枣R2R3-MYB亚家族基因鉴定及其在果实发育中的表达分析

doi:10.3864/j.issn.0578-1752.2022.06.012

摘要/Abstract

摘要：

【目的】MYB作为植物中最大的基因家族之一,在植物生长、果实发育等方面具有重要作用。本研究拟鉴定枣R2R3-MYB亚家族成员,并进行生物信息学和表达模式分析,为深入探究该类基因在枣生长发育尤其是果实发育中的功能提供参考。【方法】以拟南芥R2R3-MYB亚家族成员为探针,利用BLAST和HMMER方法,在枣全基因组数据库中筛选R2R3-MYB亚家族成员;利用植物转录因子、UniProt、Gcorn plant等数据库,采用MEGA-x、ExPASy、TBtools、PheatMap等软件进行生物信息学分析;利用转录组和实时荧光定量PCR技术分析该类基因的表达,对重点基因进行互作蛋白的预测及验证。【结果】在枣全基因组范围内,鉴定出118个R2R3-MYB亚家族成员,均具有典型的MYB超家族结构域,分为18个亚组,依次命名为ZjMYB1—ZjMYB118。理化性质分析表明,该家族基因编码的氨基酸数目介于200—400;95个成员定位到12条染色体上,其中4号染色体上分布最多;谱系分析表明该类基因进化过程中发生了直系和旁系同源事件。通过转录组和实时荧光定量PCR分析,发现一些R2R3-MYB基因参与枣果实的发育过程,并进一步筛选出了可能与果实膨大和色泽发育相关的候选基因ZjMYB59、ZjMYB104。并利用蛋白互作预测和酵母双杂试验证明ZjMYB104和ZjMPK3存在互作。【结论】鉴定出118个枣R2R3-MYB亚家族成员,结构高度保守,分布在12条染色体上,在进化中发生了直系和旁系同源事件,并筛选出了与果实发育密切相关的候选基因,为进一步的功能分析提供了线索和参考。

关键词: 枣, R2R3-MYB亚家族基因, 生物信息学分析, 表达分析, 果实发育, 蛋白互作

Abstract:

【Objective】As one of the largest gene families in plants, MYB family plays an important role in plant growth and fruit development. This study would identify the members of the R2R3-MYB subfamily of Chinese jujube and analyze their bioinformatics and expression patterns, aiming to provide the reference for further exploring their function in jujube growth, especially in fruit development.【Method】The R2R3-MYB genes of Arabidopsis thaliana were used as probes, and this subfamily members in the jujube genome were screened out by BLAST and HMMER. Based on the Plant Transcription Factors Database, Uniprot, Gcorn Plant and other databases, R2R3-MYB genes were analyzed by MEGA-X, ExPASy, TBtools, and PheatMap. Transcriptome data and real-time quantitative PCR (qRT-PCR) were used to evaluate the expression of these genes, and the interaction proteins of key candidate proteins were predicted and verified. 【Result】In this study, 118 R2R3-MYB subfamily members were identified in jujube genome, all of which had a typical MYB domain. They were divided into 18 subgroups, named ZjMYB1-ZjMYB118. The number of amino acids encoded by the genes ranged from 200 to 400. Among them, 95 genes were located on 12 chromosomes, and which on Chr. 4 was the most widely distributed. The phylogenetic analysis showed that orthologous and paralogous events occurred during the evolution of these genes. By transcriptomic and qRT-PCR analysis, some R2R3-MYB genes were involved in the fruit development. In particular, ZjMYB59 and ZjMYB104 might participate in fruit enlargement and color development. Protein interaction prediction and yeast two-hybrid test confirmed that ZjMYB104 and ZjMPK3 were interacted each other. 【Conclusion】118 members of the R2R3-MYB subfamily members were identified and distributed on 12 chromosomes. The structure of these subfamily members was highly conservative. The orthologous and paralogous events occurred in jujube genome evolution and the candidate genes closely related to fruit development were screened out. This study provided clues and references for further functional analysis of R2R3-MYB subfamily in jujube.

Key words: Chinese jujube, R2R3-MYB subfamily, bioinformatics analysis, expression analysis, fruit development, proteins interaction

李世佳,吕紫敬,赵锦. 枣R2R3-MYB亚家族基因鉴定及其在果实发育中的表达分析[J]. 中国农业科学, 2022, 55(6): 1199-1212.

