Clone hedgehog pentagar leaf-based pyrophosphate synthase GPS.

Abstract: Objective cloning the cDNA sequence of the genes of the geranyl pyrophosphatesynthase (GERanyl pyrophosphatesynthase, GPS) gene and analyzing the characteristics of the gene sequence, the level of gene expression in different organs and their correlation with the content of saponins.
method to extract the five-plus RNA, reverse transcription as cDNA.
designed a specific primer based on the unigene (c37362.graph_c0) encoded GPS in the transcription sequencing results, which amplifies the full length of the cDNA of the GPS gene through PCR.
use real-time fluorescence quantitative PCR (qRT-PCR) to analyze the expression level of GPS genes in different organs, and detect the total saponin content of the hedgehog by the photolight-splitting method.
results in cloning results in a hedgehog-5 plus GPS gene cDNA that is 1,260 bp long and encoded with 419 amino acids.
GPS protein is positioned in the mitochondria and there is no cross-membrane region.
GPS gene is expressed in all organs, with the highest amount of expression in the leaves, 5.26 times the amount expressed in the root. the relative expression of the
GPS gene and the amount of saponinshowed the trend of the same rise and fall, which showed a significant positive correlation (r-0.851, P-0.05).
conclusion that for the first time, the cloning obtained the cDNA full-length sequence of the sting five-plus GPS gene, and the positive correlation between the expression of the sting-plus GPS gene and the content of saponins was clarified.
hedgehog eleutheccussenticosus (Rupr.et Maxim.) Maxim is a five-plus-plus plant, is one of the traditional medicinal plants in China, and belongs to ginseng and 37 to the five-plus co-plants.
the root, stem and leaves of the hedgehog can be intake and have many pharmacological effects, of which triamcinolone is an important medicinal active ingredient.
triamcinolone is present in most medicinal plants and has anti-tumor, treatment of hyperlipidemia, heart protection and other functions. The synthesis pathways of triterium compounds in
plants are usually divided into mevalonateeic acid (mevalonatepathway, MVA) pathways and 2-methyl-D-erthriol-4-phosphate (methylerythritol 4-phosphate, MEP) pathways.
in the MVA pathway, after methyxamatepyride catalyzed the synthesis of isoprene-based pyriphosphate (Isopenylpyrophosphate, IPP), IPP produces the fennel-pyrophosphate synthase (geranyl pyrophosphate, GPS) catalyzed, and then the precursor of trichomoniase is produced by the catalysis of a variety of enzymes. the expression of the
GPS gene is closely related to the amount of product seisacated by it.
the use of peanut tetraoleic acid (AA) and other inducers to treat the green dushi algae Dunaliella viridis Teodoresco, with the increase in AA mass concentration, the total amount of carotenoids significantly increased;
from pepper mint Mentha x piperita L. the scented leaf-based diphosphate synthase small sub-subakey can increase the yield of monochos in genetically modified tobacco plants.
, after being cloned, the GPS gene cDNA sequence was cloned, it was proved that the expression of the GPS gene may lead to changes in cycloenoeteryeterycompounds.
there have been reports on the cloning and expression analysis of the key enzyme genes involved in the synthesis of triamcinolone, but there have been no studies on the gene of sting or GPS.
the study for the first time to clone the hedgehog-scented leaf-based comophosphate synthesis enzyme, and use bio-information to predict and analyze GPS proteins, through qRT-PCR technology to explore the expression of GPS genes in different organs and GPS gene expression and the correlation between the total amount of aporpoly plus saponins, for the follow-up further to clarify the hedgehog gene catalytic triamcine synthesis mechanism to establish an important basis.
1 material and instrument 1.1 material hedgehog samples taken from Benxi City, Liaoning Province, by the North China University of Technology Professor Yu Zhaobin identified as five plus five plus Eleutherococ cus senticosus (Rupr.et Maxim.) Maxim.
selected five-plus root, stem, leaf and leaf handle, clean water washing, filter paper to suck dry water preserved at 80 degrees C, used as a sample for subsequent extraction of hedgehog five plus total RNA and total saponins.
