Cloning And Functional Analysis Of SOC1 Homologous Gene In Pear

Pear,belonging to the subfamily pomoideae in the family Rosaceae,is one of the main fruits in the world.the production and the cultivated area are third of the world’s fruits.In our country,the cultivation area and output are second.However,the child period of pear is relatively long,generally in 4-8 years.It has been known that MADS plays an important role in the regulation of floral bud differentiation.In this study,we took’Dangshan’ pear as the research material and isolated PbrSOC1 homologous gene and cloned SOC1-L3 and SOC1-L4 genes from Dangshan pear pear.We discussed the protein sequence information,subcellular localization,and expression pattern of transcription of PbrSOCl-L3 and PbrSOC1-L4.Our results suggested that PbrSOC1-L3 and PbrSOCl-L4 have the ability to promote flowering.The main results are as follows:1.Bioinformatics analysis was carried out to construct SOC1 phylogenetic tree of SOC1 of five rose families of pear,plum blossom,peach,strawberry and apple.The pear identified 148 candidate SOC1 gene,the SOC1 gene containing MADS_box and K_box domains futher analysis of proteins,results showed that identified 34 SOC1 genes in pear,plum identified in 30,identified 27 peach,strawberry and apple were identified in 31 and 50 of SOC1 gene.The phylogenetic tree showed that SOC1 family members,SOC1 gene showed homology of Rosaceae,mainly divided into 8 subfamilies ABCDEFGH,each subfamily is relatively balanced,indicating that the gene function diversification.Among the phylogenetic trees constructed,the phylogenetic tree species PbrSOCl-L3 and PbrSOCl-L4 are related to the SOC1 in pears recently,followed by apples.The PbrSOCl-L3 and PbrSOC1-L4 gene in pears and SOC1 gene are related to flowering(including Arabidopsis,citrus and gloxinia)by phylogenetic analysis.The results suggested that PbrSOC1-L3 and PbrSOCl-L4 are associated with flowering related SOC1 genes that have been reported in a subset,indicating that PbrSOC1-L3 and PbrSOC1-L4 are similar in fuction to these genes and are associated with flowering.In order to analyze the PbrSOC1homologous gene expression patterns,we used real-time quantitative PCR organizations on the ’Dangshan pear’ were analyzed and compared to detect the distribution in different tissues,including root,stem,leaf,pollen,pistil and pulp.The results showed that the PbrSOC1 homologous gene was expressed in leaves and stems,but the relative expression in different tissues was different.2.1n order to verify the different photoperiodic conditions of PbrSOC1 homologous gene response,we used real time fluorescent quantitative PCR method to detect the short long day conditions were homologous to PbrSOCl gene expression,the PbrSOCl-L3(Pbr032787.2)and PbrSOCl-L4(Pbr032788.1)gene expression showed a great difference.Accordingly,we selected the PbrSOC1-L3 and PbrSOC1-L4 genes for further functional validation experiments to determine whether PbrSOC1-L3 and PbrSOCl-L4 were associated with flowering.From the "Dangshansuli" we cloned the Pbr032787.2 gene(PbrSOCl-L3)and Pbr032788.1(PbrSOCl-L4)gene.The CDS sequences of target genes PbrSOCl-L3 and PbrSOCl-L4 were 717 bp and 705 bp,respectively.Through the observation of PbrSOC1-L3:GFPand PbrSOCl-L4:GFP over expression of morphological and structural characteristics of plants,the leaf width of transgenic Arabidopsis plants than in control plants of GFP to narrow,smaller leaf area and plant height but early flowering time.In phenotype inheritance,the phenotypic characters of each line of T1 are about 3:1,and the phenotypic proportion is similar to that of female parent.3.In order to identify subcellular localization of PbrSOC1-L3 and PbrSOC1-L4proteins,an overexpression vector,35S:PbrSOCl-L3:GFP and 35S:PbrSOCl-L4:GFP,was constructed to transform tobacco,and GFP fluorescence was localized in the nucleus.In order to explore the molecular mechanisms of PbrSOCl:GFP promoting flowering,we examined the expression of flowering related genes including AtAGL24,AtSOC1,AtAP1,AtCO,and AtTF in transgenic Arabidopsis thaliana.The results showed that these genes related to flowering,AtAPl,AtCO and AtTF,AtAGL24,were up regulated to some extent after PbrSOC1 gene was transferred,leading to flowering ahead of time.