Cloning And Functional Analysis Of Several Genes Such As KNOPE1 That Related To Semi-dwarfing Characteristic Of Peach

Peach(Prunus persica(L.)Batsch),one of the important deciduous fruit trees,is originted from China.In China,the planting area of peach is widely.However,the pruning task is heavy and the cost of orchard management is increased because of its strong budding and the large growth of new shoots.Meanwhile,the poor economic benefits of orchard is due to its height,lacking of ventilation and light transmission,low photosynthetic efficiency,poor fruit coloring and flavor.Dwarf dense planting is the development trend of modern orchard.It has the advantages of early harvest convenient management and high fruit quality.Breeding dwarf varieties has become a hot spot in breeding.With the application of molecular biology in fruit trees,some genes related to the internode length are gradually discovered.Exploring the key factors which were controlling the internode length of peach is of great significance for dwarfing breeding.In this study,four differentially expressed genes(PpHsfB-4,PpHSP17.9,PpTFL1 and PpKNOPE1)were cloned and the transcriptome sequencing of the F1 generation of‘Huangshuimi’בZhong Youtao 14’were analyzed by bioinformatics.Tissue-specific expression analysis of PpHsfB-4,PpHSP17.9 and PpTFL1 genes was also carried out.Moreover,PpHsfB-4,PpHSP17.9 and PpTFL1 genes were overexpressed in wild Arabidopsis thaliana by Agrobacterium-mediated transformation.In addition,the 35S::PpKNOPE1-YFP subcellular localization vector was constructed to determine the subcellular localization of PpKNOPE1.The results provide a basis for revealing the semi-dwarf characteristics of ’Zhong Youtao 14’ for the molecular mechanism.The main results are as follows:1.Cloning and bioinformatics analysis of PpHsfB-4,PpHSP17.9,PpTFLl and PpKNOPE1.The 1170 bp,462 bp,519 bp,and 1170 bp coding region fragments of PpHsfB-4,PpHSP17.9,PpTFLl and PpKNOPE1 genes were successfully amplified by using the cDNA of ’Zhong Youtao 14’and‘Huangshuimi’,respectively.The sequences of four genes have no difference between ’Zhong Youtao14’and‘Huangshuimi’.The secondary structures of PpHsfB-4,PpHSP17.9,PpTFL1 and PpKNOPE1 proteins were predicted by SOPMA.The results indicated that the secondary structural compositions of PpHsfB-4,PpHSP17.9 and PpKNOPE1 were Random coil>Alpha helix>Extended strand>Beta turn,and the secondary structural compositions of PpTFL1 was Random coil>Extended strand>Alpha helix>Beta turn.The different proportion of secondary structure mayhave intimate relation to their respective functions.The physicochemical properties of proteins were predicted by Protparam.The results showed that the theoretical pI of PpHSP17.9 and PpKNOPE1 within the acidic range,and the theoretical pI of PpHsfB-4 and PpTFLlwithin the alkaline range.The total average hydrophobic index of the four proteins was negative,indicated that these four proteins are all hydrophilic proteins.2.Tissue-specific expression analysis of PpHsfB-4,PpHSP17.9 and PpTFL1.The tissue-specific expression of PpHsfB-4,PpHSP17.9 and PpTFL1 in ’Zhong Youtao 14’ was analyzed by qPCR.The results indicated that PpHsfB-4,PpHSP17.9 and PpTFL1 were all expressed in shoot tips,young leaves,mature leaves,flowers,embryos and fruits of ’Zhong Youtao 14’,while the expression levels were different.PpHSP17.9 had the highest expression in mature leaves and almost no expression in other tissues.PpHsfB-4 had the highest expression in young leaves,followed by shoot tips,and with the lowest expression in embryos and mature leaves.PpTFL1 had the highest expression at the shoot tip and was significantly higher than other tissues.There is almost no expression in other tissues,and this pattern of specific expression in vegetative organs are consistent with the function of TFL1 to maintain and prolong the vegetative growth of plants.3.Construction of overexpression vectors of PpHsfB-4,PpHSP17.9 and PpTFLl and transformation of wild type Arabidopsis.35S::PpHsfB-4、35S::PpHSP1 7.9 and 35S::PpTFL1 were transformed into wild Arabidopsis thaliana by Agrobacterium-mediated transformation.The transgenic plants were identified by PCR.The results showed that these three genes were successfully integrated into the genome of wild-type Arabidopsis.Five 35S::PpTFL1 transgenic lines were named 35S::PpTFL1#1,35S::PpTFL1#2,35S::PpTFL1#3,35S::PpTFL1#4 and 35S::PpTFL1#5,respectively.In the generation plants of T0,#1 and#2 with 35S::PpTFL1 were delayed in flowering,the number of rosette leaves and vegetative growth increased,while the transgenic plants of 35S::PpTFL1#3,35S::PpTFL1#4,and 35S::PpTFL1#5 did not flowering.After 10 d of extended light treatment(18 h light 28℃+6 h dark 26℃),all three lines have different degrees of bolting,and then placed them under normal growth condition.The transgentic line of 35S::PpTFLl#3 bolting and blossoming normally.However,the transgentic lines of 35S::PpTFLl#4 and 35S::PpTFL1#5 were normally bolting,no flowering and branches increased.Two 35S::PpHsfB-4 transgenic lines were identified by PCR,named 35S::PpHsfB-4#1 and 35S::PpHsfB-4#2,respectively.The roots of T1 transgenic seedlings with 35S::PpHsfB-4 were significantly shorter than wild-type Arabidopsis,and the 35S::PpHsfB-4#2 line showed slow-growing and dwarf.Nine 35S::PpHSP17.9 transgenic lines were identified by PCR and named 35S::PpHSP17.9#1-35S::PpHSP17.9#9,respectively.Compared to wild-type Arabidopsis,the height of T1 transgenic lines 35S::PpHSP17.9#1,35S::PpHSP17.9#6 and 35S::PpHSP17.9#9 were all increased.And the length and number of internodes were also counted.It concluded that the increasing in plant height was due to the increasing in the number of plant nodes.4.Subcellular localization analysis of PpKNOPE1.The 35S::PpKNOPE1-YFP fusion expression vector was successfully constructed and transformed into N.benthamiana epidermal cells by Agrobacterium-mediated transformation.The results showed that PpKNOPE1-YFP fusion protein located in the nucleus and the cell membrane.It indicated that PpKNOPE1 was a nuclear membrane co-expressed protein.