1.2 Instrument RNAprep Pure Plant Total RNA Extraction Kit, Fast Quant RT Kit (with gDNase), Agar sugar gel DNA Recovery Kit, pLB Zero Background Rapid Clone Kit, Talent qPCR PreMix (SYBR Green), TOP-10 Sensor Cells, 2x Taq PCR MasterMix, DL2000 DNA
ampicillin, IPTG, X-gal, etc. purchased from Beijing Byrdi Biotechnology Co., Ltd.
the rest of the chemical reagents are domestic analysis pure.
primers are synthesized by Beijing Northagenome Genomics Research Center Co., Ltd.
2 method 2.1 total RNA extraction and reverse transcription extract editing of the hedgehog pentatrice, stem, leaf and leaf handle each 0.1 g, as the material for the extraction of sting five plus RNA, according to the RNA pureprep plant total RNA extraction kit method for the hedgehog pentaplus total RNA extraction.
use 1% agarose gel electrophoresis to detect RNA integrity and reverse-record the total RNA as a template in a reverse transcript iRNA, which is stored at .80 degrees C. the cloning of the
2.2 sting five plus GPS gene is based on the results of the sequencing of the hedgehog-plus transcription group, the screening of GPS unigenes (c37362.graph_c0) design 1 pair of specific primers, the upstream primer CGPSS7:5'-ATTTTTCTCTCTCC-GGATTATAC-3';
use cDNA as a template for PCR amplification, the reaction system is 25 ?L, wherein the template is 1 ?L, the upstream and downstream primers are 1 ?L, 2 x Taq PCR MasterMix 12.5?L, ddH2O 9.5?L.
reaction conditions: pre-degeneration 94 degrees C, 3 min; denatured 94 degrees C, 30 s; annealing 50 degrees C, 30 s; extension 72 degrees C, 110 s, a total of 40 cycles, and finally supplemented extension of 5 min.
electrophoresis of PCR products using 2% agar gel, cutting down the expected DNA fragments, and using agar gel DNA recovery kit recovery, purification of DNA fragments.
connect the recycled product to the pLB carrier, and transfer the recombinant carrier into TOP-10 receptor cells, after 37 degrees C concussion culture, take 100 sl coating in a medium containing ampicillin, screening 3 to 5 positive colonies for verification.
finally sent the bacteria to Beijing Northagenome Research Center Co., Ltd. for sequencing analysis.
After the bioinformatics analysis of the 2.3 sting five-plus GPS gene was obtained according to the cloning of GPS gene cDNA sequence in "2.1" item, protparam in ExPASy was used to predict the basic properties of proteins such as the amount of protein amino acids, relative molecular mass, theory and other electrical points (PI).
analyze the protein structure functional domain in the Conserved Domain Database (CDD) online software in NCBI.
predict protein transmembrane se- with TMHMM Server v.2.0 software.
analyzed protein subcellular localization in TargetP 1.1 Server software, SignalP 3.0 Server predicted signalpeptides, and soPMA software used protein secondary structures to predict. After
, the protein three-stage structure is predicted by SWISS-MODEL.
finally, the system evolution tree of GPS proteins was built using neighbor-joining in the MEGA 6.05 software.
the expression of the 2.4 sting five plus GPS gene analysis to the cDNA of the hedgehog, stem, leaf and leaf handle as the qRT-PCR template.
designed qRT-PCR primer, upstream primer CGPSrtS3: 5'-GCCTGATGATGATGATGAGGGGGGGATGGAGGAGGAGG-3';
the method of reference to the literature, using the GAPDH gene as the endotorine gene, and designing the qRT-PCR primer, upstream primer RGS: 5'-GATTTGGCA-TTGTGAGGG-3', downstream primer RGX:5'-TGCTATC-GCCTATGAGTCC-3', with an expected amplified growth of 134 bp.
