苹果砧木‘青砧1号’叶片cDNA文库构建及MdMLO上游调控因子的筛选

Construction of cDNA Library of Apple Rootstock‘Qingzhen 1’Leaf and Screen of MdMLO Genes’Upstream Regulator

MLO(Mildew Resistance Locus O)家族基因是植物特有的“感病基因”,普遍认为它对白粉病具有负调控作用。目前在单子叶和双子叶植物中发现许多抗白粉病MLO基因,但对于其上游调控因子如何调控其表达而影响白粉病发生的研究却很少。本研究中以易感白粉病的苹果砧木‘青砧1号’叶片为材料,构建其cDNA文库,文库滴度4×10^(9)cfu·mL^(-1),重组率为100%。生物信息分析苹果MdMLO家族基因启动子区共有38种顺式作用元件,包含激素、应激防御和生长发育等响应元件。以TCA和TC-rich repeats为诱饵序列构建诱饵载体,分别命名为Bait-TCA和Bait-TC。利用酵母单杂交的方法筛选其上游调控因子,结果显示Bait-TCA具有自激活现象无法筛选,Bait-TC诱饵载体调取获得30条基因序列,其中6条参与植物抗逆防御反应,推测它们可能通过与TC-rich repeats元件互作来调控MLO的表达,进而调控苹果叶片对于白粉病的响应。

MLO is a plant specific recessive resistance gene family.It is widely believed that it has a negative regulatory effect on powdery mildew.Many powdery mildew resistance MLO genes have been found in both monocotyledon and dicotyledon.However,there are few studies on the mechanism of upstream regulator of MLO genes.In this study,a cDNA library of apple rootstock‘Qingzhen 1'leaf was constructed with a titer of about 4×10^(9)cfu·mL^(-1),and the recombination rate was 100%.Bioinformatics analysis of apple MLO gene family promoter region showed that there were 38 kinds of cis-acting elements,including hormone,stress defense response as well as growth and development response elements,etc.TCA and TC-rich repeats cis-acting elements were selected as bait sequences and constructed bait vectors named Bait-TCA and Bait-TC for yeast one-hybrid,respectively.The results showed that the Bait-TCA had self-activation and could not be used for screening.Among the Bait-TC obtained 30 sequences,six sequences were involved in plant anti-stress defense response.It suggested that they might be involved in the response of apple to powdery mildew through regulating the expression of MLO genes.