| Japanese Apricot(Prunus mume Siebold et Zucc.)originated in southeast of China that is a member of the Rosaceae family and a culturally important economic fruit tree.The ornamental value of the flowers are high,and the fruit contains amino acids which are beneficial to human metabolism.The fruit is also used for making preserved fruits and liqueurs.Compared with other deciduous fruit trees,prunus mume sprout early,and its has a broad range of chilling requirement,which is a good material to study the seasonal dormancy mechanism of fruit trees.In the GA signal transduction pathway,DELLA protein is a inhibitory factor,which can inhibit the growth and development of plants.In order to study the effect of the F-box protein encoding gene SLY1 on DELLA protein in seasonal dormancy of Japanese apricot,the PmSLY1 gene was cloned from the"Taoxingmei" located at the National Field Gene Bank for Japanese apricot.The analysis of its sequence structure and function by bioinformatics.The PmRGL2 transgenic poplars were treated with dormancy and observed its phenotypes.The qRT-PCR technique was used to analyze the expression level of PmRGL2 gene and GA-related genes in signal transduction of transgenic poplars.Simultaneously,the expression patterns of PmSLY1 at the four critical stages of dormancy in Japanese apricot was analyzed by qRT-PCR.DELLA protein lacks the typical DNA binding domain and often binds to other regulatory proteins and usually function interacting with other regulatory proteins,and regulating their growth and development.We have also constructed the expression vectors of yeast two-hybrid successfully,and that have been validated the interaction between PmSLY1 and PmRGL2 proteins by the yeast two-hybrid system.The main results of this experiment are as follows:1.Comparing with the genomic sequence mRNA of Japanese apricot by NCBI,the SLY]gene was cloned from the flower bud of ’Taoxingmei’.The complete coding region sequence of SLY1 has 1866 bp and encodes 621 amino acids.The NCBI alignment found that the sequence has a Sec1 domain and named this gene PmSLYl.The analysis of the basic physicochemical properties of PmSLY1 protein was using bioinformatics technology,the results showed that PmSLY1 protein is mainly composed of α-helix,extended chain,and random coil,is a hydrophobic protein and have not a transmembrane structure;The comparison of homologous sequences showed that PmSLYl has a high homologous with PpSLY1 of the SEC1 family transport protein.2.The transgenic poplar was used as the test material and extract the RNA from the transgenic culture seedlings.The expression patterns of PmRGL2 and GA signal pathway related genes GID1b,GA20ox2 and GA3ox1 was analyzed by qRT-PCR.Results indicated that expression of PmRGL2,GID1b,GA20ox2,and GA3ox were more highly,relative to expression levels in non-transgenic plants.That is,the expression level of GA-related genes following changes of RGL2 gene in the signal channel changes.We further allow transgenic poplars to enter the dormancy stage by low-temperature treatment and observe their phenotypes.The results showed that the transgenic and non-transgenic trees were in the environmental chamber it was observed that all of the lines of the transgenic poplar plants grew slowly,relative to wild-type trees.During the dormancy release stage,budbreak in the wild-type trees occurred earlier than in the transgenic plants.These data suggested that PmRGL2 may function as an integrator in the regulation of GA biosynthesis and metabolism.3.The expression level of PmSLYl gene in different dormancy stages was analyzed by qRT-PCR.The results showed that the expression level of PmSLYl gene down-regulated at the endodormancy.At the encodormancy,the expression level of the gene up-regulated.When it reached a certain level,the expression level began to down-regulated at the dormancy release.The expression trend of PmSLY1 gene was expressed as ’down-up-down’at the four dormancy stages.On the basis of performer studies,we can predict that the SLYl gene is associated with the degradation of DELLA in the gibberellin signaling pathway.4.The yeast two-hybrid expression vector prey vector pGADT7-PmSLYl and the bait vector pGBKT7-PmRGL2 were constructed successfully.The detection of toxicity showed that the recombinant bait plasmid pGBKT7-PmRGL2 and the recombinant prey plasmid pGADT7-PmSLY1 were transformed into Y2H Gold and Y187 yeast strains,respectively,and the single colonies grew well on the auxotrophic medium.Comparing to the empty vector,the density and size were not significantly different.Results indicated that the recombinant plasmid had no toxic to the yeast strain.The results of the self-activation detection showed that not only the prey protein PmSLYl but also the bait protein PmRGL2 could grow on the QDO medium(SD/-Leu/-Trp/-Ade/-His)which indicated that they had no transcriptional activation activity.The recombinant plasmids pGADT7-PmSLYl and pGBKT7-PmRGL2 were co-transformed into Y2H Gold yeast strains for yeast two-hybrid experiments.The results showed that PmRGL2 could interact with PmSLYl proteins. |
