茶树CHS基因结构及编码区单核苷酸多态性分析

doi:10.3864/j.issn.0578-1752.2014.01.014

摘要/Abstract

摘要： 【目的】通过试验获得茶树CHS基因（CsCHS）gDNA序列，以进一步确定CsCHS基因结构；研究CsCHS编码区单核苷酸多态性，并结合茶树多酚含量进行关联分析，寻找基因中可能存在的与茶多酚含量存在显著或极显著相关关系的SNP位点。【方法】根据NCBI数据库中已有CsCHS序列设计特异性引物，再分别以基因组DNA和cDNA为模板进行PCR扩增，经克隆、测序获得CsCHS1、CsCHS2、CsCHS3三个基因的gDNA和cDNA全长序列，通过序列比对方法确定CsCHS结构。利用Compute pI/Mw、SOPMA等软件对所得序列进行生物信息学分析，预测和比较CsCHS1、CsCHS2和CsCHS3三者蛋白质结构。以茶多酚含量差异较大的57份茶树品种为材料，分别以57份材料的cDNA为模板，用特异性引物进行PCR扩增，然后利用PCR产物直接测序法筛查CsCHS编码区序列的单核苷酸多态性。结合CsCHS编码区序列单核苷酸多态性和57份材料多酚含量，利用软件TASSEL进行关联分析，筛选基因中可能与茶多酚含量存在显著或极显著相关关系的SNP位点。【结果】试验获得CsCHS1、CsCHS2和CsCHS3的cDNA序列长度分别为1 277、1 320和1 242 bp，各自均包含一个长度为1 170 bp的开放阅读框；CsCHS1、CsCHS2和CsCHS3的gDNA序列长度分别为1 600、1 330和1 607 bp。通过gDNA序列和cDAN序列比对，结合真核生物内含子GT-AG法则，确定CsCHS1、CsCHS3分别包含2个外显子和1个内含子，内含子大小分别为323和356 bp，CsCHS2可能没有内含子。根据CsCHS1、CsCHS2和CsCHS3 cDNA序列推导三者对应氨基酸序列，比较三条氨基酸序列发现三者氨基酸同源性较高，达到92.6%—95.4%，CHS蛋白亚家族中的特征性保守位点在这三条序列中都能找到，生物信息学分析结果显示CsCHS1、CsCHS2和CsCHS3三者蛋白质结构高度相似。CsCHS1编码区序列中共发现71个SNP位点，SNP出现频率为1SNP/16.48 bp，无Indel，基因核苷酸多样性（π）值为0.01088；CsCHS2编码区序列中共发现55个SNP位点，SNP出现频率分别为1SNP/21.27 bp，CsCHS2核苷酸多样性（π）值（0.00530）明显低于CsCHS1；因扩增CsCHS3 cDNA序列的PCR反应成功率低，未对该基因遗传多样性进行分析。通过关联分析分别从CsCHS1和CsCHS2中找到2个和4个与茶叶多酚含量相关的SNP位点。【结论】CsCHS1和CsCHS3属于保守型CHS基因，且CsCHS1、CsCHS2和CsCHS3蛋白质结构高度相似，推测三者可能在茶树的不同部位或不同生长阶段发挥类似作用；CsCHS1和CsCHS2活跃，二者编码区内可能存在突变热点区。

