Isolation And Functional Analysis Of EuABP2 Gene In Eucommia Ulmoides Oliv

E.ulmoides,also known as bakelite,is a deciduous tree in Eucommia family and an unique tertiary relict plant in China.E.ulmoides is not only a valuable traditional Chinese medicine,but also an important rubber source plant.It is mainly distributed in Yunnan,Guizhou and other places in China.E.ulmoides is solc speices in the Eucommia genus,the only genus in Eucommiaceae family.Studying the growth and development related genes of E.ulmoides is helpful to the cultivation and utilization of Eucommia ulmoides.The auxin-binding protein gene(ABP)is a key gene in auxin responce which plays an important role in the regulation of plant growth and development.In this study,the ABP gene were cloned based on the transtriptome and genome database of E.ulmoides.The results are as followed:1.Cloning and sequence analysis of Eu ABP2 geneIn this study,the primers were designed according to the sequence of ABP gene from E.ulmoides transcriptome and genome database.The c DNA reverse transcripted RNA extracted from young leaves of the E.ulmoides which was planted in the labortory for over 15 years was used as a template,the full CDS of the auxin binding protein gene was cloned by PCR named Eu ABP2.The full CDS of Eu ABP2 is 609 bp long and encodes 202 amino acids.The amino acid sequence of Eu ABP2 shares the highest similarity(80%)with the auxin binding protein of Jatropha.Which contains 3highly conserved sequence of auxin binding proteins and one auxin binding site(Cupin domain).Total of 11 nucleotide different residues were found after aligendingwith Eu ABP1(Accession No: AY509875)from Genbank,which leads to 5 different amino acid residues between Eu ABP1 and Eu ABP2.Among the different amino acid residues,the fifth residues is located in Box C domain,which suggests that Eu ABP1 and Eu ABP2 may fuction differently.Bioinformatics analysis shown that the Eu ABP2 protein which has a theoretical isoelectric point of 5.17 and a molecular weight of22.7 k D.In addition,total of 19 phosphorylation sites was detected in the protein which wre throughout the polypeptide chain.The analysis also suggests that the 1st to33 th amino acid residues of the Eu ABP2 protein were likely to be a signal peptide.The Eu ABP2 protein do not contains the transmembrane domain and has a KDEL sequence on it's C terminal,which indicated the protein is probably located on the ER.The secondary structure of the protein contains alpha helix and 10 beta sheets,which were connected by 12 random coils.Phylogenetic tree analysis showen that the Eu ABP2 protein had close relationship with monocotyledonous oats and maize,but had a distant relationship with other dicotyledons,which indicats the uniqueness of Eu ABP2 in evolution.2.Spatial expression characteristics of Eu ABP2 geneTo study the expression characteristics of Eu ABP2,Real-time PCR were running to analysis Eu ABP2 gene expression in different tissues of E.ulmoides,and the EF1?were used as an internal reference.The results showen that the Eu ABP2 gene had the highest expression in the stems of E.ulmoides and the lowest expression in the male flowers of E.ulmoides.The results showen that Eu ABP2 gene had a great influence on the growth and development of Eucommia ulmoides stems in the testing period,which may be related to the secondary growth of xylem controlled by auxin.3.Construction of Eu ABP2 gene expression vector and genetic transformation of Nicotiana tobacum and E.ulmoides.The p GM626-Act1-ABt was used to as the initial vector,construetd the plant expression vector p GM626-Act1-Eu ABP2 with one-step cloning method.The geneEu ABP2 were controled by Act1 promot and a Bar gene were contained as the selectable marker gene.After PCR and sequencing validation,the vector was introduced into Agrobacterium tumefaciens LBA4404.Transformation of callus and hypocotyl transformation were used respectly to generate the transgenic plant.Total of 50 tobacco plants were obtained,and 9 of them were identified to be positive transgenic plants with GUS histochemical staining and Real-time PCR analysis.In addition,14 resistant strains of E.ulmoides were also obtained,and 12 positive plants were detected by GUS histochemical staining,which provided experimental materials for the study of Eu ABP2 gene function.4.Effect of Eu ABP2 overexpression to the development of transgenic tobacumObserving the morphology of tobacco plants transformed with Eu ABP2 and wild-type tobacco plants,it was found that the number of lateral roots of transgenic tobacco plants was significantly more than that of wild tobacco plants.It was shown that overexpression of Eu ABP2 gene resulted in the down-regulation of tobacco IAA28 gene,resulting in an increase in the number of lateral roots in transgenic tobacco plants.Real-time PCR analysis before and after exogenous IAA treatment,relative expression levels of IAA9,IAA13,IAA28,ARF8,and ARF19 were measured.In tobacco TP4 lines transformed with Eu ABP2 gene and wild type tobacco plant.The results showed that after exogenous IAA treatment,the expression of ARF19?ARF8 in transgenic tobacco was inhibited and the expression of IAA13 was promoted,indicating that when the binding of IAA to Eu ABP2 reaches a certain amount,it may affect the expression of IAA-responsive genes.5.Effect of overexpression of Eu ABP2 in the development of Eucommia ulmoides transgenic plantThe morphology charateristics were observed between Eu ABP2 transgenic E.ulmoides plant and the wild type plant after 60 days grown.The average diameter of Eucommia ulmoides root with Eu ABP2 gene overexpression(2.31 mm)wassignificantly greater than that of wild type(1.01 mm),while the length of root in transgenic Eucommia ulmoides(13.0 mm)was significantly smaller wild type(147.0mm).It showed that the increase of Eu ABP2 protein amount could affect the development of Eucommia root,and the mechanism may be related to the increase of auxin sensitivity of Eucommia root cells.