| Tomato(Solanum lycopersicum L.)belongs to the family Solanaceae,which is one of the vegetables with the largest cultivation area in China.In recent years,with the increase of multiple cropping index and facilities cultivation area,the disease of tomato root-knot nematode is becoming more and more serious,The production of tomatoes has been severely reduced or even eliminated.The breeding of anti-root-knot nematode cultivars is undoubtedly the most scientific and effective way for controlling root-knot nematode diseases.Mi-1 gene is the only root-knot nematode resistance gene that has been successfully used at present,but this gene has thermal instability.Once the soil temperature is higher than 28?,it will lose its resistance to root-knot nematodes and pose a great threat to the cultivation and production of tomatoes.In this thesis,the effects of high temperature stress and root-knot nematode infection on related physiological indexes of tomato were studied.Two candidate genes of heat-stable anti-root-knot nematode Mi-9 were cloned and obtained by Agrobacterium-mediated genetic transformation method.Tomato transgenic lines resistant to root-knot nematodes at high temperatures was obtained.The main results are as follows:1.Seedlings stage of tomato under high temperature stress and root knot nematode infection conditions,the growth of tomato lines was inhibited to a certain extent;photosynthetic electron transport process of tomato leaves was inhibited;photochemical quantum yield,photosynthetic electron transfer rate,and photochemical quenching coefficient were significantly reduced.However,The thermal stability of the root-knot nematode material '288' had the smallest decrease and the susceptible material 'Money Marker' had the largest decrease.The fluorescence parameter qN showed an increased trend after the root-knot nematode infection,but the increased range was different,and the thermal stability was stable.The growth rate of root knot nematode material '288' was the largest,and that of 'Money Marker' was the least.The difference between the two was significant.The heat-sensitive material 'VFN' was between the two;under adverse stress conditions,the leaf chlorophyll content of all the materials,dry and fresh weight decreased significantly,and root activity and root antioxidant enzyme activities significantly changed.There were significant differences among different genotypes of tomato materials.Compared with the control,the chlorophyll content and dry and fresh weight content of '288' in the heat stable and anti-root-knot nematode material had the smallest decrease.The'Money Marker' had the largest decrease in the heat-sensitive material and the 'VFN' material in the heat-sensitive material was in the middle;Under the conditions of compound stress and nematode infection,The root activity of the tomato was lower than that before inoculation,and its decreased range from high to low was 'Money Marker'>'VFN'>'288';The activities of SOD?POD and CAT in roots of heat-stable anti-root-knot nematode '288' increased significantly and the increase was higher than that of other tomato materials under compound stress conditions.The activities of SOD,POD and CAT in heat-sensitive material 'VFN' and 'Money Marker'showed a rising trend under single stress conditions,decreased significantly under combined stress conditions,and the activities of antioxidant enzymes were significantly lower than high-temperature stress.2.Using PCR technology,using the 'Motelle'gene sequence of tomato with Mi-1 gene as a template,two candidate of Mi-9 gene were cloned by homologous cloning method,which were 2157-1 and 2157-3 genes,respectively.The DNA sequence analysis showed that the homology of 2157-1 and 2157-3 was 96.35%,and the homology with the Mi-1 gene was 90.47%and 93.13%.The total length of nucleotides in 2157-1 and 2157-3 genes is 3984 bp and 4000 bp.The 2157-1 and 2157-3 genes vwere integrated with the protein sequences of the Mi-1 gene.The results showed that 2157-1 and 2157-3 had 93.16%homology,which were identical to the amino acid sequence of the Mi-1 gene(90.99%and 95.77%,respectively).The 2157-1 and 2157-3 protein consists of 1271 and 1276 amino acids respectively.Using the Mi-9 candidate gene as the target gene,the pBI121+2157-1 and pBI121+2157-3 expression vectors were successfully constructed,and the target genes 2157-1 and 2157-3 were successfully imported using Agrobacterium-mediated genetic transformation methods to cultivated tomato "Micro-tom".Through PCR detection and sequencing analysis of the transgenic plants,26 out of the 44 tomato transgenic plants amplified specific bands,including 18 of 2157-1 transgenic plants and 8 of 2157-3 transgenic plants,with an average positive rate of 59.1%. |
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