关键词: 茶 , 查尔酮合酶基因（CHS） , 单核苷酸多态性（SNP）

Abstract: 【Objective】 The objectives of this study were to determine the structures of CHS genes in Camellia sinensis (CsCHS) by obtaining gDNA sequences of these genes, and to analyze single nucleotide polymorphism. Besides, association analysis was also carried out in order to find potential SNP sites in CsCHS which would influence the polyphenol content in tea plant.【Method】 Based on CsCHS sequences uploaded to NCBI, specific primers were designed using primer 3.0 software. Genomic DNA and cDNA of tea leaves were used as templates in polymerase chain reaction (PCR) to obtain gDNA and cDNA sequences of CsCHS1, CsCHS2 and CsCHS3, respectively. Gene structures were determined through blasting gDNA and cDNA sequences. The putative amino acid sequences were analyzed by bioinformatics softwares, such as Compute pI/Mw, SOPMA, and so on. Single nucleotide polymorphism of CsCHS were analyzed in 57 cultivars with great variation polyphenol contents. In order to obtain the coding region of CsCHS genes, PCR reactions with specific primers were carried out by using cDNA of individual tea cultivars as template. TASSEL software was introduced in association analysis.【Result】 cDNA sequences of CsCHS1, CsCHS2 and CsCHS3 were 1 277 bp, 1 320 bp and 1 242 bp, respectively. And an open reading frame (ORF) of 1 170 bp was found in each CsCHS gene. gDNA sequences of CsCHS1, CsCHS2, and CsCHS3 were 1 600 bp, 1 330 bp and 1 607 bp, respectively. By comparing gDNA and cDNA sequences of each CsCHS gene, combined with GT-AG rule, it was determined that CsCHS1 and CsCHS2 have two exons and one intron, respectively. And the intron in CsCHS1 is 323 bp, compared with 356 bp in CsCHS3. While no interruption region was found in CsCHS2, this might prove there is no intron in CsCHS2. Deduced amino acid sequences analysis suggests that identity of amino acid sequences are 92.6%-95.4%. All of the conserved amino acids found in CHS protein subfamily were also found in these deduced sequences. Furthermore, bioinformatics analysis showed highly similarities among CsCHS1, CsCHS2 and CsCHS3 protein structures. There are 71 SNP sites in CsCHS1’s coding region, SNP frequency was 1SNP/16.48 bp, and no Indel was found. A total of 55 SNP sites were found in CsCHS2’s coding region, suggesting one SNP in every 21.27 bp. And the nucleotide diversity (π) in CsCHS1 (0.01088) was significantly higher than that of CsCHS2 (0.00530). By correlation analysis, two SNP sites were positioned thought to be related to polyphenol content in tea plant in CsCHS1 and 4 in CsCHS2. No further analysis referring to CsCHS3 was carried out due to low success rate of PCR reaction.【Conclusion】 Both CsCHS1 and CsCHS3 were determined to be conserved CHS genes, and bioinformatics analysis of protein structures results show that CsCHS1, CsCHS2 and CsCHS3 are similar. CsCHS1 and CsCHS3 are active, there should be hot spots of mutation in their coding regions.

Key words: tea plant (Camellia sinensis(L.)) , chalcone synthase gene (CHS) , single nucleotide polymorphism (SNP)

张丽群1, 韦康1, 王丽鸳1, 成浩1, 刘本英2, 龚武云1. 茶树CHS基因结构及编码区单核苷酸多态性分析[J]. 中国农业科学, 2014, 47(1): 133-144.

ZHANG Li-Qun-1, WEI Kang-1, WANG Li-Yuan-1, CHENG Hao-1, LIU Ben-Ying-2, GONG Wu-Yun-1. The Structure and Single Nucleotide Polymorphism Analysis of Chalcone Synthase Genes in Tea Plant (Camellia sinenesis)[J]. Scientia Agricultura Sinica, 2014, 47(1): 133-144.

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参考文献

[1]Punyasiri P A N, Abeysinghe I S B, Kumar V, Treutter D, Duy D, Gosch C, Martens S, Forkmann G, Fischer T C. Flavonoid biosynthesis in the tea plant Camellia sinensis: Properties of enzymes of the prominent epicatechin and catechin pathways. Archives of Biochemistry and Biophysics, 2004, 431(1): 22-30.

[2]马成英, 吕海鹏, 林智, 张悦, 郭丽, 谭俊峰. 茶树类黄酮O-甲基转移酶基因的克隆及原核表达分析. 中国农业科学, 2013, 46(2): 325-333.

Ma C Y, Lü H P, Lin Z, Zhang Y, Guo L, Tan J F. Cloning and prokaryotic expression of flavonoid O-methyltransferase from Camellia sinensis. Scientia Agricultura Sinica, 2013, 46(2): 325-333. (in Chinese)

[3]Frei B, Higdon J V. Antioxidant activity of tea polyphenols in vivo: Evidence from animal studies. The Journal of Nutrition, 2003, 133(10): 3275-3284.

