基于CRISPR/Cas9系统的可溶性酸性转化酶在甘蔗茎秆蔗糖积累和生长发育中的功能分析

Sugarcane is an important sugar and new type of energy crop in the world. The soluble acid invertase (SAI) is an important enzyme which will affect the sucrose metabolism and the growth of sugarcane stem. In this project, we will mutate the sugarcane genome gene by using the new plant genome editing technique which is based on CRISPR/Cas9 system. The gene of soluble acid transferase (SAI) and its promoter sequence will be edited. The transgenic sugarcane plant named TGSP-2 will be used as explant, in which the SPS gene was over expressed. After verified by antibiotics resistant selection and sequencing, we will get different mutant sugarcane plants with different mutation. After that, we will measure all the indicators related with sucrose accumulation in different types of mutants, such as sucrose content, total soluble sugar and metabolic enzymes enzyme activity. As well as plant height, stem diameter and dry matter accumulation which are related with the growth and development of sugarcane. Through the analysis of gene expression regulation, enzyme activity of metabolic enzymes and the relationship between the sucrose accumulations, we can clarify the function of SAI enzyme in the sucrose accumulation and growth of sugarcane stem.Sugarcane germplasm resources were enriched by creating sugarcane mutants with high sugar content. It will promote the creation of energy sugarcane germplasm with high sugar, high yield and high energy storage and provide important materials for the high sugar breeding and genetic research of sugarcane in our country. And also, it will enhance competitiveness of sugar and its products in the international trade. Increasing sugar content in sugarcane significantly improves the nutritional quality and has important practical significance.

甘蔗是一种重要的糖料和新型的能源作物，其茎秆中可溶性酸性转化酶（SAI）是影响蔗糖代谢和甘蔗生长发育的关键酶。本课题拟利用基于CRISPR/Cas9系统的基因组定向编辑技术，以过量表达蔗糖磷酸合成酶（SPS）基因的转基因甘蔗TGSP-2为受体材料，分别突变TGSP-2茎杆中的SAI基因及其启动子序列，经筛选和测序验证后获得不同类型的甘蔗突变体。测定突变体的蔗糖含量、总可溶性糖含量和代谢酶活性等蔗糖积累相关生理生化指标，以及株高、茎径和干物质积累量等生长发育相关的农艺性状指标。通过分析SAI的基因表达调控、酶活性变化与蔗糖积累之间的关系，阐明SAI基因在甘蔗茎杆蔗糖积累和生长发育中作用。筛选获得高蔗糖的甘蔗突变体，丰富了高糖甘蔗种质资源，促进我国高糖高产高蓄能的能源甘蔗种质创制，为我国甘蔗的高糖育种和遗传研究提供重要材料，还可以增强我国蔗糖及其制品在国际贸易中的竞争力，具有重要的现实意义。

作为一种重要的糖料和新型的能源作物，甘蔗茎秆中可溶性酸性转化酶（SAI）影响了蔗糖代谢和甘蔗生长发育。本项目优化了甘蔗SAI基因及其启动子的识别靶位点的sgRNA序列，提高了靶位点识别的特异性和专一性，降低脱靶效应，进而提高了基因编辑效率和基因突变率，建立和完善了一套用于甘蔗基因组定点编辑的CRISPR/Cas9技术体系，辅助甘蔗的遗传育种研究；利用该技术体系转化获得具有不同表达水平的可以稳定遗传的高糖突变体甘蔗材料5份，丰富了我国甘蔗高糖育种材料；同时，我们利用常规杂家育种技术，选育并在农业农村部登记高糖甘蔗新品种2个，可用于我国甘蔗的高糖遗传育种研究；通过系统研究，初步解析了SAI酶在甘蔗蔗糖积累代谢和甘蔗生长发育中的作用，为糖料作物的高糖育种提供理论依据；通过对甘蔗糖分高通量快速检测、糖分积累分子机制的系统性研究，建立了基于近红外技术的高通量甘蔗糖分品质性状评价体系，对收集的800余份国内外重要甘蔗种质的茎秆品质性状进行了系统评价；开展了甘蔗品质性状群体遗传学和甘蔗茎秆机械强度精准评价及形成机理相关研究。利用多组学联合分析揭示了甘蔗蔗糖积累和代谢调控转运网络，初步解析了甘蔗茎杆蔗糖代谢和积累相关的分子机理，为甘蔗的高糖育种研究奠定了理论基础；通过集成创新育种技术，选育出高糖高抗病的优异中蔗系列甘蔗新品种，构建超大规模抗病、分蘖和高糖甘蔗遗传群体；筛选获得高蔗糖的甘蔗突变体，丰富了高糖甘蔗种质资源，促进我国高糖高产高蓄能的能源甘蔗种质创制，为我国甘蔗的高糖育种和遗传研究提供重要材料，增强我国蔗糖及其制品在国际贸易中的竞争力，具有重要的现实意义。项目执行期内发表相关研究论文11篇，其中中文核心3篇，SCI论文8篇，出版甘蔗育种相关专著1部，部分研究结果申请了相应的知识产权3件，育成高糖甘蔗品种2个，培养从事甘蔗高糖抗病育种研究的优秀研究生3名。