闽楠中WRKY家族成员鉴定及缺磷胁迫下表达分析

doi:10.11707/j.1001-7488.20220214

摘要/Abstract

摘要：

目的: 在闽楠全基因组范围内鉴定WRKY家族成员，分析基因和蛋白序列结构特点及在闽楠幼苗缺磷胁迫下的表达特征，筛选响应缺磷逆境的WRKY基因，为进一步研究它们在楠木磷饥饿响应分子途径中的功能以及耐低磷胁迫分子辅助育种提供参考。方法: 利用WRKY蛋白序列的隐马尔科夫模型(pfam03106)，经hmmsearch在闽楠蛋白数据库中检索，筛选WRKY基因。对获得的PbWRKYs利用Protaram、GSDS2.0、MEGA、Batch CD-Search、TBtools和ClustalX等软件进行基因结构、定位分析，蛋白理化性质、序列特征分析以及进化树构建。提取3年生闽楠植株的根、韧皮部和形成层、木质部和叶的RNA，进行转录组测序，分析PbWRKYs在不同组织中的表达特点。对半年生闽楠植株进行缺磷水培处理，60天后，分别取缺磷处理和对照的叶和根进行磷含量测定和转录组测序，分析磷缺乏条件下PbWRKYs表达特征，并对差异表达基因进行qPCR验证。结果: 鉴定到68个WRKY基因，命名为PbWRKY1-68，在各染色体上均有分布，第3号染色体上数量最多，内含子数目在1~28个之间，蛋白分子质量在19.09~115.56 kDa之间。所有PbWRKY均含有WRKY结构域及其特有的锌指结构。根据WRKY结构域数量及其所含锌指结构类型，分为3个亚类：亚类Ⅰ含2个WRKY结构域，锌指结构类型为C2H2型，共14个成员；亚类Ⅱ含1个WRKY结构域和1个C2H2型锌指结构，共47个成员，进化分析表明该亚类又分为5个经典子组Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe，但其中PbWRKY37与上述子组进化上距离较远，独立成组；亚类Ⅲ含1个WRKY结构域和1个C2HC型锌指结构，共7个成员。PbWRKYs蛋白所含WRKY结构域氨基酸序列特征大部分为“WRKYGQK”型，但PbWRKY62的WRKY结构域变异为“WRKYGKK”。与其他植物相比，PbWRKYs蛋白中WRKY结构域的变异率较低。PbWRKYs的组织特异性表达模式可分为5类：韧皮部和形成层中相对表达量较低；木质部中表达量高而叶片中表达量较低；韧皮部和形成层表达量较高同时叶片中表达量较低；根中表达量相对较高；叶片中表达量高而根中表达量低。闽楠幼苗缺磷处理60天后，叶和根中表达差异达到2倍以上的PbWRKY基因共21个。它们在叶片中被强烈诱导，可能参与低磷条件下叶片中磷元素的运输及再分配过程；其中，PbWRKY52、PbWRKY55、PbWRKY56、PbWRKY65和PbWRKY66 5个基因的表达还在根中被抑制，表明它们除了参与低磷条件下叶片中磷元素再分配，还可能参与了根部磷的吸收和转运。结论: 闽楠中WRKY基因家族成员共有68个，其蛋白序列中WRKY结构域较保守。缺磷处理60天时，有21个PbWRKY基因参与磷胁迫响应，大部分基因可能主要在叶片中参与低磷条件下磷的运输和分配。PbWRKY52、PbWRKY55、PbWRKY56、PbWRKY65和PbWRKY66除了在叶片中发挥功能外，还可能参与低磷条件下根部对磷的吸收和转运。

关键词: 闽楠, WRKY转录因子, 基因表达, 缺磷

Abstract:

