茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析

Construction and Preliminary Analysis of the Suppression Subtractive Hybridization cDNA Libraries between Dormant and Sprouting Buds of Tea Plant (Camellia sinensis)

为了解茶树越冬芽休眠及其解除的分子机理,以休眠芽和萌动芽为材料,构建了茶树休眠芽与萌发芽的正、反向抑制消减杂交文库,并从两个文库各随机挑选96个阳性克隆进行序列测定和生物信息学分析。建库质量分析结果表明,所构建的正、反向消减文库有效富集了差异基因,消减效率达到要求,插入片段集中于250-750bp之间,文库质量较好。对正、反向文库随机挑选的阳性克隆测序和分析结果表明,正、反向文库在基因表达谱上存在一定的差异,萌动芽文库在亚细胞器形成、转运载体等方面的基因与休眠芽文库有较大差异,而休眠芽文库在抗氧化、翻译调控以及胁迫响应等方面的基因与萌动芽文库有较大差异。这两个文库的建立为进一步了解茶树芽休眠与解除的分子机制奠定了基础。

Bud dormancy and its release are important biological phenomena in tea plant （Camellia sinensis）. In order to understand the molecular mechanism of tea bud dormancy and its release, 2 SSH-cDNA libraries were constructed using dormant and sprouting buds. The forward library contained genes preferentially expressed in sprouting buds and the reverse library contained genes preferentially expressed in dormant buds. One hundred and ninety two positive clones were randomly selected for sequencing and bioinformatics analyzing from the two libraries. The results showed that differential express genes were enriched effectively in the forward and reverse libraries, and subtraction efficiency reached the demand. Most of the length of inserted fragments was 250~750 bp. It implied that the quality of the SSH-cDNA libraries was satisfied. The bioinformatics analysis results indicated that there were differences between forward and reverse libraries on gene expression profiles. In forward library, the percent of genes related with organelle part and transporter was higher than reverse one. And in the reverse library, the percent of genes related with antioxidant, translation regulator and response to stimulus was higher than theforward. The 2 SSH-cDNA libraries can be further used for gene isolation and provide a foundation for further investigation to explore the molecular mechanism of bud dormancy and its release in tea plant.