苹果NLP（Nin-Like Protein）转录因子基因家族全基因组鉴定及表达模式分析

doi:10.3864/j.issn.0578-1752.2019.23.014

摘要/Abstract

摘要：

【目的】探究苹果NLP转录因子全基因组特征与表达模式,以便更深入地了解其结构特点与作用机制。【方法】基于本地BLAST数据库和Pfam数据库两大数据库,运用blastp和hmmsearch两种查询策略,对苹果全基因组范围内的NLP转录因子家族成员进行鉴定。经过严格筛选与确认后,对搜索结果进一步展开分析,主要分为3部分,包括NLP蛋白分析、NLP分析与苹果NLP表达分析,其中ProtParam、Clustal Omega、MEGA7、MEME 5.0.2、SOPMA、Phyre ²、WoLF PSORT、STRING等程序或软件用于蛋白质分析,基因分析使用MG2C、GSDS 2.0、PlantCARE、psRNATarget等在线工具,对于苹果NLP表达情况利用qRT-PCR进行定量检测。【结果】从苹果全部蛋白数据库中共筛选出6个NLP成员,系统进化分析可将它们分为I、II、III三类;蛋白二级结构以无规则卷曲为主,其次是α-螺旋,占比最小的是β-转角;亚细胞定位预测均定位于细胞核中,符合转录因子的特征;染色体定位显示5个基因（MDP0000584547除外）定位于4条染色体上;启动子分析发现大量与激素和逆境响应相关的顺式作用元件,暗示它们可能参与到激素和逆境信号的调控过程中,此外还识别到一个响应氮的GCN4作用元件,进一步说明该类转录因子与氮素有密不可分的关系;通过定量检测揭示苹果NLP家族在茎、叶等组织高表达的特征模式,并且表达分析结果也证实苹果NLP响应氮饥饿、干旱胁迫等。【结论】通过对苹果全基因组的分析,共识别6个NLP转录因子,对6个基因的结构与蛋白保守域分析,发现它们之间具有极高的相似性、保守性,同时又存在差异。类比拟南芥NLP蛋白的关联网络,推测与拟南芥NLP7同源性最高的MDP0000132856可能也具有复杂的功能。

关键词: 苹果, NLP转录因子, 氮信号, 生物信息学

Abstract:

【Objective】The study was carried out to explore the whole genome characteristics and expression patterns of NLP transcription factors in apple, and further understand its structural characteristics and mechanism.【Method】Based on the local BLAST database and Pfam database, the members of NLP transcription factor family in the whole genome of apple were identified by using two query strategies with blastp and hmmsearch. Through strict filtration and confirmation, the results were used for further analysis, and analysis mainly divides into three parts, including NLP proteins analysis, analysis of NLP genes and NLP expression analysis in apple. Programs or softwares, such as ProtParam, Clustal Omega, MEGA7, MEME 5.0.2, SOPMA, Phyre ², WoLF PSORT and STRING, were used for protein analysis, online tools included MG2C, GSDS2.0, PlantCARE, psRNATarget, were used for gene analysis, and the expression of MdNLP gene was quantitatively detected by qRT-PCR.【Result】6 NLP members were identified from apple protein databases, which were classified into three categories by phylogenetic analysis: I, II and III. The protein secondary structure was dominated by random coil, followed by alpha-helix, and the smallest proportion was beta-turn. The prediction of subcellular localization was located in the nucleus, which was consistent with the characteristics of transcription factors. Chromosome localization showed that five genes (except MDP0000584547) were located on four chromosomes. Promoter analysis revealed a large number of cis-acting elements related to hormone and stress response, suggesting that MdNLP genes might be involved in the regulation of hormone and stress signals. In addition, a nitrogen-responsive GCN4 element was also identified, further indicating that such transcription factors were closely related to nitrogen. By quantitative detection, the pattern which NLP family had high expression in stem and leaf of apple was revealed. And the expression analysis results also confirmed that MdNLP genes responded to nitrogen starvation and drought stress, and so on.【Conclusion】Through the apple genome analysis, 6 NLP transcription factors were found; the analysis of structure and conserved domain of protein for 6 gene suggested that there had a very high similarity and conservation between them, at the same time, they were different in a way; the associated protein network of NLP family in Arabidopsis thaliana were used for MdNLPs, MDP0000132856, which had the highest homology with AtNLP7, might also have complicated features and functions.

