葡萄LBD基因家族的鉴定与表达分析

doi:10.3864/j.issn.0578-1752.2018.21.009

摘要/Abstract

摘要：

【目的】通过生物信息学分析明确LBD转录因子在葡萄基因组中的数量、结构和非生物胁迫差异表达,为探究LBD转录因子在葡萄非生物胁迫中的作用奠定理论基础。【方法】根据已经报道的拟南芥LBD基因,利用葡萄基因组数据库中的BLAST软件鉴定葡萄基因组中的LBD基因。采用DNAMAN5.0、Clustalx、MapInspect、MEME、GSDS2.0、ExPASy和MEGA5.0等软件进行生物信息学分析。利用PLEXdb中葡萄Affymetrix GeneChip 16K基因芯片数据绘制芯片表达谱。采用qRT-PCR方法检测VvLBD基因家族在逆境胁迫中的表达情况。【结果】从葡萄（Vitis vinifera L.）全基因组中鉴定得到30个LBD基因家族成员,可分为ClassⅠ和ClassⅡ两类,ClassⅠ分为5个亚族,分别是Ⅰa、Ⅰb、Ⅰc、Ⅰd和Ⅰe,ClassⅡ分为2个亚族,分别是Ⅱa和Ⅱb。理化性质分析表明,VvLBD基因家族编码氨基酸数目介于127—386,理论等电点分布在4.77—9.28。定位分析发现,该基因家族的30个基因分布于葡萄19条染色体中的11条染色体上,其中第13染色体上分布最多,有7个基因。多序列比对和motif分析结果表明,VvLBD基因家族具有特定的3个保守结构域,分别是类锌指结构、亮氨酸拉链结构和甘氨酸-丙氨酸-丝氨酸（GAS）结构。亚细胞定位分析表明,VvLBD基因家族主要在叶绿体、线粒体和细胞核中进行表达。VvLBD基因家族的二级结构主要以α-螺旋和不规则卷曲为主。VvLBD基因芯片表达谱分析发现,大部分基因在盐胁迫和PEG胁迫下表达量上升,并且胁迫时间的长短对该基因家族成员的表达也存在差异。qRT-PCR分析结果表明,VvLBD基因家族成员中VvLBD8、VvLBD11、VvLBD12、VvLBD15、VvLBD16、VvLBD17在400 mmol·L ^-1 NaCl处理条件下呈上调表达,表达量分别是对照的3、1、8、4、5和13倍,VvLBD12和VvLBD19在10% PEG处理条件下呈上调表达,表达量分别是对照的4和26倍。【结论】从葡萄基因组中一共鉴定出30个LBD基因家族成员,分布于11条染色体上,并且结构进化高度保守;所有成员都与逆境相关,但是该基因家族成员在不同逆境胁迫下的响应程度存在一定的差异。

关键词: 葡萄, LBD基因家族, 进化分析, 表达谱, 实时荧光定量PCR

Abstract:

【Objective】The objectives of this research are to identify the Lateral Organ Boundaries Domain(LBD) family genes from grape(Vitis vinifera) genome, to know the profile of LBD family such as gene number, gene structure and abiotic stress expression characteristics in grape, and to provide theoretical basis for exploring what roles the LBD transcription factors play in abiotic stress of grape.【Method】The LBD genes in grape genome-wide were identified by BLAST software in the grape genome database based on LBD genes have been reported from Arabidopsis. DNAMAN5.0, Clustalx, MapInspect, MEME, GSDS2.0, ExPASy and MEGA5.0 software were used for carry out various bioinformatics analysis of LBD. The expression profile chip was draw by the data comes from Affymetrix Gene Chip 16K in PLEXdb. The qRT-PCR was used to detect the expression of grape LBD gene family in abiotic stress.【Result】Total of 30 LBD genes were identified from grape genome, which can be divided into ClassⅠand ClassⅡ. The classⅠcan be divided into 5 subtribe which are namedⅠa,Ⅰb,Ⅰc,Ⅰd andⅠe. ClassⅡ can be divided into Ⅱa and Ⅱb. The number of amino acids is between 127 and 386, and the theoretical equivalence point is between 4.77 and 9.28 in the VvLBD gene family by physicochemical analysis. The analysis of the gene's location on the chromosome revealed that the 30 genes in this family were distributed on 11 of the 19 chromosomes of the grape, and the chromosome 13 contains 7 genes. Multiple sequence alignments and Motif analysis showed that the VvLBD gene family has three conserved domains, namely the sinusoidal structure, the leucine zipper structure, and the Glycine-alanine-serine (GAS) structure. The VvLBD gene family is mainly expressed in chloroplast, mitochondria and nucleus through the sub-cellular location analyzing. The alpha-helix and irregular curl are the main structure of the secondary structure in the VvLBD gene family. The expression of the most gene was increased under the salt and PEG stress, and the expression characteristic was changed by the stress time change in the chip expression profile. The qRT-PCR results showed the expression of VvLBD8, VvLBD11, VvLBD12, VvLBD15, VvLBD16, VvLBD17 were raised to the 3, 1, 8, 4, 5, and 13 times compared the control under the 400 mmol ^.L ^-1NaCl treatment. The expression of VvLBD12 and VvLBD19 was raised to the 4 and 26 times compared the control under 10% PEG treatment.【Conclusion】30 LBD gene family members were identified from the grape genome. These members distributed on 11 of the 19 chromosomes of the grape and the evolution of VvLBD is highly conservative. The LBD gene family is related to the abiotic stress, but the different expression were existed between the different members when the encountered the adversity environments.

