猕猴桃AcMED25克隆、表达分析及其与果实生长和大小的关系

Cloning and expression analysis of AcMED25 and its relationship with fruit growth and size in kiwifruit

【目的】克隆猕猴桃属(Actinidia L.)果实中介体复合物亚基25(AcMED25),分析其在美味猕猴桃‘金硕’(A.deliciosa‘Jinshuo’)果实中表达特性及其与果实生长发育的关系。【方法】根据电子克隆技术获取AcMED25保守片段;采用RT-PCR,结合RACE技术克隆AcMED25基因的全长序列并进行生物信息预测分析;在‘金硕’猕猴桃花后15 d采用CPPU浸蘸、疏果+环剥、疏果+环剥+CPPU 3种处理促进或抑制果实生长;使用Realtime PCR方法分析AcMED25在不同处理方法下果实不同发育阶段的表达特性。【结果】(1)获得AcMED25(包含保守区域)的5’cDNA序列,其对应的氨基酸序列与其他物种的一致性为65%~79%;(2)AcMED25的表达在果实发育过程中呈'低—高—低—高'变化,与果实生长速率、单果质量'快—慢—快—慢'的变化呈相反变化趋势,CPPU处理后AcMED25在果实快速生长期(花后25~30 d)为极低表达水平,对照相对高些,环剥处理(抑制果实生长)在此期的表达相对最高;(3)果实成熟期AcMED25的表达相对较高。【结论】获得猕猴桃AcMED25,包含5’全长cDNA序列和保守域序列;在花后25~30 d,AcMED25的表达水平可能影响猕猴桃果实大小,与果实生长和大小的调控有关,推测AcMED25可能抑制果实生长。

【Objective】 The aim of this study was to clone cDNA sequence of Mediator Complex Subunit 25(MED25) gene(AcMED25) from fruit of kiwifruit(Actinidia L.), and its expression in fruit of the ‘Jinshuo'kiwifruit(A. deliciosa.) during its development and its relationship with fruit growth was analyzed.【Method】 AcMED25 conserved sequence was obtained through e-clone, and the full-length cDNA of AcMED25 was cloned by the technology of rapid amplification of cDNA ends(RACE). The cDNA sequence and the deduced amino acid sequence were analyzed with bioinformatics method. CPPU and fruit thinning(1∶2 leaf-fruit ratio) and girdling treatments were used to promote or inhibit the growth of fruit in‘Jinshuo' at 15 days after anthesis. The AcMED25 expression was analyzed by qRT-PCR. 【Result】(1)The 5' full-length cDNA including conserved region of AcMED25 was obtained. Homology of deduced amino acids encoded by AcMED25 in other species were 65% to 79%.(2) It was a contrary tend in AcMED25expression and fruit growth rate and fruit weight during the progress of fruit growth; the AcMED25 expression levels in fruits treated with CPPU were extra-low during rapid growth of fruit(25 to 30 days after anthesis), while those were higher in the control, and girdling(inhibit the growth of fruit) treatments were highest.(3) The AcMED25 expression were increased at fruit ripen stage.【Conclusion】 5' full-length cDNA of AcMED25 was obtained, including its conserved domain. The expression of AcMED25 at the rapid growth time of fruit(25 to 30 days after anthesis) may affect the fruit size in kiwifruit. It is possible that AcMED25may have functions in fruit growth and regulation of fruit size, and AcMED25 may inhibit the fruit growth.