[4]Maeta K, Nomura W, Takatsume Y, Izawa S, Inoue Y. Green tea polyphenols function as prooxidants to activate oxidative-stress- responsive transcription factors in yeasts. Applied and Environmental Microbiology, 2007, 73(2): 572-580.

[5]Mukhtar H, Ahmad N. Tea polyphenols: prevention of cancer and optimizing health. The American Journal of Clinical Nutrition, 2000, 71(6): 1698-1702.

[6]成浩, 李素芳, 沈星荣. 茶树中的类黄酮物质及其生物合成途径. 中国茶叶, 1999(1): 6-8.

Cheng H, Li S F, Shen X R. Flavonoids and its biosynthetic pathway in Camellia sinensis. China Tea, 1999(1): 6-8. (in Chinese)

[7]Besseau S, Laurent H, Pierrette G, Lapierre C, Pollet B. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. The Plant Cell, 2007, 19: 148-162.

[8]Wang Y S, Gao L P, Wang Z R, Liu Y J, Sun M L, Yang D Q, Wei C L, Shan Y, Xia T. Light-induced expression of genes involved in phenylpropanoid biosynthetic pathways in callus of tea (Camellia sinensis L.) O. Kuntze). Scientia Horticulturae, 2012, 133: 72-83.

[9]牛天敏, 马会勤, 陈尚武. 大豆查尔酮合成酶(CHS)基因的克隆、表达及其在雪莲提取液中的代谢产物分析. 中国生物工程杂志, 2007, 27(2): 58-63.

Niu T M, Ma H Q, Chen S W. Cloning and expression of chalcone synthase (CHS) of Glycine max L.and analysis of it metabolize produce in the extracts from Saussurea spp.. China Biotechnology, 2007, 27(2): 58-63. (in Chinese)

[10]王金玲, 瞿礼嘉, 陈军, 顾红雅, 陈章良. CHS基因外显子2的进化规律及其用于植物分子系统学研究的可行性. 科学通报, 2000(9): 942-950.

Wang J L, Qu L J, Chen J, Gu H Y, Chen Z L. Evolution law of CHS gene exon 2 and its feasibility used for plant molecular systematics study. Chinese Science Bulletin, 2000(9): 942-950. (in Chinese)

[11]Sommer H, Saedler H. Structure of the chalcone synthase gene of Antirrhinum majus. Molecular and General Genetics, 1986, 202(3): 429-434.

[12]Ma L Q, Pang X B, Shen H Y, Pu G B, Wang H H, Lei C Y, Wang H, Li G F, Liu B Y, Ye H C. A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum. Planta, 2009, 229(3): 457-469.

[13]Jiang C, Schommer C K, Kim S Y, Suh D Y. Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens. Phytochemistry, 2006, 67(23): 2531-2540.

[14]郭小勤, 李德葆. 植物前体mRNA的选择性剪切. 农业生物技术学报, 2006, 14(5): 809-815.

Guo X Q, Li D B. Pre-mRNA alternative splicing in plants. Journal of Agricultural Biotechnology, 2006, 14(5): 809-815. (in Chinese)

[15]谢先芝, 吴乃虎. 高等植物基因的内含子. 科学通报, 2002, 47: 731-736.

Xie X Z, Wu N H. Gene intron in higher plant. Chinese Science Bulletin, 2002, 47: 731-736. (in Chinese)

[16]林玉玲. 龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究[D]. 福州: 福建农林大学, 2011.

Lin Y L. Studies on cloning, expression and regulation of SOD gene family during somatic embryogenesis in dimocarpus longan lour [D]. Fuzhou: Fujian Agriculture and Forestry University, 2011. (in Chinese)

[17]郝岗平, 吴忠义, 曹鸣庆, Georges Pelletier, Dominique Brunel, 黄丛林, 杨清. 拟南芥抗旱转录因子CBF4基因区域的核苷酸多样性及其分子进化分析. 遗传学报, 2004(12): 1415-1425.

