| Pomegranate originated in Iran,Afghanistan and other central Asia regions and is a kind of important economic fruit tree,but it is easily vulnerable to frost damage in central and northern cultivation areas in China,which seriously restricts the development of pomegranate industry in China.To solve the above problems,this study mainly carried on the work from two aspects.On the one hand,based on the comprehensive evaluation on cold tolerance of six pomegranate cultivars,we screened a reliable method to identify the cold tolerance of pomegranate cultivars.Some pomegranate cultivars used as rootstock and suitable crossing parents for new cultivars with more cold-tolerant were screed from 80 pomegranate cultivars by that identify.On the other hand,through PgCBF1 overexpressing in Arabidopsis,the function of PgCBF1 transcription factor was preliminarily analyzed,which laid a foundation for the study about the mechanism of anti-cold molecular mechanism of pomegranate,and provided the basis for the cultivation of cold-tolerant cultivars.The main contents and conclusions are as follows:1.Took annual dormant branches of six pomegranate cultivars as materials in dormancy stage.Water content including free water and bound water in branches of six pomegranate cultivars were measured.Branches were put into the cold incubator for five different low temperatures stress processing and measured the effects of low temperature stress on different physiological indexes.Last we comprehensively evaluated on cold tolerance of six pomegranate cultivars by membership function,the order was as follow: ‘Sanbai',‘Shandazi',‘Mengzi Tianlvzi',‘Qingpiruanzi',‘Tunisiruanzi',‘Israel2'.The result was the same as the order by REC,which showed that REC wsa a feasible method to briefly identify cold tolerance of pomegranate varieties.Cold tolerance of 80 pomegranate cultivars was evaluated in REC.The result showed that the most tolerant to the cold was ‘Xinjiang Hongpi' which could be as a cold-tolerant rootstock and suitable crossing parents for new cultivars with more cold tolerance.2.The relative expression level of PgCBF1 in roots,stems,leaves,flowers,skins,seeds from pomegranates were analyzed by qRT-PCR.The results indicated the transcriptional level of PgCBF1 had no significant difference.Full-length sequence of the PgCBF1 was obtained by PCR amplification,then connected the target sequence to the pEASY-Blunt Zero Cloning Vector.PgCBF1 gene sequence,homology of protein sequence and constructing the phylogenetic tree were analyzed by bioinformatics.PgCBF1 has an open reading frame(ORF)of 714 bp and encoded a protein with 237 amino acids.The molecular mass of PgCBF1 was 25.89 kDa and the isoelectric point was 5.69.It could be seen from phylogenetic tree that PgCBF1 had a high homology with several DREB1/CBF transcription factors of Arabidopsis.PgCBF1 belonged to the AP2/ERF family and was a typical DREB1/CBF transcription factors.3.Constructing an overexpression vector pBI121-PgCBF1 and pBI121-PgCBF1 recombinantplasmid was transformation into Arabidopsis by agrobacterium tumefaciens-mediated.We screened PgCBF1 overexpression Arabidopsis and observed their phenotypes.PgCBF1 overexpression Arabidopsis had a smaller leaves width,shorter petiole and more green leaves;growth speed was slow,flowering was delayed 20 days or so;inflorescence significantly shorter than WT Arabidopsis.Analying the transcriptional levels of PgCBF1 downstream cold-stress response genes(COR15A,COR15 B,RD29A,RD29 B,RD22,COR47)by qRT-PCR.The results showed that the transcription level in PgCBF1 overexpression Arabidopsis were greater than wild-type Arabidopsis under cold stress.PgCBF1 overexpression Arabidopsis and wild-type Arabidopsis were processed for 4 h under-10?,then were put into illumination incubator for 10 d to restore growth.The results showed that most leaves of wild-type Arabidopsis became withered and some plants were died,while overexpression Arabidopsis still normally grew. |
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