Cloning And Functional Analysis Of DELLA Genes Related To Dormancy Of Flower Buds In Pear

Pear(Pyrus L.)is one of the deciduous plants,which belongs to Rosaceae,Malodeae.There is a characteristics in pear that winter dormant buds’ needs accumulated low temperature to break the dormancy,in which gibberellins(GA)is an essential hormone promoting the flower bud germination.DELLA protein plays an important role in GA signal transduction during the process.As the main cultivars of pear in Fujian,Pyrus pyrifolia brings huge economic benefit for the short low temperature pear in southern China region.It is important to explore the mechanism of bud dormancy in pear in order to increase pear yield.In this study,two different chilling requirement cultivars,namely‘mixueli’and ’huanghuali’,were used to explore the DELLA genes expression and function analysis during the bud dormancy.The major results were as follows.1.Five DELLA protein-coding genes,namely PpGAI1,PpGAI2,PpGAI3,PpRGLl and PpRGL2,were isolated from both ’mixueli’ and’huanghuali’ using PCR method,as which have been registered in GenBank.The amino acid sequence similarity of PpGAIl,PpGAI2,PpGAI3,PpRGLl and PpRGL2 in these two cultivars were 99.48%,100%,99.27%,99.17%and 99.44%,respectively.The multiple sequence alignment revealed that the DELLA genes have a high conservative in the Rosaceae trees.The result of phylogenetic analysis indicated that the DELLA genes in ’mixueli’ and ’huanghuali’ were preferential clustering,and shared the similarity sequences with Pyrus x bretschneideri,Malus domestica and Pyrus betulifolia.The bioinformatics analysis showed that the properties of related proteins were similar in these two cultivars belonging to GRAS protein family.Subcellular localization analysis showed that the DELLA genes were located in the nucleus,cytoplasm and mitochondria.2.The flower buds samples from natural dormancy period and GA treated were subjected to qRT-PCR analysis.The results showed the DELLA genes in these two cultivars expressed differently during each dormant transition interval.The DELLA genes in ’mixueli’ reached the peak value during the dormancy breaking critical period,while the counterpart in ’huanghuali’ was maximum during the budding period.The results suggest that the DELLA genes involved in different mechanisms modulating flower buds dormancy breaking in these two cultivars.The flower buds samples treated with GA either from ’mixueli’or ’huanghuali’ broke dormancy in 14 days and 28 days,respectively.The qRT-PCR results showed that DELLA genes expression in GA-treated samples increased during the dormancy breaking critical period compared with that of control group indicating GA might promote the dormancy breaking by regulating the expression of DELLA proteins.3.The promoters of PpGAI1mx and PpGAI2mx from ’mixueli’,PpGAI1hh and PpRGL1hh from ’huanghuali’ were obtained by PCR method.The promoter prediction results displayed that all of the promoters contained the same elements such as the core promoter elements,light responsive elements,hormone-responsive elements and stress responsive elements.These promoters from same genes contained most of the same cis-acting elements in different cultivars.In addition,there are some responsive elements for adverse stresses in the promoters of DELLA genes,such like cooling,drought,wounded and anaerobic responsive elements indicating that DELLA genes from ’mixueli’ and’huanghuali’ involved in the regulation of bud dormancy breaking process through adverse stress response.4.Utilization of the agrobacterium infection method was applied to construct fusion expression vector in order to determine the subcellular localization of DELLA proteins.Confocal laser scanning microscopy confirmed that PpGAIlmx protein was localized in cell nucleus and the peripheral of cytomembrane;both PpGAI2mx and PpRGL2mx protein were localized in cell nucleus,cytoplasm and cytomembrane;PpGAI3mx protein was localized in cell nucleus and cytoplasm;PpRGLlmx protein was localized in the cytoplasm,cell nucleus and vacuole.The results also showed all the DELLA protein from ’mixueli’ expressed in the cell nucleus.It’s speculated that DELLA protein of pear were associated with the function of nuclear location.5.The protein of PpGAI3mx was present in prokaryotic expression system.To build the pEASY-Blunt E1-PpGAI3mx recombinant protein,we succeeded in ligating the PpGAI3mx gene to pEASY-Blunt El expression vector and transferred it into E.coli strain BL21(DE3).After the IPTG induction,the relative molecular quality 60kDa protein was detected through the SDS-PAGE and Western blotting.It turned out that the DELLA fusion protein from pear and His tag combined successfully in inducible system and the fusion protein is water soluble.6.The expression vector of PpGAI3mx-pCAMBIA1302 was transformed into tobacco by Agrobacterium through leaf disc transformation,with the processes of pre-culture,co-culture,selective culture,rooting culture,and transplanted to the sterile soil.We obtained six transgenic tobacco which were proved by molecular indentification.The observation found that PpGAI3mx gene overexpressed transgenic tobacco exhibited some features such as narrowed leaf,shorter internode and lower plant height.The results showed that DELLA protein act as a negative regulatory factor in GA signaling pathway during the growth and development process of pear.In this study,we have isolated the related genes coding DELLA protein in ’mixueli’ and ’huanghuali’ cultivars for the first time,and explored the molecular mechanism of pear flower buds dormancy regulation revealing the function of DELLA protein which could be the basis for the research of other perennial deciduous fruit species.