Cloning And Function Analysis Of EuBRI1 Gene In Eucommia Ulmoides Oliv

Eucommia ulmoides Oliver(E.ulmoides)is a traditional Chinese medicine.It has many pharmacological activities such as anti-cancer,regulating blood pressure and anti-inflammation.Therefore,it has high development value.Rubber extract from E.ulmoides is high-quality natural rubber resource because its excellent resistante ability against acid,corrosion and fatigue.Brassinosteroid is an important plant hormone.It can regulates various physiological and developmental processes of plants,such as cell elongation and division,pollen fertility,photomorphology,vascular bundle differentiation and stress resistance.At present,studies on related aspects of brassinosteroid signal transduction have made important progress in many herbs such as Arabidopsis thaliana,oilseed rape,rice and tomato.However,there are only a few studies on poplars in perennial woody plants.In this study,the brassinosteroid Insensitive 1 gene(BRI1)from E.ulmoides was cloned and analyzed.The expression characteristics of EuBRI1 was analyzed in E.ulmoides In addition,the plant overexpression vector pSH-35S-EuBRI1 was constructed and transformed into Arabidopsis brassinosteroid Insensitive 1 mutant bri1-5,in order to verify the function of EuBRI1 gene.The following results were abtained.1 Cloning and Bioinformatics Analysis of EuBRI1 gene in E.ulmoidesBased on the E.ulmoides transcriptome sequencing annotation database established by the institute,a 3612 bp gene sequence was cloned from E.ulmoides DNA and cDNA using the PCR technique.The sequencing results showed that the two sequences were identical and named as EuBRI1.The analysis shown that the complete open reading frame(ORF)of EuBRI1 contains no intron and encodes 1203 amino acid residues.Using online software analysis,the predicted EuBRI1 protein is a stable hydrophilic protein with a relative molecular weight of 131.361 kD.The theoretical isoelectric point pI is 6.31.In the secondary structure,the precentage of ?-helix,random coil and extended chain were 31.50%,55.94%,12.55%.The protein also has a conserved domain of BRI1 family kinases.EuBRI1 has a signal peptide(1-38aa)and transmembrane structure.The phylogenetic tree analysis indicates the level evolution of EuBRI1 is similarity with Solanum pimpinellifolium BRI1.2 Expression Characteristics analysis of EuBRI1 in E.ulmoidesReal-Time PCR was used to determine the expression levels of EuBRI1 gene in stems,leaves,flowers and fruit from March to September,2017.The results shown that EuBRI1 gene was expressed in stems,leaves,flowers and fruits in E.ulmoides.And there was a significant difference in expression level of different tissues.The expression level of EuBRI1 gene in stems of E.ulmoides was obvious higher than other tissues.Analyzing the expression level of EuBRI1 gene in stems and leaves from March to September,found that the expression leave of EuBRI1 gene in stems and leaves were shown a decreasing trend.Analysis the expression level of EuBRI1 in fruit during the fruiting period from April to August shown that the expression level of this EuBRI1 first decreased and then increased.In the fruiting period of E.ulmoides,the formation of fruit is the first,and then the peel begins to age but the seed begins to mature,so it's expression level in fruit appears fallen first and then increased.3 Function Identification of EuBRI1 geneTo verify the function of the EuBRI1 gene,the plant expression vectorpSH-35S-EuBRI1 was constructed,and transformed into Agrobacterium tumefaciens strain LBA4404 by freeze-melt method.The Arabidopsis BRI1 mutant bri1-5 and its corresponding wild-type Ws2(Wassilewskija)were transformed by the floral-dip method.Total of 2 Arabidopsis thaliana bri1-5 mutant transgenic lines,and 6 Ws2 transgenic lines were obtained after.Among them,the transgenic Line 2 of Ws2 has obvious BR role characteristics.The two bri1-5 transgenic lines did not restore the function of BRI1 protein.Indicating that EuBRI1 gene may not be completely identical to AtBRI1 gene function.The expression analysis of the transgenic lines revealed that the expression of EuBRI1 gene was extremely high in Ws2 line 2 with obvious BR function characteristics.Indicating that the high expression of EuBRI1 gene caused the Arabidopsis multi-branch phenotype.