CpLEA基因在蜡梅花器官抗寒中的作用研究

Late embryogenesis abundant proteins (LEA proteins) accumulated to high concentrations in plant embryo during the later stage of seed development. Based on the common amino acid sequence motifs and the conserved structural features, LEA proteins are basically divided into six groups. LEA 3, group three of LEA proteins, includes tandem repeated motifs composed of 11 amino acids. Under the condition of dehydration,LEA 3 proteins are folded into the conformation of amphipathic a-helices to protect cell membrane from damages caused by extreme stress of dehydration. .A putative wintersweet(Chimonanthus praecox) homolog of the LEA3 gene, CpLEA, with a full length of 573 bp, has been acquired in our previous study. qRT-PCR demonstrated that the expression of CpLEA was up-regulated by ABA，cold，salt，drought and hot stresses.CpLEA expressed not only in leaves but also in floral organs,especial in the stamen and pistil.During the flowering stage，the expression of CpLEA was abundant in the early stage of the flower development and then decreased until the blooming stage. Based on the results above,we speculated that the CpLEA gene may participate in floral organ cold resistance and winter flowering ..In the project, we hope to explore the function and action mode of CpLEA involved in floral organ cold resistance and flower development in Chimonanthus praecox. Experiments of CpLEA protein function analysis, heterologous expression, cold stress responses, promoter regulatory elements and activity analysis will be performed in the study. The research is significantly important to explore the unique mechanisms of floral development and stress resistance in Chimonanthus praecox and other winter flowering plants. It will also provide a theoretical basis and gene resource for developing technology of regulating Chimonanthus praecox blooming and frost prevention of forestry crops.

第3组胚胎晚期丰富蛋白LEA是一类在植物抵抗非生物胁迫中起重要作用的耐受性蛋白。前人对该组基因的研究主要针对非冬季开花植物的营养器官，对其在花器官中抗寒机理的研究相对缺乏。申请人前期从蜡梅中克隆了1个LEA3基因CpLEA，该基因除了在叶片中被低温诱导表达外，在花器官中也有表达，且在花发育早期表达量高，推测该基因可能参与花器官抗寒性和冬季开花习性的形成。在此基础上，该项目拟通过蛋白功能验证、基因表达分析、异位表达、启动子功能分析等实验，探明CpLEA蛋白的活性功能、CpLEA基因控制或影响的生物学性状、CpLEA基因表达的调控方式，进一步明确蜡梅CpLEA基因在蜡梅花器官抗寒性形成及冬季开花过程中的具体功能和作用方式。项目的完成可以加深对LEA3基因家族功能的认识、丰富植物花期抗寒理论，为木本植物开花调控和抗寒研究提供新的切入点，有望为生产上开发农林作物花期霜冻防御提供理论参考和基因资源。

该项目通过蛋白功能验证、基因表达分析、异位表达、启动子功能分析，探明了CpLEA蛋白的活性功能、CpLEA基因控制或影响的生物学性状、CpLEA基因表达的调控方式，从而进一步明确了蜡梅CpLEA基因在蜡梅花器官抗寒性形成及冬季开花过程中的具体功能和作用方式。主要结果包括：（1）获得纯化了CpLEA重组蛋白，体外实验表明CpLEA 蛋白能够保持乳酸脱氢酶LDH在低温和高温胁迫下的酶活。菌落存活数和菌液生长曲线实验结果表明，该蛋白的过表达能增强转 CpLEA 基因大肠杆菌对低温、高温、高盐、高渗透等非生物胁迫的耐受性。（2）获得了上游启动子序列1172 bp，该启动子中包含多个与植物非生物胁迫相关的响应元件。瞬时表达分析表明该启动子具有驱动下游报告基因表达的活性，拟南芥中的稳定表达分析表明，在转基因拟南芥种子萌发至植株成熟进程中该启动子均可驱动GUS基因表达，在花器官和绿色组织中均有表达，但未经过春化的种子检测不到启动子活性。GUS 基因及蛋白的定量分析结果显示，GUS基因在转基因拟南芥的根，茎，叶和花中表达，其中花器官中表达量最高，果荚表达量最低。GUS酶活在转基因拟南芥花中最高，根和果荚中较低。转基因植株经水杨酸、脱落酸和乙烯处理后，GUS酶活均有所增加。（3）对大蕾期和露瓣期切花进行低温处理，结果显示CpLEA在大蕾期切花中的表达量先升后降，在露瓣期切花中的表达量持续升高，且低温处理下大蕾期表达量的变化幅度明显大于露瓣期的表达量，推测蜡酶花中CpLEA基因的表达受低温诱导，在开花早期产生的影响可能大于开花后期。（4）对转CpLEA 基因拟南芥分析发现，转基因拟南芥株系比野生型提早抽莛、开花、结实，株高大于野生型。转基因拟南芥抗旱性、抗寒性、耐盐性相较野生型均有所增强。低温处理后，转基因拟南芥内源MYB15基因下调，CBF2基因上调，ICE1基因无明显变化。推测CpLEA基因除了通过表达抗冻蛋白提高植物抗寒性外，还可能通过参与MYB15基因的负调控和CBF2基因的正调控途径，参与植物抗寒性的形成，可能不参与ICE1基因调控途径。