番茄6号染色体上3个抗根结线虫基因Mi-HT、Mi-1和Mi-9的功能比较及应用研究

Root-knot nematodes disease is one of the most important disease in tomato production. Using resistant varieties is the most economical and effective prevention measures. To date, Mi-1 is the only gene that has been used in tomato varieties in production. However, the resistance of Mi-1 is not stable, and inactive especially when the soil temperature is above 28 °C. Developing varieties with heat-stable resistance to nematode is desirable in tomato breeding programs. After introducing and years of breeding, we finally got a inbred line ZN17 which possess resistance to Meloidogyne incognita at moderate temperature and high temperatures (32 °C). Genetic analysis and fine mapping suggested a single dominant gene Mi-HT is the resistant gene. Mi-HT, Mi-1, and another heat-stable nematode resistant gene Mi-9 were located on the same region of chromosome 6, and were proved to be homologous gene through function analysis. For clarifying the relationship of these three genes and better using of these resistant tomato lines in the production, this program focus on cloning these three genes by homologous cloning technology and comparing the differences of nucleotide sequences. Compare the differences of function through gene overexpression and gene silencing technology. Then analyze the expression level and subcellular localization of these genes in different temperature condition. This provides the theory basis for preliminarily clarifying the heat stable resistance machanisms. Meanwhile, according to the difference of the secquences, specific molecular markers was designed and used in the breeding process of new tomato lines. This lays the foundation for breeding disease resistant varieties.

根结线虫病是番茄的重要病害之一，采用抗病品种是最经济有效的防治措施。目前生产上的抗病品种都只含有Mi-1基因，但该基因的抗性并不稳定，特别是在土温高于28℃时就出现抗性丧失的现象，因此培育具有热稳定抗根结线虫病的品种是番茄育种的重要目标之一。通过引种和选育获得的栽培番茄自交系材料ZN17在常温和高温（32℃）都具有稳定的抗根结线虫能力，其抗性由单显性基因Mi-HT控制，该基因与Mi-1以及另一个热稳定抗根结线虫基因Mi-9位于6号染色体的同一区段。为明确这3个基因的关系，以便在生产上更好地利用这些抗病材料，本项目拟采用同源克隆技术获得这3个基因，比较它们核苷酸序列间的异同，通过过表达和基因沉默技术分析其功能差异，并对各基因在不同温度条件下的表达及亚细胞定位进行分析，为初步阐明热稳定抗性机理提供依据；同时，根据各基因的序列特征设计特异分子标记，并用于新材料的创制过程，为选育抗病品种奠定基础。

番茄根结线虫病是番茄重要的病害之一。种植抗病品种是最经济、有效的防治方法。但含有Mi-1基因的番茄品种，在高温条件下就丧失了对根结线虫的抗病性。发掘、利用热稳定抗根结线虫基因，对生产具有重要的应用价值。对其热稳定抗病机理的研究，不仅可以加快对热稳定抗根结线虫基因的利用，还可以为其他作物热稳定抗病性的研究提供依据。本项目对含有热稳定抗根结线虫基因Mi-HT和Mi-9的番茄材料进行病毒诱导基因沉默，证实Mi-1、Mi-9均与热稳定抗根结线虫基因Mi-HT具有同源性；以Mi-1序列信息设计全长扩增引物，在ZN17、LA2157番茄材料中对Mi-HT和Mi-9基因进行候选基因的分离，在两份材料中分别获得3个和2个与Mi-1基因同源的基因。通过对这些基因进行序列比对，筛选出其中的4个基因，将其转入感病番茄材料micro-tom中。通过对转基因株系抗性鉴定，确定ZN17中的288-41基因和LA2157中的2157-3基因在高温条件下可以赋予植株对根结线虫热稳定的抗病性。亚细胞定位证实，这两个基因均在细胞质中表达。叶绿素荧光参数测定，证实常温接种线虫处理后，抗病品种光化学效率没有显著变化，但是感病材料光化学效率下降。将根结线虫与番茄黄化曲叶病抗病基因建立多重PCR体系，实现了对抗病品种的快速选育。此外，本研究还对BTB蛋白家族基因进化进行了分析，为探索其中抗病相关基因的进一步研究奠定基础。

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