| Tulip(Tulipa gesneriana L.),a highly ornamental plant belonging to the Liliaceae family,has very broad market prospect at home and abroad due to its variety of designs and shapes.At present,tulip has the problems of no stable genetic transformation system and bulb degeneration,and the lack of water is one of the causes of bulb degeneration.Therefore,optimizing the regeneration system,exploring genetic transformation conditions,and mining tulip genes related to drought resistance have practical significance for the development of the tulip industry.In this study,we optimized the explants and the formulation of callus induction in tulip tissue culture,explored the conditions of Agrobacterium-mediated genetic transformation,and analyzed the function and regulatory network of TgDi19.The main experimental results of this study are as follows:1.Exploration of Agrobacterium-mediated genetic transformation systema.The tulip hypocotyl of ‘Regel’ could effectively induce callus on MS solid medium containing 2.0 mg/L 2,4-D + 0.5 mg/L 6-BA + 0.1 mg/L KT,and the induction rate was64.58%.In the MS medium containing 9.0 mg/L PIC + 0.5 mg/L 6-BA + 0.1 mg/L KT,the hypocotyl of ‘Verandi’ had the highest callus induction rate,but it was only 37.50%,but the efficiency was not high.b.The hypocotyl of cultivar tulip ‘Friendship’,‘Strong Gold’ and ‘Verandi’ can effectively induce adventitious buds on MS solid medium containing 0.1 mg/L NAA + 1.0mg/L 6-BA,and the proliferation coefficients are 2.70,3.29 and 2.22,respectively.c.Agrobacterium EHA105 can transfer foreign genes into the hypocotyl of ‘Regel’,some hypocotyls induced callus,and the expression of GUS reporter gene was detected in the obtained resistant callus.2.Functional study of drought-inducible protein(Di19)a.Two TgDi19 genes were screened and cloned from Tulip based on full-length transcriptome data and amino acid sequences of homologous genes from other species.Compared with Arabidopsis,the TgDi19 proteins were more closely related to rice and was named as TgDi19-2a and TgDi19-2b.Protein sequence analysis showed that encoded by TgDi19-2a and TgDi19-2b all contained zf-Di19 and Di19＿C conserved domains.b.The results of Subcellular Localization showed that TgDi19-2a and TgDi19-2b was all a nuclear membrane localization protein.Yeast transcriptional activity analysis showed that TgDi19-2a and TgDi19-2b had transcriptional activity.c.The expression pattern of TgDi19 gene in different tissues of tulip was analyzed by q RT-PCR and showed that TgDi19 was all expressed in the detected tulip tissues.The relative expression of TgDi19-2a was highest both in root.while expression of TgDi19-2b was the highest in leaf.Under abiotic stress of air drought,the expression of TgDi19-2a and TgDi19-2b genes was induced by air drought,and there were significant differences in different organs.d.The promoter of TgDi19-2a were cloned long 1803 bp.Motif analysis revealed that there was light,auxin,salicylic acid and jasmonic acid response elements.what’s more there were ABRE and MBS elements that is closely related to abiotic stress.TgDi19-2a gene may be involved in a variety of plant life activities.The result of Luciferase Assay proved that Tg MYBX could bind to TgDi19-2a promoter.e.Overexpression of TgDi19-2a and TgDi19-2b in Arabidopsis was found.Phenotype identification analysis showed that transgenic Arabidopsis reduced the sensitivity to Manntiol during stage of germination and cotyledons turning green.Compared with wildtype,transgenic Arabidopsis was less inhibited and growth better on medium containing Mannitiol.Finally,TgDi19-2a and TgDi19-2b play a positive role in drought stress and increased tolerance. |
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