甘肃地方李光桃抗寒基因挖掘及其抗寒机理

Gansu Hexi Corridor area is not suitable for the introduction of peach cultivars, but the local variety "LiGuangtao" has been able to survive in the extreme low temperature of minus 30 degrees since the history. There are few studies on genes relatived with peach cold resistance and its resistance mechanism is not clear. Using peach germplasm resources growing in cold regions, exploring cold resistance gene and the creation of new cold hardness germplasm, is the most effective way to solve the problem of low temperature damage on peach trees’ stem and branchs in winter. This project is based on the privious research of screening of cold resistance indexes for Hexi corridor local peach varieties which has the special characteristics of cold hardness, even under -30 ℃ condition. The local cultivar ‘Liguangbolicui’(cold resistant) and normal ‘Longmi 9’(cold sensitive ) will be used as materials to study mRNA expression differences at Low temperature is a limitation factor of the cultivation region for fruit trees. There are few studies on genes relatived with peach cold resistance and its resistance mechanism is not clear. Using peach germplasm resources growing in cold regions, exploring cold resistance genes and the creation of new cold hardness germplasm, is the most effective way to solve the problem of low temperature damage on peach trees’ stem and branchs in winter. This project is based on the privious research of screening of cold resistance indexes for Hexi corridor local peach varieties which has the special characteristics of cold hardness, even under -30 ℃ condition. The segregation population F1 from the parents of the ‘Liguangbolicui’ (cold resistant) and ‘Longmi 9’ (cold sensitive ) hybridization are used as materials, to screening SNP marker closely linked with the target gene, combinated with the field and laboratory cold resistance test result and the construction of molecular genetic map, the peach cold resistance gene on chromosome location, the number of genes, genetic pattern and genetic effect will be clear. The winter-shoot from local cultivar ‘Liguangbolicui’(cold resistant) and normal ‘Longmi 9’(cold sensitive ) will be used as materials to study mRNA expression differences at low temperature, find low temperature induced genes depended on the transcriptome sequencing after gradient low temperature treatment, after gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, screen differentially expressed genes associated with cold treatments, combinated with the QTL result and F1 generation cold resistant phenotype data, study the validation of SNP loci from QTL on the RNA level, identify cold resistance key genes. The objective is to reveal the mechanism of cold resistance. The related research results has significant importance to accelerate the breeding process of cold resistant peach.

甘肃河西走廊地区不适宜于引进桃品种栽培，然而当地品种“李光桃”却在零下30℃极端低温环境长期生存，其抗寒机理尚不清楚。发掘高寒区桃资源抗寒基因、培育抗寒品种是解决桃树枝干低温冻害的有效的途径。本项目利用抗寒李光桃“李光玻璃脆”与不抗寒普通桃“陇蜜9号” 杂交F1代群构建高密度遗传图谱，对抗寒性状进行表型鉴定和数据收集，定位抗寒性状紧密连锁的QTL位点及其在染色体上的位置，解析这些主效QTL与抗寒性的关系，明确桃抗寒相关基因在染色体上的位置、遗传方式、基因数量及其遗传效应；以两亲本冬季枝条为材料，经低温处理后进行转录组测序，经基因差异表达分析，对这些差异基因进行GO富集和KEGG富集分析，筛选与抗寒相关的差异基因，对其功能和表达模式进行分析，结合QTL、F1代抗寒表型数据，从RNA水平验证其QTL得到的SNP位点，确定候选基因，揭示河西李光桃抗寒机理，为加快抗寒桃遗传育种进程提供科学依据。

摘要:桃（Prunus persica L.）原产于中国，属蔷薇科（Rosaceae）李属（Prunus）桃亚属（Amygdalus），是我国的主栽果树之一。中国栽培的桃品种有1000多个，其中大多数生长在温带和亚热带地区，温度是影响桃树生长发育以及地理分布的一个重要环境因子。中国河西走廊地区平均最低温度约为−27℃，引进的桃品种无法在这种环境下生存。然而，属于当地李光桃抗寒品种群的‘丁家坝’桃品种却能在这种环境下生存并很好的适应这些环境，进化出一种高度专一的耐寒特性。本研究以抗寒型‘丁家坝’和低温敏感型‘加纳岩’为实验材料：(1)研究抗寒性鉴定的方法—电阻力法。(2)获得杂交种F1代。（3）测定4℃低温胁迫下两种桃叶片的生理生化指标，明确低温胁迫下两种桃叶片的生理生化变化。（4）利用高通量测序技术，在转录组水平对比分析抗寒型‘丁家坝’和低温敏感型‘加纳岩’桃叶片响应低温胁迫的分子机制，筛选抗寒基因，深入解析‘丁家坝’的抗寒机制。(5)对低温胁迫下的‘丁家坝’休眠枝进行转录组分析，进一步解析‘丁家坝’的抗寒机制。（6）采用生物信息学方法和qRT-PCR系统分析了桃NAC基因家族的成员、结构、功能和冷处理下的表达特征。（7）对筛选到的耐寒基因Pp4CL2进行克隆与转化，并对其功能进行初步探究。结果表明：.(1):与‘加纳岩’相比，‘丁家坝’的细胞膜不易被破坏，抗寒性强。.(2):‘丁家坝’鉴定到的569个差异基因显著富集在植物与病原菌互作、MAPK信号通路-植物和倍半萜和三萜生物合成等代谢通路；MAPK级联反应的功能酶基因MPK3/6以及WRKY22、WRKY24和WRKY33等相关基因具有较高表达。.(3):‘丁家坝’一年生枝条韧皮部的转录组分析表明，与信号转导相关、激素调控、碳水化合物和脂质代谢的DEGs显著富集。.(4):桃NAC基因家族生物信息学分析表明，桃NAC家族共有119个成员，氨基酸长度在68~862之间，分子量介于7.2~96.1kDa，等电点在4.3~9.5之间。.(5):过表达Pp4CL2基因增强了转基因拟南芥和烟草对低温的耐受性。