Isolation Of PmFRL3 Gene From Japanese Apricot And The Interaction With RGL2 And ABI5 Proteins

Japanese apricot（Prunus mume Sieb.et Zucc.）,originated in the Hengduanshan vein area of China,that is a member of Rosaceae family,is not only an ornamental plant but also an important economic fruit tree.Fruit has always been regarded as leisure food and seasoning food.Because of its strong physiological alkalinity,it is also known as healthy food.Japanese apricot is a fruit tree with early flowering and budding in deciduous fruit trees.It has many varieties and needs a wide range of cold,so it is a good material to study the mechanism of seasonal dormancy.In order to study the function of PmFRL3,a member of the Frigida-like family in Japanese apricot,in dormancy regulation,the members of the Frigida-like gene family were identified and analyzed based on the Prunus mume genome data in the NCBI database,and the flower buds of the early flowering variety‘Taoxingmei’and the late flowering variety‘Bungo’were used as experimental materials to clone the PmFRL3 gene,analyze its subcellular location,and analyze its sequence structure and function by bioinformatics.Yeast two-hybrid and bimolecular fluorescence complementary experiments were used to verify the interaction between PmFRL3,PmRGL2 and PmABI5 proteins,q RT-PCR technique was used to analyze the expression levels of PmFRL3,PmRGL2 and PmABI5 in different dormancy periods,and the effects of exogenous hormones GA₄and ABA on their expression were analyzed.The main results are as follows:1.The whole genome of Prunus mume contains 12 members of Frigida-like protein gene family,which are unevenly distributed on five chromosomes.Phylogenetic analysis of Frigida-like proteins of four Rosaceae species and Arabidopsis thaliana showed that these proteins could be divided into five subfamilies（I-V）,and subfamily I（FRI）was absent in Rosaceae species.Gene replication analysis showed that there were three tandem replication events and one fragment replication event in the PmFRL family.RT-q PCR results supported the RNA-seq results,suggesting that PmFRL genes may be involved in the regulation of dormancy in Japanese apricot.2.The FRL3 gene was cloned from the flower buds of Japanese apricot cultivars‘Taoxingmei’and‘Bungo’.The complete open reading frame of PmFRL3 is 3159 bp,encoding 1052 amino acids.After sequence alignment,it was found that although there were some base differences in the coding region of the FRL3 gene between the two varieties,the amino acid sequences of the proteins they encoded were identical.The basic physical and chemical properties of PmFRL3 protein were analyzed by bioinformatics technology.The results showed that PmFRL3 protein was mainly composed ofα-helix,extension chain,β-folding and random curl,which was an unstable hydrophilic protein without transmembrane structure.SNP polymorphism analysis showed that there were 61SNPs,near PmFRL3 gene,one of which was related to flowering time,and subcellular localization showed that PmFRL3 protein was located in nucleus.3.The yeast two-hybrid vector of PmFRL3,PmRGL2 and PmABI5 genes was constructed.The toxicity and self-activation of yeast two-hybrid recombinant plasmid were detected.The results showed that the recombinant plasmid had no toxic effect on yeast strain and had no transcriptional activation activity.The hunting material particles and bait plasmids were co-transformed into Y2H Gold yeast strain,and the yeast two-hybrid experiment was carried out.The results showed that PmFRL3 protein could interact with PmRGL2 and PmABI5 protein,but there was no interaction between PmRGL2 and PmABI5.The protein interaction was further verified by bimolecular fluorescence complementary experiment,and the results were consistent.The expression levels of PmFRL3,PmRGL2 and PmABI5 in different dormancy periods were analyzed by q RT-PCR technique.The results showed that the expression trends of PmFRL3,PmRGL2 and PmABI5 were the same.When entering the dormancy period,the expression began to increase,while when entering the ecological dormancy,the expression of the three genes reached the peak,and after entering the dormancy release period,the expression began to decrease.In addition,the expression of these genes in‘Taoxingmei’was lower than that in‘Bungo’.After treatment with exogenous hormone GA₄,the expression of PmRGL2 was down-regulated and the expression of PmABI5 was up-regulated;after treatment with exogenous hormone ABA,the expression of PmFRL3 was down-regulated and the expression of PmRGL2 was up-regulated.