| Malus hupehensis, originated in China, has shown strong resistance to various apple pathogens and is an important materials to study woody plant resistance mechanisms. In this study, we constructed the complete cDNA library of Malus hupehensis treated by SA. Sequences of MhNPR1, MhTGA2, and pathogens related proteins (Mhchitl, MhGlu, MhPR1, and MhPR5) were isolated from this library. Eexpressions of these genes were detected by real-time quantitative RT-PCR (qRT-PCR), and their functions were analyzed through overexpressing in transgenic tobacco plants. In additional, we established a technology system of plant trivalent fusion expression vector utilizing foot and mouth disease virus 2A sequence, and constructed the trivalent fusion expression vector MhTNC (MhTGA2-2A-MhNPR1-2A-Mhchitl)We preliminarily analysised its function. The main results are as follows:1. In this experiment, total RNA was extracted from leaves of Malus hupehensis treated with SA by improved CTAB method and mRNA was purified. The full-length cDNA library was constructed using the SMARTTM PCR cDNA Synthesis Kit. The results showed that the total RNA was non-degradable, non-polluting. mRNA dispersion were mainly concentrated in the 500～2000 bp, and there was no rRNA residues. Dispersion of ds cDNA was mainly distributed between the 300 and 2000bp. PCR fragment size was between 200 and 2000bp, indicating that the quality of ds cDNA synthesized was better and we successfully constructed a full-length cDNA library.2. The cDNA sequence of M.hupehensis NPR1 has an entire coding region of 1761 bp, and was named MhNPRl (GenBank sequence accession number FJ5981431). Its corresponding genomic DNA sequence was 2259 bp, and GenBank sequence accession number was GUI83100. Sequence comparison results showed that the gene contains three introns and four exons, the same with the Arabidopsis AtNPR1 and rice OsNPRl. The amino acids included 9 conserved cysteine residues, BTB and ANK_REP_REGION structure domains.1238 bp of upstream promoter sequence was isolated, and sequence analysis showed that the gene contains an intron of 601bp in 5’UTR. Promoter region of this gene contains a salicylic acid component, two ethylene response elements, two methyl jasmonate response elements, three gibberellin response elements, two heat stress response elements, an anaerobic response element, an enhanced transcription component, and a number of light response elements.3. Expression pattern of MhNPRl gene in M. hupehensis treated by plant hormones (SA, MeJA, and ACC), biotic stress (apple ring rot pathogen (Botryosphaeria berengeriana) and apple aphid(Aphis citricota)) were analyzed through qRT-PCR. The results showed that the expression level in leaf was higher than that in stem and root. S A could induce the expression of MhNPR1 gene in leaves, stems, and roots in M. hupehensis. And MeJA and ACC only induce the expression of this gene in roots. However, apple ring spot pathogens could not induce the expression at the transcription level. Apple aphid could induce the expression of this gene in leaves and stem of M. hupehensis.4. A plant binary expression vector of MhNPR1 was constructed. The vector was transformed into tobacco through the method of Agrobacterium-mediated. PCR and RT-PCR results showed that MhNPR1 gene was inserted into the genomic DNA of tobacco, and was expressed successfully in transgenic tobacco plants. Compared to wide type tobacco plants, the expression of PR1, PR3 and PR5 gene were upregulated in transgenic tobacco plants. T1 generation of transgenic tobacco plants showed strong resistance to Botrytis cinerea at the seedling stage. The expression level of osmotic stress related genes, such as NtSPS, NtSAMl, NtTOBLTP, NtERD10A, NtERD10B, NtERD10C, and NtERD10D, and antioxidant related gene such as NtCA, NtSOD, and NtRbohD were up-regulated in transgenic tobacco lines. T1 line of transgenic tobacco plants showed strong tolerance to salt at the stage of seed germination, seedling. To generation of transgenic tobacco plants, T1 generation of transgenic tobacco plants in seed germination, seedling showed strong resistance to osmotic stress. In short, MhNPR1 gene has multiple resistances to defense response in M. hupehensis.5. A bZIP transcription factor gene was cloned by RACE technology combined with in silico cloning. The full-length cDNA sequence of this gene is 1541bp, which includes 191bp of the 5’UTR, an ORF of 999bp, and 351bp of 3’UTR. The deduced amino acid sequence of MhTGA2 is 333 amino acids. MhTGA2 contain a typical bZIP domain, leucine domain, YX2RL [RQ] ALSS [LS] W structure domain at the C terminal of the protein. Phylogenetic analysis showed that MhTGA2 close with the bean TGA2.1, beans TGA2.2, grapes TGA2, and poplar TGA2.1, indicating that we isolated a bZIP transcription factor transcription factor, named MhTGA2, and GenBank accession number is FJ598138. MhTGA2 localize in the nucleus. The expression of MhTGA2 gene was higher in leaf than that in stem and root. And accumulation of MhTGA2 gene was enhanced in M. hupehensis after treatment with plant hormones SA, MeJA, and ACC. however, apple ring rot pathogen failed to induce the gene expression. PRs (NtPR1, NtPR2, and NtPR3) and stress related gene (NtSOD, NtPPO, NtPAL, NtAPX) were upregulated in tobacco plants of overexpression of MhTGA2 gene, comparing with the WT plants.6. Mhchitl, a class I chitinase gene from Malus hupehensis, was cloned, and its expression and function in seedlings were observed. Treatment with SA, MeJAand ACC resulted in the elevation of Mhchitl transcript levels in leaves, stems and roots. Infection with B. berengeriana caused an accumulation of Mhchitl transcripts, with maximum levels at 6 h post-inoculation. Mhchitl expression was also induced by the A. citricota. Transgenic tobacco plants that over-expressed Mhchitl showed enhanced resistance to B. cinerea, relative to wild type control plants, and were not susceptible to PEG. In addition, transcript levels for NtSOD, NtAPX, NtPPO and NtPAL were up-regulated in the transgenic plants. These results suggest that Mhchitl is not only involved in the SA-signal pathway, but also with the JA/ET-signal pathway. Our data support the role of Mhchitl in M. hupehensis as an important part of the plant’s defense strategy, through promotion of resistance to a number of stress abiotic and biotic factors.7. MhGlu, aβ-1,3-glucanase cDNA, was cloned from Malus hupehensis by in silico cloning and validated with RT-PCR. MhGlu has an intron and possess 84,83, and 77% nucleotide identity and 84,74, and 76% amino acid identity with Prunus persica, Prunus avium, and Vitis riparia, respectively. MhGlu genomic DNA sequence and promoter sequence including the SA motif, MeJA responsive, and ET responsive elements were isolated. MhGlu expression was detected in M.hupehensis seedlings treated with SA, MeJA and ACC. qRT-PCR revealed constitutive expression of MhGlu in leaf but not in the stem and root where it was silent and induced by SA, MeJA, and ET. This result suggests that MhGlu might be involved in the SA-and the JA/ET-signaling pathways in M. hupehensis. The expression of the gene monitored in a 96 h course after inoculation with B. berengeriana. Inoculation with B. berengeriana, up-regulated MhGlu 24 h post inoculation (PI), the expression reached to maximum at 48 h, and then decline. Moreover, A. citricota could enhance MhGlu expression in the leaf and stem compared to healthy control plants. It can be concluded from the results that MhGlu is involved in resistance to biotic stress in M. hupehensis.8. Full coding region of cDNA and genomic DNA sequence of MhPRl and MhPR5 were isolated from M. hupehensis. There were 492 bp and 741 bp of ORF and 162 and 246 amino acids for MhPRl and MhPR5, respectively. MhPRl gene has not intron, but MhPR5 contains an intron. Sequence comparing with the Apple genome showed that MhPR1, MhPR5 have 4, and 3 copies, respectively. These two genes have highly homologous and closer relationship with apple and pear. Both genes contain an N terminal signal peptide, and MhPR1, MhPR5 containing 6 and 10 conservative cysteine residues. QPCR analysis results showed that the expression of MhPRl and MhPR5 in different tissues of M hupehensis were different. The accumulation of MhPRl and MhPR5 were up regulated in leaves, stem, and roots after treatment with SA, MeJA, and ACC, suggesting that MhPRl and MhPR5 are the marker genes of SAR in M. hupehensis.9. Transformation of pMD19-T vector was carried out in this paper; then, MhTGA2, MhNPRl, and Mhchitl gene were inserted into this vector in turn, named T2AN2AC. Trivalent fusion plant expression vector, named MhTNC, was constructed successfully through digested and inserted combo of three genes into the plant expression vector. Transgenic MhTNC tobacco plants were obtained through the method of Agrobacterium-mediated. Compared with wild-type strains, transgenic tobacco lines showed early flowering, dwarfing, significantly less internode number, significantly longer. And transgenic tobacco plants were higher resistance to Botrytis cinerea and PEG6000. |
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