Key words: apple, NLP transcription factor, nitrogen signal, bioinformatics

王寻, 陈西霞, 李宏亮, 张富军, 赵先炎, 韩月彭, 王小非, 郝玉金. 苹果NLP（Nin-Like Protein）转录因子基因家族全基因组鉴定及表达模式分析[J]. 中国农业科学, 2019, 52(23): 4333-4349.

WANG Xun, CHEN XiXia, LI HongLiang, ZHANG FuJun, ZHAO XianYan, HAN YuePeng, WANG XiaoFei, HAO YuJin. Genome-Wide Identification and Expression Pattern Analysis of NLP (Nin-Like Protein) Transcription Factor Gene Family in Apple[J]. Scientia Agricultura Sinica, 2019, 52(23): 4333-4349.

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基因Gene	上游引物Forward primer (5′-3′)	下游引物Reverse primer (5′-3′)
MdNLP2	CTATGCATCGAGGAAACAGCTTG	CAATTCCCTCACCTTCCTCAAGA
MdNLP3	GAAATGGAGAAAGAGGGCTCTGA	TTCAGAAGGGCTTGGATACTTCC
MdNLP4	GTCAGTATGCTCTCGATCCTGATA	CAGTAAGATGTGTAGGATGTTGGC
MdNLP5	GCTTGCTTCTGTGGAGACATTAC	TCCAGTATGAGTGCTCTGTAAGC
MdActin	GGACAGCGAGGACATTCAGC	CTGACCCATTCCAACCATAACA

蛋白质Protein	α-螺旋α-helix	β-转角β-turn	无规则卷曲Random coil	延长链Extended strand
MdNLP1	28.69%	3.79%	53.11%	14.41%
MdNLP2	33.94%	5.57%	43.14%	17.35%
MdNLP3	23.40%	6.40%	55.89%	14.32%
MdNLP4	27.46%	5.90%	50.23%	16.41%
MdNLP5	26.52%	4.01%	55.50%	13.98%
MdNLP6	28.33%	6.27%	49.90%	15.49%

基因 Gene	登录号 Accession number	基因长度 Gene length	编码序列长度 CDS length	氨基酸数目 Size of aa	分子量 MW (D)	等电点 pI	亚细胞定位 Subcellular localization
MdNLP1	MDP0000788505	3856	2460	819	90889.49	6.25	细胞核 Nucleus
MdNLP2	MDP0000265619	9510	5121	1706	189398.44	7.9	细胞核 Nucleus
MdNLP3	MDP0000246881	9366	4152	1383	153424.77	7.88	细胞核 Nucleus
MdNLP4	MDP0000239938	8796	3990	1329	147220.18	5.5	细胞核 Nucleus
MdNLP5	MDP0000132856	4454	2949	982	107556.64	5.45	细胞核 Nucleus
MdNLP6	MDP0000584547	10212	3159	1052	117304.32	5.96	细胞核 Nucleus

GO分类GO classification	GO条目GO term	基因数目Gene number	描述Description
分子功能 Molecular Function	GO:0005524 GO:0017111 GO:0016887 GO:0000166 GO:0008270 GO:0003676	1 1 1 3 2 2	ATP结合 ATP binding 核苷三磷酸酶活性 Nucleoside-triphosphatase activity ATP酶活性 ATPase activity 核苷酸结合 Nucleotide binding 锌离子结合 Zinc ion binding 核酸结合 Nucleic acid binding
生物途径 Biological Process	GO:0006810	1	转运 Transport
细胞组分 Cellular Component	GO:0016020 GO:0016021	1 1	膜 Membrane 膜的整体成分 Integral component of membrane

基因 Gene	脱落酸 ABRE	低氧 ARE	茉莉酸甲酯 CGTCA	乙烯 ERE	赤霉素 GARE	氮素 GCN4	干旱 MBS	防御和胁迫 TC-rich repeats	水杨酸 TCA	生长素 TGA	病原菌 W-box
MdNLP1	0/1	2/0	1/1	1/1	0/1				1/0	1/0	0/1
MdNLP2	2/3	1/0	3/0				0/1	0/1	0/1
MdNLP3	0/2	0/3	0/1		0/1			0/1	0/1	1/0	0/1
MdNLP4	0/2	0/3	1/0					1/1	1/1	1/1	0/1
MdNLP5		1/1	2/0	1/2	0/1	0/1	1/0			0/1	0/1
MdNLP6	4/3	1/0	3/3	1/0							0/1