| Eggplant Verticillium wilt is a disastrous plant vascular disease causing severe yield and quality losses in eggplant. Practice shows that resistant variety is the most effective way for V. wilt control. However, little progress has been made in eggplant breeding for resistance to V. wilt. The main reasons are the lack of full understanding about the mechanism of Verticillium resistance and the rare resistant materials. Therefore, study on the mechanisms of V. wilt resistance and cloning of resistance-related genes in Solanum torvum could provide theoretical and practical basis for eggplant breeding in resistance to V. wilt.In this thesis, firstly, the physiological mechanisms in response to V. wilt were analyzed by comparing the physiological parameters of Solanum torvum and Suqi eggplant infected with V. dahliae, then the differentially expressed genes were identified using cDNA-AFLP in S. torvum infected by V. dahliae, finally, three pathogenesis-related genes were isolated by homology cloning and RACE, and their expression were analyzed by RT-PCR. The main results are as follows:1. The mechanisms of S. torvum in response to V. dahliae infection. We analyzed the resistance of S. torvum to V. dahliae, and found that the S. torvum had strong self-defense and self-repair capacity. A number of related physiological parameters in vivo analysis showed that:(1) The active oxygen scavenging system (such as:SOD, POD, CAT enzymes activity) in S. torvum was higher than in Suqi during the treatment. MDA and soluble protein content in S. torvum decreased after treatment, but increased in the Suqi. The results suggested that infection might activate active oxygen scavenging system in vivo, then producing certain defensive substances (such as lignin and chlorogenic acid et al.) quickly, while reducing the accumulation of MDA in the body, and then caused to a minimum harm. (2) The response time of the physiological parameters in S. torvum in response to V. dahliae was different:Activity of POD, PAL and content of soluble protein in S. torvum change rapidly within 12h, and then, change in activity of SOD, PPO, CAT and MDA content. Eventually, S. torvum defense against V. wilt effectively by combinations of the enzymes.2. Anaylise of differential geneexpression profiling in roots of S. torvum after V. dahliae inoculation. Optimization of cDNA-AFLP technology system applications in S. torvum. (1) Analysis of differential gene expression profiling in S. torvum after V. dahliae inoculation, a total of 118 differential transcript-derived fragments (TDFs) were isolated, which were divided into four kinds based on expression pattern: induced expression, inhibited expression, inducible up-regulated expression, and transient expression. (2) Functional analysis on showed that 50 of these TDFs belonged to function-known genes which mainly involved in seven biolgical processes:Metabolism, Cell rescue, defense and virulence, Cell cycle and DNA processing, Biogenesis of cellular components, Protein synthesis, Proteins with binding functions, and transport et al. The results showed that response of S. torvum resistance to V. wilt was a multifaceted, multi-level complex physiological process.3. Cloning and Expression of Stcyp450、Strpll6 and StDAHP from S. torvum. (1) A gene named Stcyp450 (Solanum torvum cytochrome P450) was isolated from S. torvum, the full length of the cDNA contained an open reading frame of 1536 bp coding a protein of 511 amino acids, corresponding to a 58.08 kD polypeptide with an isoelectric point of 9.17. Alignment of the amino acid sequence of Stcyp450 and that of other homologues showed that Stcyp450 had a high identity with CY77A1、CY77A3 and CY77A4 from Arabidopsis and Glycine at amino acid level. Analysis of Stcyp450 mRNA level by semi-quantity RT-PCR showed that it was induced up-regulated by V. dahliae infection, and restorated at the late infection. (2) A gene named Strp116(Solanum torvum ribosomal protein L16) was isolated from S. torvum, the full length of the cDNA contains an open reading frame of 498 bp coding a protein of 165 amino acids, corresponding to a 42.16 kD polypeptide with an isoelectric point of 5.2. Alignment of the amino acid sequence of Strpl16 and that of other homologues showed that Strpl16 had a high identity with RPL16 from five kinds of plants at amino acid level. In addition, the conserved 12-amino acid Ribosomal protein L16 signature 2 (RMGsGKGspeyW) was observed in N-terminal end of this protein. The deduced amino acid sequence and clustering analysis revealed that Strp116 was colse to RPL16 in S. tuberosum, S. lycopersicum, and C. annuum. Analysis of Strpl16 mRNA level by semi-quantity RT-PCR showed that it belongs to constitutive expression, and its expression increased during infection. (3) A gene named StDAHP (Solanum torvum 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) was isolated from S. torvum, the full length of the cDNA contains an open reading frame of 1683 bp coding a protein of 560 amino acids, corresponding to a 62.53 kD polypeptide with an isoelectric point of 9.1. Alignment of the amino acid sequence of StDAHP and that of other homologues showed that StDAHP had a high identity with DAHP from four kinds of plants at amino acid level (S. tuberosum, S. lycopersicum, N. tabacum, and O. sativa). In addition, the amino acids are more conservative near the N-terminal and C-terminal in StDAHP. Analysis of StDAHP mRNA level by semi-quantity RT-PCR showed that its expression was induced by V. dahliae infection. |
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