Objective: The WRKY transcription factor gene family members were identified in the whole genome of Phoebe bournei. Gene structure and protein sequences of them were characterized and their expression was analyzed under phosphorus deficiency. Then, PbWRKY members that were involved in phosphorus starvation response were screened. The result will give information to further study their function in the molecular mechanism of phosphorus deficiency stress and provide foundation to molecular assisted breeding for tolerance to low phosphorus in Phoebe. Method: The hidden Markov model (pfam03106) of WRKY protein sequence were used to search PbWRKY protein sequence from protein database of P. bournei. Then, the chromosome localization, gene structure, protein sequences and phylogenetic tree of PbWRKYs were analyzed by Protaram, GSDS2.0, MEGA, Batch CD-search, TBtools and ClustalX software. Tissue-specific expression was analyzed based on transcriptome sequence data of roots, phloem and cambium, xylem and mature leaves of 3-year-old P. bournei plants. And, six-month-old seedlings were planted in phosphorus deficiency solution. After 60 days, the leaves and roots of phosphorus deficiency treatment and control were harvested for phosphorus content measuring and transcriptome sequencing. The expression of PbWRKY genes under phosphorus deficiency were analyzed, and the PbWRKY genes expressed differentially were verified by qPCR. Result: 68 WRKY genes, named PbWRKY1-68, were identified in P. bournei. They were distributed on all chromosomes of P. bournei and the most were on chromosome 3. All the PbWRKYs had introns, the number of them ranged from 1 to 28. The molecular weight of them from 19.09 to 115.56 kDa. Each PbWRKY had 1 or 2 WRKY domains and a specific zinc finger motif. Based on the number of WRKY domain and the type of zinc finger structure, PbWRKYs can be divided into 3 subclasses. Subclass Ⅰ contained 2 WRKY domains and 1 C2H2 type of zinc finger, and 14 genes were included in it. There were 47 genes in subclass Ⅱ which had 1 WRKY domains and 1 C2H2 type of zinc finger. And phylogenetic analysis showed this subclass can be classified into 5 subgroups: Ⅱa, Ⅱb, Ⅱc, Ⅱd, Ⅱe, but PbWRKY37 didn't belong to any subgroup above according to the phylogenetic tree although it was a gene of subclass Ⅱ. Subclass Ⅲ contained a WRKY domain and a C2HC type of zinc finger, a total of 7 genes. Most of WRKY domain sequence in PbWRKYs were "WRKYGQK", but it was "WRKYGKK" in PbWRKY62. Tissue-specific expression analysis showed five expression patterns of PbWRKYs were found in different tissues: low expression in phloem and cambium, high expression in xylem but low expression in leaves, high expression in phloem and cambium and low expression in leaves, high expression in roots, and high expression in leaves but low expression in roots. And some of the WRKY genes which closely related had similar tissue-specific expression, suggesting their functions possibly correlated. After 60 days of phosphorus deficiency treatment, 21 PbWRKY genes expressed differently more than 2 folds in leaves and roots compared to the control. All the 21 PbWRKY genes were strongly induced in leaves, implying that they possibly participated in the transportation and distribution of phosphorus in leaves under low phosphorus condition. Among them, the expressions of PbWRKY52, PbWRKY55, PbWRKY56, PbWRKY65 and PbWRKY66 were also inhibited in roots, suggesting that they also may be involved in the transportation of phosphorus in roots simultaneously. Conclusion: There were 68 PbWRKY genes in Phoebe bournei. The WRKY domain sequence of them was very conservative and the variation was very low. 21 PbWRKY genes were involved in the response to phosphorus deficiency after 60 days of phosphorus starvation treatment, and most of them were involved in the transportation and distribution of phosphorus in leaves when plants faced phosphorus starvation. However, PbWRKY52, PbWRKY55, PbWRKY56, PbWRKY65 and PbWRKY66 possibly not only played a role in leaves, but also participated in the absorption and transportation of phosphorus in roots under phosphorus deficiency stress.

Key words: Phoebe bournei, WRKY transcription factor, gene expression, phosphorus deficiency

中图分类号:

S718.46

张苗,周生财,吴梦洁,童再康,韩潇,张俊红,程龙军. 闽楠中WRKY家族成员鉴定及缺磷胁迫下表达分析[J]. 林业科学, 2022, 58(2): 133-147.

Miao Zhang,Shengcai Zhou,Mengjie Wu,Zaikang Tong,Xiao Han,Junhong Zhang,Longjun Cheng. Identification of the PbWRKY Gene Family and Its Expression Analysis under Deficiency of Phosphorus in Phoebe bournei[J]. Scientia Silvae Sinicae, 2022, 58(2): 133-147.

