辣椒PEBP基因家族的全基因组鉴定、比较进化与组织表达分析

doi:10.16420/j.issn.0513-353x.2020-0800

摘要/Abstract

摘要：

植物磷脂酰乙醇胺结合蛋白(Phosphatidyl ethanolamine-binding protein,PEBP)分为MFT、FT和TFL1共3个亚家族,其中FT和TFL1亚家族蛋白构成了植物的成花激素—抗成花激素系统,调控开花时间和株形结构。基于辣椒(Capsicum annuum)全基因组数据,对辣椒PEBP基因家族进行鉴定,并对基因和蛋白结构、比较进化、共线性和组织表达特征等进行了分析。结果表明,辣椒基因组包含9个PEBP成员,并且9个基因均含有4个外显子和3个内含子,定位在6条染色体上,其编码蛋白质定位在细胞质和细胞核中。与番茄PEBP同源基因的比较进化分析表明,辣椒的PEBP基因受纯化选择,其中CaFT2和CaFT4在进化中较稳定,辣椒和番茄之间存在8对共线性基因。qRT-PCR分析表明CaPEBP基因在所检测的组织中明显差异表达,如CaMFT/CaTFL1-1在果实中高表达,CaFT1在叶片中相对表达量最高,CaFT3在顶端分生组织和花中表达量最高,CaTFL1-4在根中特异表达。

关键词: 辣椒, 成花素, PEBP基因家族, 全基因组鉴定, 开花调控, 基因表达

Abstract:

The plant phosphatidyl ethanolamine-binding protein(PEBP)is divided into three subfamilies:MFT-like,FT-like,and TFL1-like. The FT and TFL1 subfamilies comprise the florigen-antiflorigen system of plants that regulate flowering time and plant architecture. Here,the PEBP gene family was identified by genome-wide identification of the whole genome data of the annual pepper (Capsicum annuum),and the structures of gene and protein,comparative evolution,collinearity and tissue expression profile were analyzed. The results showed that the C. annuum genome contains nine PEBP genes,including one MFT,four FT,and four TFL1 genes. All the nine PEBPgenes contain four exons and three introns,which are located on six chromosomes. Moreover,these nine PEBP proteins are located in the cytoplasm and nucleus. Comparative evolution analysis between C. annuum and Solanum lycopersicum showed that CaPEBP genes were subjected to purification selection during evolution. Among them,CaFT2 and CaFT4 were relatively stable during evolution;there were eight pairs of collinearity genes between pepper and tomato. The qRT-PCR expression profile ofCaPEBP genes were significantly differentially expressed in the tissues tested. For example,CaMFT/CaTFL1-1 was highly expressed in fruits,and CaFT1 had the highest relative expression level in leaves;the expression of CaFT3 was the highest in shoot apical meristem and flower,while CaTFL1-4 was specifically expressed in roots.

Key words: Capsicum annuum, florigen, PEBP gene family, genome-wide analysis, flowering regulation, gene expression

中图分类号:

S641.3

牛西强, 罗潇云, 康凯程, 黄先忠, 胡能兵, 隋益虎, 艾昊. 辣椒PEBP基因家族的全基因组鉴定、比较进化与组织表达分析[J]. 园艺学报, 2021, 48(5): 947-959.

NIU Xiqaing, LUO Xiaoyun, KANG Kaicheng, HUANG Xianzhong, HU Nengbing, SUI Yihu, AI Hao. Genome-wide Identification,Comparative Evolution and Expression Analysis of PEBP Gene Family from Capsicum annuum[J]. Acta Horticulturae Sinica, 2021, 48(5): 947-959.

