以福鼎大白茶为试验材料,用荧光标记的mRNA差异显示技术,研究外源诱导物ID1在诱导提高茶树新梢EGCG含量过程中基因转录水平的变化,分离相关基因片段。从诱导茶树芽叶及对照中分离出18个差异显示的cDNA片段,反向Northern杂交显示其中5个片段可能是来自差异表达基因的转录产物,其中编号DD2、DD9、DD12、DD16为诱导表达上调片段,编号DD3为诱导表达下调片段。将这5个片段克隆于pGEM T-Easy载体,酶切鉴定结果显示载体以及插入的特异片段。序列测定表明,DD12的cDNA片段出现未识别码。通过NCBI网站（http:/www.ncbi.nlm.nih.gov）作Blast序列同源性分析。结果表明,DD2的差异cDNA片段与烟草的硫氧还蛋白过氧化物酶的基因序列同源;DD3的差异cDNA片段,是茶树一个RAPD产物（AJ516005.1）的一段序列,功能未知;DD9的差异cDNA片段与拟南芥、莲子的NADH脱氢酶具有很高的同源性;DD16的差异cDNA片段未能找到与之同源的基因序列,可能为新基因。
In order to study the variation of gene transcription in tea plant (Fuding Dabaicha cultivar) tender shoots during the increasing of EGCG content by ID1 exogenous induction, some gene fragments were isolated by the technique of fluorescent messenger RNA differential display reverse transcription-PCR (DDRT-PCR). 18 differentially displayed cDNA fragments were obtained from induced tea shoots and normal shoots by DDRT-PCR. Reverse northern hybridization showed that 5 of them might derive from transcripts of differentially expressed genes. Among them, the expression levels of differential cDNA sequences of DD2、DD9、DD12 and DD16 in induced tea shoots were higher than the control, and the expression level of differential cDNA sequences of DD3 was reversed. They were cloned into pGEM T-Easy vector, and the positive clones were confirmed by EcoRⅠ digestion. Their sequences were analyzed and DD12 appeared many unknown codes. BLAST was used to search the NCBInr-database at http://www.ncbi.nlm.nih.gov. The results were as follows: DD2 shows 86% homology with the thioredoxin peroxidase gene from Nicotiana tabacum. The function of DD3, a fragment of RAPD Marker (AJ516005.1) from Camellia sinensis, was unknown. DD9 shows 80% homology with aldehyde dehydrogenase (NADH) gene from Lotus corniculatus and Arabidopsis thaliana. DD16 could not be found homologous with any sequences, thus, might be a new gene.
[1] 林金科, 郑金贵. 诱导提高茶树EGCG含量过程对鲜叶品质生化成分的影响[J]. 福建农林大学学报(自然科学版), 2004, 33(1): 65~68.
[2] Liang P, Pardee A B.Differential display of eukaryotic by means of the polymerase chain reaction[J]. Science, 1992, 257: 967~971.
[3] Sturtevant V.Applications of differential-display reverse transcription-PCR to molecular pathogenesis and medical mycology[J]. Clinical Microbiology Reviews, 2001, 13(3): 408~427.
[4] 王夏. 快速、有效筛选新的功能基因—差异显示技术[J].微生物学通报, 2003, 30(1): 103~105.
[5] 陈亮, 杨亚军. 茶树分子标记和基因克隆研究进展[J]. 茶叶文摘, 1999, 13(5): 1~6.
[6] Liang Y R, Takeda Y, Tanaka Z. Molecular cloning and sequencing of a Camellia sinensis cDNA encoding S-adenosylmethionine synthetase[A].Proceeding of2001 lnternational conference on tea culture and science (2001.10), Shizuoka, Japan, 58~61.
[7] 赵丽萍, 陈亮, 高其康. 茶树基因克隆、遗传转化研究进展及EST在茶树功能基因研究中的应用前景[A]. 海峡两岸茶业科技学术讨论会论文集[M]. 长沙: 2003, 292~300.
[8] 王丽鸳, 成浩, 周健. 茶树DNA分子标记及基因工程研究进展[J]. 茶叶科学, 2004, 24(1): 12~17.
[9] 韦朝领, 江昌俊, 陶汉之,等. 茶树紫黄素脱环氧化酶基因的cDNA 克隆及其生物信息学分分析[J]. 南京农业大学学报, 2003, 26(1): 14~19.
[10] 赵东, 刘祖生, 奚彪. 茶树多酚氧化酶基因的克隆及其序列比较[J]. 茶叶科学, 2001, 21(2): 94~98
[11] Kato M, Mizuno K, Crozier A, et al.Caffeine synthase gene form tea leaves[J]. Nature, 2000, 406(6799): 956~957.
[12] Mizutani M, Nakanishi H, Ema J, et al Cloning of B-Primeverosidase form tea leaves,a key enzyme in tea aroma formation[J]. Physiol., 2002, 130(4): 2164~2176.
[13] 林金科, 开国银.茶树总RNA提纯与鉴定[J]. 福建农林大学学报(自然科学版), 2003, 32(1): 70~73
[14] F.萨姆布鲁克, E.F.弗里奇, T.曼尼阿蒂斯著.分之克隆实验指南(第二版)[M]. 金冬雁, 黎孟枫译. 北京: 科学出版社, 1999, 362~371.
[15] J.萨姆布鲁克,D.W.拉塞尔著.分之克隆实验指南(第三版)[M]. 黄培堂等译. 北京: 科学出版社, 2002, 96~99.
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