含有MALECTIN结构域的类受体蛋白激酶调控板栗淀粉积累的分子机理

Accumulation of starch determines chestnut seeds yield and quality. How starch is synthesized and accumulated in chestnut represents a core question. This research will study the temporal and spatial expression of genes in different organs. We will identify the receptor-like protein kinases containing the MALECTIN domain (MRLKs) that are in response to ABA and sugar signaling and shown to control starch accumulation in Castanea mollissim. Interactors of CmMRLKs will also be studied. Previously we have shown that CmMRLK38/39 interact with GAPDH through a yeast two hybrid assay. In this study, we will test whether this interaction occurs in vitro and in vivo by using BiFC, Pull-Down and iTC methods. Silencing and overexpression of CmMRLK38/39 mutants will be obtained via a callus transgenic system that established in our lab. Transcriptome profiling, to be specific the starch biosynthesis genes, in WT and CmMRLK38/39 mutants will be compared upon ABA and/or sugar treatment. Meanwhile, we will measure the enzymatic activity of GADPH and CmMRLKs, and quantify starch accumulation in WT and CmMRLK38/39 mutants. This could answer whether CmMRLKs interact with GADPH to regulate starch accumulation and shed light on genetic modification to improve chestnut yield and quality.

板栗坚果中主要以积累淀粉为主，淀粉的生物合成直接决定其产量和品质特征。本研究通过不同组织和坚果发育期的时空表达分析，筛选确定响应ABA或蔗糖信号且与淀粉积累相关的类受体蛋白激酶CmMRLKs，挖掘候选互作候选互作蛋白，利用双分子荧光互补、Pull-Down 和等温滴定微量热技术，验证CmMRLK38/39与GAPDH在体内体外的互作；通过构建CmMRLK38/39 超表达和沉默载体转化板栗愈伤组织，经ABA和蔗糖处理，检测转基因愈伤和植株中CmMRLK38/39基因和淀粉合成相关基因的表达、CmMRLK38/39与GAPDH酶活性和淀粉含量等。确定CmMRLKs与GAPDH互作调控淀粉积累的分子机制。为调控板栗产量形成、品质调控和分子改良育种奠定理论基础。

本研究围绕板栗淀粉积累与调控机理的关键问题，在板栗基因组中分析鉴定出70个的含有MALECTIN结构域的类受体蛋白激酶家族成员。板栗MRLK基因在板栗坚果发育不同时期均有表达。利用优化后的板栗愈伤组织瞬时转化体系对CmMRLKs进行RNAi干扰验证，发现干扰Cm11G01237后，总淀粉和支链淀粉含量极显著增加，Cm11G01237基因负调控板栗淀粉积累。进一步分析Cm11G01237-RNAi转录组，推测下游靶基因为相关淀粉合成关键酶基因和WRKY、bZIP及AP2-ERF部分转录因子。利用优化后的瞬时转化体系对淀粉合成关键酶基因进行功能验证，结果表明沉默干扰CmSS1、CmSS2和CmSBE2后，支链淀粉含量极显著性降低，总淀粉含量极显著性降低。沉默干扰CmGBSS1后，直链淀粉含量极显著降低为0.294%，支链淀粉和总淀粉含量没有明显变化，其他淀粉合成关键酶沉默后淀粉含量变化不明显，进一步通过RT-PCR方法验证CmSS1、CmSS2、CmSBE2在板栗高支链中品种中表达量明显升高，主要参与板栗支链淀粉合成，而CmGBSS1在高直链品种中表达量明显升高，主要参与板栗直链淀粉合成。同时，发现外源ABA处理后，Cm11G01237有明显的响应，AP2/ERF家族转录因子在ABA处理后的83 d子叶中，有63个基因响应，AP2/ERF家族在坚果发育中响应ABA信号与坚果的发育及淀粉物质的积累有关，可作为淀粉合成酶的上游调控因子进行互作验证。本研究为进一步解析了类受体蛋白激酶对板栗淀粉合成分子机理的调控，为板栗产量形成、品质调控和分子改良育种提供理论基础。项目资助发表核心论文6篇，SCI收录2篇。培养硕士生4名，其中4名已经取得硕士学位.