图/表 10

表1

组织表达与缺磷处理的转录组数据质量特征"

数据库 Database	样本 Sample	下机reads数 Raw data reads	有效reads数 Valid data reads	有效reads所占比例 Valid reads ratio(%)	质量值≥20的碱基占比 Quality value≥20 base ratio(%)	GC含量 GC content(%)
组织部位转录组 Tissue site transcriptome	根_1 Root_1	53 265 418	52 314 540	98.21	97.58	47.0
	根_2 Root_2	60 367 530	59 258 996	98.16	97.55	47.0
	根_3 Root_3	67 986 088	66 925 916	98.44	97.33	46.5
	韧皮部+形成层_1 Phloem+Cambium_1	47 044 618	46 460 688	98.76	97.41	47.5
	韧皮部+形成层_2 Phloem+Cambium _2	54 879 794	54 150 716	98.67	97.76	47.5
	韧皮部+形成层_3 Phloem+Cambium _3	49 863 640	49 150 792	98.57	97.49	47.5
	木质部_1 Xylem_1	56 535 724	55 919 236	98.91	97.54	47.5
	木质部_2 Xylem_2	68 749 458	67 967 894	98.86	97.83	47.5
	木质部_3 Xylem_3	58 523 758	57 910 268	98.95	97.40	47.5
	叶_1 Leaf_1	64 149 258	62 985 490	98.19	97.34	46.5
	叶_2 Leaf_2	49 853 644	48 988 614	98.26	97.49	46.5
	叶_3 Leaf_3	52 759 766	51 818 374	98.22	97.40	46.0
缺磷处理转录组 Phosphorus deficient transcriptome	对照_叶_1 Control_leaf_1	48 886 672	47 529 702	97.22	99.98	46.0
	对照_叶_2 Control_leaf_2	46 207 606	45 054 036	97.50	99.98	46.0
	对照_叶_3 Control_leaf_3	48 291 502	46 983 802	97.29	99.97	46.0
	处理_叶_1 Treatment_leaf_1	54 114 780	52 660 520	97.31	99.98	45.5
	处理_叶_2 Treatment_leaf_2	51 402 300	49 945 948	97.17	99.98	46.0
	处理_叶_3 Treatment_leaf_3	51 530 670	50 450 068	97.9	99.97	46.0
	对照_根_1 Control_root_1	51 965 850	50 815 942	97.79	99.98	46.0
	对照_根_2 Control_root_2	46 113 970	44 381 266	96.24	99.98	46.0
	对照_根_3 Control_root_3	54 147 580	52 605 564	97.15	99.98	46.5
	处理_根_1 Treatment_root_1	52 197 938	50 873 872	97.46	99.97	45.5
	处理_根_2 Treatment_root_2	53 236 180	52 050 506	97.77	99.97	45.5
	处理_根_3 Treatment_root_3	53 060 194	51 829 528	97.68	99.97	46.0

表1

表2

表3

闽楠WRKY基因及编码蛋白特性"