图/表 10

表1 辣椒PEBP基因家族基因表达分析的实时荧光定量PCR引物

Table 1 qRT-PCR primers for expression analysis of PEBP in Capsicum annuum

基因 Gene	上游引物序列(5′-3′) Forward primer sequence	下游引物序列(5′-3′) Reverse primer sequence
CaMFT	GCTCCAAGTCCAAGTGAACC	CCGAGATCAAGATGCTGTGC
CaFT1	CAATGCGCCAGACATAATTG	AGACGACGACCACCAGTACC
CaFT2	GGTTCATATTGGAGGCAACG	TGCTGGGATATCTGTGACCA
CaFT3	TGGTACTGATTTGAGGCCTTCT	CTCAAATTTGGGTTGCTAGGAC
CaFT4	TGGTACAAATTTGAGGCCTTCT	GCATTTCCAAAGGTTACTCCTG
CaTFL1-1	ACCTCCACTGGATTGTGACAG	CATTCTCTGCAGCAAACCTTC
CaTFL1-2	TAGAGGGGCTTGTGAACCAC	TCTTCACCCCCAATCTCAAC
CaTFL1-3	ACGGACATTCCAGGCACTAC	CAGAATCGATCCCTGGAGAG
CaTFL1-4	AGATCCTGATGTTCCTGGCC	CTGAACTCCGACGTTTCTGC
CaUBI3	TGTCCATCTGCTCTCTGTTG	CACCCCAAGCACAATAAGAC

表2 辣椒(Capsicum annuum)PEBP基因家族信息

Table 2 The information of PEBP gene family in Capsicum annuum

基因 Gene	基因登录号 GenBank ID	染色体 Chr. No.	开放阅读框长度/bp ORF	氨基酸数/aa Amino acid	分子量/kD Molecular weight	等电点 pI	亚细胞定位 Subcellular location
CaMFT	PHT88651.1	Chr03	522	173	18.98	9.20	细胞质和细胞核 Cytoplasmic and nuclear
CaFT1	PHT82416.1	Chr05	531	176	19.69	6.82	细胞质和细胞核 Cytoplasmic and nuclear
CaFT2	PHT82541.1	Chr05	528	175	19.81	7.76	细胞质和细胞核 Cytoplasmic and nuclear
CaFT3	PHT65068.1	Chr12	534	177	19.98	8.64	细胞质和细胞核 Cytoplasmic and nuclear
CaFT4	PHT61065.1	Sca. 2174	531	176	19.71	6.73	细胞质和细胞核 Cytoplasmic and nuclear
CaTFL1-1	PHT94378.1	Chr01	534	177	19.92	9.01	细胞质和细胞核 Cytoplasmic and nuclear
CaTFL1-2	PHT87214.1	Chr03	528	175	19.54	9.07	细胞质和细胞核 Cytoplasmic and nuclear
CaTFL1-3	PHT80015.1	Chr06	528	175	19.93	8.64	细胞质和细胞核 Cytoplasmic and nuclear
CaTFL1-4	PHT71587.1	Chr09	519	172	19.55	7.97	细胞质和细胞核 Cytoplasmic and nuclear

图1 拟南芥、番茄和辣椒PEBP氨基酸多重序列比对三角形表示决定FT-like和TFL1-like功能的关键氨基酸,方框表示[FYL]-x-[LVM]-[LIVF]-x-[TIVM]-[DC]-P-D-x-P-[SNG]-x(10)-H和G-X-H-R保守基序。At:拟南芥;Sl:番茄;Ca:辣椒。

Fig. 1 Multiple amino acid sequence alignment of PEBP proteins from Arabidopsis thaliana,Solanum lycopersicum and Capsicum annuum The red triangles represent the key amino acids that determine the functions of FT-like and TFL1-like,the rectangles represent [FYL]-x-[LVM]-[LIVF]-X-[TIVM]-[DC]-P-d-x-P-[SNG]-x(10)-H and G-X-H-R conserved motif. At:Arabidopsis thaliana;Sl:Solanum lycopersicum;Ca:Capsicum annuum.

图2 CaPEBP进化树分析、辣椒PEBP基因外显子-内含子结构和motif分析分支上的数值表示自展值;标尺表示分支长度。

Fig. 2 Phylogentic tree of CaPEBP,gene intron/exon structure and conserved motif analysis of the PEBP genes in Capsicum annuum The number in the tree represents boots value. The scale bar represents branch length.