LI ShiJia,LÜ ZiJing,ZHAO Jin. Identification of R2R3-MYB Subfamily in Chinese Jujube and Their Expression Pattern During the Fruit Development[J]. Scientia Agricultura Sinica, 2022, 55(6): 1199-1212.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.06.012

https://www.chinaagrisci.com/CN/Y2022/V55/I6/1199

图/表 10

表1

表2

表3

枣R2R3-MYB亚家族成员信息"

基因名称 Gene name	NCBI登录号 NCBI reference	染色体位置Chromosomes locus	阅读框长度 ORF (bp)	氨基酸数量 Amino acids size (aa)	分子量 Mw (Da)	等电点 pI	组 Group	外显子数目 Exon number
ZjMYB1	XM_016020618.2	1	1125	374	42125.68	6.41	III	3
ZjMYB2	XM_016019589.2	1	1107	368	41303.37	6.36	XI	2
ZjMYB3	XM_016026021.2	1	969	322	35915.26	6.37	III	3
ZjMYB4	XM_016031375.2	1	1098	365	41463.99	5.74	I	3
ZjMYB5	XM_016033817.2	1	1068	355	40263.36	6.46	XI	3
ZjMYB6	XM_016041113.2	1	1092	363	40879.74	7.12	I	3
ZjMYB7	XM_016044244.2	1	1551	516	56216.27	7.38	XIV	3
ZjMYB8	XM_016047554.2	1	918	305	34305.95	5.88	VIII	3
ZjMYB9	XM_016047610.2	1	918	305	34205.92	6.27	VIII	3
ZjMYB10	XM_025071868.1	1	633	210	24181.15	9.41	X	4
ZjMYB11	XM_016010160.2	1	975	324	36742.03	6.23	VIII	3
ZjMYB12	XM_016011494.2	1	600	199	22085.17	9.86	XVIII	1
ZjMYB13	XM_025070775.1	2	1545	514	56159.92	6.51	XVI	3
ZjMYB14	XM_016019055.2	2	1014	337	37964.79	6.44	III	3
ZjMYB15	XM_016020065.2	2	843	280	31740.73	8.87	II	3
ZjMYB16	XM_016020395.2	2	1182	393	43572.07	7.07	XI	2
ZjMYB17	XM_016020396.2	2	1392	463	52118.77	9.06	XI	2
ZjMYB18	XM_025072101.1	3	1068	355	40263.36	6.46	XI	5
ZjMYB19	XM_025072033.1	3	1674	557	60498.13	5.26	XIV	6
ZjMYB20	XM_016020719.2	3	1182	393	43139.24	6.24	I	3
ZjMYB21	XM_016021844.2	3	1269	422	45817.40	5.76	XVIII	2
ZjMYB22	XM_016021930.2	3	600	199	22085.17	9.86	XVIII	1
ZjMYB23	XM_025071932.1	3	540	179	19445.06	7.87	XVIII	2
ZjMYB24	XM_016022267.2	3	930	309	35021.56	5.70	XV	3
ZjMYB25	XM_016022405.2	3	483	160	18529.47	9.63	XII	2
ZjMYB26	XM_016022551.2	3	912	303	34188.03	6.80	XI	3
ZjMYB27	XM_016022613.2	3	912	303	34188.03	6.80	XI	3
ZjMYB28	XM_016022841.2	3	570	189	21194.98	4.51	XII	3
ZjMYB29	XM_016022861.2	3	816	271	30610.07	5.28	XII	2
ZjMYB30	XM_016023053.2	3	1152	383	42646.60	6.57	I	3
ZjMYB31	XM_016023353.2	4	1350	449	49829.67	6.73	VII	2
ZjMYB32	XM_016023411.2	4	918	305	35111.19	5.44	XII	3
ZjMYB33	XM_025072351.1	4	918	305	35111.19	5.44	XII	3
ZjMYB34	XM_025073253.1	4	1635	544	61347.96	8.53	XVI	3
ZjMYB35	XM_016023591.2	4	600	199	22857.66	6.66	XIII	3
ZjMYB36	XM_025072429.1	4	1038	345	38887.27	6.07	III	3
ZjMYB37	XM_025073248.1	4	1635	544	61386.09	8.53	XVI	3
ZjMYB38	XM_016023883.2	4	1053	350	38719.09	6.47	III	3
ZjMYB39	XM_016024167.2	4	996	331	37360.48	7.37	IV	3
ZjMYB40	XM_016024719.2	4	816	271	24261.55	8.65	IV	4
ZjMYB41	XM_016025498.2	4	822	273	31374.58	5.52	II	4
ZjMYB42	XM_016025511.2	4	741	246	28267.43	6.01	II	3
ZjMYB43	XM_016025514.2	4	1080	359	40269.26	5.36	I	3
ZjMYB44	XM_016025643.2	4	1647	548	59274.73	6.78	XIV	3
ZjMYB45	XM_016026187.2	5	1089	362	41025.21	5.88	XI	2
ZjMYB46	XM_016026978.2	5	1170	389	43445.01	5.40	XI	3
ZjMYB47	XM_016027029.1	5	849	282	32365.59	5.74	XVI	3
ZjMYB48	XM_016027102.2	5	801	266	30479.45	7.08	IX	3
ZjMYB49	XM_016027959.2	5	1026	341	38533.86	6.18	I	4
ZjMYB50	XM_016027963.2	5	930	309	34855.98	6.76	I	3
ZjMYB51	XM_016027995.2	5	930	309	34855.98	6.76	I	3
ZjMYB52	XM_025074262.1	5	687	228	27115.88	9.98	XVII	3
ZjMYB53	XM_016028420.2	5	747	248	29081.60	8.59	XVII	3
ZjMYB54	XM_016028861.2	6	942	313	34430.54	7.63	XVIII	1
ZjMYB55	XM_016028962.2	6	600	199	23299.35	9.36	X	3
