月季切花失水胁迫MYB类转录因子分离及功能鉴定

Rose (Rosa hybrida) is one of the most important ornamental plants worldwide. It is known that water dehydration is the main limiting factor that terminates the ornamental value of the flower, resulting in flower wilts and abnormal flower opening. However, the molecular mechanisms underlying the dehydration tolerance are still unclear. It is important to investigate the candidate transcription factor genes and their biological functions involved in response to dehydration tolerance in cut rose flowers. With the help of suppression subtractive hybridization and cDNA microarray methods, 52 MYB type transcription factors were found in rose flower dehydration library, and six RhMYBs were closely related to dehydration tolerance in rose petals. We want to isolated and analyzed the key RhMYBs in rose petals under dehydration conditions. In addition, the gene silencing, transforming to model plants and functional complementation methods were also used to investigate the candidate genes biological function. Moreover, expression analysis, transactivation and yeast-one hybrid analysis were also used to investigate the transcription regulaton of RhMYBs. Therefore, our study will uncover the physiology and molecular mechanism of the RhMYBs regulatory network in the dehydration tolerance in rose petals at the transcription level. These analysis of RhMYBs will provide a rational scientific and practice basis for the utilization of them and enrich the rose plant species. Moreover, the selected genes are informative for the genetic crop improvement by non-conventional methods.

月季是全球第一大切花。已经明确，失水胁迫是导致月季切花采后流通损耗的主要影响因素，极易造成花朵畸形、开放异常，严重影响观赏和使用价值。但是，在月季花瓣失水胁迫过程中的转录调节机理尚不明确。筛选和鉴定参与花瓣失水胁迫的重要转录因子便成为解析转录调节机理的一个重要课题。申请者前期已通过抑制性差减杂交和芯片技术，从花瓣失水胁迫文库中筛选到52个RhMYBs参与花瓣失水胁迫进程，初选出6个与花瓣失水胁迫关系密切的RhMYBs。本课题拟对参与失水胁迫的关键RhMYBs进行分离和鉴定，通过过表达拟南芥和月季体内的基因沉默技术对候选基因的功能进行鉴定；开展RhMYBs的生化特性和转录活性分析；通过酵母单杂交技术筛选出RhMYBs的上游基因，确定其转录调控机制。本项目将从转录水平上阐明RhMYBs参与切花月季失水胁迫耐性的转录调节机制，为月季的抗逆分子育种及遗传改良提供坚实的理论基础和基因资源。

月季是重要的观赏作物之一，水分胁迫是造成月季遗传品质和商品价值降低的关键环境因子，解析水分胁迫下参与月季失水胁迫的MYB转录因子是重要的科学问题。项目在前期的高通量解析月季花朵失水胁迫响应基因表达谱的基础上，分离获得12个参与失水胁迫过程的RhMYBs，对其在不同失水时间点的表达特性进行了检测，获得参与失水胁迫过程的关键成员RhMYB96(登录号为MF185658)，该基因开放阅读框包含1065 bp，编码354个aa，分子量为39.49 kDa，等电点为6.89，公式C1693H2655N519O548S14。蛋白序列多重比对发现，RhMYB96在N端具有保守的R2-MYB和R3-MYB结构域，为典型的螺旋-转角-螺旋结构；构建RhMYB96与不同植物R2R3-MYB蛋白C端氨基酸序列的系统发育树， RhMYB96与拟南芥AtMYB96、AtMYB94和AtMYB30聚为一类，属于S1亚组R2R3-MYB类型转录因子；荧光定位结果显示RhMYB96定位在细胞核内；采用qRT-PCR的方法检测了RhMYB96的表达模式，RhMYB96参与了月季花朵开放过程，月季不同器官中在花瓣中的表达量最高；高盐、失水和乙烯处理能够诱导RhMYB96的转录本表达；12 h盐胁迫和脱落酸处理诱导了RhMYB96的表达水平，盐胁迫下施加脱落酸也能诱导RhMYB96的转录本表达。构建了包含RhMYB96全长等6个不同片段长度的酵母表达载体，利用酵母单杂交技术分析了RhMYB96的转录激活活性， RhMYB96是转录激活子，N端没有自激活性，C端具有明显的自激活性。RhMYB96过表达在拟南芥中，在种子萌发阶段和幼苗生长阶段提高了对高盐的适应性，转基因株系具有更长的初生根根长和生物量的积累；同时，转基因株系表现出更强的活性氧清除能力；ABA处理下，转基因株系在种子萌发阶段对ABA超敏；非生物胁迫和ABA相关的基因在转基因株系中表达量上调； 上述结果获得了月季中关键转录因子RhMYB96，明晰了其生物学功能，初步揭示了RhMYB96的转录调控机制，为今后月季的分子育种研究提供了理论参考。