Key words: Vitis vinifera, LBD gene family, evolutionary analysis, expression profile, qRT-PCR

何红红,马宗桓,张元霞,张娟,卢世雄,张志强,赵鑫,吴玉霞,毛娟. 葡萄LBD基因家族的鉴定与表达分析[J]. 中国农业科学, 2018, 51(21): 4102-4118.

HongHong HE,ZongHuan MA,YuanXia ZHANG,Juan ZHANG,ShiXiong LU,ZhiQiang ZHANG,Xin ZHAO,YuXia WU,Juan MAO. Identification and Expression Analysis of LBD Gene Family in Grape[J]. Scientia Agricultura Sinica, 2018, 51(21): 4102-4118.

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https://www.chinaagrisci.com/CN/Y2018/V51/I21/4102

图/表 11

表1

VvLBD基因家族表达分析的实时荧光定量引物"

基因Gene	上游引物Forward primer (5′-3′)	下游引物Reverse primer (5′-3′)
VvLBD1	CAGCAGGAGACTGACAGCATGTTC	GATAGAAGCCAAGAGTGAGTTCCAACC
VvLBD2	GCCGTGGATGCAGTGCTCAG	GCTTGGACTTAGAAGAACGCTTGAATG
VvLBD3	TGGCCTTCATCTCCACCGTACC	CCGCTCCGAACACAGGATTCAC
VvLBD4	GAGCTAGTGAACATGCAATGCCAAC	GCTGTCATCGAGGAAGAAGGAGTTAC
VvLBD5	AGAGGAGCCTGCCTTACCAACTC	AGCTGAGGTAGTGTTGAAGGTTGTTG
VvLBD6	TCCTCCACCTCCTCTACCTCTACC	CCTGTTGTGCTTGAAGGCAGTATTG
VvLBD7	GCTGCGGTTACCATTGCTTATGAAG	TGTAGATTAACAACCTGCTGCTGGAG
VvLBD8	GCCTTGCAGCAGCAGGTAGC	TGCGGCGATACTGAGTTCATGTAAC
VvLBD9	TTCGCTAATGTCCACAAGGTCTTCG	ACGGCATCTTCTCGATGTGCAAC
VvLBD10	TTCGTTAATGTCCACAAGGTCTTCGG	AATCAGACCAACACAGCCATAGACG
VvLBD11	GTCCTTCATCTCTGCTGTTCCTGAG	TCACGGTCCGGCCACACG
VvLBD12	AGCAGCAGCAGCAGCATCAG	TCTCTTCCTCGTCTTCGTCCTCATC
VvLBD13	AGTTCTCCATCGTTCACAAGGTCTTC	GCAGGTATGATATGGCTCCGACAC
VvLBD14	CAGCACAGCCACCAGCACAG	GGAGGAAGAGGAGTTAGGAGGAAGAG
VvLBD15	CTCAACCAACAGGCTCTGCTACTATC	AAGGAGGTGGCTGTTCATTCATTAGG
VvLBD16	AGCAGCAGCAGCCACAATATCAG	TCCATGACAAGATTGCCATCCTCAAG
VvLBD17	GATGCCTTCTCCTTCTCTTCCAACTG	GTTCCTGCAACGCCTTGACAATG
VvLBD18	CTTGCAGCTCTGAGGATGGAATACG	AGCTAGAATACAACTCTCATCGCATCG
VvLBD19	ATTCGTGATCCAGTCTATGGTTGTGC	ATCTGTCCAGGAAGAAGCTTGCATTAG
VvLBD20	GCCAATGCCACCGTCTTCCTC	CACAAGCCTCGTAGAGAAGAGAACTG
VvLBD21	GCCACCAATGCTGACCTGATCC	CTCCACCTCCACCTCCATGACTC
VvLBD22	GATGGTAACGCCGCCTTCACTC	ACCTCTGGAGCCACCGAACTG
VvLBD23	CCTGTCTATGGCTGTGTTGCTCATG	GCGGTGCCAGAGAAGGAATGC
VvLBD24	GTCCGTCAGCGATGGCACATG	ATAAGGAATGACCGACGATGAAGAAGG
VvLBD25	CCTTCTTCCAACTTCAACAACTCCTTG	GGTCGGATGGAGAGCGGTAGAG
VvLBD26	TGCGGATGAGTTGTAATGGCTGTC	GCGAGGAAGACGGTGGCATTG
VvLBD27	TCGTTACGCCTGCAATGAGATGTC	GCTTCCTCCTCCTTCATTACCAATCC
VvLBD28	GCAGCGGTGGACACAGTTCTC	CAGCGAGTATGCGTCACGTAGC
VvLBD29	TAGGAGAAGATGTGCTCAGGACTGTG	TGCTTCATACACCATGCTACTCACTG
VvLBD30	CCAACTCCGCCAACTCCAGATG	CTGTGGATGGTGGTGGTGATTGTG
UBI	GCTCGCTGTTTTGCAGTTCTAC	AACATAGGTGAGGCCGCACTT