表3 辣椒PEBP蛋白氨基酸保守序列

Table 3 Conserved motifs of the PEBP proteins in Capsicum annuum

基序 Motif	长度/aa Length	氨基酸保守序列 Amino acid conserved sequence
motif 1	29	DPLVVGRVIGDVVDPFTPSVKMSVVYNNR
motif 2	31	YNGHELRPSQIAAQPRVEIGGNDLRTFYTLI
motif 3	50	MTDPDAPSPSBPYLREYLHWJVTDIPGTTDVSFGNEIVCYESPRPSIGIH
motif 4	11	RYVFVLFRQLG
motif 5	41	VYAPTWRDHFNTRDFAEENNLGSPVAAVYFNCQRETATRRR

表4 辣椒和番茄PEBP同源基因的Ka/Ks

Table 4 Ka/Ks values of PEBP genes between Capsicum annuum and Solanum lycopersicum

辣椒基因 Gene ID in C. annuum	番茄基因 Gene ID in S. lycopersicum	基因模型 Gene model	非同义突变频/Ka	同义突变频率/Ks	Ka/Ks
CaMFT	SlMFT	MFT-like	0.07	0.44	0.17
CaFT1	SlSP5G	FT-like	0.03	0.39	0.09
CaFT2	SlSP6A	FT-like	0.06	0.24	0.27
CaFT3	SlSP5G3	FT-like	0.03	0.29	0.11
CaFT4	SlSP5G1	FT-like	0.18	0.48	0.37
CaTFL1-1	SlCEN1.3	TFL1-like	0.06	0.56	0.11
CaTFL1-2	SlCEN1.1	TFL1-like	0.04	0.57	0.08
CaTFL1-3	SlSP	TFL1-like	0.03	0.22	0.15
CaTFL1-3	SlCEN1.3	TFL1-like	0.26	1.16	0.22
CaTFL1-4	SlSP9D	TFL1-like	0.05	0.36	0.13

图3 番茄和辣椒的PEBP基因的共线性分析红色线条表示种间PEBP共线性关系;灰色线条代表种间所有基因成员的共线性关系。

Fig. 3 Collinearity analysis of orthologous PEBP gene pairs betweenSolanum lycopersicum andCapsicum annuum The red lines representPEBP collinearity between species,and the gray lines are collinearity of all gene members between species.

图4 辣椒根、茎、叶、顶端分生组织(SAM)、花和果(果实发育的第6时期)中PEBP基因表达的qRT-PCR分析

Fig. 4 Expression analysis of CaPEBP genes by qRT-PCR in roots,stems,leaves,shoot apical meristems(SAM),flowers and fruit(6th stage of fruit development)of pepper

图5 辣椒果实发育的6个时期

Fig. 5 Six developmental stages of fruits in Capsicum annuum

图6 辣椒果实发育(果1 ~ 果6分别表示果实发育的不同阶段)中4个CaPEBP基因的表达特征

Fig. 6 Expression profiles of four CaPEBPgenes at different developmental stages(Fruit 1-6)in fruit of Capsicum annuum

