草莓miR390基因及其启动子的鉴定与表达分析

Identification and expression analysis of miR390 gene and its promoter from strawberry

【目的】明确草莓miR390基因的表达特性,为进一步揭示其与草莓白粉病抗性的关系奠定基础。【方法】采用PCR和qRT-PCR的方法,从栽培草莓‘全明星’中克隆了miR390基因及其启动子序列,系统研究了高盐、低温、不同培养方式、IAA处理等对草莓miR390基因表达的影响。【结果】克隆到的418 bp的草莓miR390基因序列中,包含97 bp的茎环结构及侧翼序列,并且高度保守的21 nt的成熟miR390序列位于茎环的5′端臂上。利用生物信息学软件分析草莓miR390基因的启动子序列发现,除了具有TATA/CAAT-box外,还含有spl、HSE等特异作用元件。定量RT-PCR结果表明,高盐能明显抑制草莓miR390的表达,而低温对其表达影响不大,相同条件下固体培养基比液体培养基更能促进草莓miR390的表达;与GA3等激素相比,IAA能显著增加草莓miR390的表达。【结论】采用IAA质量浓度为0.01 mg·L-1的固体培养基,最能增加草莓微繁殖苗miR390的表达量。

【Objective】 The aim of this study was to clarify the expressing character of miR390 gene of strawberry, and lay foundation for further study on the relationship between miR390 and the strawberry resistance to powdery mildew. 【Method】 The gene of miR390 and its promoter were obtained from strawberry(Fragaria×ananassa ‘Allstar') with the method of PCR. The expression level of miR390 gene under some treatments including NaCl, low temperature, different methods of tissue-culture and IAA was studied by qRT-PCR. 【Result】 The cloned sequence of 418 bp contained a 97 bp stem-loop structure and flanking sequence, and the highly conserved mature miR390 with 21 nt was at the 5' end of stem-loop. In addition to the TATA/CAAT-box, its promoter identified by bioinformatic analyses also contained some specific regulatory elements such as spl, HSE and etc. The results of qRT-PCR showed that the expression level of strawberry miR390 was decreased in high concentration of NaCl, few change in low temperature treatment, and it was also higher in solid-medium than that in liquid-medium. The expression level of miR390 was notably increased in IAA treatment contrast to other hormone treatment for example GA3.【Conclusion】 It was best to increase the expression level of miR390 in micropropagated strawberries with the solid-medium involving 0.01 mg·L-1IAA.