柑橘果实高表达基因CsMT3的功能解析

Plant Metallothioneins（MTs）are postulated to involve in some important physiological processes, such as stress response, embryo development and fruit ripeness. So far, such roles are mostly supported by gene expression profiles not by direct evidence. In our previous study, we explored the mechanism of lycopene accumulation in the red-fleshed sweet oranges via EST and MPSS analysis. A type III metallothionein-like homolog（CsMT3）was found to be the most abundant transcripts in citrus fruits. The expression of CsMT3 was relatively lower in fruits of mutants than that of their respective wild-type, suggesting the gene was down-regulated by the mutant allele and may play important roles in the fruit development. Here the functions of CsMT3 in stress response, plant development and fruit quality formation were further investigated. The study is mainly presented as follows: the expression profiles of CsMT3 will be analyzed by quantitative real time RT-PCR analysis; in vitro assay of recombinant CsMT3 protein will be detected; the over-expression and RNAi manipulation of CsMT3 will be performed using short-juvenile germplasm 'Shanjingan' as explants. Then the phenotypic characteristics and gene microarray analysis of transgenic lines will be carried out to verify the CsMT3's functions and metabolic pathways regulated by CsMT3. The study will provide a direct evidence for the important applications of CsMT3 in Citrus improvement and germplasm innovation.

目前对植物金属硫蛋白（MT）的功能研究多局限于根据其表达行为所进行的推测，认为其可能参与了许多重要的生理过程如抗逆反应、胚胎发育及果实成熟衰老等。我们前期在对柑橘红肉突变体番茄红素积累机制的研究中，分离到了一个在果实中高丰度表达的MT基因（CsMT3），该基因在突变体及其野生型果实中差异表达，受红肉位点负调控，说明其极有可能在柑橘果实发育过程中发挥着某些重要的作用。本项目拟进一步对CsMT3在柑橘抗性反应、植株发育尤其是果实品质形成中的作用进行深入探讨。主要包括通过实时荧光定量PCR技术分析CsMT3的时空及诱导表达特性；原核表达检测CsMT3重组蛋白的体外活性；构建CsMT3基因的超量表达和干涉载体，转化柑橘短童期种质山金柑，观察转基因株系表型，用基因芯片检测基因表达变化，进而获知CsMT3所参与的生理过程和生化反应路径，为未来利用该基因进行柑橘种质创新和品种改良提供重要理论依据。

本项目重点研究了柑橘果实中高丰度表达的CsMT3基因的基因组结构、表达模式以及生物学功能，并在全基因组水平分离鉴定了柑橘CsMT基因家族所有成员。研究表明，CsMT3蛋白是一个编码68aa的小分子蛋白，具有type 3 MT蛋白Cys分布的典型特征。该基因在柑橘所有组织中均表达，在果实中的表达量极高；其在果实发育过程中的表达趋势与ABA含量变化趋势一致，在果实转色期（150DAFB）的表达量在红肉突变体和野生型间有显著差异，表明该基因可能对于果实转色具有重要作用，受到红肉位点的负调控；进一步的ABA和胡萝卜素环化酶抑制剂CPTA处理实验发现，该基因表达可受低浓度ABA的诱导，而CPTA处理增加了番茄红素的含量，显著抑制了CsMT3基因的表达，表明番茄红素和CsMT3基因表达之间存在负向调控关系。体外活性实验表明，CsMT3具有保护核内DNA免受自由基氧化损伤的作用；同时构建了CsMT3超量和RNA干涉表达载体，分别转化短童期种质早实枳和山金柑，获得超量表达株系8个、干涉株系3个，相关生理生化指标测定以及抗逆性评价因植株弱小未开花结果尚未完成。此外，从柑橘基因组中鉴定了四种类型的MT蛋白，其中CsMT1和CsMT4为前人未报道过的柑橘MT基因；4个CsMT基因具有不同的组织器官优先表达特性，CsMT1在花和叶中表达最为丰富，CsMT2主要在根中表达，CsMT3 和CsMT4在果实中表达量高，表明不同的CsMT基因在不同组织器官的生长发育过程中发挥作用。相关研究结果为全面解析植物MT蛋白的生物学功能提供了理论依据。