| The cultivation and application of cold-tolerant germplasm has always been the focus of research in the botany field,but the research objects are mainly limited to food crops and major economic crops.The research methods are mainly focused on traditional breeding techniques,there are few in-depth researches which are using modern breeding techniques such as genetic engineering on formulating new cold-tolerant germplasms of ornamental flowers.Petunia hybrida belongs to the family Solanaceae（Petunia）and is widely used in flower beds and open landscaping.It is also used as a model plant in molecular biology,but it is sensitive to low temperature,when the temperature is lower than 15℃,it will lead to late flowering or non-flowering.Meanwhile,it is difficult to overwinter in northern region under the freezing stress,which hinders its promotion and application.Lilium spp.belongs to the the family Liliaceae（Lilium）and is one of the top ten cut flowers in the world.It is a high-value ornamental,edible,medicinal and commercial flower.Among them,Lilium longiflorum is an important wild flower in lily breeding,it can tolerate low temperature around 0℃during germination,however if it encounters a low temperature below 12℃,it is easy to cause blind flowers after flower bud differentiation is completed,which seriously affects the quality of cut flowers.In order to break the geographical limitations on the application of Petunia hybrida and Lilium longiflorum,as well as achieve year-round supply of flowers,it is urgent to formulate new cold-resistant germplasms.At present,transgenic technology is considered to be the most effective breeding method on the directed improvement of plant cold tolerance.Among the candidate genes related to cold tolerance,DREB1/CBF transcription factor（Dehydration responsive element binding protein 1/C-repeat binding factor）plays a key role in the low-temperature signaling network of model plants.It belongs to the DREB subfamily of the AP2/ERF family and can activate the expression of a series of cold-regulated genes downstream.Therefore,Petunia axillaris and Lilium longiflorum were used as research objects in this study.Based on the corresponding genomic and transcriptome data,DREB1s/CBFs genes from different species were selected and cloned,a series of expression analysis,transgene and cold tolerance identification experiments were carried out.It was found that the overexpression of DREB1/CBF homologous genes from Petunia axillaris and Lilium longiflorum could improve basic cold tolerance in Arabidopsis.At the same time,overexpressing a part of DREB1/CBF genes from Petunia axillaris could not only improve the cold tolerance,but also change the horticultural traits of petunia seedlings.Besides,new cold-tolerant germplasms of petunia would have potential application value.The main findings are as follows:1.According to the sequence information in Petunia axillaris genome and Lilium longiflorum stress-related transcriptome,specific primers were designed,then 11 and 5DREB1/CBF homologous genes were isolated from Petunia axillaris and Lilium longiflorum,respectively.The g DNA and c DNA of the above 16 genes have identical base sequences,no introns have been found,and the c DNA can encode amino acids with a length ranging from 201 to 242.Sequence analysis indicated that these 16DREB1s/CBFs transcription factors all contained typical conserved sequences,such as AP2 binding domain,nuclear localization signal,DSAWR and LWSY.Phylogenetic analysis indicated that these 16 DREB1s/CBFs transcription factors belonged to A-1subgroup branch of the DREB subfamily.Most DREB1s/CBFs members of Petunia axillaris and Lilium longiflorum are located at the top or bottom of the the A-1 subgroup branch,respectively,and their evolutionary status is quite different;2.Semi-quantitative RT-PCR was used to analyze the spatiotemporal expression patterns of DREB1s/CBFs homologous genes from Petunia axillaris and Lilium longiflorum under different abiotic stress.The results showed that in different tissues of Petunia axillaris,most members of Pa DREB1s/CBFs were expressed in leaves,flower buds and fruits after low temperature treatment,except for the Pa DREB1K gene,which only responded to low temperature in root.However,when it came to different abiotic stresses,Pa DREB1D,Pa DREB1F,Pa DREB1G,Pa DREB1I and Pa DREB1J genes could only respond to low temperature in a single way,while Pa DREB1A,Pa DREB1B and