柑橘U-box基因家族的鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2019.11.009

摘要/Abstract

摘要：

目的通过生物信息学分析U-box在柑橘基因组中的分布、结构及进化,研究家族成员在不同组织中的表达特异性以及对非生物胁迫和激素的响应,解析柑橘U-box基因家族的生物学功能。方法根据已经报道的拟南芥U-box基因,利用Phytozome数据库中的BLASTp工具鉴定柑橘基因组中的U-box基因。采用MEGA6.0、Cello、SMART、GSDS2.0、ExPASy和MapChart等软件构建系统进化树、亚细胞定位预测、预测蛋白的相对分子质量与等电点等理化性质、绘制家族成员Scaffold定位图等,分析U-box基因家族在低温胁迫下表达模式,利用实时荧光定量PCR技术（qRT-PCR）检测柑橘U-box基因家族部分成员在NaCl、PEG6000和不同激素处理下的表达情况。结果从柑橘克里曼丁（Citrus clementina）全基因组中鉴定出56个CcPUBs基因家族成员,可将其分为7类,即U-box only、U-box+ARM-1、U-box+WD40-1、TPR+U-box、Kinase+U-box、U-box+ARM-2和U-box+WD40-2。该家族蛋白理论等电点分布在5.19—9.14,编码的氨基酸数目介于281—1 441;亚细胞定位预测结果显示该基因家族成员位于细胞不同位置,主要位于细胞核或叶绿体,少数位于质膜;聚类分析发现,柑橘U-box与单子叶植物水稻的亲缘关系较远,柑橘中具有相同结构域的成员和拟南芥具有相同结构域的成员聚在一起,表明柑橘U-box成员具有不同生物学功能;Scaffold定位分析发现,56个U-box成员分布在1—9 Scafflod上且呈不均分布。在冷胁迫下的RNA-Seq分析结果表明,U-box基因家族参与植物对冷胁迫的应答,并表现出4种不同的应答模式;从柑橘U-box基因家族的5个不同簇上分别选取1个代表基因进行qRT-PCR,分析结果表明,CcPUB48在金柑的各个组织中均有表达,CcPUB9和CcPUB4主要在茎和花中表达,CcPUB10主要在花、茎和幼果中表达,CcPUB41主要在叶和茎中表达,体现了不同U-box成员的组织特异性的表达差异。CcPUB4、CcPUB10和CcPUB41在NaCl胁迫下表达均上调,而CcPUB48在盐胁迫下的表达无明显变化。CcPUB4在Na₂CO₃处理下表达明显上调,与NaCl处理存在一致的表达趋势,而CaCl₂处理下的表达与NaCl处理下的表达趋势却存在差异。在PEG6000的处理条件下,CcPUB4的表达量呈现先上升后下降的趋势,CcPUB9、CcPUB41和CcPUB48在PEG6000的处理下无明显变化。在赤霉素（GA₃）处理下,CcPUB4在3 h时明显上调,在生长素（IAA）和脱落酸（ABA）下呈现无规律变化,CcPUB10在ABA处理下表达量表现出逐渐上升。结论从柑橘克里曼丁全基因组上鉴定出56个U-box基因成员,各成员均含有U-box保守结构域,并位于细胞的不同位置。U-box基因家族参与植物对冷胁迫的应答并表现出4种不同的应答模式,在NaCl、PEG6000和激素处理下,CcPUB4和CcPUB10有不同程度的响应,而CcPUB9、CcPUB41和CcPUB48响应不明显或未响应。

关键词: 柑橘, CcPUB基因家族, 激素, 胁迫, 基因表达

Abstract:

【Objective】 The objectives of this study were to analyze the distribution, structure and evolution of U-box in citrus genome by bioinformatics, to study the expression specificity of family members in different tissues and their responses to abiotic stress and hormones, and to investigate the biological function of U-box gene family in citrus. 【Method】 According to the reported U-box gene of Arabidopsis thaliana, the U-box gene in citrus genome was identified by BLASTp tool in Phytozome database. Phylogenetic tree, subcellular localization prediction, relative mass and isoelectric point and other physical and chemical properties, and scaffold location of U-box member were analyzed with MEGA6.0, Cello, SMART, GSDS2.0, ExPASy and MapChart, respectively. The expression pattern of U-box gene family under low temperature stress was analyzed, and the expression of some members of U-box gene family in citrus treated with NaCl, PEG6000 and different hormones was detected by real-time fluorescence quantitative PCR (qRT-PCR). 【Result】 Fifty-six members of CcPUBs gene family were identified from the whole genome of Citrus clementina, and they could be divided into 7 categories, namely as U-box only, U-box+ARM-1, U-box+WD40-1, TPR+U-box, Kinase+U-box, U-box+ARM-2 and U-box+WD40-2. The theoretical isoelectric point of the family protein was ranged from 5.19 to 9.14, and the number of amino acids encoded was 281 to 1 441. The results of subcellular localization prediction showed that the members of the gene family were located in different positions of the cell, mainly in the nucleus or chloroplast, and a few in the plasma membrane. Cluster analysis showed that citrus U-box was closely related to monocotyledonous rice, and members with the same domain in citrus and Arabidopsis thaliana were clustered together. The results showed that U-box members of citrus had different biological functions, and scaffold localization analysis showed that 56 U-box members were unevenly distributed on citrus 1-9 scaffold. The results of RNA-Seq analysis under cold stress showed that U-box gene family was involved in the response of plants to cold stress and showed four different response patterns. One representative gene was selected respectively from five different clusters of citrus U-box gene family for qRT-PCR analysis. The results showed that CcPUB48 was expressed in all tissues of kumquat, and CcPUB9 and CcPUB4 were mainly expressed in stems and flowers, CcPUB10 was mainly expressed in flowers, stems and young fruits, and CcPUB41 was mainly expressed in leaves and stems. It reflected the tissue-specific expression difference of different U-box members. The expression of CcPUB4, CcPUB10 and CcPUB41 was up-regulated under NaCl stress, but the expression of CcPUB48 had no significant change under salt stress. The expression of CcPUB4 was up-regulated under Na₂CO₃ treatment, which was consistent with that of NaCl treatment, but the expression trend of CaCl₂ treatment was different from that of NaCl treatment. Under the treatment of PEG6000, the expression of CcPUB4 increased at first and then decreased, while CcPUB9, CcPUB41 and CcPUB48 did not change significantly under the treatment of PEG6000. Under gibberellin (GA3) treatment, CcPUB4 was significantly up-regulated at 3 h, while under auxin (IAA) and abscisic acid (ABA), the expression of CcPUB10 showed irregular changes, and the expression of CcPUB10 increased gradually under gibberellin (gibberellin) treatment.【Conclusion】 Fifty-six members of U-box gene were identified from the whole genome of Citrus clementina. All members contained U-box conserved domain and were located in different positions of cells. U-box gene family was involved in the response of plants to cold stress and showed four different response patterns. Under NaCl, PEG6000 and hormone treatment, CcPUB4 and CcPUB10 had different degrees of response, but CcPUB9, CcPUB41 and CcPUB48 had no obvious or no response. This experiment provided a theoretical guidance for the further study of U-box gene family in citrus stress resistance and growth and development mechanism.

Key words: citrus, CcPUB gene family, hormone, stress, gene expression

李秋月,张亚飞,彭洁,王旭,张志强,戴祥生,江东. 柑橘U-box基因家族的鉴定及表达分析[J]. 中国农业科学, 2019, 52(11): 1942-1960.