used the above 2 pairs of primers to perform a qRT-PCR reaction, each sample repeated 3 times.
total reaction system is 10?L, upstream and downstream primers 0.3 s, cDNA templates 0.5?L, 50 x ROX Reference Dye 1?L, RNase-Free ddH2O 2.9?L, 2 x Talent qPCR PreMix 5?L.
reaction conditions: pre-degeneration 95 degrees C, 3 min; denatured 95 degrees C, 5 s; annealing 55 degrees C, 10 s; extension 72 degrees C, 15 s, a total of 40 cycles.
the relative expression of GPS genes in each sample, the method of reference literature.
method of 2.5 sting five plus total saponin content and correlation analysis reference literature, and the total saponins of sting five-plus roots, stems, leaves and leaf shanks were measured.
also used this method to screen out the high and low content groups with significant differences in sapnucleoside content, and 3 strains in each group, and analyzed the saponin content and GPS gene expression.
used SPSS 18.0 software to analyze the correlation between GPS gene expression and saponin content in five plus six leaf samples.
3 results and analysis of 3.1 sting five plus GPS gene cloning and sequence analysis to sting five plus cDNA as a template, design a pair of specific primers CGPSS7, CGPSX7, after PCR amplification to obtain about 1,200 bp DNA fragments.
connected to the pLB carrier, sequencing resulted in a strip length of 1,260 bp (Figure 1), consistent with the expected amplified growth.
after BLAST software comparison, confirmed that the sequence is the whole length of cDNA of the hedgehog five plus GPS gene, through DNASTAR and BLAST pair confirmation open reading box (ORF) length of 1,260 bp, coding 419 amino acid proteins, the starting cryptoon at ATG, termination crypt is TAA.
3.2-plus-GPS gene bioinformatics analysis predicted by Protparam software in ExPASy: protein relative molecular mass is 46 329.16; PI is 6.32; leucine has the highest proportion of all amino acids, reaching 12.6%, the total number of negatively charged amino acid residues (Asp-Glu): 46;
in in vitro mammalian net-woven red blood cells, the predicted half-life is 30 h; The
hydrophilic average is 0.040 and belongs to hydrophilic protein.
uploaded amino acid sequences to SOPMA online software, the results showed that GPS proteins had 233 alpha-helix, accounting for 55.61 percent; 30 extension strands), accounting for 7.16 percent, 22 beta-coils, accounting for 5.25 percent, and 134 irregular curls (random curls) accounting for 31.98 percent. The results of the
protein structure functional domain show that GPS protein is one of the super family members of isoprene (Isoprenoid_Biosyn_C1.
GPS protein has four Trans_IPPS_HT (in 102-417 amino acids), IspA (80-419), polyprenyl_synt (104-364), prenyl_cyano (82-417) 4 domains, of which the Trans_IPPS_HT domain contains substrate binding pockets, substrate-Mg2 plus binding sites, chain length decision area, catalytic residuals, tiandong rich area I 7 conservative points.
also contains Trans_IPPS_HT domain in the reported green dushi algae , Kawasi etresanda , which also contains the Trans_IPPS_HT domain , indicating that the GPS protein of the hedgehog belongs to the isoprene-like super-family.
the results of the TMHMM Server v.2.0 software prediction results show that the GPS protein does not exist in the transmembrane region, and TargetP1.1 Server shows that the GPS protein is positioned in the mitochondria with a high degree of reliability.
in signalP 3.0 Server software predicts that GPS proteins contain signal peptides, location may be in the 13th to 14th amino acids. The prediction results of the three-stage structure of the
sting five plus GPS protein are shown in Figure 2.
3.3 hedgehogs plus GPS protein system evolutionary tree analysis based on blast amino acid sequence comparison results, 18 species and hedgehogs.