基因名称 Gene name	基因编号 GenBank No.	染色体定位 Chromosome localization	基因长度 Gene length/bp	氨基酸残基数 Number of amino acid residue	分子质量 Molecular weight/kDa	理论等电点 Theoretical pI
PbWRKY1	MW473791	chr01	3 676	589	64.20	7.27
PbWRKY2	MW473811	chr01	13 733	653	70.94	5.84
PbWRKY3	MW473826	chr01	6 178	472	52.39	9.03
PbWRKY4	MW473810	chr01	3 528	471	52.04	8.86
PbWRKY5	MW473792	chr01	9 790	519	56.33	7.28
PbWRKY6	MW473800	chr02	14 628	740	80.05	6.02
PbWRKY7	MW473780	chr02	8 311	571	62.38	6.25
PbWRKY8	MW473787	chr02	4 298	488	54.21	6.69
PbWRKY9	MW473812	chr02	22 288	397	43.15	7.99
PbWRKY10	MW473832	chr02	6 191	728	78.47	5.98
PbWRKY11	MW473835	chr03	3 043	169	19.09	9.66
PbWRKY12	MW473824	chr03	1 212	305	33.55	5.84
PbWRKY13	MW473833	chr03	4 349	352	39.48	9.67
PbWRKY14	MW473816	chr03	2 028	316	34.94	7.08
PbWRKY15	MW473828	chr03	1 346	303	33.55	6.34
PbWRKY16	MW473823	chr03	1 990	335	37.23	5.61
PbWRKY17	MW473790	chr03	2 814	393	43.24	5.67
PbWRKY18	MW473782	chr03	8 358	563	61.31	6.35
PbWRKY19	MW473802	chr03	3 391	442	48.77	8.66
PbWRKY20	MW473834	chr03	3 135	182	20.88	9.37
PbWRKY21	MW473784	chr03	2 924	353	39.40	9.65
PbWRKY22	MW473778	chr03	8 769	683	74.51	5.70
PbWRKY23	MW473831	chr03	3 119	247	27.73	6.05
PbWRKY24	MW473829	chr03	11 753	623	67.55	5.80
PbWRKY25	MW473794	chr03	3 969	373	41.33	6.12
PbWRKY26	MW473786	chr04	2 048	343	37.17	6.08
PbWRKY27	MW473838	chr04	8 136	325	36.52	9.76
PbWRKY28	MW473819	chr04	3 849	331	37.06	9.72
PbWRKY29	MW473806	chr05	3 759	415	45.36	5.28
PbWRKY30	MW473821	chr05	5 549	193	21.99	9.51
PbWRKY31	MW473817	chr05	2 788	430	47.37	6.53
PbWRKY32	MW473840	chr05	2 648	336	36.90	8.53
PbWRKY33	MW473776	chr05	5 068	183	20.97	9.58
PbWRKY34	MW473801	chr05	2 430	421	45.83	5.17
PbWRKY35	MW473815	chr05	1 243	293	31.58	9.33
PbWRKY36	MW473809	chr05	2 214	302	33.51	8.88
PbWRKY37	MW473799	chr05	2 981	312	34.39	5.23
PbWRKY38	MW473818	chr06	1 018	267	29.21	5.88
PbWRKY39	MW473779	chr06	1 494	290	31.72	9.81
PbWRKY40	MW473805	chr06	3 260	212	23.90	5.10
PbWRKY41	MW473798	chr06	9 328	225	25.80	9.69
PbWRKY42	MW473774	chr07	29 115	282	31.72	9.11
PbWRKY43	MW473836	chr07	2 599	477	52.92	5.68
PbWRKY44	MW473789	chr07	5 008	195	22.03	8.97
PbWRKY45	MW473785	chr07	1 251	328	36.70	5.84
PbWRKY46	MW473777	chr07	9 866	202	23.26	9.08
PbWRKY47	MW473808	chr08	10 829	227	24.21	5.04
PbWRKY48	MW473822	chr08	1 561	238	27.24	5.70
PbWRKY49	MW473788	chr08	4 588	588	63.55	5.67
PbWRKY50	MW473803	chr08	2 602	520	57.39	7.27
PbWRKY51	MW473841	chr08	2 604	520	57.70	9.06
PbWRKY52	MW473783	chr09	1 931	277	30.50	8.65
PbWRKY53	MW473839	chr09	1 508	272	29.51	5.02
PbWRKY54	MW473820	chr09	2 379	593	63.78	5.62
PbWRKY55	MW473795	chr09	1 882	318	35.77	5.66
PbWRKY56	MW473793	chr09	2 097	313	34.95	6.97
PbWRKY57	MW473797	chr09	1 917	343	39.00	5.10
PbWRKY58	MW473827	chr09	7 292	501	54.42	8.92
PbWRKY59	MW473825	chr09	3 967	499	54.41	6.84
PbWRKY60	MW473814	Chr10	26 131	276	30.65	9.49
PbWRKY61	MW473837	Chr10	2 401	530	58.00	5.95
PbWRKY62	MW473775	Chr10	1 337	202	22.94	7.00
PbWRKY63	MW473830	Chr11	1 829	236	26.79	6.51
PbWRKY64	MW473804	Chr12	39 719	986	111.56	5.16
PbWRKY65	MW473781	Chr12	1 723	314	34.31	6.71
PbWRKY66	MW473807	Chr12	1 887	324	35.88	8.65
PbWRKY67	MW473813	Chr12	1 755	317	35.26	8.51
PbWRKY68	MW473796	Scaffold 356	4 517	192	22.26	8.81