LI QiuYue,ZHANG YaFei,PENG Jie,WANG Xu,ZHANG ZhiQiang,DAI XiangSheng,JIANG Dong. Genome Wide Identification and Expression Analysis of the U-box Gene Family in Citrus[J]. Scientia Agricultura Sinica, 2019, 52(11): 1942-1960.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.11.009

https://www.chinaagrisci.com/CN/Y2019/V52/I11/1942

图/表 12

表1

表2

柑橘基因组中的U-box基因"

基因名称 Gene name	基因组登录号 Gene accession No.	蛋白质大小 Protein length (aa)	分子量 Molecular mass (KD)	等电点 Isoelectric point (PI)	box位置 U-box domain location	细胞定位 Subcellular localizations
CcPUB1	Ciclev10007255m	1380	152.75	5.46	449-513	细胞核 Nuclear
CcPUB2	Ciclev10007527m	769	85.86	6.02	700-762	叶绿体 Chloroplast
CcPUB3	Ciclev10018144m	1088	121.83	6.16	613-677	细胞核 Nuclear
CcPUB4	Ciclev10015253m	444	49.84	8.1	36-100	叶绿体 Chloroplast
CcPUB5	Ciclev10014584m	636	69.48	6.56	257-320	叶绿体 Chloroplast
CcPUB6	Ciclev10014430m	715	78.42	7.01	291-354	叶绿体 Chloroplast
CcPUB7	Ciclev10019643m	537	58.77	6.17	28-91	叶绿体 Chloroplast
CcPUB8	Ciclev10019147m	683	75.51	8.48	279-342	叶绿体 Chloroplast
CcPUB9	Ciclev10032341m	281	31.97	6.03	207-270	细胞质 Cytoplasmic
CcPUB10	Ciclev10024300m	1441	160.62	5.78	506-570	细胞核 Nuclear
CcPUB11	Ciclev10024335m	433	47.91	5.77	79-142	细胞质 Cytoplasmic
CcPUB12	Ciclev10018951m	775	85.47	6.91	239-302	细胞核 Nuclear
CcPUB13	Ciclev10018671m	1008	112.36	5.75	265-328	细胞核 Nuclear
CcPUB14	Ciclev10018795m	888	99.09	5.89	821-884	细胞核 Nuclear
CcPUB15	Ciclev10033932m	687	75.23	6.63	284-347	质膜 Plasma
CcPUB16	Ciclev10030962m	627	68.42	5.49	249-312	叶绿体 Chloroplast
CcPUB17	Ciclev10031496m	456	50.85	8.39	75-138	细胞质 Cytoplasmic
CcPUB18	Ciclev10030608m	1019	113.74	6.24	233-300	细胞核 Nuclear
CcPUB19	Ciclev10030759m	775	86	5.57	282-345	细胞核 Nuclear
CcPUB20	Ciclev10030837m	713	79.13	5.19	220-283	细胞核 Nuclear
CcPUB21	Ciclev10030762m	775	86	5.57	282-345	细胞核 Nuclear
CcPUB22	Ciclev10001406m	395	44.04	6.57	6-69	细胞核 Nuclear
CcPUB23	Ciclev10001380m	398	44.51	8.54	13-76	叶绿体 Chloroplast
CcPUB24	Ciclev10000413m	728	80.97	6.79	248-311	细胞核 Nuclear
CcPUB25	Ciclev10000389m	744	82.67	6.62	264-327	细胞核 Nuclear
