Clone Of CBF And ICE1 Homologue Genes And Analysis Of Expression Profilings During Cold Stress In Prunus Mume

Prunus mume Sieb.et.Zucc.,also known as Mei flower in China,is a small tree native to the mountain area of Southwest China.It is welcomed by people for its beautiful flowering in winter and its graceful tree form.Mei flower is one of the ten traditionally famous flowers in China.In Chinese literature,Mei flower symbolized for lofty and unyielding character,and nobleness.Mei flower has now been cultivated widely in the south area of Qinling Mountains in China for its ability to confer multiple resistances.Nevertheless,its planting area is restricted in northern China for its incompetence to survive the frosty in winter,and cold and dry condition in spring.To explore the molecular mechanism of hardiness and cold respondance in Mei flower is a quick and necessary aspect in Mei flower hardiness breeding.In this paper,we cloned CBF/DREB1 and ICE1 homologs from P.mume ’Xue Mei’,and analysed the differential expression genes of ’Xue Mei’ leaves under a 0℃ treatment using RNA-seq.The main achievements are as follows:1.Using homology cloning strategy and Tibet Mei genome as a reference,we got 5 new PmCBFs,named PmCBFc～PmCBFg,respectively.Sequence analysis showed that CBF/DREB1 homologs in Mei flower were conservative and were tandemly arrayed in genome.The position between CBF genes in P.mume ’Xue Mei’ and Tibet Mei genome was highly conserved.The phylogenetic analysis showed that all seven PmCBFs belonged to dicotyledon branch in CBF/DREB1 family,while one was under CBF subgroup and six were under DDF subgroup.In genus Prunus,CBF/DREB1 homologs could be distributed to seven classes represented by seven PmCBFs,respectively.2.The promoter prediction and binding sites analysis of the sequence between PmCBFd-PmCBFc showed that PmCBFc could be regulated by multiple factors including ICE1 transcription factor,light and CBF/DREB1 itself.3.The cold acclimation at 2℃ was performed using P.mume ’Xue Mei’ seedlings.The expression analysis of PmCBFa～PmCBFc via qRT-PCR showed that PmCBFs were cold-regulated genes,and their expression levels quickly increased in an exponential growth rate and reached a peak after 8 h～12 h of cold acclimation;besides,PmCBFs had constitutive expression characterizations with a kind of regular fluctuation under normal conditions.4.Through double digestion and plasmid recombination,PmCBFd～PmCBFg overexpression vectors were obtained.Transgenic Arabidopsis lines overexpressed these genes were gained though Agrobacterium-mediated transformation using floral dip method.5.Using RACE and homology cloning technology,three PmICE genes namely PmICEa～PmICEc were got from ’Xue Mei’ genome.Seuqences analysis showed that PmICEa and PmICEb were highly conserved in gene structure and predicted amino acid sequence with AtICE1.PmICEc was a homolog with PmICEb with no introns,while its amino acid sequence was lack of bHLH conservative domain and C-terminal region.The phylogenetic analysis of PmICEs with other ICE1 homologs showed that the evolutionary divergence between PmICEb and PmICEa was earlier than Prunus species,which was just the contrary between PmICEb and PmICEc.6.The twigs of P.mume ’Xue Mei’ with unfold leaves were collected from 3 year old tree and cultured in 0℃ water for chilling treatment.The RNA extracted from the leaves was for RNA-seq.The sequencing was performed at Hiseq 2000 with 50 bp long read.Under a criteria of |Log2(Fold change)|>=1,q-value<0.05,4705 genes were thought to be differentially expressed genes(DEGs).3,678 of them were defined as cold-responsive DEGs including cold up-regulated and cold down-regulated groups,except one with fluctuated expression.According to the heatmap of DEGs and h-cluster results,the treatment was seperated into three phases:Phase Ⅰ(0 h～12 h),named cold prelimilary phase,Phase Ⅱ(12 h～24 h),named cold midterm phase,and Phase Ⅲ(24 h～48 h).In PlantTFDB database,208 transcription factor(TF)genes were screened from all the DEGs.They were predicted to coding 36 kinds of TFs.Thirty-one genes among 208 TFs were ERF,the most one in these TFs.GO enrichment analysis showed that hormone signal pathway responsed at 12 h.KEGG pathway enrichment showed that cross-talk between cold and water stress was significant at Phase Ⅱ,and at 48 h the interaction of cold stress with biotic stress was notable.7.The physiological indexes of the chilling treated materials were detected.Results showed that soluble sugar and proline content of the leaves significantly increased at 24 h,while MDA content only significantly enhanced at 12 h,SOD enzyme activity was significantly improved at 12 h with stable activity until the end of the treatment,but POD enzyme activity was not obviously changed.All the results above are important for Mei flower hardiness research and cultivar breeding.