表1

图1

表2

葡萄中LBD转录因子家族信息"

基因 Gene	基因登录号 Gene accession No.	染色体定位 Chromosome location	长度 Length of CDS (bp)	基因全长 Full length genomic (bp)	分子量 Molecular weight (kD)	等电点 pI	氨基酸 Amino acid	外显子 Exon	cDNA全长 FL-cDNA (bp)	拟南芥同源基因Arabidopsis homologous gene
基因 Gene	基因登录号 Gene accession No.	染色体定位 Chromosome location	长度 Length of CDS (bp)	基因全长 Full length genomic (bp)	分子量 Molecular weight (kD)	等电点 pI	氨基酸 Amino acid	外显子 Exon	cDNA全长 FL-cDNA (bp)	登录号 Accession No.	名称 Gene name	E值 E-value
VvLBD1	GSVIVT01011895001	Chr.01:3210000..3211373	1158	1374	43.30087	8.96	386	3	1158	AT1G68510	LBD42	7e-05
VvLBD2	GSVIVT01011896001	Chr.01:3202459..3205665	726	1076	26.97486	9.28	242	2	998	AT1G68510	LBD42	7e-05
VvLBD3	GSVIVT01037853001	Chr.03:7098160..7099838	615	1679	22.87014	6.73	205	3	714	AT4G37540	LBD39	1e-09
VvLBD4	GSVIVT01025128001	Chr.06:4180823..4182134	609	1312	22.02887	4.77	202	2	953	AT2G28500	LBD11	8e-14
VvLBD5	GSVIVT01024662001	Chr.06:7971774..7972539	492	766	18.36397	6.40	164	2	644	AT1G31320	LBD4	0.003
VvLBD6	GSVIVT01024592001	Chr.06:8584134..8585865	690	1735	24.92138	8.58	229	3	1529	AT2G30340	LBD13	6e-18
VvLBD7	GSVIVT01028294001	Chr.07:5837981..5838834	699	854	25.72175	5.87	232	2	750	AT2G42430	LBD16	0.018
VvLBD8	GSVIVT01028295001	Chr.07:5846731..5847540	588	810	21.46149	7.54	195	2	588	AT2G42430	LBD16	0.018
VvLBD9	GSVIVT01003547001	Chr.07:14517830..14519654	426	1825	15.69194	8.60	142	2	513	AT5G66870	LBD36	2e-09
VvLBD10	GSVIVT01003548001	Chr.07:14524923..14526703	663	1781	23.96578	9.16	220	2	765	AT5G66870	LBD36	2e-09
VvLBD11	GSVIVT01000141001	Chr.07:15589316..15590604	687	1289	24.96159	8.30	229	2	1167	AT4G37540	LBD39	0.005
VvLBD12	GSVIVT01029979001	Chr.08:2636746..2653598	777	16853	27.99185	5.85	259	4	777	AT3G03760	LBD20	7e-27
VvLBD13	GSVIVT01032752001	Chr.13:1022036..1023024	381	989	14.19636	8.80	127	3	417	AT2G30130	LBD12	4e-09
VvLBD14	GSVIVT01032714001	Chr.13:1310001..1311298	675	1298	24.53103	8.99	225	3	956	AT2G30340	LBD13	2e-11
VvLBD15	GSVIVT01016500001	Chr.13:3355781..3356736	660	955	24.29509	5.08	219	2	849	AT2G42430	LBD16	8e-08
VvLBD16	GSVIVT01016335001	Chr.13:4853462..4854800	594	1339	21.75785	5.67	197	2	677	AT2G28500	LBD11	3e-16
VvLBD17	GSVIVT01016332001	Chr.13:4961089..4962007	498	919	18.35293	8.21	166	2	498	AT2G28500	LBD11	3e-16
VvLBD18	GSVIVT01016329001	Chr.13:4999327..4999957	444	631	16.23288	8.72	148	2	505	AT3G11090	LBD21	8e-04
VvLBD19	GSVIVT01016328001	Chr.13:5001203..5002266	420	1064	15.73712	8.50	140	2	462	AT3G11090	LBD21	8e-04
VvLBD20	GSVIVT01031035001	Chr.14:21210990..21212344	564	1355	20.29311	8.98	187	3	1124	AT1G68510	LBD42	7e-05
VvLBD21	GSVIVT01032415001	Chr.14:27152774..27157752	606	4979	22.07197	8.52	201	3	720	AT3G27650	LBD25	0.003
VvLBD22	GSVIVT01027621001	Chr.15:14983162..14985661	795	2500	28.19431	8.12	264	3	924	AT2G45420	LBD18	2e-21
VvLBD23	GSVIVT01027620001	Chr.15:14992324..14994786	585	2463	21.17114	6.50	195	2	585	AT2G42430	LBD16	1.1
VvLBD24	GSVIVT01018486001	Chr.16:14419741..14420915	672	1175	24.58742	7.65	223	2	672	AT5G66870	LBD36	3e-11
VvLBD25	GSVIVT01010625001	Chr.16:15894465..15895136	462	672	17.36371	9.08	154	2	462	AT3G26620	LBD23	7e-19
VvLBD26	GSVIVT01008284001	Chr.17:3660239..3661508	684	1270	24.77241	8.05	228	3	1035	AT3G02550	LBD41	0.005
VvLBD27	GSVIVT01007677001	Chr.17:10578569..10581053	468	2485	16.93792	6.81	156	3	670	AT5G63090	LOB	3e-22
VvLBD28	GSVIVT01009360001	Chr.18:7746214..7747271	669	1058	24.10543	8.54	222	2	939	AT3G49940	LBD38	3e-07
VvLBD29	GSVIVT01013631001	Chr.Un:1949856..1952420	528	2565	19.30889	7.68	176	2	1646	AT1G16530	LBD3	2e-10
VvLBD30	GSVIVT01006269001	Chr.Un:24370759..24373006	651	2248	23.99094	8.25	217	1	1243	AT1G65620	LBD6	3e-47