参考文献 35

[1]	Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T. 2005. FD,a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science, 309(5737):1052-1056. doi: 10.1126/science.1115983 URL
[2]	Ahn J H, Miller D, Winter V J, Banfield M J, Lee J H, Yoo S Y, Henz S R, Brady R L, Weigel D. 2006. A divergent external loop confers antagonistic activity on floral regulators FT and TFL1. The EMBO Journal, 25(3):605-614. doi: 10.1038/sj.emboj.7600950 URL
[3]	Bernier I, Jolles P. 1984. Purification and characterisation of a basic 23 kDa cytosolic protein from bovine brain. Biochimica et Biophysica Acta, 790(2):174-181.
[4]	Cao K, Cui L, Zhou X, Ye L, Zou Z, Deng S. 2016. Four tomato FLOWERING LOCUS T-like proteins act antagonistically to regulate floral initiation. Frontiers in Plant Science, 6:1213.
[5]	Carmel-Goren L, Liu Y S, Lifschitz E, Zamir D. 2003. The SELF-PRUNING gene family in tomato. Plant Molecular Biology, 52:1215-1222. doi: 10.1023/B:PLAN.0000004333.96451.11 URL
[6]	Carmona M J, Calonje M, Martínez-Zapater J M. 2007. The FT/TFL1 gene family in grapevine. Plant Molecular Biology, 63(5):637-650. doi: 10.1007/s11103-006-9113-z URL
[7]	Chardon F, Damerval C. 2005. Phylogenomic analysis of the PEBP gene family in cereals. Journal of Molecular Evolution, 61(5):579-590. doi: 10.1007/s00239-004-0179-4 URL
[8]	Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools:an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13(8):1194-1202. doi: 10.1016/j.molp.2020.06.009 URL
[9]	Elitzur T, Nahum H, Borovsky Y, Pekker I, Eshed Y, Paran I. 2009. Co-ordinated regulation of flowering time,plant architecture and growth by FASCICULATE:the pepper orthologue of SELF PRUNING. Journal of Experimental Botany, 60(3):869-880. doi: 10.1093/jxb/ern334 URL
[10]	Eshed Y, Lippman Z B. 2019. Revolutions in agriculture chart a course for targeted breeding of old and new crops. Science, 366(6466):eaax0025. doi: 10.1126/science.aax0025 URL
[11]	Hanzawa Y, Money T, Bradley D. 2005. A single amino acid converts a repressor to an activator of flowering. Proceedings of the National Academy of Science of the United States of America, 102(21):7748-7753.
[12]	Hedman H, Källman T, Lagercrantz U. 2009. Early evolution of the MFT-like gene family in plants. Plant Molecular Biology, 70(4):359-369. doi: 10.1007/s11103-009-9478-x URL
[13]	Ibiza V P, Blanca J, Cañizares J, Nuez F. 2012. Taxonomy and genetic diversity of domesticated Capsicum species in the Andean region. Genetic Resources and Crop Evolution, 59(6):1077-1088. doi: 10.1007/s10722-011-9744-z URL
[14]	Jaeger K E, Wigge P A. 2007. FT protein acts as a long-range signal in Arabidopsis. Current Biology, 17(12):1050-1054. pmid: 17540569
[15]	Kaneko-Suzuki M, Kurihara-Ishikawa R, Okushita-Terakawa C, Kojima C, Nagano-Fujiwara M, Ohki I, Tsuji H, Shimamoto K, Taoka K I. 2018. TFL1-Like proteins in rice antagonize rice FT-Like protein in inflorescence development by competition for complex formation with 14-3-3 and FD. Plant & Cell Physiology, 59(3):458-468.
[16]	Karlgren A, Gyllenstrand N, Källman T, Sundström J F, Moore D, Lascoux M, Lagercrantz U. 2011. Evolution of the PEBP gene family in plants:functional diversification in seed plant evolution. Plant Physiology, 156(4):1967-1977. doi: 10.1104/pp.111.176206 URL
[17]	Kim S, Park J, Yeom S I, Kim Y M, Seo E, Kim K T, Kim M S, Lee J M, Cheong K, Shin H S, Kim S B, Han K, Lee J, Park M, Lee H A, Lee H Y, Lee Y, Oh S, Lee J H, Choi E, Choi E, Lee S E, Jeon J, Kim H, Choi G, Song H, Lee J, Lee S C, Kwon J K, Lee H Y, Koo N, Hong Y, Kim R W, Kang W H, Huh J H, Kang B C, Yang T J, Lee Y H, Bennetzen J L, Choi D. 2017. New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication. Genome Biology, 18(1):210. doi: 10.1186/s13059-017-1341-9 URL
[18]	Kim S, Park M, Yeom S I, Kim Y M, Lee J M, Lee H A, Seo E, Choi J, Cheong K, Kim K T, Jung K, Lee G W, Oh H K, Bae S Y, Kim S Y, Jo Y D, Yang H B, Kang W H, Yu Y, Bark B S, Kim R Y, Choi I K, Choi B S, Lim J S, Less Y H, Choi D. 2014. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nature Genetics, 46(3):270-279. doi: 10.1038/ng.2877 URL
