过表达钙调素类似蛋白基因CML25-like诱导黄瓜单性结实的研究

Study on cucumber parthenocarpy induced by over-expressing of calmodulin-like protein gene CML25-like

[目的]钙调素类似蛋白(calmodulin-like protein,CML)是一类含有EF-hand结构域的钙受体蛋白,与植物细胞的分裂及形态建成密切相关。本文基于前期克隆的一个黄瓜果实特异性表达基因CML25-like,研究其调控单性结实的机制。[方法]采用RT-qPCR方法研究CML25-like表达的时空特点;构建植物表达载体pLP100-35S-CML25-like,利用农杆菌介导子叶节法转化黄瓜自交系CCMC,并研究转基因植株的表达特点;观察转基因植株的表型,采用RT-qPCR方法检测其坐果期激素相关基因的表达变化。[结果]CML25-like在黄瓜幼叶和雌花中表达量最高,在雄花中表达量最低,而且CML25-like在果实发育过程中上调表达,在果实败育过程中下调表达;性状统计发现过表达CML25-like植株的单性结实率最高可达到98.25%,比对照CCMC提高了86%,而且单性结实果实与对照授粉果实的长度和横径均无明显差异。RT-qPCR检测结果显示,在转基因植株坐果期22个激素相关基因中有16个显著上调表达。[结论]CML25-like基因的活跃表达与果实发育密切相关,而且CML25-like能够诱导黄瓜单性结实,促进果实膨大,推测CML25-like通过上调弱单性结实黄瓜子房中激素相关基因表达而诱导单性结实。

[Objectives]Calmodulin-like protein(CML),a class of Ca 2+binding proteins containing the EF-hand domain,are closely related to the division and morphogenesis of plant cells.Based on the cloned CML 25-like gene which expressed specifically in the cucumber fruit,we studied its mechanism of regulating parthenocarpy.[Methods]RT-qPCR method was used to study the temporal and spatial characteristics of CML 25-like expression.Plant expression vector pLP100-35S-CML 25-like was constructed,the cucumber inbred line CCMC was transformed by Agrobacterium-mediated transformation method using cotyledon as explants,and the expression characteristics of the transgenic plants were studied.The phenotypes of transgenic plants were observed,and the expression changes of hormone-related genes in fruit-set period of transgenic plants were detected using RT-qPCR method.[Results]In transgenic plants,the expression level of CML 25-like was the highest in young leaves and female flowers of cucumber,and the lowest in male flowers.Moreover,CML 25-like was up-regulated expression during the fruit development,and down-regulated expression during the fruit abortion.Statistical results of traits showed that the parthenocarpy rate of transgenic plants of over-expressing CML 25-like could reach to 98.25%,which increased 86%compared with control CCMC.And there were no significant differences in length and transverse diameter between the parthenocarpic fruits and control pollinated fruits.The results of RT-qPCR showed that 16 of the 22 hormone-related genes were significant up-regulated expression during the fruit setting of transgenic plants.[Conclusions]The active expression of CML 25-like was closely related to fruit development,and CML 25-like could induce parthenocarpy of cucumber and promote fruit enlargement.We inferred that CML 25-like induced parthenocarpy by promoting the up-regulated expression of hormone-related genes in the ovaries of weakly parthenocarpic cucumber.