Cloning And Expressions Of Rehmannia Glutinosa Genes RgVP, RghKAT, RglKAT And RghBNG

So far, some functional genes related to plant organ size and biomass,yield-enhancing genes,have been obtained,for example, Auxin-regulated gene involved in organ size,(ARGOS）,（ArabidopsisVacuolar H+-pyrophosphatase Gene，AVP）and so on.. In this study, a pair of degenerate primers weredesigned using Software CLUSTAL2.1and DNAMAN4.0. according to the conserved regions of fiveknown plants′ARGOS base sequences from GenBank Nucleic acid database in NCBI to clone RehmanniaGlutinosa functional genes by RT-PCR and RACE techniques, by which four Rehmannia glutinosa f.hueichingensis (Chan et Sehih)Hsiao functional genes such as RgVP, RghKAT, RglKAT and RghBNG,which is a unknown gene and homologous with B12D, were cloned. Based on them, bioinformaticstechnologies and RT-qPCR were used to predict their functions and analyze their expression patterns inthirteen tissues of Rehmannia glutinosa f. hueichingensis (Chan et Sehih)Hsiao plants at threedevelopmental phases, respectively. And RgKAT and RghBNG were cloned into prokaryotic expressionvector and expressed in E.coli using genetic engineering technique. The results were the following:1. RgVP was495bp in length. BLAST sequence analysis indicated that its base sequence washomologous to that of seven known plant V-PPase genes from GenBank Nucleic acid database in NCBIwith identity of83%to86%, showing that it is V-PPase cDNA in Rehmannia glutinosa f. hueichingensis(Chan et Sehih)Hsiao. RghKAT （GenBank accession number:JX290369）was1713bp in length,contains a1395bp open reading frame encoding a464amino acid protein, Sequence alignment and phylogeneticanalysis revealed that RghKAT shares high nucelotide sequence identity to that of Vitis vinefera (84%),Solanum lycopersicum(82%),Populus trichocarpa (82%),Arabidopsis thaliana (79%) andTriticum aestivum (73%). RglKAT （GenBank accession number:983188）was1640bp in length,contains a1395bp openreading frame encoding a464amino acid protein. Sequence alignment and phylogenetic analysis revealedthat RglKAT shares high nucelotide sequence identity to that of Vitis vinefera (82%), Solanumlycopersicum(81%), Populus trichocarpa (81%) and Arabidopsis thaliana (79%). Meanwhile, RghKAT andRglKAT do high amino acid sequence identity to that of Petunia x hybrida （88%），Vitis vinefera (88%),Cucumis satiuus (86%), Arabidopsis thaliana (87%) and Triticum aestivum (78%).The phylogenetic tree ofRgKAT was consistent with the evolutionary relationship among species.The physical and chemicalproperties of RgKAT indicated that it was a slightly alkaline and stable pritein. RgKAT shows a secondarystructure made of α-helixes, random coil,β-sheets and β-turns,and a tertiary structure characteristic ofthiolases. RghBNG （GenBank accession number:290370）was548bp in length,contains a282bp openreading frame encoding a93amino acid protein, Sequence alignment and phylogenetic analysis revealedthat RghBNG shares without high nucelotide sequence identity. However, RghBNG do high amino acidsequence identity to B12D of Beta vulgaris （80%）, Camellia sinensis (79%), Arabidopsis thaliana (79%),Lpomoea batatas (74%), Castanea sativa (73%) and Wolffia arrhiza (72%). And it do high amino acidsequence identity to unknown gene amino acid sequences of Populus trichocarpa （86%）, Glycine max(84%), Ricinus communis (82%), Lotus japonicus (81%), Medicago truncatula (79%) and Arabidopsislyrata subsp (79%). The phylogenetic tree of RghBNG was consistent with the evolutionary relationshipamong species.The physical and chemical properties of RghBNG indicated that it was a alkaline andunstable pritein. RghBNG shows a secondary structure made of α-helixes, random coil,β-sheets andβ-turns,and without signal peptide.2. Expression patterns anylysis of RghKAT、RglKAT and RghBNG in thirteen tissues ofRehmannia glutinosa f. hueichingensis (Chan et Sehih)Hsiao plants at three developmental phases by RT-qPCR demonstrated that RghKAT and RglKAT at seedling stage with the highest expression level in thestem, at blooming stage with the highest one in the petal and at maturation stage with the highest one in theleaf. At different stages, the expression level in the root increased firstly and then decreased, the expressionlevel in the petal decreased continuously up to without expression at the maturation and the expressionlevel in the leaf increased continuously. RghBNG at different stages and tissues was expressed. With thehighest expression level in the petal at blooming stage and then receptacle, low one in the root at bloomingstage and stem at maturation stage. Overall the expression level change of these three genes is obvious atdifferent stages and tissues.3. According to the full length sequences of RghKAT、RglKAT and RghBNG, we chose pET-32aas the prokaryotic expression vector, and BamHI and XhoI as restriction enzyme of upstream anddownstream. We inserted the ORF of RghKAT、RglKAT and RghBNG to expression vector pET-32a toconstructed prokaryotic expression vectors: pET-32a-RghKAT、pET-32a-RglKAT and pET-32a-RghBNG.These vectors transformed E.coli BL21（DE3） to obtain transformant: pET-RghKAT-BL21、pET-RglKAT-BL21and pET-RghBNG-BL21for studying protein characteristics of RghKAT、RglKAT andRghBNG. It had constrcted prokaryotic expression transformant of RghKAT、RglKAT and RghBNG andwere induced to express proteins by IPTG successfully.Thses results will lay the fundation for further structural and functional researches of RehmanniaGlutinosa genes, and its molecular breeding and the development of its gene products by geneticengineering.