应用cDNA-AFLP (Amplified Fragment Length Polymorphism)技术分析了结实率差异显著的龙井43和大叶乌龙两个茶树品种在花蕾发育过程中基因表达的差异。从获得的差异图谱中,克隆得到一个与茶树花发育相关的钙依赖蛋白激酶基因片段,然后用RCAE方法扩增获得其cDNA全长序列,命名为茶树TCK(Camellia sinensis calcium-dependent protein kinase)基因,GenBank登录号EU732607。该基因cDNA序列全长2 281 bp,编码760个氨基酸。用RT-PCR方法进一步研究该基因的功能,检测其表达特异性,结果表明该基因只在茶树花蕾发育后期特异表达,在叶、花蕾发育早期均无表达,提示TCK基因可能在茶树花发育过程中发挥重要作用。
To have an insight into the comprehensive molecular characteristics relating to the flower development mechanisms of tea plant [Camellia sinensis (L.)O.Kuntze], cDNA-AFLP (Amplified Fragment Length Polymorphism) was performed within two tea plant cultivars, Longjin43 and Daye Wulong. A cDNA fragment encoding a calcium-dependent protein kinase gene was isolated and identified. The complete cDNA sequence was cloned by RACE (rapid amplification of cDNA ends), named TCK(Camellia sinensis calcium-dependent protein kinase)gene. GenBank accession number is EU732607. To identify differential expression of TCK gene, RT-PCR experiments were performed in six samples including leaves, small flower buds and big flower buds of Longjing 43 and Daye Wulong. Results showed that the expect band exists in the big flower buds, no band in leaves and small flower buds of these two cultivars. It hinted that TCK might play a key role during flower development of tea plant.
[1] 冯艳飞, 梁月荣. 茶树S-腺苷甲硫氨酸合成酶基因的克隆和序列分析[J]. 茶叶科学, 2001, 21(1): 21~25.
[2] 马春雷, 赵丽萍, 张亚丽, 等. 茶树查耳酮异构酶基因克隆及序列分析[J]. 茶叶科学, 2007, 27(2): 127~132.
[3] Li YY, Jiang CJ, Wan XC, et al. Purification and partial characterization of β-glucosidase from fresh leaves of tea plant[J]. Acta Biochimica et Biophysica Sinica, 2005, 37(6): 363~370.
[4] 董丽娟. 茶树孕性研究[J]. 茶叶科学, 1989, 9(1): 35~38.
[5] 萨姆布鲁克, D W 拉塞尔. 分子克隆实验指南(第三版)[M]. 北京: 科学技术出版社, 2002: 96~99.
[6] Bachem C W B, Hoeven R S, Van D E, et al. Visualization of Differential Gene Expression Using a Novel Method of RNA Finger printing Based on AFLP: Analysis of Gene Expression during Potato Tuber Development[J]. Plant J, 1996, 9: 745~753.
[7] 王永勤, 余小林, 曹家树. 白菜小孢子发育相关基因Bc2MF3的分离及序列分析[J]. 遗传学报, 2004, 31(11): 1302~1308.
[8] 余梅, 江昌俊, 叶爱华, 等. 利用cDNA-AFLP技术研究茶树花蕾发育基因差异表达片段[J]. 茶叶科学, 2007, 27(3): 259~264.
[9] Fang WP, Jiang CJ, Yu M, et al. Differentially expression of Tua1, a tubulin-encoding gene, during flowering of Tea plant [Camellia Sinensis(L.)O.Kuntze] using cDNA-AFLP[J]. Acta Biochimica et Biophysica Sinica, 2006, 38(9): 653~662.
[10] Snedden W A, Fromm H.Calmodulin as a versatile calcium signal transducer in plants[J]. New Phytol 2001, 151: 35~66.
[11] 刘茜, 孙健, 李莉云, 等. 5个水稻蛋白激酶的克隆、表达及活性研究[J]. 中国农学通报, 2007, 23(6): 83~88.
[12] Cheng S H, Willmann M R, Chen H C, et al. Calciumsignalling through protein kinases The Arabidopsis calcium-dependent protein kinase gene family[J]. Plant Physiol 2002, 129: 469~485.
[13] Sanders D, Pelloux J, Brownlee C, et al. Calcium at the crossroads of signaling[J]. Plant Cell [Suppl] 2002, 14:401~417.
[14] Hrabak E M.Calcium-dependent protein kinases and their relatives[J]. Adv Bot Res 2000, 32:185~223.
[15] Romeis T, Piedras P, Jones J D.Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defence response[J]. Plant Cell 2000, 12: 803~816.
[16] Lee S S, Cho H S, Yoon G M, et al. Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26 S proteasome in Nicotiana tabacum[J]. Plant J, 2003, 33: 825~830.
[17] Raıces M, Gargantini P R, Chinchilla D, et al. Regulation of CDPK isoforms during tuber development[J]. Plant Mol Biol, 2003, 52: 1011~1024.
[18] Ludwig A A, Romeis T, Jones J D.CDPK-mediated signalling pathways: specificity and cross-talk[J]. J Exp Bot 2004, 55: 181~188.
[19] Roe J L, Rivin C J, Sessions R A, et al. The tousled gene in Arabidopsis thaliana encodes a protein homolo that is required for leaf and flower development[J]. Cell, 1993, 75: 939~950.
[20] Liu Z, Xia M, Poovaiah B W.Chimeric calcium/calmodulin-dependent protein kinase in tobacco: Differential regulation by calmodulin isoforms[J]. Plant Mol Biol, 1998, 38: 889~897.
[21] Poovaiah B W, Xia M, Liu Z.Development regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers[J]. Planta, 1999, 209: 161~171.
浙公网安备 33019902000101号 