表3

图1

图2

图3

图4

表4

图5

图6

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基因名称 Gene name	引物序列 Primer sequences(5’—3’)
PbWRKY1	F: TTCAAAGGACAACAGCAGCCR; R: TGAGGAGGTTGAGTGAGCTG
PbWRKY8	F: GCTGCAAATTCACACCAAGC; R: CCCAAGATTCGACCCAGTTC
PbWRKY14	F: GCTGTGGGAGAAGAGGATGT; R: CTCGCTCTTGGTCATGAACG
PbWRKY16	F: TGGGTAGCTTTTCTCCTCCA; R: GAAAATCCAAGTCCCCCAAT
PbWRKY20	F: TCCAAAATCAGATGCCAACA; R: CACAGCCTTCTGCCCATATT
PbWRKY25	F: ACTGAATCCCCATCACCCAG; R: AAATATCCATGGCCTGCGTG
PbWRKY26	F: GGTGCAAATGTGGGAGAGTG; R: CCTCCTCCTCCGTAGCTATT
PbWRKY32	F: AGGGGAGAAGAGGCAAAGAG; R: ACTGAGCACTTCTGGCTGGT
PbWRKY38	F: GGCAAGATGAGTGACACGTC; R: TGCATCTGTAGTAGCCCCTG
PbWRKY43	F: TGCCCAAGAGCCTACTATCG; R: ACAGGCAATGGATGGTTGTG
PbWRKY50	F: CTCCATTCCCCACTGTCACT; R: GAATTGGGTGGCTTCAGTGT
PbWRKY51	F: CTCCATTCCCCACTGTCACT; R: GAATTGGGTGGCTTCAGTGT
PbWRKY52	F: GGGTCAAACCCAAACAGAGA; R: AACCTGGAGCTGTGGAACAC
PbWRKY55	F: AGACCGACGAAGAGGCTACA; R: AGCCCTGATCATTTCTGTGG
PbWRKY56	F: CAATCTATCGCGGGTGCTAT; R: AGAGCAATCTCCACGCTGTT
PbWRKY59	F: AGCGAAGAGAACCAGAAGCT; R: GTTGATCTTCCCACCTTCCT
PbWRKY61	F: CCACACTCCCAACCCATTAC; R: TGGCTGCACTCACACTATCC
PbWRKY62	F: AGGGGAGAAGAGGCAAAGAG; R: ACTGAGCACTTCTGGCTGGT
PbWRKY65	F: CAGATTCGGACCAATCCATC; R: CTCCCCCTCATATGTGGCTA
PbWRKY66	F: TCAAATGCTCCTTTGCTCCT; R: CCACTCGAACAAGGTTTGGT
PbWRKY67	F: TGTTTTGAAGGCGGAGTTGG; R: CATCTCGGTCCTCCTCCTTC
PbEF1α	F: CATTCAAGTATGCGTGGGT; R: ACGGTGACCAGGAGCA

基因名称 Gene name	叶 Leaf				根 Root
基因名称 Gene name	CK	Treatment	log₂(FoldChange)	P	CK	Treatment	log₂(FoldChange)	P
PbWRKY1	18.80	83.55	2.15	0.00	63.86	51.11	-0.32	0.08
PbWRKY8	2.31	20.67	3.16	0.00	1.72	2.65	0.62	0.15
PbWRKY14	0.32	1.42	2.15	0.00	63.86	51.11	-0.32	0.08
PbWRKY16	10.21	139.75	3.78	0.00	39.87	21.81	-0.87	0.00
PbWRKY20	3.28	10.94	1.74	0.01	17.89	9.75	-0.88	0.04
PbWRKY25	0.26	1.30	2.31	0.00	0.41	0.13	-1.72	1.00
PbWRKY26	0.33	4.21	3.69	0.00	2.57	1.82	-0.49	0.17
PbWRKY32	0.94	2.89	1.63	0.00	33.32	25.03	-0.41	0.03
PbWRKY38	0.90	2.26	1.33	0.01	98.33	81.89	-0.26	0.11
PbWRKY43	0.35	1.07	1.61	0.00	9.80	10.13	0.05	0.99
PbWRKY50	19.84	44.19	1.16	0.00	33.15	26.59	-0.32	0.09
PbWRKY51	21.10	46.09	1.13	0.00	31.53	22.78	-0.47	0.01
PbWRKY52	8.71	42.86	2.30	0.00	50.52	7.88	-2.68	0.00
PbWRKY55	20.01	194.96	3.28	0.00	292.50	92.15	-1.67	0.00
PbWRKY56	8.07	44.42	2.46	0.00	26.10	6.82	-1.94	0.00
PbWRKY59	0.04	0.44	3.46	0.00	20.88	16.00	-0.38	0.05
PbWRKY61	0.68	8.79	3.69	0.00	6.64	9.31	0.49	0.07
PbWRKY62	2.60	26.94	3.37	0.00	17.52	11.34	-0.63	0.01
PbWRKY65	0.76	4.71	2.62	0.00	1.94	0.88	-1.14	0.02
PbWRKY66	6.81	124.64	4.19	0.00	57.24	5.20	-3.46	0.00
PbWRKY67	9.96	37.79	1.92	0.00	20.59	23.51	0.19	0.56

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