互花米草NAC转录因子家族的鉴定与表达分析

doi:10.16288/j.yczz.19-250

遗传 ›› 2020, Vol. 42 ›› Issue (2): 194-211.doi: 10.16288/j.yczz.19-250

互花米草NAC转录因子家族的鉴定与表达分析

王涛涛^1,²(), 杨勇^1,², 魏唯², 林辰涛², 马留银²()

^1. 福建农林大学林学院,福州 350002
^2. 福建农林大学基础林学与蛋白质组学研究中心,福州 350002

收稿日期:2019-08-27 修回日期:2019-12-26 出版日期:2020-02-21 发布日期:2020-02-08
基金资助:
福建农林大学林学高峰学科建设项目(71201800725);福建农林大学林学高峰学科建设项目(71201800773);福建农林大学优秀博士学位论文基金项目(324-1122yb043)

Identification and expression analyses of the NAC transcription factor family in Spartina alterniflora

Wang Taotao^1,²(), Yang Yong^1,², Wei Wei², Lin Chentao², Ma Liuyin²()

^1. College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
^2. Basic Forestry and Proteomics Research Center, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Received:2019-08-27 Revised:2019-12-26 Online:2020-02-21 Published:2020-02-08
Supported by:
Peak Subject Construction Project of Fujian Agricultural and Forestry University(71201800725);Peak Subject Construction Project of Fujian Agricultural and Forestry University(71201800773);Scientific Research Foundation of the Graduate School of Fujian Agriculture and Forestry University(324-1122yb043)

摘要/Abstract

摘要：

互花米草(Spartina alterniflora)作为一种海岸带盐生植物,高度耐盐胁迫,但因为缺少参考基因组,其耐盐的分子机制却尚未见报道。NAC家族蛋白是植物特有的转录因子,调控植物的生长发育和胁迫应答。为了鉴定互花米草NAC蛋白(SaNAC)并探究它们与互花米草生长发育及胁迫响应之间的关系,本研究以互花米草三代全长转录组数据为参考,通过与水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和玉米(Zea mays)NAC蛋白序列进行比对,并结合保守功能域进一步筛选,最终找到62个SaNAC蛋白。从蛋白序列比对、进化、motif预测、同源性比较、亚细胞定位、组织表达以及非生物胁迫下的基因差异表达等方面分别对互花米草NAC家族成员进行分析,结果发现SaNAC蛋白均含有保守的NAM结构域,且在进化上与水稻NAC家族具有一定的相似性;SaNAC家族中的两个蛋白SaNAC9和SaNAC49在细胞核表达;另外,本研究还发现互花米草SaNAC基因表达具有高度组织和胁迫应答差异性。这些结果表明互花米草NAC转录因子家族不仅具有保守的功能域,而且在调控互花米草的生长发育和非生物胁迫响应过程中具有重要的作用。

关键词: 互花米草, NAC, 转录因子, 基因家族, 基因表达

Abstract:

As a coastal halophyte, Spartina alterniflora has high salt tolerance. However, the mechanism at the molecular level has not been widely studied due to the absence of a reference genome. The proteins of NAC families are plant-specific transcription factors that regulate the growth, development and stress response in plants. To identify the NAC family and explore the relationship between NAC proteins and the growth, development and stress response of Spatina alterniflora, full-length transcriptome data of Spartina alterniflora by the third generation sequencing technology was used as reference sequences in this study to blast with the NAC protein sequences from Oryza sativa, Arabidopsis thaliana and Zea mays. Finally, 62 SaNAC proteins were found in Spartina alterniflora by deep analysis on conserved domains. Then we analyzed sequence alignment, evolution, motif prediction, homology comparison, subcellular localization, tissue and abiotic stress-induced gene differential expression profile on the NAC family members in Spartina alterniflora. As a result, all SaNAC proteins were found containing a conserved NAM domain and having certain evolutionary similarity with rice; two family proteins, SaNAC9 and SaNAC49, were expressed in the nucleus; moreover, SaNAC genes were identified to have distinct expressional profiles in different tissues and stress response of Spartina alterniflora. These results indicated the SaNAC transcription factor family not only had conserved functional domains but also played important role in the regulation of growth, development and abiotic stress response.

