木薯果胶甲基酯酶抑制因子基因MePMEI1在延缓块根采后腐烂变质中的作用

The postharvest physiological deterioration (PPD) of cassava storage roots easily take place after harvest, thus delaying root PPD is one of the main target of cassava breeding. Pectin methylesterase (PME) control the degree of pectin methylesterification, regulating the rigidity of plant cell walls, which associates with plant resistance. Its activity is controlled by the inhibitor-PMEI. In previous studies, we obtained a high PPD resistance cassava germplasm RYG-1, in which the expression of MePMEI1 gene in its postharvest root is significantly increased than in the control of SC8, while PMEs are significantly decreased. It is speculated that these genes may be related to resistance to PPD in RYG-1. The aims of this project will focus on 1) Clone of the 4 genes (MePMEI1、MePME1 -3) from cassava; 2) expression of them in the Yeast of Pichia pastoris, purification, and analysis of their enzymatic properties and PMEI-PME interaction; 3) Through analysis the expressing pattern of MePMEI1, MePME1~3, PME enzyme activity, the degree of methylesterification of pectin and the change of cell structure in cell wall in cassava storage roots after harvest, to investigate the relationship between PPD and PMEs and PMEI1 in cassava storage roots; 4) Overexpression or RNAi inhibit expression of MePMEI1 in cassava, to further identify the relationship between root PPD and PMEI, and regulation. The results will provide a new way to improve the tolerance to PPD in cassava breeding.

木薯块根极易发生采后变质PPD，延缓块根PPD是木薯育种的主要目标之一。果胶甲基酯酶PME控制果胶的甲酯化程度，调控植物细胞壁的刚性与植物的抗性相关，其活性受抑制因子PMEI控制。前期研究我们获得了一个高抗PPD木薯品种RYG-1，发现其块根MePMEI1基因表达量较华南8号木薯显著提高，而PMEs显著下降。推测这些基因可能与RYG-1抗PPD有关。本项目拟克隆这4个基因，在毕赤酵母中表达并分离纯化，分析其酶学特性及PMEI-PME互作关系；通过分析木薯块根采后MePMEI1、MePME1~3的表达及活动规律、PME酶活性分析、细胞壁中果胶甲基酯化程度和细胞结构变化，探讨木薯块根PPD与PMEI的关系；通过MePMEI1基因在木薯块根中过量或 RNAi抑制表达，进一步验证块根PPD与PMEI的关系，探讨MePMEI1的作用以及调控机制，为改良木薯种质的耐储性提供新思路。

果胶甲基酯酶-PME控制果胶的甲酯化程度，调控植物细胞壁的刚性与植物的抗性相关，其活性受抑制因子-PMEI控制。前期研究我们获得了一个高抗PD木薯品种RYG-1，发现其块根MePMEI1基因表达量较华南8号木薯显著提高，而PMEs显著下降。推测这些基因可能与RYG-1抗PD有关。本项目克隆了MePMEI1, MePME1~3四个基因，利用qRT-PCR 验证了木薯块根采后MePMEI1、MePME1~3的表达及活动规律。通过酵母双杂交点对点互作实验发现MePMEI与MePME1~3均没有直接互作关系；通过酵母双杂交筛选木薯cDNA文库获得了37个互作蛋白，其中2个互作蛋白（VDAC2和Gly2）经回转验证和双分子荧光实验得到了确认。测定了木薯块根采后PME酶活性。通过透射电镜观察了块根采后细胞壁的结构变化。获得了MePMEI1过表达的木薯转基因株系，CRISPR-Cas9介导的MePMEI1敲除木薯株系。通过比较转基因木薯块根采后变质情况，证明了MePMEI1在延缓木薯采后变质中起了一定的作用。本研究结果为改良木薯种质的耐储性提供新思路。