茶叶是世界上最流行的无酒精饮品之一,含有许多有价值的次生代谢产物,如儿茶素类物质。分离和克隆重要的茶树功能基因,对利用基因工程技术进行茶树遗传调控具有重要意义。本文采用EST测序技术和T4RNA连接酶介导的5′RACE技术,获得了一个茶树儿茶素代谢中的重要基因—查尔酮异构酶(CHI)基因,在GenBank登录(DQ904329),其序列全长1 163 bp,其中开放阅读框长723 bp,编码240个氨基酸,3′端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为26.4 kD,理论等电点为5.19。序列分析表明它与番茄CHI基因序列的亲缘关系比较近。
Tea is one of the most popular non-alcoholic healthy beverages in the world, which possesses great value as a source of secondary metabolic products, such as catechins. Isolation and cloning of important functional genes of tea plant (Camellia sinensis) is of crucial significance for using biotechnology method to regulate the metabolism of tea plant. In this paper, the chalcone isomerase gene, which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by using EST sequencing and RACE (rapid amplification of cDNA ends) approaches. The full-length cDNA of chalcone isomerase gene is 1 163 bp (GenBank Accession No. DQ904329), containing a 723bp open reading frame (ORF) encoding a 240 amino acid protein, and its 3′ untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 26.4 kD and its theoretical isoelectric point was 5.19. Sequence analysis result showed that it is closely related with that of Lycopersicon esculentum.
[1] 王锡洪, 奚彪. 略谈茶叶抗癌作用的研究与应用[J]. 中国茶叶, 2003(3): 20~21.
[2] Jane VH, Balz F. Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions[J]. Food Science and Nutrition, 2003, 43: 89~143.
[3] Muir SR, Collins GJ, Robinson S, et al. Over expression of petunia chalcone isomerase in tomatoresults in fruits containing increased levels of flavonols[J]. Nature Biotechnology, 2001, 19: 470~474.
[4] Punyasiri PA, Abeysinghe IS, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: Properties of enzymes of the prominent epicatechin and catechin pathway[J]. Archives of Biochemistry and Biophysics, 2004, 431: 22~30.
[5] Furukawa T, Eshima A, Kouya M, et al. Coordinate expression of genes involved in catechin biosynthesis in Polygonum hydropiper cells[J]. The Plant Cell, 2002, 21: 385~389.
[6] 马春雷, 陈亮. 茶树功能基因分离克隆研究进展[J]. 分子植物育种, 2006, 4(3S): 16~22.
[7] 赵丽萍, 高其康, 陈亮, 等. 茶树基因芯片的研制和初步应用[J]. 茶叶科学, 2006, 26(3): 166~170.
[8] Chen L, Zhao LP, Gao QK. Generation and analysis of expressed sequence tags from the tender shoot cDNA library of tea plant (Camellia sinensis)[J]. Plant Science, 2005, 168: 359~363.
[9] Mamati EG, Liang YR. Genetic control studies to exploit flavonoids synthesis pathway in tea biochemical[J]. Journal of Tea Science, 2005, 25(2): 81~89.
[10] Tunen AJV, Koes RE, Spelt CE, et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes[J]. The EMBO Journal, 1988, 7(5): 1257~1263.
[11] Krol AVD, Lenting PE, Vennstra J, et al. Antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation[J]. Nature, 1988, 333: 866~869.
[12] Krol AVD, Mur LA, Beld M, et al. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression[J]. The Plant Cell, 1990, 2: 291~299.
[13] Aida R, Yoshida K, Kondo T, et al. Copigmentation gives bluer flowers on transgenic torenia plants with the anti-sense dihydroflavonol1-4-reductase gene[J]. Plant Science, 2000, 160: 49~56.
[14] Zuker A, Tzfira T, Hagit BM, et al. Modification of flower color and fragrance by antisense suppression of the flavanone 3- hydroxylase gene[J]. Molecular Breeding, 2002, 9: 33~41.
[15] Verhoeyen ME, Bovy A, Collins G, et al. Increasing antioxidant levels in tomatoes through modification of the flavonoid biosynthetic pathway[J]. Journal of Experimental Botany, 2002, 53: 2099~2106.