表2

图2

图3

图4

图5

表3

表4

图6

图7

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名称Name	α-螺旋Alpha helix (%)	β-转角Beta turn (%)	不规则卷曲Random coil (%)
VvLBD1	39.38	6.74	36.01
VvLBD2	20.66	6.20	53.72
VvLBD3	37.56	8.78	39.02
VvLBD4	45.05	2.97	41.58
VvLBD5	41.46	1.83	39.63
VvLBD6	49.34	3.49	37.12
VvLBD7	48.28	6.90	38.79
VvLBD8	39.49	7.69	38.97
VvLBD9	33.80	8.45	30.28
VvLBD10	56.36	4.55	27.37
VvLBD11	32.89	11.84	40.35
VvLBD12	33.98	3.47	44.02
VvLBD13	52.76	0.00	26.77
VvLBD14	43.11	4.44	44.89
VvLBD15	47.95	6.85	33.33
VvLBD16	29.95	5.58	41.62
VvLBD17	39.76	2.41	42.77
VvLBD18	37.84	6.76	41.22
VvLBD19	45.71	6.43	37.14
VvLBD20	27.81	11.23	38.50
VvLBD21	31.34	9.95	42.79
VvLBD22	33.71	6.82	49.24
VvLBD23	51.79	7.69	33.33
VvLBD24	39.91	8.97	32.29
VvLBD25	51.30	6.49	28.57
VvLBD26	32.46	10.96	38.60
VvLBD27	37.82	7.69	45.51
VvLBD28	41.11	12.96	33.33
VvLBD29	42.61	4.55	42.05
VvLBD30	49.77	5.99	36.41