Cloning Of LpSVP Gene From Lilium Pumilum And Transformation Of Tobaccos

Lilium pumilum is a widely distributed,wild flower with anti-roll petals and bright color,which is strongly adaptable to the environment,and is highly resistant to cold,drought as well as salt.Lilium pumilum can beautify the landscape environment,but also is a valuable material for cut flowers.In this study,the high expression and complete open reading frame with conserved domain was selected from the MADS-box transcription factor family of Lilium pumilum cDNA library.The full length of this gene was cloned by homologous sequence,which encodes 224 amino acids.Sequence homology and phylogenetic analysis showed that this gene belonged to SVP-like MADS-box genes,and named LpSVP.Real-time PCR analysis showed that the expression of LpSVP increased firstly and then decreased continuously during flower development,which suggesting that the regulatory function of LpSVP on Lilium pumilum flower development.The main findings of this study are as follows:1.According to morphological observation of flower buds,flower bud differentiation occurred during the low temperature storage of Lilium pumila bulbs and deepened with the prolongation period of low temperature storage.QRT-PCR analysis showed that the relative expression of LpSVP gene decreased with the prolongation of low temperature storage.It is speculated that LpSVP acts as a repressor for flower meristem identification,and the down-regulated expression of this gene responds to the process of flower bud differentiation.2.The full-length of ORF was 675bp with a highly conserved MADS region and a semi-conserved K region,belonging to the MADS-box gene family.The sequence has 99%homology with the SVP protein of Lilium formosanum x Lilium longiflorum.Bioinformatics analysis showed that the relative molecular mass of the LpSVP gene was 25.783kDa,the theoretical isoelectric point was 5.62,and it was hydrophilic protein with no transmembrane structure.3.Tissue-specific expression analysis of LpSVP was performed by RT-PCR.The result showed that LpSVP was expressed in bulb,leaf,pistil,stamen and petal,but varied widely in the level of expression.4.Five stages of Lilium pumilum flower development,namely bud Ⅰ,bud Ⅱ,bud Ⅲ,bud end and full bloom were selected for qRT-PCR analysis.The expression level of LpSVP firstly increased and then decreased,which was the highest in bud Ⅱ and the lowest in full bloom.5.The plant expression vector pBIl21-LpSVP-GFP was constructed and the fusion protein was transferred into wild tobacco via agrobacterium-mediated transformation of leaf disc,the results showed that transgenic plants delayed flowering,increased inflorescence branch and slightly evacuated inflorescence compared with the wild-type.