CcPUB26	Ciclev10003715m	643	71.66	5.37	274-337	叶绿体 Chloroplast
CcPUB27	Ciclev10000306m	813	89.10	5.38	32-100	细胞核 Nuclear
CcPUB28	Ciclev10011217m	683	74.80	9.14	276-339	叶绿体 Chloroplast
CcPUB29	Ciclev10011844m	416	46.12	8.38	9-74	叶绿体 Chloroplast
CcPUB30	Ciclev10011847m	414	46.93	8.16	12-78	叶绿体 Chloroplast
CcPUB31	Ciclev10011415m	546	59.20	8.42	45-108	叶绿体 Chloroplast
CcPUB32	Ciclev10010958m	1049	118.38	5.48	954-1017	叶绿体 Chloroplast
CcPUB33	Ciclev10011388m	564	64.68	5.22	469-532	细胞质 Cytoplasmic
CcPUB34	Ciclev10011387m	564	64.68	5.22	469-532	细胞质 Cytoplasmic
CcPUB35	Ciclev10011113m	779	88.36	5.43	684-747	叶绿体 Chloroplast
CcPUB36	Ciclev10025062m	679	75.18	5.65	210-273	叶绿体Chloroplast
基因名称 Gene name	基因组登录号 Gene accession No.	蛋白质大小 Protein length (aa)	分子量 Molecular mass (KD)	等电点 Isoelectric point (PI)	box位置 U-box domain location	细胞定位 Subcellular localizations
CcPUB37	Ciclev10024987m	737	81.58	5.84	268-331	叶绿体Chloroplast
CcPUB38	Ciclev10024899m	828	90.51	5.92	239-302	叶绿体Chloroplast
CcPUB39	Ciclev10025589m	450	49.00	7.01	69-132	细胞质Cytoplasmic
CcPUB40	Ciclev10025214m	597	65.21	6.98	39-101	叶绿体Chloroplast
CcPUB41	Ciclev10024952m	760	85.82	6.21	688-751	细胞核Nuclear
CcPUB42	Ciclev10024909m	808	91.47	6.18	736-799	叶绿体Chloroplast
CcPUB43	Ciclev10025637m	435	48.23	8	13-76	质膜Plasma
CcPUB44	Ciclev10028003m	643	71.65	6.54	272-335	叶绿体Chloroplast
CcPUB45	Ciclev10028522m	418	45.74	6.21	17-80	细胞核Nuclear
CcPUB46	Ciclev10028557m	407	45.49	8.78	9-76	叶绿体Chloroplast
CcPUB47	Ciclev10028564m	405	45.68	8.92	9-74	叶绿体Chloroplast
CcPUB48	Ciclev10027966m	661	71.37	5.43	261-324	叶绿体Chloroplast
CcPUB49	Ciclev10028111m	564	60.47	5.46	164-227	叶绿体Chloroplast
CcPUB50	Ciclev10027788m	887	99.83	8.08	820-883	细胞核Nuclear
CcPUB51	Ciclev10029840m	1048	115.56	6.56	265-333	叶绿体Chloroplast
CcPUB52	Ciclev10004235m	1012	112.79	5.91	261-324	叶绿体Chloroplast
CcPUB53	Ciclev10004724m	526	56.90	6.07	1-66	叶绿体Chloroplast
CcPUB54	Ciclev10004393m	757	84.621	5.93	692-754	细胞核Nuclear
CcPUB55	Ciclev10004338m	810	90.50	6.31	745-807	细胞核Nuclear
CcPUB56	Ciclev10006472m	780	88.56	6.55	713-776	叶绿体Chloroplast