Hao G P, Wu Z Y, Cao M Q, Georges P, Dominique B, Huang C L, Yang Q. Nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana and its molecular evolution analyses. Acta Genetica Sinica, 2004(12): 1415-1425. (in Chinese)

[18]Takeuchi A, Matsumoto S, Hayatsu M. Chalcone synthase from Camellia sinensis: Isolation of the cDNA and the organ-specific and sugar responsive expression of the genes. Plant Cell and Physiology, 1994, 35(7): 1011-1018.

[19]Kaundun S, Matsumoto S. Development of CAPS markers based on three key genes of the phenylpropanoid pathway in tea, Camellia sinensis (L.) O. Kuntze, and differentiation between assamica and sinensis varieties. Theoretical and Applied Genetics, 2003, 106(3): 375-383.

[20]刘本英. EST-SSR和ISSR分子标记在云南茶树资源中的应用研究[D]. 北京: 中国农业科学院, 2009.

Liu B Y, Application studies of EST-SSR and ISSR markers in tea germplasms (Camellia spp.) from Yunnan [D]. Beijing: Chinese Academy of Agricultural Sciences, 2009. (in Chinese)

[21]Lerner M R, Boyle J A, Mount S M, Wolin S L, Steitz J A. Are snRNPs involved in splicing? Nature, 1980, 283: 220-224.

[22]Suh D Y, Fukuma K, Kagami J, Yamazaki Y, Shibuya M, Ebizuka Y, Sankawa U. Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases. Biochemical Journal, 2000, 350(1): 229.

[23]Batschauer A, Ehmann B, Schäfer E. Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression. Plant Molecular Biology, 1991, 16(2): 175-185.

[24]卢其能, 杨清, 却志群, 黄友明. 马铃薯野生种CHS基因cDNA的克隆与表达分析. 华北农学报, 2008(5): 17-22.

Lu Q N, Yang Q, Que Z Q, Huang Y M. cDNA cloning and expression analysis of CHS gene from wild potato. Acta Agriculturae Boreali-Sinica, 2008(5): 17-22. (in Chinese)

[25]周晨阳. 茶树次黄嘌呤核苷酸脱氢酶基因克隆及其与咖啡碱含量的关联分析[D]. 北京: 中国农业科学院, 2012.

Zhou C Y. Molecular cloning of Inosine-5’-monophosphate Dehydrogenease gene and association analysis of the gene with caffeine content in tea plant [D]. Beijing: Chinese Academy of Agricultural Sciences, 2012. (in Chinese)

[26]王兰. RNA编辑研究进展. 中国农学通报, 2011, 27(5): 308-311.

Wang L. The advances of RNA editing. Chinese Agricultural Science Bulletin, 2011, 27(5): 308-311. (in Chinese)

[27]陈吉宝, 景蕊莲, 员海燕, 卫波, 昌小平. 小麦TaDREB1基因的单核苷酸多样性分析. 中国农业科学, 2005, 38(12): 2387-2394.

Chen J B, Jing R L, Yuan H Y. Wei B, Chang X P. Single nucleotide polymorphism of TaDREB1 gene in wheat germplasm. Scientia Agricultura Sinica, 2005, 38(12): 2387-2374. (in Chinese)

[28]夏楠. 苦荞CHS基因多样性与黄酮含量表型多样性分析[D]. 太原: 山西大学, 2012.

Xia N. Analysis of phenotypic diversity of tartary buckwheat CHS gene diversity and flavonoid content [D]. Taiyuan: Shanxi University, 2012. (in Chinese)

[29]Fusari C M, Lia V V, Hopp H E, Heinz R A, Paniego N B. Identification of single nucleotide polymorphisms and analysis of linkage disequilibrium in sunflower elite inbred lines using the candidate gene approach. BMC Plant Biology, 2008, 8: 7.

[30]Yang J B, Tian X, Li D Z, Guo Z H. Molecular composition and evolution of the chalcone synthase (CHS) gene family in five species of camellia (theaceae). Acta Botanica Sinica, 2003, 45(06): 659-666.