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作物学报 ›› 2019, Vol. 45 ›› Issue (8): 1146-1157.doi: 10.3724/SP.J.1006.2019.84124

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜AVP1、VHA-a2和VHA-a3基因的鉴定及功能性研究

姚珺玥1,2,华营鹏1,2,周婷1,2,王涛1,2,宋海星1,2,官春云3,张振华1,2,*()   

  1. 1 湖南农业大学资源环境学院, 湖南长沙 410128
    2 南方粮油作物协同创新中心, 湖南长沙 410128
    3 国家油料作物改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2018-09-20 接受日期:2019-04-15 出版日期:2019-08-12 网络出版日期:2019-07-16
  • 通讯作者: 张振华
  • 作者简介:E-mail: yjy950606@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2018YFD0200901);本研究由国家重点研发计划项目(2018YFD0200906);国家现代农业产业技术体系建设专项资助(CARS-13)

Identification and function analysis of AVP1, VHA-a2, and VHA-a3 genes in Brassica napus L.

YAO Jun-Yue1,2,HUA Ying-Peng1,2,ZHOU Ting1,2,WANG Tao1,2,SONG Hai-Xing1,2,GUAN Chun-Yun3,ZHANG Zhen-Hua1,2,*()   

  1. 1 College of Resource and Environment, Hunan Agricultural University, Changsha 410128, Hunan, China
    2 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China
    3 Hunan Branch, National Oil Crops Improvement Center, Changsha 410128, Hunan, China
  • Received:2018-09-20 Accepted:2019-04-15 Published:2019-08-12 Published online:2019-07-16
  • Contact: Zhen-Hua ZHANG
  • Supported by:
    This study was supported by the National Key R&D Program of China(2018YFD0200901);This study was supported by the National Key R&D Program of China(2018YFD0200906);the China Agriculture Research System(CARS-13)

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摘要：

液泡是调控植物细胞分化、生长发育的重要部位, AVP1、VHA-a2和VHA-a3基因调控植物液泡内外离子平衡、离子运输以及能量供应。本研究利用功能已知的拟南芥AVP1、VHA-a2和VHA-a3基因为参考序列在甘蓝型油菜全基因组数据库、NCBI植物基因组注释数据库等鉴定并筛选出9个BnaAVP1、3个BnaVHA-a2和4个BnaVHA-a3, 并分析基因拷贝数变异、分子特征、跨膜结构域、保守基序、染色体定位、系统进化树构建、蛋白二级结构及三维结构预测、高通量转录组测序等。发现甘蓝型油菜BnaAVP1和BnaVHA-a3的基因数量明显多于甘蓝和白菜; 甘蓝型油菜AVP1、VHA-a2和VHA-a3蛋白属于由酸性氨基酸组成的稳定蛋白; 系统进化选择能力的分析表明, 甘蓝型油菜AVP1、VHA-a2和VHA-a3家族基因与甘蓝、白菜关系相近。转录组测序表明, 低氮处理后, BnaAVP1s基因主要在地上部表达, 且低氮3 h后地上部表达下调, 低氮处理72 h根中表达量上调; BnaVHA-a2s和BnaVHA-a3s基因在地上部和根中均有表达, BnaVHA-a2s在低氮处理72 h后表达量基本呈上调趋势, BnaVHA-a3s在低氮3 h后基本呈下调趋势。低磷处理后, BnaAVP1s根中大部分基因表达上调, 地上部表达基本无差异; BnaVHA-a2s表达基本无差异; BnaVHA-a3s地上部和根中均基本为上调趋势。该结果为进一步研究甘蓝型油菜AVP1、VHA-a2和VHA-a3基因生物学功能及AVP1、VHA-a2和VHA-a3蛋白水解ATP提供能量供植物代谢的分子机制奠定基础, 为已知大量数据的其他物种家族基因生物信息学研究提供参考。

关键词: 甘蓝型油菜, AVP基因, VHA基因, 生物信息学

Abstract:

Vacuoles play an important role in regulating plant cell differentiation and growth. AVP1, VHA-a2, and VHA-a3 genes are important carrier elements for regulating the acid-base balance inside and outside the vacuole of plants and the energy provided by ion transport. In this study, nine BnaAVP1, three BnaVHA-a2, and four BnaVHA-a3 genes were identified in Brassica napus genome database and NCBI plant genome annotation database by using the known AVP1, VHA-a2, and VHA-a3 genes of Arabidopsis thaliana as reference sequences. Bioinformatics studies on copy number variation, molecular characteristics, transmembrane domain, conserved motifs, chromosome localization, phylogenetic tree construction, secondary structure and three- dimensional structure prediction of proteins and high-throughput transcriptome sequencing were carried out, indicating that the number of genes of BnaAVP1 and BnaVHA-a3 was significantly higher than that of B. oleracea and B. rapa; the AVP1, VHA-a2 and VHA-a3 proteins of B. napus belonged to a stable protein composed of acidic amino acids. The analysis of evolutionary selection ability showed that after low nitrogen treatment, BnaAVP1 genes were mainly expressed in shoots, and the expression of BnaAVP1 genes was down-regulated in shoots after three hours of low nitrogen treatment, and up-regulated in roots after 72 hours of low nitrogen treatment. BnaVHA-a2 and BnaVHA-a3 genes were expressed in both shoots and roots, while BnaVHA-a2 genes were up-regulated after 72 hours of low nitrogen treatment, and BnaVHA-a3 genes were down-regulated after three hours of low nitrogen treatment. After low phosphorus treatment, the expression of most BnaAVP1 genes was up-regulated in roots, and no difference in shoots. The expression of BnaVHA-a2 genes was almost no difference, and that of BnaVHA-a3 genes up-regulated in both shoots and roots. This result lays a foundation for further study on the biological functions of AVP1, VHA-a2, and VHA-a3 genes in B. napus and the molecular mechanism that AVP1, VHA-a2, and VHA-a3 proteins hydrolyze ATP to provide energy for plant metabolism, and provides references for other species family genetic bioinformatics studies that are known to have large amounts of data.

Key words: Brassica napus L, AVP gene, VHA gene, bioinformatics

图1

甘蓝型油菜、甘蓝、白菜AVP1、VHA-a2和VHA-a3基因的拷贝数变异 柱状图顶部数值为该物种拷贝的基因数目。"

表1

甘蓝型油菜AVP1、VHA-a2和VHA-a3基因成员信息"

基因名称
Gene name		 基因库编号
Database No.		 基因编号
Gene ID		 基因长度
CDS (bp)		 氨基酸长度
Amino acids (aa)		 分子量
MW (kD)		 理论等电点
pI		 亲水性指数
GRAVY
BnaAVP1-1 GSBRNA2T00010424001 BnaA09g56670D 2409 802 83.97 5.06 0.620
BnaAVP1-2 GSBRNA2T00033628001 BnaA06g38620D 1821 606 64.61 5.00 0.281
BnaAVP1-3 GSBRNA2T00136659001 BnaC08g38730D 3525 1174 126.46 6.29 0.269
BnaAVP1-4 GSBRNA2T00139755001 BnaC08g16730D 2310 769 80.59 5.03 0.611
BnaAVP1-5 GSBRNA2T00068861001 BnaC06g40530D 2310 769 80.61 5.00 0.574
BnaAVP1-6 GSBRNA2T00054245001 BnaC05g11930D 2316 771 80.83 5.08 0.618
BnaAVP1-7 GSBRNA2T00103071001 BnaA08g23780D 2310 769 80.52 5.03 0.616
BnaAVP1-8 GSBRNA2T00146278001 BnaA07g35600D 2310 769 80.70 5.01 0.572
BnaAVP1-9 GSBRNA2T00062272001 BnaA06g10340D 2316 771 80.83 5.08 0.621
BnaVHA-a2-1 GSBRNA2T00137010001 BnaC08g35720D 2466 821 92.97 5.61 0.060
BnaVHA-a2-2 GSBRNA2T00108093001 BnaC04g33510D 2472 823 92.96 5.61 0.056
BnaVHA-a2-3 GSBRNA2T00090255001 BnaA04g12160D 2460 819 92.80 5.56 0.059
BnaVHA-a3-1 GSBRNA2T00061106001 BnaC03g60340D 2463 820 92.85 5.86 0.041
BnaVHA-a3-2 GSBRNA2T00075109001 BnaC01g00150D 2457 818 92.50 5.80 0.080
BnaVHA-a3-3 GSBRNA2T00150888001 BnaA08g16770D 2460 819 92.79 5.75 0.043
BnaVHA-a3-4 GSBRNA2T00073949001 BnaA01g05300D 2454 817 92.32 5.66 0.071

图2

甘蓝型油菜中BnaAVP1、BnaVHA-a2和BnaVHA-a3蛋白的同义突变频率和非同义突变频率"

