Clone Vernalization Releted Gene BoVIN3from Cabbage And Analysis

Cabbage (Brassica oleracea var. captata L.)belongs to green vernalizatio plants,young plants need to grow to certain sizes then can accept low temperature and go through vernalization.The pr-ocess of long colding time promoting plant flowering known as vernalization, playing an important role in transition to flowering, in recent years had made considerable progress in vernalization res-earch,especially the molecular mechanism discussion highlights its natural gradually.Although the understanding of the vernalization mechanism show some outline,but the molecular control process is not clear. According to related reports in the arabidopsis and brassica,this work focusing on core gene FLC in flowering regulation way and its upper key gene VIN3in vernalization to research,first isolate vernalization related gene VIN3from cabbage by Homology cloning, employ the biological software to analyze BoVIN3gene and deduced amino acid sequence,the expression patterns of the BoVIN3genes were investigated by semi-quantity RT-PCR, in an attempt to explore the relation be-tween its expression and vernalization, provide base theory for cabbage vernalization.The main conclusions of this study are as follows:In this experiment, the research of morphological characters of the cabbage plants can accept low temperature growth under natural condition showed that medium maturing varieties of "GUANG RONG YI HAO", with13leaves and12.83±0.14mm in stem diameter, begin to be sensitive to vernalization,low temperature treatment48d vernalization complete, transition to flower bud differentiation. Cabbage plants only accept cold inducement reached saturation level to activate the corresponding gene expression,so this work must employ cabbage plants can feel low temperature to vernalize, prepared to following cloning the VIN3gene.According to AtVIN3gene mRNA sequence (NM-125121.3) ORF coding region,design gene specific primer,take cabbage RNA and DNA as template to amplified BoVIN3gene.Two vernalization-related genes were isolated by RT-PCR. Sequencing analysis results showed that their ORF were both1680bp in length, which encoded560amino acids, and GenBank accession number are JQ394927and JQ394928respectively.Their nucleotide sequences shared85.55%and79.95%identity with AtVIN3respectively, and the deduced amino acid sequence showed homology to AtVIN3with80.72%and72.96%. At the same time we isolated BoVIN3part DNA segment,was1405bp, GenBank accession number is JQ394929.Take advantage of DNAMAN software and on line analyze Bo VIN3gene and their coding protein.Result indicated BoVIN3protein560AA,molecular weigh is62.116KD and62.434KD respectively,PI is6.56and5.63respectively; Phosphorylation site analysis of BoVIN3,both have many Phosphorylation site; analysis of protein conserved domains of BoVIN3showed that they have Zn-finger domains.The expression patterns of the BoVIN3genes were investigated by semi-quantity RT-PCR, results showed that BoVIN3gene could express induced by low temperature treatment, and expressed in the stem apex specifically, transcript levels increased with increasing treatment time.The transcription reach the peak at42d of vernalization. At the same time the expression of FLC was suppressed after BoVIN3was induced by low temperature treatment,results suggest that both genes maybe involved in the biological process of flowering regulation in vernalization. However both BoVIN3could not be induced by GA3and KT treatment.It indicated that cabbage BoVIN3was specific vernalization genes, whose transcripts could specially response to vernalization.Employ pB1121plasmid and BoVIN3-l gene segment to construct anti-sense expression vector. Insert the BoVIN3-1anti-sense gene segment at Xbal and Smal restriction enzymes site between CaMV355promoter and GUS gene,construct plasmid pBI35S-BoVIN3-1.Transform it to cabbage flower bud by means of micro-injection,observed that after vernalization treatment the transgenic plant vernalization response be delayed at certain extent, the result further indicated that BoVIN3-1gene maybe part in vernalization directly.