ZjMYB56	XM_016028941.2	6	918	305	34060.09	6.71	VIII	3
ZjMYB57	XM_016029606.2	6	945	314	34389.41	6.24	III	3
ZjMYB58	XM_016029902.2	6	1041	346	38761.22	6.30	I	3
ZjMYB59	XM_016029928.2	6	693	230	25510.24	8.78	XVIII	1
ZjMYB60	XM_016030857.2	6	864	287	32390.60	8.71	IV	4
ZjMYB61	XM_016032123.2	7	891	296	33380.87	5.06	XV	3
ZjMYB62	XM_016032124.2	7	891	296	33380.87	5.06	XV	3
ZjMYB63	XM_016032235.2	7	891	296	34314.38	8.99	XIII	3
ZjMYB64	XM_016032377.2	7	822	273	30832.44	5.07	II	4
ZjMYB65	XM_016033142.2	7	999	332	36740.26	6.08	I	4
ZjMYB66	XM_016033215.2	8	882	293	33694.74	8.99	VI	2
ZjMYB67	XM_025076686.1	8	876	291	31728.46	8.32	III	3
ZjMYB68	XM_025076102.1	8	831	276	31421.30	5.45	X	2
ZjMYB69	XM_016033873.2	8	786	261	29525.12	5.81	X	3
ZjMYB70	XM_016034776.2	8	1011	336	36075.90	9.18	XVIII	1
ZjMYB71	XM_016034941.2	8	1020	339	37865.24	7.58	III	3
ZjMYB72	XM_016035211.2	8	1185	394	43392.16	6.34	XI	3
ZjMYB73	XM_016035688.2	8	831	276	31676.43	9.33	XVII	2
ZjMYB74	XM_016035781.2	9	870	289	33214.83	5.70	II	3
ZjMYB75	XM_016036214.2	9	846	281	32204.33	6.26	XIII	3
ZjMYB76	XM_016036355.2	9	1257	418	46922.01	4.78	V	3
ZjMYB77	XM_016037555.2	9	867	288	32264.01	5.84	XVIII	1
ZjMYB78	XM_016039031.1	10	771	256	29181.57	5.93	XII	3
ZjMYB79	XM_016039426.2	10	1437	478	53487.56	6.22	XVI	13
ZjMYB80	XM_016039546.2	10	1368	455	49832.18	6.19	XVII	3
ZjMYB81	XM_016039548.2	10	1215	404	44681.25	6.83	I	3
ZjMYB82	XM_016040154.2	10	936	311	35286.65	7.73	XIII	3
ZjMYB83	XM_016040524.2	11	894	297	33864.66	5.68	VI	2
ZjMYB84	XM_016041018.1	11	996	331	37414.53	6.00	XI	3
ZjMYB85	XM_016040967.2	11	1089	362	40382.56	5.40	XI	3
ZjMYB86	XM_016041507.2	11	1071	356	40987.76	9.49	XV	4
ZjMYB87	XM_025079477.1	11	1218	405	43892.18	7.07	XVIII	2
ZjMYB88	XM_016042019.2	11	1092	363	40468.92	5.42	I	3
ZjMYB89	XM_016042953.2	12	717	238	27832.61	5.11	X	4
ZjMYB90	XM_016042930.2	12	1032	343	38266.45	8.25	III	3
ZjMYB91	XM_016043881.2	12	972	323	35595.67	8.20	VII	3
ZjMYB92	XM_016044239.2	12	999	332	37141.53	6.52	III	3
ZjMYB93	XM_016044250.2	12	1188	395	43529.46	7.65	XVII	3
ZjMYB94	XM_025066858.1	12	1323	440	48646.07	8.76	V	3
ZjMYB95	XM_016044619.2	12	1044	347	38753.66	6.30	XIII	3
ZjMYB96	XM_016046006.2	Un	753	250	29105.62	7.82	IX	3
ZjMYB97	XM_016046190.2	Un	654	217	25294.42	6.95	IX	3
ZjMYB98	XM_016046427.2	Un	1434	477	53467.56	6.22	XVI	13
ZjMYB99	XM_025067712.1	Un	990	329	37401.37	4.95	XII	7
ZjMYB100	XM_016010432.2	Un	1011	336	36075.90	9.18	XVIII	1
ZjMYB101	XM_016010643.2	Un	1191	396	45085.59	5.37	II	3
ZjMYB102	XM_025068375.1	Un	750	249	28737.43	8.37	XIII	3
ZjMYB103	XM_016011368.2	Un	930	309	35021.56	5.70	XV	3
ZjMYB104	XM_016011429.2	Un	855	284	31104.15	8.98	IV	2
ZjMYB105	XM_025068805.1	Un	840	279	32350.20	5.97	IX	3
ZjMYB106	XM_016011795.2	Un	1044	347	38753.66	6.30	XIII	3
ZjMYB107	XM_016012645.2	Un	633	210	24195.17	9.41	X	4
ZjMYB108	XM_016012644.2	Un	975	324	36762.97	6.16	VIII	3
ZjMYB109	XM_016013201.2	Un	1050	349	39860.17	9.32	XVII	2
ZjMYB110	XM_016013430.2	Un	1467	488	53445.69	7.85	XI	4
ZjMYB111	XM_016013592.2	Un	669	222	26206.36	6.09	IX	3
ZjMYB112	XM_016015024.2	Un	1014	337	37307.21	6.72	III	4
ZjMYB113	XM_016015356.2	Un	2016	671	75206.30	8.84	XVI	8
ZjMYB114	XM_016016480.2	Un	747	248	29081.60	8.59	XVII	3
ZjMYB115	XM_016017001.2	Un	1467	488	53445.69	7.85	XI	3
ZjMYB116	XM_016017183.2	Un	1467	488	53445.69	7.85	XI	3
ZjMYB117	XM_016017439.1	Un	1221	406	43823.66	6.05	XI	1
ZjMYB118	XM_016017473.1	Un	1221	406	43823.66	6.05	XI	1

