利用cDNA-AFLP技术进行了茶树低温胁迫处理的基因表达差异分析,获得低温诱导后特异表达的差异片段TDF11（transcript derived fragment, TDF）。经BLAST比对发现该片段与白菜、拟南芥、烟草的抗寒基因CBF（C-repeat binding factor）分别有94%、84%、81%的同源性。通过3’/5’RACE的方法获得该片段cDNA全长序列（GenBank,登录号EU857638）。所得序列全长981bp,其开放阅读框编码275个氨基酸。该基因与白菜、烟草、拟南芥中的CBF基因编码的氨基酸序列分别有74%、57%、57%的同源性。用RT-PCR的方法对其在低温处理过程中的表达进行分析,结果表明,茶树CBF基因在低温诱导4 h开始表达,在8 h左右表达量最高,然后开始下降,但仍维持在一个较高的水平。结果提示CBF基因可能在茶树抗寒分子机制的形成过程中发挥重要的作用。
The cDNA-AFLP was applied to identify genes expressed differentially in cold acclimatized tea plant. A cDNA fragment, TDF11 encoding a CBF (C-repeat binding factor) protein was isolated and identified. The cDNA fragment had 94%、84%、81% homology with CBF genes from Brassica napus, Arabidopsis thaliana, Nicotiana tabacum by BLAST on NCBI. The complete cDNA sequence was cloned by RACE (rapid amplification of cDNA ends), named Camellia sinensis CBF gene. Its full cDNA sequence was 981bp (GenBank accession number EU857638) and contained an open reading frame encoding a polypeptide of 275 amino acid residues. Analysis of the nucleotide sequence and deduced amino acid sequence showed 74%、57%、57% homology with CBF genes from Brassica napus, Nicotiana tabacum, Arabidopsis thaliana. To identify differential expression of CBF gene, RT-PCR experiments were performed in six samples including leaves cold-induced for different treatments. The results showed that tea CBF gene expressed after cold induced for 4 hours and up to a maximum when cold induced for 8 h, and then decreased. It hinted the CBF gene might play a key role during the development of cold resistant molecular mechanism in tea plant.
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