Pa DREB1E genes could respond to low temperature,drought and high salinity stresses,Pa DREB1H gene could respond to low temperature and high salinity stresses,Pa DREB1C gene could respond to cold,heat,drought and ABA treatments,Pa DREB1K gene could respond to drought,high salinity and H2O2 stresses.Meanwhile,in different tissues of Lilium longiflorum,most members of Ll DREB1s/CBFs were expressed in leaves and bulblets after cold treatment,while Ll DREB1A,Ll DREB1B and Ll DREB1E genes could also respond to low temperature in root.However,when it came to different abiotic stresses,all five Ll DREB1s/CBFs genes from Lilium longiflorum could be stimulated by at least three different abiotic stresses,but none of them could respond to cold stress only.It indicated that compared with Ll DREB1s/CBFs members,the expression patterns among different Pa DREB1s/CBFs members were more diverse.Meanwhile,in order to further detect the maximum expression of the above 16 genes under cold stress,q RT-PCR was carried out.Compared with Pa DREB1s/CBFs members,more Ll DREB1s/CBFs members could be induced by low temperature for a longer period,and the slower decline in expression levels had been found at the late stage of cold stress,as well as different peak values of DREB1s/CBFs genes among various species might affect their sustained expression time under cold stress;3.Based on the results of expression patterns and phylogenetic analysis,we selected four genes from 16 DREB1s/CBFs genes which have significantly different cold response patterns or special evolutionary status,the overexpression vectors of 35S::Pa DREB1E,35S::Pa DREB1H,35S::Pa DREB1J and 35S::Ll DREB1B were constructed and transformed into Arabidopsis thaliana and Petunia hybrida.By observing the phenotype of transgenic Arabidopsis,combining with the measurement of physiological indexes and q RT-PCR analysis of At CORs regulons,it was found that Pa DREB1E,Pa DREB1H,Pa DREB1J and Ll DREB1B transgenic lines had significantly enhanced basal freezing tolerance than wild type.It indicated that the four selected DREB1s/CBFs genes were all suitable for improving cold tolerance in plants and could be used as the target genes for the subsequent formulation of the new cold-tolerant germplasms in Petunia hybrida and Lilium longiflorum.In addition,phenotypic observations of petunia plants in non-acclimated and cold-acclimated groups were also carried out,combined with physiological indexes measurement and q RT-PCR analysis of downstream components Ph DHN2 and Ph DHN10.It was found that Pa DREB1E and Pa DREB1J genes could enhance the basal cold tolerance of petunia,and the contents of proline and carbohydrate in transgenic lines were significantly higher than that of wild type,which revealed that Pa DREB1E and Pa DREB1J genes might have functional redundancy in improving basal cold tolerance of petunia.Besides,overexpression of Pa DREB1E,Pa DREB1H,Pa DREB1J and Ll DREB1B genes could significantly up-regulate the expression levels of Ph DHN2 and Ph DHN10 genes in the non-acclimated group（Except for the Ph DHN2gene in the 35S::Ll DREB1B transgenic lines）.At the same time,overexpression of Pa DREB1J gene in the cold-acclimated group could enhance the acquired cold tolerance of petunia.Compared with the wild type,the contents of carbohydrate significantly increased in transgenic lines,but the contents of proline did not increase significantly.Besides,only Pa DREB1J gene overexpressed could significantly up-regulate the expression of Ph DHN2 and Ph DHN10 genes in the cold-acclimated group;4.Thin layers of bulblets from Lilium longiflorum were considered as transforming receptor material,the Ll DREB1B gene was introduced into Lilium longiflorum by Agrobacterium-mediated method,as a result,a total of 20 resistant plants were obtained.After preliminary detection by PCR,12 of them may be positive.Besides,semi-quantitative RT-PCR showed that the expression of Ll DREB1B gene in transgenic lines was significantly higher than that of wild type;5.Significant delays in flowering were observed in most petunia plants constitutively expressing Pa DREB1E,Pa DREB1H,Pa DREB1J and Ll DREB1B genes,with a significant decrease in the number of branches and visible flower buds.QRT-PCR analysis revealed that expression levels of Ph FT1 and Ph MADS17-like genes,which were associated with the delayed flowering,were significantly higher in the transgenic lines than the wild type. |
