茶树miR156a靶基因SPL6和SPL9的克隆及表达分析

摘要/Abstract

摘要： MicroRNA156a-SQUAMOSA promoter binding-like protein（miR156a-SPLs）参与植物生长阶段转变、叶片形态建成和逆境胁迫等复杂的生理过程。基于茶树转录组数据,从茶树中克隆出2个SPL家族成员——CsSPL6和CsSPL9。CsSPL6 cDNA全长2β318βbp,ORF框长1β668βbp,编码555个氨基酸;CsSPL9 cDNA全长1β954βbp,ORF框长1β116βbp,编码371个氨基酸;CsSPL6和CsSPL9都有典型SBP结构域和Csn-miR156a识别位点。时空表达特性分析表明,Csn-miR156a在芽中表达量最高,第7叶中最低,与CsSPL6和CsSPL9呈负调控关系;不同品种（系）表达特性分析表明,Csn-miR156a的表达模式与新梢生长量、光合指标结果一致,与CsSPL6和CsSPL9表达模式相反,说明Csn-miR156a、CsSPL6和CsSPL9参与茶树生长过程,Csn-miR156a和CsSPLs可作为初步判断品种生长势强弱的分子手段;高温处理4个品种（系）表达特性表明Csn-miR156a与CsSPL9呈负相关,推断茶树在高温胁迫下Csn-miR156a可能通过负调控CsSPL9来增加耐高温性。Csn-miR156a负调控CsSPLs在茶树生长发育、逆境胁迫中发挥作用,为茶树生长和抗逆机制提供理论依据。

关键词: 茶树, 生长势, 高温, miR156a, SPL

Abstract: MicroRNA156a-SQUAMOSA promoter binding-like protein（miR156a-SPLs）play important roles in growth process, formation of leaves and response to stress in tea plant (Camellia sinensis). Based on transcriptome data, two cDNA CsSPL6 (2β318βbp) and CsSPL9 (1β954βbp) were cloned from tea plant. The nucleotide and amino acid sequences, biological information, phylogenetic tree and molecular expression models of the CsSPL6 and CsSPL9 were analyzed. Sequence analysis showed that the open reading frames of CsSPL6 and CsSPL9 are 1β668βbp and 1β116βbp, encoding 555 and 371 amino acids respectively. Both genes contain typical SBP domain and the recognition sites of microRNA156a. Quantitative real-time PCR analysis showed that the expression profile of Csn-miR156a was the highest in the buds, and the lowest in the 7th leaves, which was negatively correlated with CsSPL6 and CsSPL9. For different varieties(strains), the expression of Csn-miR156a was consistent with the growth of new shoots and the value of Photosynthesis index, but negatively correlated with the expression of CsSPL6 and CsSPL9, indicating. Csn-miR156a,CsSPL6 and CsSPL9 were involved in the growth of tea plant and can be used as markers to assess growth abilities among varieties. In addition, the expression of CsSPL9 was negatively correlatedwith Csn-miR156a under heat stress in four varieties (strains), which might be associated with the enhanced heat tolerance ability. The results showed that the expression of CsSPLs were negatively regulated by Csn-miR156a,which played an important role in the growth, development of tea plant and the stress resistance, providing a theoretical basis for understanding the growth and resistance mechanism of tea plant.

Key words: Camellia sinensis, growth potential, high temperature, miR156a, SPL

中图分类号:

TS272.2
Q52

刘亚芹, 田坤红, 孙琪璐, 潘铖, 李叶云, 蒋家月, 江昌俊. 茶树miR156a靶基因SPL6和SPL9的克隆及表达分析[J]. 茶叶科学, 2017, 37(6): 551-564.

LIU Yaqin, TIAN Kunhong, SUN Qilu, PAN Cheng, LI Yeyun, JIANG Jiayue, JIANG Changjun. Cloning and Expression Analysis of miR156a-targeted Genes SPL6 and SPL9 in Camellia sinensis[J]. Journal of Tea Science, 2017, 37(6): 551-564.