图3

甘蓝型油菜AVP1、VHA-a2和VHA-a3基因染色体定位"

图4

甘蓝型油菜、拟南芥、白菜以及甘蓝的AVP1、VHA-a2和VHA-a3蛋白家族成员的保守基序分布"

图5

甘蓝型油菜、拟南芥、白菜以及甘蓝的AVP1、VHA-a2和VHA-a3基因外显子-内含子结构"

图 6

甘蓝型油菜BnaAVP1、BnaVHA-a2和BnaVHA-a3蛋白的进化分析 蓝色代表BnaAVP1家族, 绿色代表BnaVHA-a3家族, 红色代表BnaVHA-a2家族。"

图7

甘蓝型油菜BnaVHA-a2-1蛋白二级结构分析 GOR4 (A)和PSIPRED (B)分别是进行二级结构分析的2种不同的工具。"

图8

甘蓝型油菜BnaAVP1、BnaVHA-a2和BnaVHA-a3蛋白三级结构预测"

图9

在低氮条件下甘蓝型油菜BnaAVP1、BnaVHA-a2和BnaVHA-a3家族的转录组分析。 S: 地上部; R: 根; 0、3、72 (h)代表缺氮胁迫的时间。图中结果表示3次生物学重复的平均值。*代表有显著差异。"

图10

在低磷条件下甘蓝型油菜BnaAVP1、BnaVHA-a2和BnaVHA-a3家族的转录组分析 S: 地上部; R: 根。CK代表正常条件, LP代表低磷胁迫。图中结果表示3次生物学重复的平均值。*代表有显著差异。"

