| As a kind of cytokine, Chicken interferon-gamma(ChIFN-?) has a broad spectrum of high antiviral activity, antibacterial activity and anti-parasite activity in vivo and so on. The Chicken ChIFN-? resistance mechanism and immune regulation mechanism have been investigated clearly yet. It was sure that ChIFN-? can significantly improve insect resistance in tobacco. Transgenic ChIFN-? increased obviously in density of glandular trichome and the weight of the exudates per unit area, and it speculatled that characters improved resistance to herbivores attack of tobacoo. And many differentially expressed genes was screening by gene expression microarry of transgenic ChIFN-?. CyP71, LBM1 and Isp H were screening from those genes through GO and KEGG Pathway analysis and identified by fluorescence quantitative PCR. Cy71, Lbm1 and Isp H might play an important role in plant trichome development. So this work focused on the function of three genes, to investigate overexpression of genes by gene cloning and the genetic transformation of Arabidopsis Thaliana.With a one-step RT-PCR method, Nicotiana tabacum CyP71, LBM1 and Isp H gene has been successfully cloned. The full-length CyP71 DNA sequences contained 1545 bp, which includes 1545 bp ORF and encodes 514 amino acids residues, highest content of leucine(11.9%). Protein was predicted molecular mass of 58.11 k Da and an isoelectric point(p I) of 8.49 by biological information science. Protein sequence analysis showed there was no signal peptide sequence and transmembrane helix structure in CyP71. This protein was a stable protein which has a instability index of 38.04. Blast analysis indicated that the CyP71 nucleotide has a highest homology beyond 88% with Solanum tuberosum. The phylogenetic tree analysis showed that CyP71 was conservative in the evolution process.The length of LBM1 DNA sequence was 846 bp, as its ORF was. LBM1 encoded a 281 amino acids residues peptide, among which serine content is the highest(9.6%). Protein was predicted molecular mass of 31.98 k Da and an isoelectric point(p I) of 5.08 by biological information science. The instability index of this protein is 48.07, it's an unstable protein. There was no signal peptide sequence and transmembrane helix structure in LBM1. Protein structure domain analysis found that LBM1 hit a SANT protein- protein interactions domain, and it belongs to the MYB transcription factors.Sequence analysis indicated that the open reading frame length of Nicotiana tabacum Isp H gene was 1386 bp and would encode a 461 amino acids resudes peptide, among which the content of glutamate and lysine were 8.7% and 8.5% respectively. Protein was predicted molecular mass of 51.66 k Da and an isoelectric point(p I) of 5.59 by biological information science. It was a stable protein and had predicted target signal peptide into mitochondria or chloroplasts. The amino acids sequence homology among Solanum tuberosum, Solanum lycopersicum and Nicotiana tabacum were beyond 92%. Protein structure domain analysis results showed that Isp H belongs to Lyt B super family, which were the catalytic for the last reaction of MEP pathway.Plant overexpression vector pSH737-Isp H, pSH737-CyP71 and pSH737-LBM was constructed, and transformed into Arabidopsis by Agrobacterium-mediated method. Seeds was collected and screened after genetic transformation of Arabidopsis, then many resistant plants that Cy P71 for 38,LBM1 for 35,Isp H for 30 were screened. Three genes transformation rates were all over 5%. The result of genome PCR rapid identification and GUS staining showed that Isp H was transforred into Arabidopsis thaliana successfully. Compared with wild Arabidopsis thaliana, transgenic Arabidopsis thaliana had deeper color and growed better. The results of GUS staining showed that all GUS reporter gene in trichomes cells of transgenic Arabidopsis thaliana was promoted and expressed. Trichome cells were blue with microscope, indicating that Isp H was related to development of trichome. The size and distribution of trichome had no significant difference betweent wild and transgenic Arabidopsis thaliana, while the latter had more nubmber. Statistical analysis showed that the average number of trichome in wild and transgenic Arabidopsis thaliana were 19.6 and 9.6 respectively. The number of trichome in transgenic Arabidopsis thaliana had more than doubled, indicating that the expression of Isp H and the development of trichome were positively associated. Resistant plants LBM1 and CyP71 were still young and showed no significance difference on young stage in trichome between wild transgenic Arabidopsis thaliana. And their function needed further observation and investigation. This paper laid the foundation for explaining the mechanism of ChIFN-? regulating development of tobacoo trichome. |
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