Cloning And Functional Analysis Of Taraxacum Kok-saghyz Rodin Small Rubber Particle Protein Gene And Its Promoter

Object:Natural rubber plays an important role in the national economy and science and technology.The quality of rubber produced in the roots of Taraxacum kok-saghyz Rodin is comparable to that of Hevea brasiliensis,due to its short reproductive cycle and easy genetic transformation.The advantages are the ideal model plants for studying the mechanism of rubber production in natural rubber.Small Rubber Particle Protein(SRPP)is an important component of rubber particle membrane protein,which plays a role in rubber solidification and rubber production.Therefore,the function of SRPP gene in rubber grass is studied and the expression of SRPP gene is controlled.The regulatory elements lay the theoretical foundation for studying the molecular mechanism of natural rubber biosynthesis.Method:In this study,we used Taraxacum kok-saghyz Rodin as experimental material to analyze the expression pattern of TkREF/SRPPs gene family by fluorescence quantitative method.The TkSRPP3 gene was used as the research object to analyze its spatiotemporal expression and prokaryotic expression pattern.The transgenic technology was used to obtain the Taraxacum kok-saghyz Rodin plant with TkSRPP3 gene.Based on the genomic data of Taraxacum kok-saghyz Rodin,the PTkSRPP3 promoter was cloned,and the P0/P1/P2/P3/P4::GUS plant expression vector was constructed by genetic engineering technology.The activity of the deleted promoter was analyzed by histochemical staining;hormone and injury.The P0/P1/P2/P3/P4::GUS transgenic tobacco was treated,and the change of GUS reporter gene expression was detected by GUS enzyme activity assay,and the influence of promoter deletion elements on environmental changes was analyzed.Result:(1)The expression levels of TkSRPP1 and TkSRPP3 in TkREF/SRPPs family were significantly up-regulated after treatment with methyl jasmonate(MeJA)in TkREF/SRPPs gene family.After treatment with abscisic acid(ABA),the expression level of TkSRPP3 in TkREF/SRPPs family was significantly up-regulated;(2)The TkSRPP3 gene was cloned,the open reading frame was 690 bp,encoding 229 amino acids,and the TkSRPP3 protein was 24.431 kDa.It was an acidic protein with a REF conserved domain and was a member of the REF/SRPPs family.Phylogenetic tree analysis indicated that TkSRPP3 was Taraxacum brevirostre TbSRPP3 has high homology;spatiotemporal expression analysis showed that TkSRPP3 gene showed the highest expression in root tissues of June age;prokaryotic expression analysis TkSRPP protein size was 24.3kDa,and induced at 1mM IPTG for 6h.Good induction conditions,a large number of proteins were obtained;pCAMBIA2300-TkSRPP3 plant expression vector was constructed,and 5 transgenic TkSRPP3 gene Taraxacum kok-saghyz Rodines were successfully obtained;(3)The PTkSRPP3 promoter was cloned and the sequence length was 2188 bp.Sequence analysis indicated that the promoter has basic promoter elements,and also contains tissue-specific expression elements and cis-acting elements in response to light,hormones,damage,heat,etc.A plant expression vector incorporating the GUS reporter gene,histochemical staining confirmed that the promoter has promoter activity and no tissue specificity;construct P0(-2188bp)/P1(-1592bp)/P2(-1274bp)/P3(-934bp)/P4(-450bp)promoter deletion fragment was fused to the plant expression vector of the GUS reporter gene.GUS staining analysis showed that the promoter deletion fragment was active,and the deletion promoter activity decreased with the truncation of the fragment;(4)Tobacco plants stably expressing GUS gene by ABA,salicylic acid(SA),MeJA and injury treatment showed that the expression of GUS enzyme showed P0(-2188bp)containing abscisic acid response element./P1(-1592bp)/P2(-1274bp)was positively regulated by ABA treatment;P2(-1274bp)containing jasmonic acid response element was positively regulated by MeJA treatment;P1(-1592bp)containing salicylic acid response element)/P2(-1274bp)is positively regulated by SA treatment;P3(-934bp)containing damage response elements is positively regulated by injury treatment.Conclusion:(1)The TkREF/SRPPs gene family responded to the expression patterns of MeJA and ABA.The TkSRPP1-6 gene responded differently under the treatment of MeJA and ABA hormones.The TkSRPP3 gene was simultaneously affected by two types of hormones.Strongly induced expression;(2)The bioinformatics analysis of TkSRPP3 gene indicated that TkSRPP3 belongs to the REF/SRPPs family and has no transmembrane domain.The spatiotemporal expression analysis of TkSRPP3 gene showed that the expression level of TkSRPP3 was the highest in the root tissues of June age Taraxacum kok-saghyz Rodin.(3)The PTkSRPP3 promoter has a promoter activity and has no tissue expression specificity;P0(-2188bp)/P1(-1592bp)/P2(-1274bp)containing the ABA response element in the PTkSRPP3 deletion promoter is positively regulated by a ABA treatment;P2(-1274bp)of JA response element was positively regulated by MeJA treatment;P1(-1592bp)/P2(-1274bp)containing SA response element was positively regulated by SA treatment;P3(-934bp)was positively regulated by injury treatment.