苹果MdTMT1和MdTMT2在果实糖代谢与积累中的作用及其调控分析

Sweentness as a major factor in Fruit quality, is mainly contributed to accumulation of soluble sugars in vacuole. TMT located on tonoplast may play an important role in stweetness formation. Previous works found that expression abundances of MdTMT1 and MdTMT2 were realted with sugar content in apple fruit. In this program, to understand function and regulation mechanism of MdTMT1 and MdTMT2 for sugar accumulation in apple fruit, the following works will be done: (1) detect their expression characteristics via subcellular localization, qRT-PCR and in situ hybridization technique, (2) investigate their capability to transport Fru and other sugars as well as their characteristics in yeast system, (3) over-express or silence MdTMT1and MdTMT2 respectively in cellus of apple fruit to study infulence of both expression level to sugar concentrations and absorbing capbilities in cellus of apple; (4) over-express two genes into tomato to analyze their effect on growth, sugar metabolism and stress tolerence; (5) isolate the promoters of MdTMT1 and MdTMT2 from apple to invesitigate controling characteristics of MdTMT1 and MdTMT2 expression，and using yeast single hybrid method to hunt the transcript factor involved in expression regulation for both genes and confirm them；(6) investigate the relationship of both sugar metabolism and TMT expression under different water or N conditions or daily temperature. This study aim is to understand the functions of MdTMT1 and MdTMT2 and relationship of them with sugar accumulation in fruit of apple, and gives new insights into mechanism of sugar transportation and accumulation. These results would serve to improvement and breeding for sweetness.

甜度取决于糖在液泡中的积累，是评价果实品质的主要指标。果实甜度形成主要依赖于液泡糖转运蛋白。前期基因筛选结果表明MdTMT1和 MdTMT2与苹果果实中果糖（Fru）和蔗糖（Suc）的积累相关。为探明MdTMT1和 MdTMT2与果实糖积累的关系，本项目拟通过亚细胞定位、qPCR、免疫原位杂交研究二者在苹果果实中的表达特性；酵母系统中研究它们对Suc、Fru等糖的转运能力及特性；在苹果愈伤组织中过量表达或干扰，分析表达量改变对Fru、Suc等糖含量及吸收能力的影响；在番茄中过量表达，研究过量表达对番茄生长发育及果实糖代谢的影响；分离二者启动子，分析其表达调控特性与环境和糖信号的关系；同时研究水分、N素供应及昼夜温差对苹果果实糖积累调控与MdTMT表达的关系。目的在于探明MdTMT1和 MdTMT2与果实糖积累的关系及调控机制，为认识果实中糖积累及调控机制和甜度品质改良与育种奠定理论基础

为探明苹果MdTMT1和MdTMT2在果实糖积累中的功能及糖在液泡中的积累调控途径，项目组按照研究计划和实施细则，首先利用qRTPCR、蛋白质组、启动子分析等研究发现MdTMT1/2表达在苹果中与糖含量显著相关，且受糖浓度、干旱、低温等诱导；通过拟南芥原生质体和苹果免疫原位杂交确定MdTMT1/2定位于液泡膜。在酵母缺失体EBY.VW-4000中表达证实，MdTMT1/2对Suc、Fru、Glc均有转运活性，其中MdTMT1对Suc转运活性高于己糖，而MdTMT2对Fru和Glc的转运活性高，且不同糖之间存在竞争性。在苹果愈伤组织中，MdTMT1过量表达能显著增加Suc、Glc和Fru含量，而干扰抑制了生长，降低了糖含量。在MdTMT1/2 GL3转基因苹果和番茄叶片中，MdTMT1和MdTMT2过量表达均增加了Suc含量50%以上，但Glc和Fru含量降低，而转基因番茄果实中的SSC增加了60%以上，且Suc、Glc和Fru含量均增加了近1倍。对转基因苹果和番茄叶片糖代谢和转运的研究发现，MdTMT的过量表达增加了叶片中FRK2和ERD6.7的表达，抑制了转基因植物叶片中Glc和Fru的积累。为进一步探明FRK2和ERD6.7与TMT表达及糖积累的关系，项目组对二者开展了相关研究，发现FRK2控制着胞质中Fru及Glc和Suc的C流，影响着TMT的表达及其向液泡中转运Fru的潜力；而EDR6.7可通过将液泡中Glc的外排，产生胞质与液泡间瞬间Glc化学势梯度，诱导TMT的表达，增加液泡中糖的积累。通过本项目的研究，建立了苹果等果实中糖积累的调控途径，完善了糖调控理论，为水果糖酸改良提供方法和基因资源。项目已发表相关论文5篇，其中SCI论文3篇，中文核心2篇，投出再审文章2篇，培养已毕业硕士研究生5名，在读博士2名。同时本项目的研究为进一步探明苹果糖稳态调控机制奠定了材料和方法基础。