Key words: Spartina alterniflora, NAC, transcription factor, gene family, gene expression

王涛涛, 杨勇, 魏唯, 林辰涛, 马留银. 互花米草NAC转录因子家族的鉴定与表达分析[J]. 遗传, 2020, 42(2): 194-211.

Wang Taotao, Yang Yong, Wei Wei, Lin Chentao, Ma Liuyin. Identification and expression analyses of the NAC transcription factor family in Spartina alterniflora[J]. Hereditas(Beijing), 2020, 42(2): 194-211.

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基因名称	引物序列(5′→3′)	引物用途
SaNAC1	F: AACACACTATCCTGCCTGCT	qRT-PCR
SaNAC1	R: AGCTAGGTTCAAAGGACGCT	qRT-PCR
SaNAC5	F: TCCATCCTTCTGACGCTGAA	qRT-PCR
SaNAC5	R: TGTTGCCCTGTTTGATCTGC	qRT-PCR
SaNAC9	F: CGAGGAGCTCATCACGTACT	qRT-PCR
SaNAC9	R: TTAGTGGCACGGTTTGTTCG	qRT-PCR
SaNAC11	F: ACTGCCACCACAAAATCGAC	qRT-PCR/RT-PCR
SaNAC11	R: TAACATATGCCGTCCTCCCC	qRT-PCR/RT-PCR
SaNAC15	F: CAAGAAGGTGGTCAACGAGC	qRT-PCR
SaNAC15	R: TCGCCTTCCAGTATCCAGTC	qRT-PCR
SaNAC17	F: CCTCTACAAGTTCGACCCGT	qRT-PCR/RT-PCR
SaNAC17	R: GACGAGCGCCTTCTTGATG	qRT-PCR/RT-PCR
SaNAC18	F: CTTGGTTCCATACAGCAGCC	qRT-PCR
SaNAC18	R: GCTCTTCGCCTTGACATCTG	qRT-PCR
SaNAC19	F: ATCATGCACGAGTACAGGCT	qRT-PCR
SaNAC19	R: GCGCGTTCTTGTTGTTCTTG	qRT-PCR
SaNAC22	F: AACTGGGTCATGCACGAGTA	qRT-PCR/RT-PCR
SaNAC22	R: TCATCCTCCTCCTCTTCCCA	qRT-PCR/RT-PCR
SaNAC24	F: GGCGAGAAGGAGTGGTACTT	qRT-PCR
SaNAC24	R: CTCGTGCATGATCCAGTTGG	qRT-PCR
SaNAC25	F: TCAAGGTTCGAACGAGACCA	qRT-PCR
SaNAC25	R: TTCATAGTGCCATCCCGACA	qRT-PCR
SaNAC26	F: ATCCACATACCCCACCCAAG	qRT-PCR
SaNAC26	R: CCGGAAGAAGACGACGAGTA	qRT-PCR
SaNAC28	F: GGTGAGGAGGAACAGAACGA	qRT-PCR/RT-PCR
SaNAC28	R: CCTGCCCTTGTAGTACACCA	qRT-PCR/RT-PCR
SaNAC30	F: AGTGGTACTTCTTCTCGCCG	qRT-PCR
SaNAC30	R: CTCGTGCATGATCCAGTTGG	qRT-PCR
SaNAC31	F: GATCGTCTCGCACTACCTCA	qRT-PCR/RT-PCR
SaNAC31	R: TTGTCCTTTCCGGTAGCCTT	qRT-PCR/RT-PCR
SaNAC37	F: AAGAACGAGTGGGAGAAGGC	qRT-PCR/RT-PCR
SaNAC37	R: TAGCTGAGGTCGACGAACAG	qRT-PCR/RT-PCR
SaNAC38	F: AGTCTCTCCGTGCTTCAACA	qRT-PCR
SaNAC38	R: CTCTAGAAGCTCCTGGTCCG	qRT-PCR
SaNAC43	F: CTCCTCCTGGCTAACTCGAC	qRT-PCR/RT-PCR
SaNAC43	R: TCCCCACGTTAGGATGATGG	qRT-PCR/RT-PCR
SaNAC45	F: GAGGAGCTCATCACGCACTA	qRT-PCR
SaNAC45	R: AAGATCTCCCTGTCCTTGCC	qRT-PCR