[19]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7):1870-1874. doi: 10.1093/molbev/msw054 URL
[20]	Li Chao, Zhang Yan-nan, Liu Huan-long, Huang Xian-zhong. 2015. Identification of PEBP gene family in Gossypium arboreum and Gossypium raimondii and expression analysis of the gene family in Gossypium hirsutum . Acta Agronomica Sinica, 41(3):394-404. (in Chinese) doi: 10.3724/SP.J.1006.2015.00394 URL
	李超, 张彦楠, 刘焕龙, 黄先忠. 2015. 亚洲棉和雷蒙德氏棉PEBP家族基因的鉴定及该家族基因在陆地棉组织中表达分析. 作物学报, 41(3):394-404.
[21]	Li Q, Fan C, Zhang X, Wang X, Wu F, Hu R, Fu Y. 2014. Identification of a soybean MOTHER OF FT AND TFL1 homolog involved in regulation of seed germination. PLoS ONE, 9(6):e99642. doi: 10.1371/journal.pone.0099642 URL
[22]	Lifschitz E, Eshed Y. 2006. Universal florigenic signals triggered by FT homologues regulate growth and flowering cycles in perennial day-neutral tomato. Journal of Experimental Botany, 57(13):3405-3414. doi: 10.1093/jxb/erl106 URL
[23]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25(4):402-408. pmid: 11846609
[24]	Molinero-Rosales N, Latorre A, Jamilena M, Lozano R. 2004. SINGLE FLOWER TRUSS regulates the transition and maintenance of flowering in tomato. Planta, 218(3):427-434. pmid: 14504922
[25]	Nakamura S, Abe F, Kawahigashi H, Nakazono K, Tagiri A, Matsumoto T, Utsugi S, Ogawa T, Handa H, Ishida H, Mori M, Kawaura K, Ogihara Y, Miura H. 2011. A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination. The Plant Cell, 23(9):3215-3229. doi: 10.1105/tpc.111.088492 pmid: 21896881
[26]	Navarro C, Abelenda J A, Cruz-Oró E, Cuéllar C A, Tamaki S, Silva J, Shimamoto k, Prat S. 2011. Control of flowering and storage organ formation in potato by FLOWERING LOCUS T. Nature, 478(7367):119-122. doi: 10.1038/nature10431 URL
[27]	Qi Shiming, Liang Yan. 2020. Advances in research of the SELF-PRUNING gene family and plant architecture regulatory functions in tomato . Acta Horticulturae Sinica, 47(9):1705-1714. (in Chinese)
	祁世明, 梁燕. 2020. 番茄SELF-PRUNING基因家族及株形调控功能研究进展. 园艺学报, 47(9):1705-1714.
[28]	Qin C, Yu C, Shen Y, Fang X, Chen L, Min J, Cheng J, Zhao S, Xu M, Luo Y, Yang Y, Wu H, Wu Z, Mao L, Zhou H, Lin H, Tang X, Zhao M, Huang Z, Zhou A, Yao X, Cui J, Li W, Chen Z, Feng Y, Niu Y, Yang X, Li W, Cai H. 2014. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proceedings of the National Academy of Science of the United States of America, 111(14):5135-5140.
[29]	Rozas J, Ferrer-Mata A, Sánchez-DelBarrio J C, Guirao-Rico S, Librado P, Ramos-Onsins S E, Sánchez-Gracia A. 2017. DnaSP 6:DNA sequence polymorphism analysis of large datasets. Molecular Biology and Evolution, 34(12):3299-3302. doi: 10.1093/molbev/msx248 URL
[30]	Wan H, Yuan W, Ruan M, Ye Q, Wang R, Li Z, Zhou G, Yao Z, Zhao J, Liu S, Yang Y. 2011. Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper(Capsicum annuumL.). Biochemical and Biophysical Research Communications, 416(1-2):24-30. doi: 10.1016/j.bbrc.2011.10.105 URL
[31]	Wang Y, Tang H, DeBarry J D, Tan X, Li J, Wang X, Lee T H, Jin H, Marler B, Guo H, Kissinger J C, Paterson A H. 2012. MCScanX:a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research, 40(7):49.
[32]	Wang Y D, Chen G J, Lei J J, Cao B H, Chen C M. 2020. Identification and characterization of a LEA-like gene,CaMF5,specifically expressed in the anthers of male-fertileCapsicum annuum. Horticultural Plant Journal, 6(1):39-48. doi: 10.1016/j.hpj.2019.07.004 URL
[33]	Wickland D P, Hanzawa Y. 2015. TheFLOWERING LOCUS T/TERMINAL FLOWER 1 gene family:functional evolution and molecular mechanisms. Molecular Plant, 8(7):983-997. doi: 10.1016/j.molp.2015.01.007 pmid: 25598141
[34]	Xi W, Liu C, Hou X, Yu H. 2010. MOTHER OF FT AND TFL1 regulates seed germination through a negative feedback loop modulating ABA signaling inArabidopsis. The Plant Cell, 22(6):1733-1748. doi: 10.1105/tpc.109.073072 URL
[35]	Yadav C B, Bonthala V S, Muthamilarasan M, Pandey G, Khan Y, Prasad M. 2015. Genome-wide development of transposable elements-based markers in foxtail millet and construction of an integrated database. DNA Research, 22(1):79-90. doi: 10.1093/dnares/dsu039 URL