The Related Functional Gene Analysis Of Iterpenoid Biosynthesis Pathway In Rabdosia Rubescenss (Hems1.) Hara

Objective:1.Selecting suitable materials of rubescenss for the transcriptome technical analysis.2.Based on the pathway for diterpenoid biosynthesis,the candidate genes DXR?IdI?ISPF?ISPH were selected for E. coli cloning and sequencing analysis.to get the right gene sequence information.3. Based on the gene cloning sequencing information of genes DXR etc, analyze their biological information.4.The expression pattern of these genes in different tissue was analyzed by real-time fluorescence quantitative PCR.5.Analysis of the prokaryotic expression,to verify the the theoretical protein weight and lay the foundation of Protein function analysis.Method:1.First domesticate the Rabdosia rubescenss aseptic seedling of lower Oridonin and Ponicidin, to make sure they can grow in natural environment.Then respectively spray different concentrations of methyl jasmonic acid on the surface of rubescenss leafs and measure the content of oridonin of different rubescenss leafs collected after Oh?6h?12h?24h?48h? 72h to determine the time of highest content2.Afte rubescenss transcriptome data obtained,according to Rabdosia rubescenss pathway of diterpenoid biosynthesis,the candidate genes (1-deoxy-D -lxyluloses-phosphatereduetoisomerase,DXR)?(isopentenyl diphosphate isomerase,IdI)?(2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,ISPF)?(4-hydroxy-3-methylbut-2-enyl diphosphate reductase,ISPH) were selected for further analysis and design the primers of them to do E. coli cloning sequencing analysis.3.Based on the sequencing result,BLAST homologous gene sequences to confirm the ORF of these gene sequences and use online software to analyze the biological information.4.Designing qRT-PCT primers, the expression pattern of the gene in different tissue was analyzed by real-time fluorescence quantitative PCR.5.Designing the primers of prokaryotic expression for DXR?IDI?ISPF? ISPH?HMGS?AACT?DXS,use PET-28a vector and BL21(DE3) Competent Cell to do prokaryotic expression analysis.Result:1.After adding different concentration of stimulants, the change of oridonin content was not outstanding, so based on the result of Rabdosia rubescenss different sources quality evaluation, select the highest oridonin (DLC-G) and lowest oridonin(DLC-D) samples for transcriptome sequencing.2.High quality RNA were extracted from leaves of Rabdosia rubescens samples(DLC-G,DLC-D). mRNA was used as a templated to obtain cDNA library. The cDNA library as materials for transcriptome sequencing. After performing the paired-end Illumina sequencing,50934276 and 61561674 reads were obtained from Rabdosia rubescens samples(DLC-G;DLC-D).Guanine and Cytosine nucleotides (GC) percentage (proportion of guanidine and cytosine nucleotides among the total nucleotides) were 48.06% and 48.13%, respectively. In addition, a total of 44,626 unigenes were yielded.1298 differentially expressed genes were found between DLC-G and DLC-D, among them 339 genes were up-regulated and 959 down regulated genes.3.After E. coli cloning sequencing analysis,got the DXR?IDI?ISPF?ISPH gene sequences.Through online BLAST homologous gene analysis,Finally settled the sequence information of DXR?IDI?ISPF?ISPH4.Through online software, the length of DXR ORF is 1422bp,coding 473 amino acids,Protein molecular weight 51390.2,soelectric point 6.09;the length of IDI ORF is 906bp,coding 301 amino acids,Protein molecular weight 27444.3,soelectric point 5.06;the length of ISPF ORF is 708bp,coding 196 amino acids,Protein molecular weight 20870.1, soelectric point 6.75;the length of ISPH ORF is 1389bp,coding 462 amino acids,Protein molecular weight 52094.0,soelectric point 5.82.5.The expression of different tissues show that the expression of DXR is 2 times higher than in stems than in other tissues.The expression of IDI in leafs is higher than in stems and roots, but the result in stems and roots is essentially same.The rsult of ISPF in leafs is highest,almost 5 times higher than in roots,and the expression in stems is 3.8 times higher than in roots.For the expression of ISPH,it is basically the same in roots,stems and leafs; For stress expression analysis,Methyl jasmonic acid promote the expression of DXR? IDI?ISPF?ISPH,just express quantity is not obvious.While activol?ATP? Rare earth element lanthanum and cerium inhibit the expression of DXR,etc.6.After by IPTG induction expression for the night,by protein SDS-page analysis,induced the DXR?ISPF?DXS protein.