基因名称	引物序列(5′→3′)	引物用途
SaNAC46	F: GGCGAGAAGGAGTGGTACTT	qRT-PCR/RT-PCR
SaNAC46	R: CTCGTGCATGATCCAGTTGG	qRT-PCR/RT-PCR
SaNAC51	F: GCTCGTCAAATCCTACCTGC	qRT-PCR/RT-PCR
SaNAC51	R: TTGGATTTGGCCTCGTTGTG	qRT-PCR/RT-PCR
SaNAC56	F: TCGACATGACCACCTCCTAC	qRT-PCR/RT-PCR
SaNAC56	R: ATGCTCTGGATGTCGTCGAA	qRT-PCR/RT-PCR
SaNAC57	F: GAAGAGCTGGTGGTGCAGTA	qRT-PCR
SaNAC57	R: CCGGATCGCGAAGAAGTACT	qRT-PCR
SaNAC59	F: CATGATGTTGGACTGGGTGC	qRT-PCR/RT-PCR
SaNAC59	R: ATGGAACTGGTGGTGATCGT	qRT-PCR/RT-PCR
SaNAC60	F: AGGGCGAGTGGTACTTCTTC	qRT-PCR/RT-PCR
SaNAC60	R: CTTCTTGACGCCGATCATGG	qRT-PCR/RT-PCR
SaACTIN	F: AGGGCAGTTTTCCCTAGCAT	qRT-PCR/RT-PCR
SaACTIN	R: CTCTCTTGGACTGTGCCTCA	qRT-PCR/RT-PCR
SaNAC9	F: TGACCTCGAGACTAGTATGAGTACGGAAGGGTCAGG	亚细胞定位载体构建
SaNAC9	R: AGGTGGAGGTCCCCCGGGCACCTGGTAACCAGCAGCA	亚细胞定位载体构建
SaNAC49	F: TGACCTCGAGACTAGTATGGAGATGGAGCAGGATCTC	亚细胞定位载体构建
SaNAC49	R: AGGTGGAGGTCCCCCGGGGTAGAGCAGATTGGCCAGGGT	亚细胞定位载体构建

转录本序列号	蛋白名称	氨基酸数量(aa)	分子量(kDa)	等电点	预测的蛋白定位
Cluster275-001	SaNAC1	445	49.6	4.56	细胞核
Cluster1229-001	SaNAC2	430	61.2	4.3	细胞外
Cluster1867-001	SaNAC3	187	21.4	9.91	细胞核
Cluster2112-001	SaNAC4	747	82.7	4.71	细胞外
Cluster4261-001	SaNAC5	359	39.3	5.31	细胞核
Cluster4343-001	SaNAC6	858	93.2	4.47	细胞外
Cluster5577-001	SaNAC7	569	62.1	5.62	细胞核
Cluster6349-001	SaNAC8	438	48.9	4.59	细胞核
Cluster7422-001	SaNAC9	361	39.1	8.93	细胞核
Cluster9525-001	SaNAC10	731	80.7	4.86	细胞外
Cluster10144-001	SaNAC11	405	45.6	8.48	细胞核
Cluster12101-001	SaNAC12	479	52.8	5.8	细胞核
Cluster13102-001	SaNAC13	518	56	5.55	细胞核
Cluster14225-001	SaNAC14	685	75.8	4.85	细胞外
Cluster15400-004	SaNAC15	186	21.2	9.57	细胞核
Cluster15711-001	SaNAC16	710	77.7	4.61	细胞外
Cluster16418-001	SaNAC17	320	34.8	5.92	细胞核
Cluster17526-001	SaNAC18	653	71.4	4.73	细胞外
Cluster17869-001	SaNAC19	322	35.2	6.01	细胞核
Cluster18322-001	SaNAC20	190	21.5	9.94	细胞核
Cluster18506-001	SaNAC21	487	53.4	4.46	细胞核
Cluster18550-001	SaNAC22	636	69.1	4.53	细胞外
Cluster18579-001	SaNAC23	638	69.4	4.5	细胞外
Cluster19615-001	SaNAC24	280	31.2	8.74	细胞核
Cluster20092-001	SaNAC25	312	34.2	4	细胞外
Cluster20280-001	SaNAC26	288	31.9	6.92	细胞核
Cluster21221-001	SaNAC27	601	67	5.76	细胞外
Cluster23463-001	SaNAC28	345	39.8	6.25	细胞核