参考文献 41

[1]	Klein J, Saedler H, Huijser P.A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA[J]. Mol Gen Genet, 1996, 250(1): 7-16.
[2]	Yu S, Galvao V C, Zhang Y C, et al.Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA promoter binding-like transcription factors[J]. Plant Cell, 2012, 24(8): 3320-3332.
[3]	Wu G, Poethig R S.Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3[J]. Development, 2006, 133(18): 3539-3547.
[4]	Wang J W, Czech B, Weigel D. miR156-Regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[J]. Cell, 2009, 138(4): 738-749.
[5]	Schwarz S, Grande A V, Bujdoso N, et al.The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis[J]. Plant Molecular Biology, 2008, 67(1/2): 183-195.
[6]	Jung J H, Seo P J, Kang S K, et al.miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions[J]. Plant Molecular Biology, 2011, 76(1/2): 35-45.
[7]	Eriksson M, Moseley J L, Tottey S, et al.Genetic dissection of nutritional copper signaling in chlamydomonas distinguishes regulatory and target genes[J]. Genetics, 2004, 168(2): 795-807.
[8]	Yang Z, Wang X, Gu S, et al.Comparative study of SBP-box gene family in Arabidopsis and rice[J]. Gene, 2008, 407(1/2): 1-11.
[9]	Rhoades M W, Reinhart B J, Lim L P.Prediction of plant microrna targets[J]. Cell, 2002, 110(4): 513-520.
[10]	Gandikota M, Birkenbihl R P, Höhmann S, et al.The miRNA156/157 recognition element in the 3' UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings[J]. Plant Journal for Cell & Molecular Biology, 2007, 49(4): 683-693.
[11]	Zhang Y, Schwarz S, Saedler H, et al.SPL8, a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis[J]. Plant Molecular Biology, 2007, 63(3): 429-439.
[12]	Wang J W, Schwab R, Czech B, et al.Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana[J]. Plant Cell, 2008, 20(5): 1231-1243.
[13]	Shikata M, Koyama T, Mitsuda N, et al.Arabidopsis SBP-box genes SPL10, SPL11 and SPL2 control morphological change in association with shoot maturation in the reproductive phase[J]. Plant & Cell Physiology, 2009, 50(12): 2133-2145.
[14]	Cui L, Shan J, Shi M, et al.The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants[J]. Plant Journal for Cell & Molecular Biology, 2014, 80(6): 1108-1117.
[15]	Stief A, Altmann S, Hoffmann K, et al.Arabidopsis miR156 regulates tolerance to recurring environmental stress through spl transcription factors[J]. The Plant Cell, 2014, 26(4): 1792-1807.
[16]	Arshad M, Feyissa B A, Amyot L, et al.MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13[J]. Plant Science, 2017, 258:122-136.
[17]	Wang M, Wang Q, Zhang B.Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.)[J]. Gene, 2013, 530(1): 26-32.
[18]	Lei K J, Lin Y M, An G Y. miR156 modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis[J]. Journal of Plant Research, 2016, 129(2): 275-284.
[19]	宋长年, 钱剑林, 房经贵, 等. 枳SPL9和SPL13全长cDNA克隆、亚细胞定位和表达分析[J]. 中国农业科学, 2010, 43(10): 2105-2114.
[20]	Miura K, Ikeda M, Matsubara A, et al.OsSPL14 promotes panicle branching and higher grain productivity in rice[J]. Nature Genetics, 2010, 42(6): 545-549.
[21]	Li M, Zhao S Z, Zhao C Z, et al.Cloning and characterization of SPL-family genes in the peanut (Arachis hypogaea L.)[J]. Genetics and Molecular Research, 2015, 14(1): 2331-2340.
[22]	Zhang X, Dou L, Pang C, et al.Genomic organization, differential expression, and functional analysis of the SPL gene family in Gossypium hirsutum[J]. Molecular Genetics and Genomics, 2015, 290(1): 115-126.
[23]	赵晓初, 李贺, 代红艳, 等. 草莓miR156靶基因SPL9的克隆与表达分析[J]. 中国农业科学, 2011, 44(12): 2515-2522.
[24]	李磊. 不同肥料处理对茶树生长和茶叶品质的影响[D]. 泰安: 山东农业大学, 2010.
[25]	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.
[26]	Erika V G.Stem-Loop qRT-PCR for the detection of plant microRNAs [M]. Totowa N J: Humana Press, 2016.
[27]	谢小芳. 茶树miRNA靶基因的鉴定及在低温胁迫下的表达分析[D]. 合肥:安徽农业大学, 2016.
[28]	张永福, 任禛, 莫丽玲, 等. 葡萄品种生长势差异的相关机制研究[J]. 北方园艺, 2015 (2): 1-5.
[29]	杨雨华, 宗建伟, 杨风岭. 不同生长势马尾松光合日变化研究[J]. 中南林业科技大学学报(自然科学版), 2014, (8): 25-29.
[30]	Yamasaki K, Kigawa T, Inoue M, et al.A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors[J]. Journal of Molecular Biology, 2004, 337(1): 49-63.
[31]	Birkenbihl R P, Jach G, Saedler H, et al.Functional dissection of the plant-specific sbp-domain: overlap of the dna-binding and nuclear localization domains[J]. Journal of Molecular Biology, 2005, 352(3): 585-596.
[32]	Yang L, Conway S R, Poethig R S.Vegetative phase change is mediated by a leaf-derived signal that represses the transcription of miR156[J]. Development, 2011, 138(2): 245-249.
[33]	Yoshikawa T, Ozawa S, Sentoku N, et al.Change of shoot architecture during juvenile-to-adult phase transition in soybean[J]. Planta, 2013, 238(1): 229-237.
[34]	Gou J Y, Felippes F F, Liu C J, et al.Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor[J]. Plant Cell, 2011, 23(4): 1512-1522.
[35]	Salinas M, Xing S, Höhmann S, et al.Genomic organization, phylogenetic comparison and differential expression of the SBP-box family of transcription factors in tomato[J]. Planta, 2012, 235(6): 1171-1184.
[36]	冯圣军. MicroRNA156调控烟草发育阶段转变的功能研究[D]. 临安: 浙江农林大学, 2014.
[37]	虞莎, 王佳伟. miR156介导的高等植物年龄途径研究进展[J]. 中国科学, 2014, 59(15): 1398-1404.
[38]	Xin M M, Wang Y, Yao Y Y, et al.Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat[J]. BMC Plant Biology, 2010, 10: 123-133.
[39]	Yu X, Wang H, Lu Y Z, et al.Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa[J]. Journal of Experimental Botany, 2012, 63(2): 1025-1038.
[40]	Arshad M, Gruber M Y, Wall K, et al.An insight into microrna156 role in salinity stress responses of alfalfa[J]. Frontiers in Plant Science, 2017, 8(658):356-370.
[41]	Yu Z X, Wang L J, Zhao B, et al.Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and patchouli (Pogostemon cablin) by the miR156-Targeted SPL transcription factors[J]. Molecular Plant, 2014, 8(1): 98-110.