.87%	55.46%
RpMYB31	Isoform0031429	309	34 015.51	8.9	-0.638	34.23	76.31	47.25%	8.41%	3.56%	40.78%
RpMYB32	Isoform0032110	303	32 954.93	8.78	-0.656	47.02	61.88	24.09%	7.92%	3.63%	64.36%
RpMYB33	Isoform0033061	290	31 756.23	6.16	-0.932	53.13	65.97	36.21%	5.52%	4.83%	53.45%
RpMYB34	Isoform0033819	274	30 662.11	8.79	-0.498	45.09	66.2	39.85%	8.21%	4.63%	47.31%
RpMYB35	Isoform0034193	355	38 644.09	7.14	-0.704	65.31	61.66	30.42%	5.07%	1.13%	63.38%
RpMYB36	Isoform0036039	305	33 516.15	9.09	-0.505	43.44	72.52	43.28%	8.85%	3.61%	44.26%
RpMYB37	Isoform0036743	310	345 36.43	8.61	-0.464	48.9	71.42	22.58%	8.06%	3.87%	65.48%
RpMYB38	Isoform0037363	301	33 028.59	9.09	-0.502	45.84	73.49	46.18%	7.64%	3.65%	42.52%
RpMYB39	Isoform0037744	289	31 634.73	9.42	-0.741	38.65	66.99	47.06%	7.61%	2.08%	43.25%
RpMYB40	Isoform0038308	293	31 243.52	6.41	-0.696	46.86	59.32	19.45%	12.29%	3.75%	64.51%
RpMYB41	Isoform0039688	303	32 998.98	8.78	-0.656	47.02	62.18	24.09%	7.92%	3.63%	64.36%
RpMYB42	Isoform0039877	283	31 021.6	8.5	-0.576	53.12	67.84	20.49%	12.01%	1.77%	65.72%
RpMYB43	Isoform0039969	282	30 351.76	9.04	-0.411	63.8	74.65	19.86%	14.54%	4.26%	61.35%
RpMYB44	Isoform0041222	266	28 533.8	7.94	-0.486	56.9	68.98	15.41%	15.41%	2.63%	66.54%
RpMYB45	Isoform0042536	331	34 629.55	8.71	-0.4	58.08	65.5	10.88%	12.99%	2.42%	73.72%
RpMYB46	Isoform0043169	275	30 598.69	7.55	-0.699	51.93	74.62	46.18%	5.45%	3.64%	44.73%
RpMYB47	Isoform0043290	260	28 148.4	9.39	-0.492	74.89	70.12	16.92%	21.54%	3.85%	57.69%
RpMYB48	Isoform0044981	269	28 623.89	6.4	-0.384	43.88	68.25	32.71%	8.18%	5.20%	53.90%
RpMYB49	Isoform0048867	260	28 134.38	9.39	-0.492	74.89	70.12	16.54%	16.15%	4.23%	63.08%

Gene symbol

Sequencing ID

Protein length (aa)

Molecular weight (Da)

