两个荔枝果肉低表达基因启动子克隆与功能鉴定

Litchi (Litchi chinensis Sonn.) is one of the important fruit trees in southern China. However, the development of litchi industry is restricted by the severe reduction of fruit quality during storage, resulted from the postharvest diseaseases, pericarp dehydration and browing. The new diseases-resistant and/or long shelf-life litchi varieties should be obtained using transgenic technology to integrate the exogenous target genes into litchi genome, which express highly in non-edible tissues but have no expression in pulp. That would be a practical scheme for improve productivity and quality of litchi fruit. The key to the scheme is to identify the specific promotors which regulate the low expression of genes in pulp . In our former research, we identified that two genes from litchi , i.e., thaumatin-like protein gene (LcTHL) and mannose/glucose specific-lectin gene (LcLEC), showed markedly lower expression level in pulp than other litchi tissues. And their expression levels in pericarp were all increased after the fruit being harvested. The expressions of the two genes were also inhibited by the water loss of pericarp and the low storage temprature, but were induced by the infection of fungus. The expression pattern of the two genes indicated that their promoters should have important potential value for litchi breeding..The objective of this project is to clone the promotors of the two genes and analyse their characters of core regulation elements and so on. Then the temporal and spatial rules of gene expression regulalted by the promotors will be identified. The results will provide a basis for further study on the use of these two genes and their promoters, and provide a basis for litchi transgenic breeding.

荔枝是华南重要果树之一，但果实采后病菌滋生、果皮褐变、失水变干等严重降低了果实品质，制约着荔枝贸易和产业的发展。采用转基因技术导入在果肉中不表达而在果皮等非食用组中高表达的功能基因，是在不影响食用安全和食用品质前提下培育果实抗病虫和耐贮的新品种，进而提高荔枝产量与品质的可行途径，寻找并鉴定果肉低表达基因的特异启动子是其中的关键。本研究组前期研究中鉴定了两个果肉低表达基因，即荔枝类甜蛋白基因与甘露糖/葡萄糖特异凝集素基因。表达分析表明，这两个基因在果肉中表达量极显著低于其它组织，均在采后荔枝果皮中上调表达，且其表达受果皮失水及低温抑制，受真菌侵染诱导。这种表达规律说明这两个基因的启动子具有重要的潜在育种应用价值。.本项目拟克隆这两个基因的启动子，并分析其结构特征，研究确定其核心调控元件，鉴定其调节基因表达的时空特征，为进一步研究利用这两个基因及其启动子提供依据，并为荔枝转基因育种提供基础。

采用转基因技术导入在果肉中不表达而在果皮等非食用组织中高表达的功能基因，是在不影响食用安全和食用品质前提下培育果实抗病虫和耐贮的新品种，进而提高荔枝产量与品质的可行途径，寻找并鉴定果肉低表达基因的特异启动子是其中的关键。本研究首先克隆了前期研究中鉴定的两个果肉低表达基因的启动子片段，即荔枝类甜蛋白基因上游的2817bp启动子序列与甘露糖/葡萄糖特异凝集素基因上游的1072bp启动子序列，通过生物信息学分析了两者的调控元件以及预测了两者的转录起始位点。然后，分别构建了荔枝类甜蛋白基因和凝集素基因启动子缺失片段正、反向植物表达载体，用农杆菌介导转化荔枝胚性愈伤组织和拟南芥，并且用基因枪轰击荔枝不同组织，结合GUS组织化学染色，证实了这两个启动子对GUS基因的表达调控均具有组织特异性，为进一步研究利用这两个基因及其启动子提供了依据。在上述研究基础上，我们还克隆了两个叶片特异表达基因FKBP16-2和GRX启动子，证实了这两个启动子不能介导GUS基因在果肉中表达。建立了农杆菌注射转化荔枝果实不同组织验证基因表达的技术体系，建立和完善了荔枝花粉管通道法转基因技术体系，为后续研究打下了更坚实的基础。