表3

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图3

图4

图5

图6

图7

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
ACT-F	AGCCTTCCTGCCAACGAGT
ACT-R	TTGCTTCTCACCCTTGATGC
ZjMYB59-F	GGGTTTTGACCCGTCTTGTTG
ZjMYB59-R	CCTGTTACCATACCTGGCGT
ZjMYB104-F	CACCACCACCTCAGGAAACA
ZjMYB104-R	ACGACGAGGTCCTTTCCAAC

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
ZjMYB104-F	ATGGGAAGGTCTCCATGTTGT
ZjMYB104-R	TTATTTCATTTCCAAGCCTCTATAATCC
AD1-F	GTACCAGATTACGCTCATATGGGAAGGTCTCCATGTTGT
AD1-R	ACTGGCCTCCATGGCCATTTATTTCATTTCCAAGCCTCTATAATCC
BD1-F	TCAGAGGAGGACCTGCATATGGGAAGGTCTCCATGTTGT
BD1-R	TTCGGCCTCCATGGCCATTTATTTCATTTCCAAGCCTCTATAATCC
ZjMPK3-F	ATGTCGGGTGCTAACCAGAC
ZjMPK3-R	TCAGGCATACTCCGGATTAAGTG
AD2-F	GCCATGGAGGCCAGTGAAATGTCGGGTGCTAACCAGAC
AD2-R	ATGCCCACCCGGGTGGAATCAGGCATACTCCGGATTAAGTG
BD2-F	ATGGCCATGGAGGCCGAAATGTCGGGTGCTAACCAGAC
BD2-R	ACGGATCCCCGGGAATTCTCAGGCATACTCCGGATTAAGTG