库尔勒香梨萼片脱落与宿存分子机理研究

There are two types of calyx leaving fruit and calyx persistence fruit in Korla Fragrant Pear fruit. This can cause poor fruit uniformity and seriously affect product quality and food quality of pear fruit, and result in a declining competitiveness in the market ultimately. Currently, the point of view of "calyx persistent fruit inferiors to calyx leaving fruit in appearance and taste" has been formed in the minds of consumers. If the problem of calyx persistent in Fragrant Pear fruit dose not be solved, the sustained and healthy development of the Fragrant Pear industry will be greatly affected. In the present study, PCR and hiTAIL-PCR methods are used to obtain MYB-like genes and its upstream regulatory sequences, and some related bioinformation are analyszed. Further, the differential expression genes related to leaving and persistence of calyx are screened between the calyx leaving fruits and the calyx persistence fruits in Korla Fragrant Pear using transcriptomic sequencing technique. On this basis, the expression pattern of MYB genes in the period of inflorescence exposure to calyx leaving as well as the influence of physical and chemical measures on the expression are also investigated using the real-time fluorescence quantitative PCR method, and the biological function of major genes are identified by the RNAi technique. Our results will make for clearing the molecular mechanism of leaving and persistence of calyx and providing a theoretical basis for the effective control of calyx leaving in Korla Fragrant Pear fruit. Thus, this research has an important value in theory and practice.

库尔勒香梨有萼片脱落与宿存两种果实，造成果实均一性差，严重影响香梨的商品品质和食用品质，致使市场竞争能力下降。目前在广大消费者心中已经形成"宿萼果既不如脱萼果好看，也不如脱萼果好吃"的观点。如不解决香梨宿萼问题，香梨产业的持续健康发展将会受到很大影响。本研究拟通过PCR和hiTAIL-PCR等技术，获得库尔勒香梨MYB同源基因及其上游调控序列，分析其生物信息；通过对库尔勒香梨脱萼组样品和宿萼组样品的转录组测序，比较样本间的表达水平差异，进一步筛选与库尔勒香梨萼片脱落和宿存相关的差异表达基因。利用实时荧光定量PCR技术，研究MYB等基因在花序显露至萼片脱落期间的表达规律以及化学和物理措施对其表达的影响，并利用RNAi技术研究其干扰效果，鉴定其生物学功能，进而明确萼片脱落与宿存的分子机理，为有效研制库尔勒香梨萼片脱落新技术提供理论依据，具有重要的理论和应用价值。

，致使市场竞争能力下降。目前在广大消费者心中已经形成“宿萼果既不如脱萼果好看，也不如脱萼果好吃”的观点。如不解决香梨宿萼问题，香梨产业的持续健康发展将会受到很大影响。本研究通过PCR和hiTAIL-PCR等技术，获得了库尔勒香梨MYB、SPL同源基因及其上游调控序列，并发现启动子序列中有基本转录元件TATA-box、CAAT-box和一些响应激素等的顺式作用元件。利用实时荧光定量PCR检测了与萼片脱落和宿存相关主要基因MYB和SPL在不同阶段（初花至萼片脱落期间）和花、果不同部位的表达状况，以及喷施PBO、PP333、GA、IAA、ABA等不同药剂对其表达的影响，发现在盛花期MYB表达量与萼片脱落和宿存密切相关，高表达有利于宿存，低表达易引发脱落；通过转录组测序从库尔勒香梨脱萼组和宿萼组样品间获得了大量差异表达基因，其中包括细胞壁代谢、植物激素、SPL和MYB、胁迫应对、锌指蛋白、脂转移蛋白等相关基因，进一步分析差异基因的功能和代谢路径，发现库尔勒香梨萼片脱落是一个受多种与细胞壁代谢相关基因的诱导，并由多种与果实脱落相关的植物激素调控因子共同参与完成的细胞程序性衰老、死亡过程。通过小RNA测序从脱萼组和宿萼组中得到了大量差异表达的 miRNA 基因，预测出 8 个可能与库尔勒香梨萼片脱落相关的 miRNA 基因，它们可能参与离层形成，植物激素信号转导，细胞壁降解，钙离子途径的等过程；克隆鉴定了9种新的miRNA和NAC转录因子。成功构建了MYB转录因子的RNAi表达载体，采用注射法和浸泡法将干扰载体导入子房，通过检测分析，发现RNA干扰载体对MYB表达有一定干扰效果。本研究为进一步阐明库尔勒香梨萼片脱落和宿存的调控网络奠定了良好的基础，具有重要的理论和应用价值。