高温诱导RCA1可变剪接介导海南杜鹃耐热性机制研究

There are extremely rich of Rhododendron germplasm resources in China, but only few of them were exploited and utilized in landscaping. Lack of heat resistant varieties of Rhododendron is the major factor that restricts their wide application in urban landscaping. Transcriptome of R. hainanense was analyzed in previous study and the heat-induced Rubisco activase 1 (RCA1) seems to play important role in photosynthetic response to heat stress. Moreover, RCA1 could generate five alternative spliced transcripts that coding four types of protein isoforms via post-transcriptional regulation under high temperature. In order to investigate the biological significances of generating these alternative spliced transcripts, functions of different RCA1 isoforms mediating heat tolerance in R. hainanense will be further studied. Expression characteristics, subcellular localization and distribution, and differences of binding to Rubisco will be investigated to illuminate function patterns of different isoforms in response to heat. In addition, alternative spliced transcripts that coding different isoforms will be transformed into R. hainanense to study their differences in conferring heat tolerance. Functional differences and commonalities of RCA1 isoforms will be comprehensively analyzed to illuminate the entire gene function of RCA1 in response to heat stress. This research will provide candidate genes and enrich the theoretical basis of heat tolerance molecular breeding of Rhododendron, and have great importance in development and utilization of valuable genes from our unique germplasm resources of Rhododendron.

我国杜鹃花（Rhododendron L.）种质资源非常丰富，但园林应用率较低，耐热品种缺乏是影响杜鹃花园林应用的主要限制因子。前期对耐热种质海南杜鹃（R. hainanense）进行转录组分析，发现热诱导型Rubisco活化酶（RCA1）可能在海南杜鹃热响应中发挥重要作用，并且高温胁迫下RCA1能够通过转录后调控产生5个可变剪接转录本，编码4类蛋白亚基。在此基础上，本项目拟进一步研究RCA1不同亚基响应热胁迫的功能差异。通过研究不同亚基的热响应表达特性、蛋白定位与分布、Rubisco结合差异等，明确各亚基响应高温的作用模式；将编码不同亚基的转录本转化海南杜鹃，研究各亚基介导海南杜鹃耐热性差异；最后，综合不同亚基的功能差异及共性，整体阐明海南杜鹃RCA1基因响应高温胁迫的作用机制。该研究将为杜鹃花耐热育种提供候选基因和理论基础，对我国特有杜鹃花种质资源优良基因的开发利用具有重要意义。

高温是影响杜鹃花园林应用的主要因素，随着全球气候变暖形势的加剧，耐热杜鹃花品种培育更是成为亟需解决的关键产业问题。本项目以耐热种质海南杜鹃为材料，研究了热诱导RCA1基因通过可变剪接编码4种亚基的生物学特性，从蛋白结构、热响应表达模式、蛋白亚细胞定位、介导耐热性功能等方面解析了不同可变剪接的功能差异，并且鉴定了RCA1上游调控的转录因子。结果表明，RCA1受高温信号诱导表达，都定位于叶绿体，在海南杜鹃中，X1表达丰度最高，是主效转录本；异源过表达显示各个转录本都能够互补拟南芥RCA功能，并能够介导耐热性；RCA1上游热诱导调控因子为热激转录因子HSFA2，HSFA2通过结合RCA1启动子上HSE-like元件促进RCA1转录。海南杜鹃RCA1的热诱导性和稳定性决定了其抵御高温胁迫的特异性，是高温特定环境下表达的逆境响应功能基因；RCA1的可变剪接特性，使得其能够产生大量转录本编码不同亚基，形成了功能多样性和叠加效应，加强了应对高温胁迫的能力，是介导海南杜鹃耐热性的重要因子。本项目的研究为杜鹃花耐热性基因工程育种和分子辅助育种提供了候选基因和位点，对于推动杜鹃花品种改良具有重要价值。