木薯碱性/中性转化酶MeNINV1的酶活性调节机制研究

Cassava leaf has a highest rate of photosynthetic carbon assimilation, however the accumulated starches in tuberous roots are far reaches its yield potentials. Thus, the increase of the conversion efficiency of assimilation product into starch will be helpful to improve cassava starch yield. Our previous studies have found that: the activity of alkaline/neutral invertase is closely related to the starch accumulation in tuberous roots of cassava, and their activities are post-translationally regulated. MeNINV1 has been found to be highly expressed in tuberous roots during starch accumulation, and is the key gene of sucrose metabolism in tuberous roots. However, catalytic sites which catalytic decomposition of sucrose in plant alkaline/neutral invertase is not clear, and the regulatory mechanisms of α group members activity is lack of study. Based on this, the project will focus on: 1) Researches the catalytic characterization and sites of the MeNINV1 decomposition of sucrose through prokaryotic expression and yeast mutants. 2) Researches its regulatory mechanisms of MeNINV1 by screening its interacted proteins with MeNINV1 through yeast two-hybrid; identifying these proteins how to regulate the activity of MeNINV1 in vitro; expressing the genes of the interacted proteins into cassava roots through root-specific promoter or RNAi. The results will be genetic bases for breeding high starch varieties of cassava.

木薯叶片具有极高的光合碳同化效率，但是块根中积累的淀粉却低于潜在产量，提高同化物在块根转化成淀粉的效率将有助于提高木薯的产量。我们前期研究发现：碱性/中性转化酶活性与木薯块根淀粉积累密切相关，且存在着翻译后调节机制; MeNINV1（属于α组）在木薯块根淀粉积累各时期高表达，是木薯块根蔗糖分解代谢的关键基因。但是，植物碱性/中性转化酶催化蔗糖分解的催化位点并不清楚，α组成员的酶活性调节机制缺乏研究。本项目将利用原核表达技术和酵母突变体研究MeNINV1催化蔗糖分解的酶学特性及其催化位点；利用酵母双杂交筛选MeNINV1的互作蛋白，体外检测它们对MeNINV1酶活性的影响，探讨MeNINV1酶活性的调节机制；利用块根过量表达和RNAi技术，在木薯中进一步研究MeNINV1的互作蛋白对MeNINV1酶活性的调节机制及在块根淀粉积累过程中的作用，为高淀粉木薯品种的选育提供理论基础。

木薯叶片具有极高的光合碳同化效率，但是块根中积累的淀粉却低于潜在产量，提高同化物在块根转化成淀粉的效率将有助于提高木薯的产量。碱性/中性转化酶催化蔗糖分解成葡萄糖和果糖，其活性与木薯块根淀粉积累密切相关。本项目在转化酶酵母突变体SEY2102中验证了木薯块根关键碱性/中性转化酶MeNINV1 催化蔗糖分解成葡萄糖和果糖的功能；进一步研究发现MeNINV1催化活性位点为D298，最适pH值为6.5，最适反应温度为40℃，200 mM以内的蔗糖浓度酶活性没有明显抑制作用，却被其产物果糖抑制；利用酵母双杂交筛选获得MeNINV1的互作蛋白MeCSN5B，逐段截短MeNINV1序列与MeCSN5B进行酵母双杂交发现，它们互作的区域位于MeNINV1的80-285氨基酸位置；利用CRISPR/Cas9基因编辑技术编辑MeCSN5B基因，获得杂合突变体；突变体木薯的碱性\中性转化酶活性下降，蔗糖含量升高，表明MeCSN5B正调控MeNINV1的酶活性；MeCSN5B突变体的叶片变小、无裂叶、叶柄变短、根系增长、节间距紧凑，表明MeCSN5B参与调控木薯的器官的形态建成。综上所述，本项目解析了MeNINV1的酶学特性及其调控机制，研究结果将为为高产、高淀粉木薯的分子设计育种提供了新的思路。