Cloning And Expression Analysis Of EuABCF1 Gene In Eucommia Ulmoides Oliv.

Eucommia ulmoides Oliver?E.ulmoides?is a genus of E.ulmoides.It is a unique and valuable medicinal material in China.It has a long history of medicinal use and has a wide range of clinical applications.E.ulmoides has high economic value and scientific research value,but currently it has a high economic value and scientific research value.The mechanism of growth and development is not fully understood.ABC transporter,also known as ATP-binding cassette transporters,is the main carrier of transmembrane transport of various substances in plants,and is an essential component of living cell metabolism.It is divided into eight sub-clusters A-H and is one of the largest and oldest known protein families.In order to reveal the regulation of the growth and development of E.ulmoides ABC transporter gene and the expression and function of ABC transporter F subfamily gene,this study used the E.ulmoides transcriptome database constructed in our laboratory to clone the ABC transporter gene EuABCF1 by PCR.The bioinfor-matics and expression characteristics analysis was carried out,and the following main results were obtained.1 Cloning and Bioinformatics Analysis of EuABCF1 gene in E.ulmoidesBased on the E.ulmoides transcriptome database constructed in the laboratory,the ABC transporter homologous sequence was screened.The specific primers were designed according to the encoded cDNA sequence,and the full-length 1416 bp ORF sequence was cloned by polymerase chain reaction?PCR?using E.ulmoides cDNA as template.Sequencing and comment comparison,the sequence is an ABC transporter gene sequence designated EuABCF1.Bioinformatics analysis showed that the sequence encoded 471 amino acids,The predicted protein molecular weight is 54 kDa and its theoretical isoelectric point?pI?is 5.81,it was an unstable hydrophilic protein,the subcellular localization was predicted in the nucleus,with two transmembrane domain,without the transmembrane domain or signal peptide,which belongings to the ABC transporter F subfamily.Secondary structure analysis showed that Alpha helix accounted for 53.50%,Beta turn accounted for 5.94%,Random coil accounted for25.69%,and Extended strand accounted for 14.86%.Phylogenetic analysis indicated that the EuABCF1 protein of E.ulmoides was closely related to the EuABCF1 protein of the ABC transporter F subfamily of Rosa chinensis during the evolution process.2 E.ulmoides EuABCF1 gene expression analysisThe expression of EuABCF1 in roots,stems,leaves and male flowers of E.ulmoides was analyzed by qRT-PCR.The expression of EuABCF1 gene in E.ulmoides was analyzed.The results showed that EuABCF1 gene was expressed in all parts,and the expression level had significant differences.It shows tissue specificity in male flowers.The result speculated that this gene plays an important role in flower development.The expression levels of EuABCF1 gene in juvenile roots,young stems and leaves of E.ulmoides seedlings and adult E.ulmoides were analyzed.The results showed that adult young stems were significantly higher than young seedlings?P<0.01?,?P<0.01?.The expression of leaf in adult leaves and seedlings was not significantly different?P>0.01?,and the roots of adult plants were significantly higher than the young roots of seedlings?P<0.01?.At the same developmental stage,the expression of EuABCF1 gene was significantly higher in young leaves than in young steams and young roots,and was significantly higher in adult plants than in the leaves of adult plants?P<0.01?.It is speculated from the above results that EuABCF1 gene plays an important role in the growth and development of different parts of E.ulmoides at different stages,especially in flower development.3 The induction EuABCF1 gene by exogenous treatmentTo investigate whether EuABCF1 gene was induced by 400?mol·L^-1 IAA,120?mol·L^-1GA₃,100?mol·L^-1MeJA and 4?,the experiment used exogenous IAA,GA₃,MeJA and low temperature stress E.ulmoides seedlings.The expression of EuABCF1gene was detected by Real-time PCR,and the function of EuABCF1 protein was preliminarily estimated.The results showed that the relative expression of EuABCF1gene in E.ulmoides leaves increased under the induction of 400?mol·L^-1 IAA and 100?mol·L^-1MeJA compared with pretreatment.In the 4? induction test,the expression level of EuABCF1 gene in E.ulmoides leaves increased first and then decreased.;while120?mol·L^-1GA₃ treatment had no significant effect on the relative expression of EuABCF1 gene in E.ulmoides leaves?P>0.01?.The results showed that the expression of EuABCF1 gene was induced by 400?mol·L^-1 IAA,100?mol·L^-1 MeJA and 4?,and it was speculated that EuABCF1 protein may directly or indirectly participate in Methyl Jasmonate and auxin signal transduction and involved in regulation of plant growth and development of E.ulmoides and the anti-cold process of E.ulmoides.In summary,the EuABCF1 gene was induced by the experimental concentrations of MeJA and IAA,and may have a significant effect on the growth and development of different parts of E.ulmoides at different stages and the response to cold stress.