茶树花蕾14-3-3蛋白基因的分子克隆及差异表达分析  

Cloning and Analysis of Differential Expression of a 14-3-3 Protein Gene from Tea Flower Bud

作　　者：余梅[1] 江昌俊[1] 房婉萍[2] 叶爱华[1] 王朝霞[3] 李叶云[1] 朱林[1] 

机构地区：[1]安徽农业大学茶叶生物化学和生物技术教育部、农业部重点实验室,合肥230036 [2]南京农业大学园艺学院,南京210095 [3]合肥师范学院生物系,合肥230061

出　　处：《中国农业科学》2008年第10期2983-2991,共9页Scientia Agricultura Sinica

基　　金：安徽省教育厅自然科学研究计划重点项目(2004KJ137ZD)

摘　　要：【目的】从分子水平研究茶树花蕾发育阶段的相关基因表达,了解茶树花蕾的发育机理,利用分子生物学技术抑制茶树的生殖生长、促进营养生长。【方法】利用cDNA-AFLP技术对茶树花蕾发育早期和晚期的差异表达进行分析。采用RACE技术克隆到花蕾发育晚期阶段特异表达的、在花蕾发育中可能起重要作用的14-3-3基因,通过RT-PCR和Western-blot方法研究该基因转录水平和蛋白质表达水平的特点。【结果】在测定的大约1110个茶树花蕾cDNA片段中,122个(10.9%)是差异性表达的,其中87个在发育晚期特异表达,包括茶树14-3-3蛋白基因(GenBank登录号:DQ444463),其全长为1072bp,包含1个由783个核苷酸组成的ORF,编码260个氨基酸,5′非翻译区和3′非翻译区分别有67和222个核苷酸,该基因与其它植物的14-3-3蛋白基因在核苷酸序列和推导的氨基酸序列方面均有着比较高的相似性。RT-PCR和Western-blot分析表明该基因在茶树的晚期发育花蕾中特异表达。【结论】14-3-3蛋白仅存在于茶树发育晚期的花蕾中。cDNA-AFLP技术能用于茶树花蕾不同发育时期的基因分离研究。[ Objective]This study was conducted to analyze differential gene expression pattern during the early and late stages of flower bud at molecular levels. [Method] cDNA-amplified fragment length polymorphism （cDNA-AFLP） was employed to analyze the gene expression pattern during different developmental stages of flower bud. The complete cDNA sequence of 14-3-3 gene was isoslated from flower bud at the late stage using rapid amplification of cDNA ends （RACE）. The expression of 14-3-3 protein was analyzed through reverse transcription polymerase chain reaction （RT-PCR） and Western-blot. [Result] One hundred and twenty-two （10.9%） from approximately 1 100 cDNA fragments analyzed were differentially expressed. The full-length cDNA sequence of 14-3-3 gene is 1 072 bp （GenBank accession number DQ444463）. It contained a 780-nucleotides-long open reading frame starting at position 68 and terminated by a stop codon （TAA） at position 848-850, and a 193-nucleotide-long 3＇-untranslated region plus a 29-monomers-long poly （A）-tail. The encoded protein of 260 amino acids in length had a predicted molecular mass of 29.4 kD. Homology searches with the deduced 260 amino acid residues indicated that 14-3-3 of tea （Camellia sinensis） had a high similarity to other plant 14-3-3 proteins. RT-PCR and Western-blot analyses showed that the 14-3-3 gene and protein could be found in the flower buds at the late stage, but not at the early stage, root, stem, and leaf. [Conclusion] The protein of 14-3-3 expressed especially in tea flower bud at the late stage. It further indicated that cDNA-AFLP technique is a useful tool for study of gene isolation during tea flower bud development.

关 键 词：茶树 差异表达基因 14-3-3蛋白 CDNA-AFLP WESTERN-BLOT 

分 类 号：S571.1[农业科学—茶叶生产加工]