采用基于热不对称交错式 PCR 的基因组步移法,从藤稔葡萄( Vitis vinifera×V. labrusca ‘ Fujiminori’)的基因组总 DNA 中扩增花发育相关基因 FT、FLC、AP3 和 AG 的启动子上游序列片段,并通过序列分析及 PlantCARE 在线预测对 FT、FLC 和 AP3 启动子片段的序列及顺式调控作用元件和功能进行了分析。 扩增结果表明:AG 的第 1 轮扩增产物无明显条带,第 2 轮扩增产物条带弥散,不能用于序列分析及顺式调控作用元件和功能分析;FT、FLC 和AP3 基因 2 轮扩增产物均有特异条带,FT、FLC 和 AP3 启动子序列的实际长度分别为 1 470、1 698 和 1 061 bp, GenBank 登录号分别为 HM192806、 HM192805 和 HM192804。 PlantCARE 在线预测结果表明:FT、FLC 和 AP3 启动子片段中均含有 TATA-box、CAAT-box 等启动子的特异表达元件及光响应元件、真菌刺激响应元件、MYB 结合位点等多个顺式调控作用元件,共同受真菌刺激、MYB 和光等因素的调控;FT、FLC 和 AP3 均可能在花的分生组织中表达,且 FT 和 AP3 还可能在胚乳中表达。 根据研究结果推测:FT、FLC 和 AP3 基因的启动子序列片段中均存在多种诱导响应元件,可能均为诱导型启动子。

 

Using genome walking method based on thermal asymmetric interlaced PCR, upstream sequence fragments of promoters of flower development related genes FT, FLC, AP3 and AG were amplified from the total genomic DNA of Vitis vinifera×V. labrusca ‘Fujiminori’, and sequence and cis- acting regulatory elements and functions of promoter fragments of FT, FLC and AP3 were analyzed by methods of sequence analysis and PlantCARE on-line prediction. The amplification results show that bands of the first round amplification products of AG are not obvious while those of the second round amplification products are dispersed, so it can not be used for analyses of sequence and cis-acting regulatory element and function. And the two round amplification products of FT, FLC and AP3 all have special bands, actual length of promoter sequences of FT, FLC and AP3 is 1 470, 1 698 and 1 061 bp, and GenBank accession number is HM192806, HM192805 and HM192804, respectively. The results of PlantCARE on-line prediction show that promoter fragments of FT, FLC and AP3 all have promoter specific elements such as TATA-box and CAAT-box, etc, and cis-acting regulatory elements such as light responsive element, fungal elicitor responsive element, MYB binding site, etc. And their expression may be controled by fungal elicitor, MYB, light and other factors. FT, FLC and AP3may express in floral meristem, and FT and AP3 may also express in endosperm. According to the results, it is conjectured that promoter fragments of FT, FLC and AP3 all contain many induction-responsive elements, which may be inducible promoters.