Copine蛋白是一类包含2个C2（N端）和1个vWA（C端）保守域的Ca2+依赖蛋白或磷脂结合蛋白,在胞内信号转导中发挥重要作用。基于序列相似性分析,从茶树转录组数据库中筛选出1条与Copine家族基因高度同源的EST序列。经测序验证该序列包含1β746βbp的完整ORF,编码581个氨基酸。同源比对显示该基因与拟南芥AtBON3序列相似度最高（65%）,将其命名为CsBON3（GenBank登录号为KY435900）。生物信息学分析显示,CsBON3蛋白分子量为63.66βkD,理论等电点为5.48;具有Copine家族蛋白特有的保守结构域;属亲水性蛋白,无信号肽位点,非分泌性蛋白,无跨膜结构域。表达分析表明,CsBON3在茶树花和根系中表达量最高,茎干和成熟叶中表达量最低。低温（4℃）处理茶树1βd后,其表达被显著上调;在生长阶段,该基因表达量高于休眠阶段;同时在接种炭疽菌的茶树叶片中,该基因也被快速上调,表明该基因可能与茶树低温、生长发育及抗病相关。
Abstract
Copine protein, a kind of Ca2+-dependent protein or phospholipid-binding protein containing two C2 (N-terminal) and one vWA (C-terminal) conserved domain, plays an important role in intracellular signal transduction. Based on the sequence identity analysis, an EST sequence showing high homology to Copine family genes was selected from our previous tea plant transcriptome database. The sequence was verified to contain 1β746βbp complete ORF, encoding 581 amino acids. Homologous alignment showed that the gene had the highest similarity with Arabidopsis AtBON3 (65%) and was named as CsBON3 (GenBank accession number: KY435900). Bioinformatic analysis showed that the molecular weight of CsBON3 protein was 63.66βkD and the theoretical isoelectric point was 5.48. It possesses the conserved domain unique to Copine family protein, which is a hydrophilic protein, no signal peptide site, non-secretory protein and no transmembrane domain. Expression analysis showed that CsBON3 had the highest expression levels in tea flowers and roots, and the lowest in stems and mature leaves. It was significantly up-regulated in tea plants treated with low temperature (4℃) for one day, and its expression was suppressed at dormant stage and up-regulated at growth stage. It was also up-regulated rapidly in the leaves with inoculation of Colletotrichum fructicola. The results indicated that the CsBON3 plays important roles in tea plant growth in response to low temperature and disease resistance.
关键词
BON基因 /
Copine家族 /
表达分析 /
茶树 /
逆境胁迫响应 /
休眠
Key words
BON gene /
Copine family /
dormancy /
expression analysis /
stress response /
tea plant
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参考文献
[1] Creutz C E, Tomsig J L, Snyder S L, et a1. The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans[J]. Journal of Biological Chemistry, 1998, 273(3): 1393-1402.
[2] Rizo J, Südhof T C.C2-domains, structure and function of a universal Ca2+-binding domain[J]. Journal of Biological Chemistry, 1998, 273(26): 15879-15882.
[3] Nalefski E A, Falke J J.The C2 domain calciumbinding motif: structural and functional diversity[J]. Protein Science, 1996, 5(12): 2375-2390.
[4] Tomsig J L, Creutz C E.Copines: a ubiquitous family of Ca2+-dependent phospholipid-binding proteins[J]. Cellular and Molecular Life Sciences, 2002, 59(9): 1467-1477.
[5] Tomsig J L, Snyder S L, Creutz C E.Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif[J]. Journal of Biological Chemistry, 2003, 278(12): 10048-10054.
[6] Williams S C, Hinshelwood J, Perkins S J, et al.Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B[J]. Biochemical Journal, 1999, 342(3): 625-632.
[7] Zou B, Hong X, Ding Y, et al.Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant specie[J]. Genome, 2016, 59(8): 565-573.
[8] Yang S,Yang H, Grisafi P, et al.The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis[J]. Plant Journal, 2006, 45(2): 166-179.
[9] Wang X, Zhao Q, Ma C, et al.Global transcriptome profiles of Camellia sinensis during cold acclimation[J]. BMC Genomics, 2013, 14(1): 415.
[10] 岳川, 曹红利, 周艳华, 等. 茶树谷胱甘肽还原酶基因CsGRs的克隆与表达分析[J]. 中国农业科学, 2014, 47(16): 3277-3289.
[11] 曹红利, 岳川, 郝心愿, 等. 茶树胆碱单加氧酶CsCMO的克隆及甜菜碱合成关键基因的表达分析[J]. 中国农业科学, 2013, 46(15): 3087-3096.
[12] Hao X, Horvath D, Chao W, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[13] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[J]. Methods, 2001, 25(4): 402-408.
[14] Tamura K, Peterson D, Peterson N, et al.MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.
[15] Gastegier E, Hoogland C, Gattuker A, et al.The proteomics protocols handbook[M]. Biochemistry, 2005: 571-607.
[16] Petersen T N, Brunak S, von Heijne G, et al. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nature Methods, 2011, 8(10): 785-786.
[17] Zhou X, Lu P, Wulf G, et al.Phosphorylation-dependent prolyl isomerization: a novel signaling regulatory mechanism[J]. Cellular and Molecular Life Sciences , 1999, 56(9/10): 788-806.
[18] Emanuelsson O, Nielsen H, Brunak S, et al.Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[J]. Journal of Molecular Biology, 2000, 300(4): 1005-1016.
[19] Hardie G, Hanks S.The protein kinase factsbook[M]. London: Academic, 1995: 402-404
[20] Thompson G A, Okuyama H.Lipid-linked proteins of plants[J]. Progress in Lipid Research, 2000, 39(1): 19-39.
[21] Li Y, Gou M, Sun Q, et al.Requirement of calcium binding, myristoylation, and protein-protein interaction for the Copine BON1 function in Arabidopsis[J]. Journal of Biological Chemistry, 2010, 285(39): 29884-29891.
[22] Hua J, Grisafi P, Cheng S H, et al.Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes[J]. Genes Development, 2001,17(15): 2263-2272.
[23] Yang S, Hua J.A haplotype-specific resistance gene regulated by BONZAI1 mediates temperature-dependent growth control in Arabidopsis[J]. Plant Cell, 2004, 16(4): 1060-1071.
基金
国家自然科学基金（31370690）、国家茶叶产业技术体系（CARS-19）资助
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