小G蛋白是一类重要的信号转导蛋白,参与植物的各项生命活动,但目前在茶树中的研究尚少。本研究以茶树品种龙井长叶为实验材料,克隆获得1个小G蛋白基因,命名为CsRAC5。序列分析显示,CsRAC5含有597βbp,编码198个氨基酸,具有Rho蛋白的保守结构域;序列多重比对发现,该序列与其他物种序列的一致性高达95.96%。CsRAC5蛋白质的相对分子质量为21.79βkDa,为亲水性蛋白;烟草瞬时表达实验显示,CsRAC5定位于细胞膜和细胞核中。荧光定量PCR结果显示,CsRAC5在茶树中的表达具有明显的组织特异性,其中在叶片中的表达最高,在花粉中的表达最低;低温（4℃）胁迫抑制茶树叶片中CsRAC5的表达。
Abstract
Small GTPase binding proteins are a kind of important signal transduction proteins, which are involved in various life activities of plants. However, few relative studies were reported in tea plants (Camellia sinensis). Here, a small GTPase binding protein named CsRAC5 was cloned by using a cDNA template from tea cultivar ‘Longjingchangye’. The results showed that the length of its open reading frame (ORF) is 597βbp, encoding 198 amino acids. It has a conserved Rho domain which belongs to ROP family. Multiple alignment of CsRAC5 with homologue genes in other plant species showed that their identity could reach 95.96%. CsRAC5 is a hydrophilic protein with the theoretical relative weight of 21.79βkDa. The subcellular assay showed that CsRAC5 was localized in the nuclear and membrane. In addition, the results of RT-PCR analysis showed that the highest expression level of CsRAC5 was in leaves but the lowest in pollen. The expression level of CsRAC5 was decreased under cold stress.
关键词
CsRAC5 /
表达分析 /
茶树 /
低温胁迫 /
生物信息学分析
Key words
CsRAC5 /
bioinformatics analysis /
Camellia sinensis /
cold stress /
expression analysis
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参考文献
[1] Bischoff F, Molendijk A, Rajendrakumar C S V, et al. GTP-binding proteins in plants[J]. Cellular and molecular life sciences: CMLS, 1999, 55(2): 233-256.
[2] Xu T, Wen M, Nagawa S, et al.Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[J]. Cell, 2010, 143(1): 99-110.
[3] 汪云刚, 矣兵, 冉隆珣, 等. 云南茶树种质资源的抗性鉴定和评价[J]. 中国农学通报, 2011, 27(13): 86-91.
[4] 杨新国. 茶树冻害发生原因及防御补救措施[J]. 现代农业科技, 2011(21): 198-198.
[5] Ma Y, Dai X, Xu Y, et al.COLD1 Confers chilling tolerance in rice[J]. Cell, 2015, 160(6): 1209-1221.
[6] Xiong L, Ishitani M, Lee H, et al.The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression[J]. The Plant cell, 2001, 13(9): 2063-2083.
[7] Lee B H, Lee H, Xiong L, et al.A mitochondrial complex I defect impairs cold-regulated nuclear gene expression[J]. Plant Cell, 2002, 14(6): 1235-1251.
[8] Bloch D, Lavy M, Efrat Y, et al.Ectopic expression of an activated RAC in Arabidopsis disrupts membrane cycling[J]. Molecular Biology of the Cell, 2005, 16(4): 1913-1927.
[9] Lin D, Ren H, Fu Y.ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana[J]. Chinese Journal of Plant Ecology, 2015, 57(1): 31-39.
[10] Huang J B, Liu H, Chen M, et al.ROP3 GTPase contributes to polar auxin transport and auxin responses and is important for embryogenesis and seedling growth in Arabidopsis[J]. Plant Cell, 2014, 26(9): 35013518.
[11] Xin Z, Zhao Y, Zheng ZL.Transcriptome analysis reveals specific modulation of abscisic acid signaling by ROP10 small GTPase in Arabidopsis[J]. Plant physiology, 2005, 139(3): 1350-1365.
[12] Tao L Z, Cheung A Y, Wu H M.Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression[J]. Plant Cell, 2002, 14(11): 2745-2760.
[13] Hong D, Jeon B W, Kim S Y, et al.The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis[J]. New Phytologist, 2016, 209(2): 624-635.
[14] Baxterburrell A, Yang Z, Springer P S, et al.RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance[J]. Science, 2002, 296(5575): 2026-2028.
[15] Berken A.ROPs in the spotlight of plant signal transduction[J]. Cellular & Molecular Life Sciences Cmls, 2006, 63(21): 2446-2459.
[16] Huang S, Robinson R C, Gao L Y, et al.Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization[J]. Plant Cell, 2005, 17(2): 486-501.
[17] 陈暄, 彭英, 郝姗, 等. 茶树花粉的离体萌发研究[J]. 江苏农业科学, 2010(6): 233-235.
[18] Wang W, Sheng X, Shu Z, et al.Combined cytological and transcriptomic analysis reveals a nitric oxide signaling pathway involved in cold-inhibited Camellia sinensis pollen tube growth[J]. Frontiers in Plant Science, 2016, 7(342): 456. doi: 10.3389/fpls.2016.00456.
[19] Sparkes I A, Runions J, Kearns A, et al.Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J]. Nature Protocols, 2006, 1(4): 2019-2025.
[20] 孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45(5): 579-587.
[21] 史成颖, 李正国, 徐乾, 等. 茶愈伤组织实时定量PCR分析中内参基因的选取[J]. 安徽农业大学学报, 2014, 41(6): 905-910.
[22] Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Research, 2001, 29(9): e45.
[23] Zheng Z L, Yang Z B.The Rop GTPase: an emerging signaling switch in plants[J]. Plant Molecular Biology, 2000, 44(1): 1-9.
[24] Nibau C, Wu H M, Cheung A Y.RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants[J]. Trends in Plant Science, 2006, 11(6): 309-315.
[25] Yang Z.Small GTPases: versatile signaling switches in plants[J]. The Plant cell, 2002, 14(Sl): S375-S388.
[26] Molendijk A J, Bischoff F, Rajendrakumar C S V, et al. Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth[J]. Embo Journal, 2001, 20(11): 2779-2788. DOI: 10.1093/emboj/20.11.2779.
[27] 梁卫红, 李辉, 李佳佳, 等. 非生物胁迫和植物激素对与水稻OsRac5结合的含CC域蛋白编码基因OsMY1和OsMY2表达的影响[J]. 中国生物化学与分子生物学报, 2013, 29(4): 368-376.
[28] Jin W W, Xu C J, Xian L, et al.Expression of ROP/RAC GTPase genes in postharvest loquat fruit in association with senescence and cold regulated lignifications[J]. Postharvest Biology & Technology, 2009, 54(1): 9-14.
基金
国家自然科学基金（31770733,31370014）、连云港市科技计划项目（SF1502）、南京市科技计划项目（201608068）
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