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根据CBF(C-repeat binding factor)AP2/EREBP保守区设计1对简并引物,采用PCR方法对月季‘寒锦4号’(Rosa hybrida‘Hanjin4’)CBF转录因子基因中间片段进行克隆;再根据中间片段区域设计了两对特异引物,采用反向PCR方法对该基因的5’和3’端的序列进行克隆,将中间片段与反向PCR产物拼接得到CBF基因全长序列,命名为RhCBF,GenBank注册编号为EF582843;该基因序列长758bp,ORF为603bp,编码200个氨基酸;同时,根据基因序列设计1对特异引物,利用荧光定量PCR分析月季CBF在不同逆境胁迫下的表达情况。结果显示低温和盐均可以诱导RhCBF的表达,而干旱处理不能诱导其表达。
A pair of degenerate primers was designed based on the CBF (C-repeat binding factor) AP2 / EREBP conserved region. The PCR fragment was cloned from the CBF transcription factor gene of Rosa hybrida’Hanjin4 ’ Two pairs of specific primers were designed in the middle fragment region. The 5 ’and 3’ end sequences of the gene were cloned by reverse PCR. The full length sequence of the CBF gene was obtained by splicing the intermediate fragment with the reverse PCR product and named as RhCBF, GenBank The sequence of the gene was 7558bp and the ORF was 603bp, encoding a protein of 200 amino acids. At the same time, a pair of specific primers was designed based on the gene sequence. The expression of CBF in different stress conditions was analyzed by fluorescence quantitative PCR. The results showed that both low temperature and salt could induce RhCBF expression, but drought treatment could not induce its expression.