The Watermelon Wilt Induced Suppression Subtractive Hybridization CDNA Library And EST Analysis

In this study, watermelon variety with high resistance to Fusarium wilt race1Calhoun Gray’was used as materials, the root tissue after inoculation with Race1and distilled water0,6,12,24,48,72h were sampled as Tester and Driver, and the SSH-cDNA forward library was constructed by SSH technology. The sequences of EST in the SSH library were analyzed by bioinformatic measure and RT-PCR. The main results are as follows:1、The light intensity of28S is two times of18S in Total RNA, the figure of A260/A280is1.8～2.0. Double strained cDNA scattered and distributed from0.2kb to4kb. Restriction fragments distributed from0.1kb to4kb, mainly focuse on200bp-750bp. suppression subtractive fragments distributed from150bp to800bp, mainly focuse on500bp. Cloning efficiency reached more than80%. The forward SSH library contained more than1400clones.2、Partial clones were randomly picked to test false positive by reverse Northern dot-blot, the results show that more than85%clones were positive clones.3、Three hundred positive clones were sequenced randomly, and a total of259differentially expressed sequences were screened out, and167of them were quite similar to known genes, accounting for65.5%. Sixty-four genes related to disease resistance and defense, accounting for24.7%. Homologous genes involved in resistance signaling proteins, disease resistance protein, HR (hypersensitive reaction) and SAR (Systemic acquired resistance) related proteins, transcription factors, defense responses and secondary metabolite biosynthesis protein, plant cells protective protein.4、According to the sequences of Peroxidase and Aquaporin gene in this library, the gene expression of peroxidase and aquaporin were analyzed by RT-PCR. The results showed that Aquaporin and Peroxidase both increased after inoculation with fusarium wilt physiological race1, suggesting that peroxidase enzyme and aquaporin enzyme were related to the anti-wilt character of "Calhoun Gray".