黄瓜WD-repeat蛋白CsTTG1与Tu互作调控果瘤形成的研究

The cucumber (Cucumis sativus L.) fruit, as economic organs, is covered with spines and tubercules. When spines are combined with tubercules, cucumber fruits have a characteristic warty (Wty) trait, which is an important external quality trait and affects the market value with respect to wart size and density. However, to date, the regulatory mechanisms underlying the development of fruit wart are largely unknown. Our previous analysis found that CsTTG1 regulate the formation of fruit spines and tubercules, moreover, CsTTG1 can positively regulate the expression level of Tu, which is required for the Wty fruit phenotype, but the molecular mechanisms of CsTTG1 regulating tubercules formation remains elusive. In this project, to identify the interaction protein of CsTTG1 with Yeast two-hybrid cDNA library. We found that CsTTG1 interacts physically with Tu, suggesting that CsTTG1 may form a complex with Tu to regulate the formation of tubercules. To further verify this hypothesis, Co-IP (Co-Immunoprecipitation) and Pull-down assay will be performed to examined the interaction between CsTTG1 and Tu; exploring the genetic relationship between CsTTG1 and Tu; illuminating the phenotype of transgenic plant that CsTTG1 is knockout; screening the downstream genes of CsTTG1 by Digital Gene Expression Profile (DGE), and to find directly target genes using yeast one-hybrid and EMSA, moreover, to verify the function of this gene through the cucumber genetic transformation experiment. Furthermore, to provide important insights into the role of CsTTG1 in the regulatory network regulating fruit tubercules formation of cucumber. We hope the results presented here will be helpful to reveal more about the genetic regulatory mechanism of cucumber wart, and to improve breeding and enhance the economic value of cucumber production.

刺瘤（warts）是黄瓜果实重要外观品质，且其大小和密度直接影响果实的商品性，但目前人们对黄瓜果实刺瘤发育的分子机制及其调控途径知之甚少。申请人前期研究发现CsTTG1基因调控黄瓜果实刺瘤形成，且正调控果瘤基因Tu的表达，但其调控果瘤形成的作用机理尚不清楚。本项目利用CsTTG1进行酵母双杂筛库，发现CsTTG1与Tu直接互作，推测它们可能通过互作影响黄瓜果瘤的形成。为验证这一假说，我们拟通过免疫共沉淀和Pull-down等技术进一步验证二者的互作，并解析CsTTG1与Tu基因的遗传关系，明确CsTTG1基因缺失突变体的表型，利用数字基因表达谱分析筛选差异表达基因，采用酵母单杂和EMSA找到直接下游靶基因，并通过黄瓜遗传转化实验验证该基因的功能，从而解析CsTTG1调控黄瓜果瘤形成的内在机制及调控途径。研究结果对于指导黄瓜以品质改良为目标的分子育种也具有重要参考价值。

刺瘤（warts）是黄瓜果实重要外观品质，且其大小和密度直接影响果实的商品性。申请人前期研究发现CsTTG1基因调控黄瓜果实刺瘤形成，但其调控刺瘤形成的作用机理尚不清楚。本项目揭示了CsTTG1调控黄瓜果实刺瘤形成的分子机制。通过数字基因表达(Digital Gene Expression)分析比较35S:CsTTG1转基因植株和对照植株的子房表皮转录组，鉴定到1241个差异表达基因。CsTTG1基因过量表达使果刺数目增加，果刺基座和刺杆细胞变大且细胞数目增加导致果刺的变大和变长。在35S:CsTTG1转基因植株中，调控细胞扩张、伸长的XTH23和Cyclin家族基因被显著激活，表明这些基因可能参与调控果刺的大小。进一步的分析证实，激素GA和CTK也参与了黄瓜果刺发育起始的调控。果刺对果瘤性状具有隐性上位性，有刺是有瘤的前提。因此，CsTTG1可能通过调控GA和CTK途径影响黄瓜果刺的数量，进而影响果瘤的形成。另一方面，我们通过酵母双杂试验、亚细胞共定位及双分子荧光互补试验、免疫共沉淀等技术证实了CsTTG1与果瘤基因Tu编码蛋白的互作；酵母单杂和荧光素酶试验表明Tu可结合下游靶基因Cs6M的启动子，激活其表达。对Cs6M基因进行功能验证，发现该基因可调控拟南芥果实长度和根长；其在黄瓜上的功能需进一步验证。基于以上结果我们推测CsTTG1可能通过该互作激活Tu的表达，进而调控下游靶基因Cs6M从而调控果瘤的形成。但是在Tu缺失的无果瘤材料中过表达CsTTG1基因，在黄瓜果实上也会形成果瘤，表明CsTTG1调控果瘤形成并不仅仅依赖于Tu，可能还存在其他可调控果瘤形成的基因。该研究重点阐明了CsTTG1参与调控黄瓜果实刺瘤形成的调控途径，为解析黄瓜刺瘤形成的分子调控网络奠定了基础。