枣抗裂果实性状与抗裂基因的鉴定研究

Shaanxi is one of the important production area of jujube in China. But the phenomenon of fruit cracking was seriously in the mature stage in jujube. The early work had identified 2 variations with high crack resistant in main planting variety “Muzao”. The correlation between shape and crack resistance of jujube fruit was determined. In this study, in order to screen the fruit characters related to crack resistance, the development process of fruit was be observed, and the fruit characteristic including crack resistance, characteristics of pulp flesh, fruit shape and nutrient content was measured. Then, the cDNA library related to difference fruit characteristic was constructed using RNA sequencing (RNA-Seq), and the genes related to difference fruit characteristic was filtering through comparison of transcriptome in fruit. Further, the transgenic material was acquired through agrobacterium-mediated transformation. This project can provide scientific and practical basis for breeding of new variety based on crack resistance genes and the solution of the cracking problem in jujube.

陕北是我国重要的枣产区之一，枣裂果现象严重。前期工作发现并鉴定了陕北主栽品种木枣及其2个高抗裂变异系，确定了枣果形状与抗裂的相关性。因此，本项目拟通过木枣及其高抗裂变异系的比较研究，观察枣果发育的全动态过程，检测外部果形、果皮果肉、营养成分等果实特性，筛选抗裂相关的果实性状，确定裂果发生的脆熟期与抗裂的相关性。利用RNA-Seq技术构建果实性状差异cDNA文库，通过枣果转录组高通量测序，筛查果实差异性状相关基因，鉴定抗裂相关基因，构建农杆菌质粒，获得抗裂转基因枣苗。项目将为基于抗裂基因的枣树新品种选育和裂果防治提供科学与实践依据。

枣裂果现象普遍严重，抗裂机理探讨和抗裂品种培育是解决枣裂果问题的根本途径。为鉴定枣抗裂相关果实性状和基因，基于木枣抗裂变异系的发现，以木枣、变异系V1、V2和狗头枣为材料，通过果实性状测定和抗裂性分析筛选到木枣优良抗裂变异系V2。品种的抗裂性与果形、肉核比、梗洼宽度、枣果还原糖、可溶性总糖、纤维素含量有关。枣果不同部位果皮厚度一致、果皮细胞紧实，抗裂性好。采用RNA-Seq技术构建枣抗裂相关转录组文库，木枣与V2间筛选出50个差异表达基因，26个基因表达上调、24个基因表达下调。木枣未裂果与裂果间筛选出12个差异表达基因，8个基因表达上调、4个基因表达下调。GO富集分析生物学过程在品种抗裂性中发挥重要作用。KEGG富集分析大多数基因分布在代谢相关通路中，尤其是淀粉和糖代谢、嘌呤和嘧啶代谢等通路，个别分类在遗传信息处理通路中，且都未被分类在细胞过程、环境信息处理过程等通路中。qRT-PCR定量结果与测序后基因表达定量结果具有较高的一致性。对于所验证的基因，V2中26-Z1681（PHD finger蛋白）基因相对表达量最高，39-C9451（纤维素合成酶D5）基因相对表达量最低，几乎无表达。裂果后54-C5274（bc1复合蛋白激酶）相对表达量最高，62-C7532（羧酸酯酶12）基因相对表达量最低。依据基因的表达量差异和代谢通路分析，采用RT-PCR技术克隆了2-C5027（受体蛋白质Cf-9）、15-C1327（半胱氨酸蛋白酶抑制剂）、24-C6179（组蛋白乙酰化酶）等基因的cDNA序列，并进行了时空表达模式分析。利用同源重组方法构建植物过表达载体，以Micro-Tom番茄受体，采用农杆菌侵染法分别将3个基因导入，获得16、10、14株阳性苗，移栽后进行基因功能验证。枣抗裂果实性状和抗裂基因的鉴定，可为抗裂机理揭示和抗裂品种培育提供科学和实践依据。