GRAVY

Instability index

Fatty coefficient

Alpha helix

Extended strand

Beta turn

Random coil

RpMYB1

Isoform0001686

720

78 633.76

6.07

-0.781

52.08

60.39

24.03%

5.42%

2.22%

68.33%

RpMYB2

Isoform0002152

720

78 556.62

6.12

-0.79

52.35

60.39

25.97%

6.25%

2.64%

65.14%

RpMYB3

Isoform0002173

658

74 424.35

8.34

-0.974

55.08

62.07

56.84%

5.47%

4.26%

33.43%

RpMYB4

Isoform0002641

659

74 547.59

8.34

-0.955

54.14

63.31

57.21%

5.46%

4.25%

33.08%

RpMYB5

Isoform0002840

720

78 575.66

6.12

-0.782

52.08

60.39

24.44%

7.22%

2.22%

66.11%

RpMYB6

Isoform0004774

635

69 756.57

5.17

-0.664

48.11

71.67

24.57%

11.34%

3.15%

60.94%

RpMYB7

Isoform0005245

614

68 171.04

8.92

-0.643

58.12

72.07

21.99%

12.38%

3.75%

61.89%

RpMYB8

Isoform0005378

690

75 187.64

8.34

-0.592

50.34

75.42

17.68%

12.61%

3.48%

66.23%

RpMYB9

Isoform0006986

640

72 243.68

6.29

-0.957

55.89

63.05

54.37%

5.62%

3.91%

36.09%

RpMYB10

Isoform0010223

670

75 137.03

8.69

-1.165

72.4

62.15

39.85%

8.21%

4.63%

47.31%

RpMYB11

Isoform0010351

514

55 678.65

6.07

-0.625

46.78

62.72

21.98%

6.23%

2.14%

69.65%

RpMYB12

Isoform0010794

608

68 280.64

9.51

-0.926

60.17

67.38

36.68%

4.11%

2.30%

56.91%

RpMYB13

Isoform0012565

470

53 946.03

6.23

-0.785

49.73

80.26

52.98%

2.34%

1.49%

43.19%

RpMYB14

Isoform0014170

432

46 499.83

5.58

-0.506

51.11

66.67

34.95%

5.09%

0.93%

59.03%

RpMYB15

Isoform0014609

514

55 610.57

6.13

-0.613

45.69

62.92

22.76%

6.23%

2.53%

68.48%

RpMYB16

Isoform0015432

405

44 703.66

8.45

-0.577

54.01

76.25

18.27%

11.60%

1.98%

68.15%

RpMYB17

Isoform0015860

374

41 792.19

9.44

-0.693

59.47

65.45

20.32%

12.83%

2.41%

64.44%

RpMYB18

Isoform0019461

402

44 436.33

8.45

-0.586

54.34

75.85

22.14%

10.20%

1.49%

66.17%

RpMYB19

Isoform0021823

402

44 463.36

8.45

-0.593

54.34

75.85

18.66%

11.69%

2.24%

67.41%

RpMYB20

Isoform0022646

293

31 216.49

6.41

-0.687

47.52

59.32

20.14%

12.29%

4.10%

63.48%

RpMYB21

Isoform0023114

399

44 513.38

9.53

-0.972

51.18

67.09

60.90%

1.75%

3.26%

34.09%

RpMYB22

Isoform0023982

370

40 697.27

5.9

-0.823

60.57

68.57

30.00%

5.68%

1.62%

62.70%

RpMYB23

Isoform0024607

427

46 837.45

9.44

-0.928

44.17

55.76

34.19%

4.22%

2.11%

59.48%

RpMYB24

Isoform0024646

375

40 075.85

6.7

-0.592

61.63

56.99

13.07%

9.87%

2.13%

74.93%

RpMYB25

Isoform0025237

370

40 628.16

5.77

-0.813

59.9

68.57

28.92%

6.76%

2.43%

61.89%

RpMYB26

Isoform0025261

353

38 441.88

7.14

-0.71

65.39

61.73

31.16%

7.37%

1.70%

59.77%

RpMYB27

Isoform0025338

307

33 080.65

6.01

-0.386

52.66

72.77

44.95%

7.49%

3.91%

43.65%

RpMYB28

Isoform0027902

306

33 609.99

8.3

-0.593

42.34

64.41

28.10%

6.86%

4.25%

60.78%

RpMYB29

Isoform0027961

333

34 781.7

8.71

-0.413

59.52

64.23

7.21%

17.12%

3.00%

72.67%

RpMYB30

Isoform0029831

348

39 259.05

7.36

-0.818

41.14

74.28

32.18%

9.48%

2.87%

55.46%

RpMYB31

Isoform0031429

309

34 015.51

8.9

-0.638

34.23

76.31

47.25%

8.41%

3.56%

40.78%

RpMYB32

Isoform0032110

303

32 954.93

8.78

-0.656

47.02

61.88

24.09%

7.92%

3.63%

64.36%

RpMYB33

Isoform0033061

290

31 756.23

6.16

-0.932

53.13

65.97

36.21%

5.52%

4.83%

53.45%

RpMYB34

Isoform0033819

274

30 662.11

8.79

-0.498

45.09

66.2

39.85%

8.21%

4.63%

47.31%

RpMYB35

Isoform0034193

355

38 644.09

7.14

-0.704

65.31

61.66

30.42%

5.07%

1.13%

63.38%

RpMYB36

Isoform0036039

305

33 516.15

9.09

-0.505

43.44

72.52

43.28%

8.85%

3.61%

44.26%

RpMYB37

Isoform0036743

310

345 36.43

8.61

-0.464

48.9

71.42

22.58%

8.06%

3.87%

65.48%

RpMYB38

Isoform0037363

301

33 028.59

9.09

-0.502

45.84

73.49

46.18%

7.64%

3.65%

42.52%

RpMYB39

Isoform0037744

289

31 634.73

9.42

-0.741

38.65

66.99

47.06%

7.61%

2.08%

43.25%

RpMYB40

Isoform0038308

293

31 243.52

6.41

-0.696

46.86

59.32

19.45%

12.29%

3.75%

64.51%

RpMYB41

Isoform0039688

303

32 998.98

8.78

-0.656

47.02

62.18

24.09%

7.92%

3.63%

64.36%

RpMYB42

Isoform0039877

283

31 021.6

8.5

-0.576

53.12

67.84

20.49%

12.01%

1.77%

65.72%

RpMYB43

Isoform0039969

282

30 351.76

9.04

-0.411

63.8

74.65

19.86%

14.54%

4.26%

61.35%

RpMYB44

Isoform0041222

266

28 533.8

7.94

-0.486

56.9

68.98

15.41%

2.63%

66.54%

RpMYB45

Isoform0042536

331

34 629.55

8.71

-0.4

58.08

65.5

10.88%

12.99%

2.42%

73.72%

RpMYB46

Isoform0043169

275

30 598.69

7.55

-0.699

51.93

74.62

46.18%

5.45%

3.64%

44.73%

RpMYB47

Isoform0043290

260

28 148.4

9.39

-0.492

74.89

70.12

16.92%

21.54%

3.85%

57.69%

RpMYB48

Isoform0044981

269

28 623.89

6.4

-0.384

43.88

68.25

32.71%

8.18%

5.20%

53.90%

RpMYB49

Isoform0048867

260

28 134.38

9.39

-0.492

74.89

70.12

16.54%

16.15%

4.23%

63.08%