Functional Analysis Of The HMGR Gene Encoding The Key Enzyme Involved In Rubber Biosynthesis Of Eucommia Ulmoides

China is the largest natural rubber consuming country in the world,while the dependence of imported natural rubber is more than 80%,and it has surpassed Chinese national strategic security cordon.In face of shortage of natural rubber,a new species for producing rubber is eager to be found.At present,the Eucommia rubber(EU-rubber)is regarded as the most potential development nature rubber,while,EU-rubber content in leaves and pericap of Eucommia ulmoides(E.ulmoides),which are the main raw materials for extracting the gutta-percha,is relatively low,this leading to production cost increasing and become the bottleneck of developing the Eu-Rubber industry.Therefore,revealing the mechanism of gene regulation in EU-rubber biosynthesis will contribute to get the ways to improve content of Eu-Rubber.In this study,the gene expression patterns and function of EuHMGR were analyzed by using gene cloning,Real-time PCR,sub-cellular localization,over-expression,cloning promoter and express active validation,besides,the system of tissue culture of E.ulmoides was established.These work lay the foundation of revealing the molecular mechanism of EuHMGR gene in the EU-rubber biosynthesis process.The main results in this study are as follows:(1)The full-length cDNA sequence of EuHMGR was cloned,and was analyzed by bioinformatics tools.These results showed that length of EuHMGR gene's open reading frame(ORF)was 1770 base-pairs,encoded 590 amino acids,and the relative molecular mass of that coding product was 63 k Da,isoelectric point was 6.89.EuHMGR contained two HMG-Co A binding sites(EMPVGYVQIP and TTEGCLVA),and two NADP(H)binding sites(DAMGMNM and GTVGGGT).In addition,alignment of the deduced protein sequences of EuHMGR revealed more than 80% identity with the HMGR from other known plants.(2)The correlation of the EuHMGR gene expression pattern and the synthesis of EU-rubber was analyzed.EuHMGR expression of leaves and pericarp at different periods in E.ulmoides was quantified,the results showed that EuHMGR was highly expressed exclusively in pericarp during the young fruit stage(from April to May),whereas,it was present at highest levels of leaves in the middle of July.The correlation analysis of EuHMGR expression patterns and accumulation rate of Eu-Rubber in leaves and pericarp at different stages suggested that expression of EuHMGR correlated with the accumulation rate of Eu-Rubber in pericarp of E.ulmoides and might be responsible for it.By contrast,the expression level in leaves was not correlated with the accumulation rate of Eu-Rubber..(3)The sub-cellular location of the EuHMGR gene coding product was determined.The fusion expression vector of the EuHMGR gene and YFP was constructed and was transisent expressed in the tobacco leaves to analysis the sub-cellular localization of EuHMGR gene.The result showed that the EuHMGR was targeted in the endoplasmic reticulum,which was consistent with that MVA pathway was located in the cytoplasm.(4)The functional verification of EuHMGR.By constructing 35S::EuHMGR over-expression vector,the recombined plasmid was introduced into EuTIDS5 transgenic tobacco via Agrobacterium-mediated transformation,and a total of 28 transgenic tobacco were obtained.The expression level of EuTIDS5 was detected and that was increased at different levels.Then,the trans-polyisoprene content of the positive plants were determined,the result showed that,the trans-polyisoprene in EuHMGR gene excessive expression plants increased 4-13%,which indicate that over-expressing EuHMGR gene was helpful to the expression of EuTIDS5 and improved the synthesis and accumulation of trans-polyisoprene.(5)EuHMGR gene promoter sequence was cloned and analyzed,and its function was verified.The fragment length 2104 bp upstream EuHMGR gene was cloned,and the sequence analysis showed that EuHMGR gene promoter sequences contained a lot of conservative transcription elements,such as like TATA box,CAAT box.Besides,it also contained many cis-acting elements involve in plant growth and development,hormone response,biotic and abiotic stress.The plant recombinant expression vector p EuHMGR::GUS was constructed and transisent expressed in tobacco.GUS histochemical assay showed that EuHMGR gene promoter could activate the GUS reporter gene normally transcription,which suggested that the fragment had the function of promoter.(6)Using The hypocotyl segments of E.ulmoides aseptic seedling were chose as explant to preliminary establish the system of E.ulmoides tissue culture technology.MS(Murashige-Skoog,inorganic salt)supplemented with B5(organics),30 g/L sucrose and.6.0 g/L agar was chose as the basal medium,the hormone formula of callus induction was 6-BA(1.0 mg/L)and NAA(1.0 mg/L),and the formula of callus transgenerational was 6-BA(0.5 mg/L)and NAA(0.5 mg/L),the rate of callus induction was up to 90%.The hormone formula of differential medium was 6-BA(1.0 mg/L),TDZ(1.0 mg/L)and NAA(0-0.15 mg/L),the differentiation rate was approximately 20%.The rooting medium used half-strength MS medium with 15 g/L sucrose and 6.0 g/L agar,and those in best growth form were the end of incision treated by 300 mg/L sterile ABT for 5seconds.