转录本序列号	蛋白名称	氨基酸数量(aa)	分子量(kDa)	等电点	预测的蛋白定位
Cluster24200-001	SaNAC29	441	49.8	7.43	细胞外
Cluster24596-001	SaNAC30	333	36.5	5.75	细胞核
Cluster24844-001	SaNAC31	350	38.6	5.97	细胞核
Cluster24971-001	SaNAC32	401	43.7	6.2	细胞核
Cluster25024-001	SaNAC33	397	42.6	6.39	细胞核
Cluster25479-001	SaNAC34	346	38.2	6.03	细胞核
Cluster25519-001	SaNAC35	376	40.4	6.27	细胞核
Cluster25968-001	SaNAC36	280	31.7	7.62	细胞核
Cluster26335-001	SaNAC37	296	33.3	5.84	细胞核
Cluster26479-001	SaNAC38	367	40.3	8.97	细胞核
Cluster26810-001	SaNAC39	471	53	8.25	细胞核
Cluster26901-001	SaNAC40	314	34.7	8.68	细胞核
Cluster27315-001	SaNAC41	399	43.3	6.59	细胞核
Cluster27759-001	SaNAC42	357	40	5.59	细胞核
Cluster27876-001	SaNAC43	379	41	8.26	细胞核
Cluster27938-001	SaNAC44	419	46.8	5.18	细胞核
Cluster28031-001	SaNAC45	350	37.8	6.4	细胞核
Cluster28253-001	SaNAC46	194	21.7	9.97	细胞核
Cluster28923-001	SaNAC47	181	20.1	10.4	细胞核
Cluster28941-001	SaNAC48	402	44.1	6.12	细胞核
Cluster28982-001	SaNAC49	313	35.2	6.72	细胞核
Cluster29067-001	SaNAC50	361	40.2	6.79	细胞核
Cluster29514-001	SaNAC51	331	35.6	6.12	细胞外
Cluster29630-001	SaNAC52	373	40.5	8.26	细胞核
Cluster29846-001	SaNAC53	243	26.8	9.78	细胞核
Cluster30123-001	SaNAC54	285	31.3	8.43	细胞核
Cluster30128-001	SaNAC55	357	39.5	9.36	细胞核
Cluster30156-001	SaNAC56	282	31.5	8.69	细胞核
Cluster30326-001	SaNAC57	215	23.2	9.96	细胞核
Cluster30465-001	SaNAC58	306	33.9	8.63	细胞核
Cluster30667-001	SaNAC59	349	38.8	6.35	细胞核
Cluster30676-001	SaNAC60	288	31.7	8.92	细胞核
Cluster31570-001	SaNAC61	229	24.4	10.15	细胞核
Cluster32036-001	SaNAC62	271	30.6	7.03	细胞核

互花米草NAC转录因子家族的鉴定与表达分析

Identification and expression analyses of the NAC transcription factor family in Spartina alterniflora

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