番茄同源GLKs转录因子调控果实叶绿体发育的功能性冗余研究

Previously, researchers had found that over-expression GLK transcription factors in tomato leaded to develop the number of chloroplast in immature fruit significantly, which affected soluble solid content and lycopene level in ripe fruit. However, functional redundancy of two GLKs transcription factors for chloroplast development regulation in tomato fruit is not illustrated clearly. In this study, we will develop four constructs and transform them in the TILLING mutant background by typical Agrobacteria constructs, moreover acquire four kinds of transgenic lines. Using the qPCR, RNA-seq, electron microscopy and metabolic compounds analysis to complete the research of functional redundancy of homologous GLK transcription factors for chloroplast development regulation in tomato fruit. The purpose of this research was to identify whether expression of solely SlGLK1 in fruit is sufficient to increase fruit chloroplast capacity, determine whether expression of solely SlGLK2 in leaves is sufficient to rescue the mutant phenotype (light green leaves and fruit), and determine whether the up-stream 2.5kb of promoter sequence for each GLK is sufficient to specify SlGLK expression in fruit shoulders (the SlGLK2 promoter) or solely in leaves (the SlGLK1 promoter). The results will illustrate regulatory mechanism of tomato quality enhancing by GLK transcription factors and provide scientific theory of genetic improvement for high quality tomato variety. Moreover, the results will also increase the breeding level for processing tomato.

GLKs转录因子在番茄中的表达显著影响了叶片及未成熟果实叶绿体的数量和形成、进而影响了成熟果实中的可溶性固形物含量和番茄红素水平。但，两个同源GLKs转录因子的功能性冗余特性一直研究的不够深入。本研究拟通过农杆菌介导技术，以Tilling双突变体为背景，将GLKs及其启动子转入双突变体，并运用qPCR技术、RNA-seq技术、细胞组织结构显微观察技术和代谢产物分析技术，完成GLKs转录因子调控番茄果实叶绿体发育的功能性冗余研究，研究结果将进一步明确是否单个SlGLKs在叶片及果实中表达就能足以增加果实叶绿素含量，从而提高果实品质。明确是否每个GLKs上游2.5kb启动子足以能驱使SlGLK在果肩（SlGLK2启动子）或单独在叶片中（SlGLK1启动子）的表达，并阐明GLKs转录因子提高番茄品质的调控分子机制，为高品质番茄品种遗传改良奠定基础，提升加工番茄分子育种水平。

GLKs转录因子能够调控番茄叶片和果实中叶绿体的发育，增强叶片及果实中的光合效率，从而提高果实品质。GLKs转录因子有两个同源基因，其功能冗余特性一直研究的不够深入。本项目以Tilling双突变体为研究背景材料，通过GLKs转录因子的农杆菌转化，获得稳定遗传的基因过表达植株。通过表型观察，在Tilling突变体中过表达SlGLK1能够明显提高叶片中叶绿素的含量，而对果实中叶绿素的含量影响不明显；过表达SlGLK2能够同时提高叶片和果实中叶绿素的含量。在MT中进行了GLKs启动子功能的检测，结果表明不同GLKs转录因子的启动子不能改变叶片及果实表型。通过全转录组测序发现，SlGLK1仅有少数基因受到调控参与到光系统反应中，而SlGLK2在果实中过表达后能够引起果实中更多基因参与到光合反应、次生代谢和糖原代谢的过程中，为果实品质的提升提供了分子基础。ATAC-Seq的结果与转录组相似，过表达SlGLK2的果实染色质开放的富集区域含有多个光合反应所需的叶绿素a/b结合位点及糖转运蛋白结合位点。通过透射电镜对叶片和果实叶绿体的观察，SlGLK1和SlGLK2均能够使叶片中的叶绿体数量增多，叶绿体的基粒层变厚，仅SlGLK2使果实中的叶绿体基粒层变厚。本项目从细胞生物学和分子生物学角度明确了SlGLK1仅能够提高叶片中叶绿体的数量及叶绿素的合成，而SlGLK2不仅在叶片中发挥作用，还能够通过增加果实细胞中叶绿体的基粒层来提高光合作用，同时在糖转运蛋白的协同作用下提高果实中可溶性固形物的含量。