[1] Sze H, Schumacher K, Müller M L, Padmanaban S, Taiz L . A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H +-ATPase. Trends Plant Sci, 2002,7:157-161.
[2] Sze H, Li X, Palmgren M G . Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. Plant Cell, 1999,11:677-689.
[3] Schumacher K . pH in the plant endomembrane system: an import and export business. Curr Opin Plant Biol, 2014,22:71-76.
doi: 10.1016/j.pbi.2014.09.005
[4] Drozdowicz Y M, Rea P A . Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci, 2001,6:206-211.
doi: 10.1016/S1360-1385(01)01923-9
[5] Maeshima M . Tonoplast transporters: organization and function. Annu Rev Plant Biol, 2001,52:469-497.
doi: 10.1146/annurev.arplant.52.1.469
[6] Bayer P E, Hurgobin B, Golicz A A, Chan C K K, Yuan Y, Lee H, Zou J . Assembly and comparison of two closely related Brassica napus genomes. Plant Biotechnol J, 2017,15:1602-1610.
doi: 10.1111/pbi.2017.15.issue-12
[7] Altschul S F, Gish W, Miller W, Myers E W, Lipman D J . Basic local alignment search tool. J Mol Biol, 1990,215:403-410.
doi: 10.1016/S0022-2836(05)80360-2
[8] Finn R D, Bateman A, Clements J, Coggill P, Eberhardt R Y, Eddy S R, Sonnhammer E L . Pfam: the protein families database. Nucl Acids Res, 2013,42:D222-D230.
[9] Letunic I, Doerks T, Bork P . SMART: recent updates, new developments and status in 2015. Nucl Acids Res, 2014,43:D257-D260.
[10] Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel R D, Bairoch A . ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucl Acids Res, 2003,31:3784-3788.
doi: 10.1093/nar/gkg563
[11] Bailey T, Lelka N . Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol, 1994,4:28-36.
[12] Aiyar A . The use of CLUSTAL W and CLUSTAL X for multiple sequence alignment. Methods Mol Biol, 2000,132:221-241.
[13] Kumar S, Nei M, Dudley J, Tamura K . MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform, 2008,9:299-306.
doi: 10.1093/bib/bbn017
[14] Krzywinski M I, Schein J E, Birol I, Connors J, Gascoyne R, Horsman D, Marra M A . Circos: an information aesthetic for comparative genomics. Genome Res, 2009,19:1639-1645.
doi: 10.1101/gr.092759.109
[15] Combet C, Blanchet C, Geourjon C, Deleage G . NPS@: network protein sequence analysis. Trends Biochem Sci, 2000,25:147-150.
doi: 10.1016/S0968-0004(99)01540-6
[16] Jones D T . Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol, 1999,292:195-202.
doi: 10.1006/jmbi.1999.3091
[17] Buchan D W, Minneci F, Nugent T C, Bryson K, Jones D T . Scalable web services for the PSIPRED Protein Analysis Workbench. Nucl Acids Res, 2013,41:W349-W357.
doi: 10.1093/nar/gkt381
[18] Mezulis S, Sternberg M J, Kelley L A . PhyreStorm: A web server for fast structural searches against the PDB. J Mol Biol, 2016,428:702-708.
doi: 10.1016/j.jmb.2015.10.017
[19] Hoagland D R, Arnon D I . The water culture method for growing plants without soil. Calif Agric Exp Stn Circ, 1950,347:4-32.
[20] Morin R D, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh T J, Marra M A . Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques, 2008,45:81-94.
doi: 10.2144/000112900
[21] Hua Y, Feng Y, Zhou T, Xu F . Genome-scale mRNA transcriptomic insights into the responses of oilseed rape (Brassica napus L.) to varying boron availabilities. Plant Soil, 2017,416:205-225.
[22] Eisen M B, Spellman P T, Brown P O, Botstein D . Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA, 1998,95:14863-14868.
doi: 10.1073/pnas.95.25.14863
[23] Kohl M, Wiese S, Warscheid B . Cytoscape: software for visualization and analysis of biological networks. Methods Mol Biol, 2011,696:291-303.
doi: 10.1007/978-1-60761-987-1
[24] Nekrutenko A, Makova K D, Li W H . The KA/KS ratio test for assessing the protein-coding potential of genomic regions: an empirical and simulation study. Genome Res, 2002,12:198-202.
[25] Segami S, Nakanishi Y, Sato M H, Maeshima M . Quantification, organ-specific accumulation and intracellular localization of type II H+-pyrophosphatase in Arabidopsis thaliana. Plant Cell Physiol, 2010,51:1350-1360.
doi: 10.1093/pcp/pcq096
[26] Li J, Yang H, Peer W A, Richter G, Blakeslee J, Bandyopadhyay A, Krizek B . Arabidopsis H+-PPase AVP1 regulates auxin- mediated organ development . Science, 2005,310:121-125.
doi: 10.1126/science.1115711
[27] Gaxiola R A, Palmgren M G, Schumacher K . Plant proton pumps. Febs Lett, 2007,581:2204-2214.
doi: 10.1016/j.febslet.2007.03.050
[28] Beyenbach K W, Wieczorek H . The V-type H+ ATPase: molecular structure and function, physiological roles and regulation . J Exp Biol, 2006,209:577-589.
doi: 10.1242/jeb.02014
[29] Narasimhan M L, Binzel M L, Perez-Prat E, Chen Z, Nelson D E, Singh N K, Hasegawa P M . NaCl regulation of tonoplast ATPase 70-kilodalton subunit mRNA in tobacco cells. Plant Physiol, 1991,97:562-568.
doi: 10.1104/pp.97.2.562
[30] Binzel M L, Dunlap J R . Abscisic acid does not mediate NaCl-induced accumulation of 70-kDa subunit tonoplast H+-ATPase message in tomato . Planta, 1995,197:563-568.
[31] Yamashita K, Kasai M, Ezaki B, Shibasaka M, Yamamoto Y, Matsumoto H, Sasakawa H . Stimulation of H+ extrusion and plasma membrane H+-ATPase activity of barley roots by ammonium-treatment . Soil Sci Plant Nutr, 1995,41:133-140.
doi: 10.1080/00380768.1995.10419566
[32] 周金泉, 张明超, 魏志军, 胡军, 朱毅勇 . 高粱分泌硝化抑制物对羟基苯丙酸与质子泵的关系研究. 土壤学报, 2015,52:620-627.
Zhou J Q, Zhang M C, Wei Z J, Hu J, Zhu Y Y . Relationship between exudation of nitrification inhibitor MHPP and plasma membrane proton pump of sorghum root. J Soil, 2015,52:620-627 (in Chinese with English abstract).
[33] De Angeli A, Monachello D, Ephritikhine G, Frachisse J M, Thomine S, Gambale F, Barbier-Brygoo H . The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature, 2006,442:939-942.
[34] 黄海涛, 张振华, 宋海星, 刘强, 荣湘民, 彭建伟, 官春云 . 基于液泡膜质子泵的硝态氮再利用研究进展. 生态学杂志, 2012,31:731-737.
Huang H T, Zhang Z H, Song H X, Liu Q, Rong X M, Peng J W, Guan C Y . Nitrate nitrogen reutilization based on proton pump of vacuole membrane: a review. J Ecol, 2012,31:731-737 (in Chinese with English abstract).
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