表2

图1

图2

图3

图4

表3

PlantCARE预测CcPUB基因家族启动子区顺式作用原件"

顺式元件 Cis-element	典型序列 Typical sequence	特性 Characteristic	基因 Gene
G-box	CACGT	光响应 Cis-acting regulatory element involved in light responsiveness	CcPUB1、CcPUB3—CcPUB11、CcPUB13—CcPUB17、CcPUB21— CcPUB23、CcPUB25、CcPUB26、CcPUB28—CcPUB37、CcPUB39、CcPUB42— CcPUB45、CcPUB47—CcPUB56
ATCT-motif	AATCT	光响应 Part of a conserved DNA module involved in light responsiveness	CcPUB7、CcPUB18、CcPUB22、CcPUB23、CcPUB28、CcPUB30、CcPUB31、CcPUB39、CcPUB53、CcPUB55、CcPUB56
ACE	AAAACGTTTA	光响应 Cis-acting element involved in light responsiveness	CcPUB3、CcPUB10、CcPUB21、CcPUB26、CcPUB32、CcPUB39
LTR	CCGAAA	低温反应 Cis-acting element involved in low- temperature responsiveness	CcPUB2、CcPUB5、CcPUB8、CcPUB10、CcPUB16、CcPUB17、CcPUB23、CcPUB25、CcPUB26、CcPUB28、CcPUB29、CcPUB32、CcPUB38、CcPUB39、CcPUB42—CcPUB44、CcPUB46、CcPUB56
TGACG-motif	TGACG	MeJA响应 Cis-acting regulatory element involved in the MeJA-responsiveness	CcPUB1—CcPUB6、CcPUB8、CcPUB9、CcPUB11、CcPUB13— CcPUB18、CcPUB23、CcPUB27、CcPUB29、CcPUB31—CcPUB38、CcPUB40—CcPUB44、CcPUB46、CcPUB47、CcPUB50、CcPUB52、CcPUB55、CcPUB56
ABRE	GACACGTGGC TACGTG	脱落酸响应 Cis-acting element involved in the abscisic acid responsiveness	CcPUB1—CcPUB11、CcPUB13—CcPUB17、CcPUB22、CcPUB23、CcPUB26、CcPUB28—CcPUB30、CcPUB32—CcPUB37、CcPUB39、CcPUB42—CcPUB45、CcPUB47—CcPUB53、CcPUB55、CcPUB56
GARE-motif	TCTGTTG, AAACAGA	赤霉素响应 Gibberellin-responsive element	CcPUB2、CcPUB5、CcPUB6、CcPUB8、CcPUB9、CcPUB13、CcPUB15、CcPUB16、CcPUB27、CcPUB31、CcPUB45、CcPUB47、
MBS	TAACTG	MYB结合位点参与干旱诱导 MYB binding site involved in drought- induction	CcPUB1、CcPUB4—CcPUB9、CcPUB11、CcPUB14、CcPUB16、CcPUB17、CcPUB21、CcPUB23、CcPUB25—CcPUB27、CcPUB31— CcPUB37、CcPUB39、CcPUB44、CcPUB50、CcPUB51、CcPUB55、CcPUB56
DRE	TGGCCGAC	冷和脱水 Regulatory element involved in cold- and dehydration-responsiveness	CcPUB4、CcPUB5、CcPUB7、CcPUB8、CcPUB13、CcPUB21、CcPUB23、CcPUB26、CcPUB29、CcPUB37、CcPUB40—CcPUB42、CcPUB44、CcPUB46、CcPUB51、CcPUB56
ERE	ATTTTAAA	乙烯响应 Ethylene responsive element	CcPUB2—CcPUB9、CcPUB11、CcPUB12、CcPUB14、CcPUB17、CcPUB18、CcPUB21—CcPUB23、CcPUB25、CcPUB26、CcPUB28、CcPUB29、CcPUB31、CcPUB32、CcPUB37—CcPUB42、CcPUB44、CcPUB45、CcPUB47、CcPUB50—CcPUB53、CcPUB56

表3

图5

图6

图7

图8

图9

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引物名称 Primer name	正向引物序列 Forward primer sequence	反向引物序列 Reverse primer sequence
CcPUB4	CCGGTGACTTTATCCACTGGG	GAATGGGTTCAAAACTCGTCAGG
CcPUB9	GCCAGCTGAGGTCCCGGATT	TGCCTTGTATGCCCAACCATGT
CcPUB10	CAAAACCCGTAGTCCGAAAA	AATTCACCATCCGAGTCTGC
CcPUB41	ACGCCGTCCAGTATCCTTC	AATTCCAGCATTCCCATCCA
CcPUB48	CTGAGCGAAGGTACCAGAG	AGTGTTCATCCACCATTCC
β-Actin	CCCCATCGTTACCGTCCAG	CGCCTTGCCAGTTGAATATCC