CN105886638A - Method for detecting salidroside synthetase gene expression activity by using dual reference genes - Google Patents
Method for detecting salidroside synthetase gene expression activity by using dual reference genes Download PDFInfo
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- CN105886638A CN105886638A CN201610316147.0A CN201610316147A CN105886638A CN 105886638 A CN105886638 A CN 105886638A CN 201610316147 A CN201610316147 A CN 201610316147A CN 105886638 A CN105886638 A CN 105886638A
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention provides a method for detecting salidroside synthetase gene expression activity by using dual reference genes. Specifically, a primer which is obtained through design and screening is adopted to amplify to obtain glyceraldehydes phosphate dehydrogenase and phytochelatin synthetase genes, and correct and detect the gene expression activity of three key enzymes, namely, tyrosine decarboxylase, tyrosine transaminase and phenylalanine ammonialyase of synthesized salidroside in rhodiola crenulata tissue culture seedlings by using the gene as the dual reference genes. The method comprises the following steps: extracting total RNA of a rhodiola crenulata plant; synthesizing a cDNA first chain; and by using a fluorescent quantitative PCR technique, amplifying by using a given primer so as to obtain the dual reference genes of GAPDH and PCS, and correcting and detecting the expression activity of synthesized salidroside key enzyme genes TyDC, TAT and PAL in the rhodiola crenulata tissue culture seedlings. The method has the advantages of rapidness, accuracy, practicability, stability and the like in aspects such as production quality control and product activity detection on rhodiola crenulata tissue culture seedlings, cell suspension culture, hairy root culture and the like.
Description
Technical field
The invention belongs to the key enzyme activity detection of reactive compound in plant, particularly relate to use PAL, tyrosine decarboxylase and the method for TAT activity of gene expression in double reference gene combination correction detection rhodiola.
Background technology
Rhodiola (Rhodiola crenulata) is under the jurisdiction of the rare medicinal plant of Crassulaceae (Crassulaceae) rhodiola (Rhodiola), for perennial meat draft, it is distributed mainly on that the southwest China height above sea level strong ultraviolet more than 3000m, the temperature difference be big, arid, high and cold, the mountain area of anoxic, being unique Original plant of specifying of the Rhodiola that People's Republic of China's " pharmacopeia " includes, Ye Shi China high mountain people use the autonomic drug among the people of nearly one thousand years.It is mainly used in treating altitude sickness and air hunger, is described as " plateau ginseng ".Its main active is rhodioside, has the former activity of adaptation, has good curative effect at antifatigue, radioresistance, the aspect such as anti-oxidant, is widely used in the production of cosmetics, specific function health products, food etc..
The market demand of rhodiola root and rhodioside rises year by year, and supply falls short of demand for wild resource, and people attempt multiple production method and improve the output of rhodioside.People have carried out numerous studies at aspects such as artificial cultivation, tissue cultures, suspension cell culture, fungi growth-promotings, achieve relatively much progress.
People use and change environmental condition, interpolation precursor or the accumulation ability of intermediate change rhodioside at present, and the main method to the detection of rhodioside metabolin is to use HPLC to detect rhodioside yield；Also having scholar to pass through clone, transgenosis, the simple way raising rhodiola root yield improving some key gene in rhodiola root, afterwards by yield of method detection rhodioside such as HPLC etc..But there is the shortcomings such as time-consuming length, the most notable, the quantitative inaccuracy of effect in these methods.It addition, for utilizing endogenetic fungus resource and host's symbiosis, promote the practice of the accumulation of rhodioside in rhodiola plant by the way rebuilding microorganism or its group, be the method for emerging solution problem, still there is no the report that metabolic pathway key enzyme activity detects.
Summary of the invention
It is an object of the invention to provide the amplimer of 5 rhodiola root genes for rhodiola root study metabolic pathways, including 2 reference genes [Triose phosphate dehydrogenase (GAPDH), phytochelatin synthase (PCS)] and the primer of 3 key genes [PAL (PAL), tyrosine decarboxylase (TyDC) and TAT (TAT)].
Further object is that the above-mentioned primer amplification of employing, and then obtain with GAPDH and PCS three rhodioside synthesis key gene TyDC of double reference gene correction detection rhodioside, the method for TAT, PAL expression activity.
The primer that the present invention provides, is specifically shown in Table 1.
2 reference genes and the PCR primer of 3 rhodioside key synthase genes in table 1. rhodiola root
The method of a kind of pair of reference gene detection rhodioside synthase gene expression activity that the present invention provides, step is as follows:
1) method for tissue culture is used to obtain rhodiola plantlet in vitro.Inoculated fungi elicitor, hot-house culture (intensity of illumination 30 μm ol m-2·s-1；Light cultivates 14h, temperature 28 DEG C；Light culture 10h, temperature 22 DEG C；Humidity 70%～75%), periodically (4d, 6d, 8d, 10d, 12d, 14d, 16d) gathers rhodiola root plantlet in vitro；The plantlet in vitro do not inoculated is as comparison (CK).
2) Total RNAs extraction: use conventional method to extract the total serum IgE of rhodiola plantlet in vitro, and synthesize cDNA the 1st chain.
3) primer provided with table 1, with cDNA the 1st chain as template, 5 genes of quantitative pcr amplification.Amplified reaction program is: 94 DEG C of denaturations 30s, 95 DEG C of sex change 5s, and 56 DEG C of annealing 15s, 72 DEG C extend 10s, 45 circulations.
4) geNorm is used, Normfider, the stability of 2 reference gene GAPDH, PCS expression of Bestkeeper software analysis and CT value etc., result shows, GAPDH and PCS good stability, can be as three rhodioside synthesis key gene TyDC of double reference gene combination corrections detection rhodioside, TAT, PAL expression activity.
5) key gene expression activity is analyzed: utilizes excel software to calculate each sample TyDC, the relative expression quantity of TAT, PAL respectively, and using GAPDH and PCS as double internal references, carries out experimental error rectification.Utilize 2- ΔΔ CTThe method of [Δ Δ CT=(the ct value of the ct value of to be measured group of key gene-to be measured group of reference gene)-(the ct value of the ct value-control group reference gene of control group key gene)] calculates the TyDC of symbiosis seedling, TAT, PAL activity of gene expression.Result shows that the method can three key gene expression activities of detection rhodioside stable, accurate, efficient.
Described step 1) in fungi be preserving number be the fungi Phialocephala fortinii of CGMCC No.9347.The depositary institution of this bacterial classification: Chinese microorganism strain administration committee common micro-organisms center (CGMCC), preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, the preservation time: on July 10th, 2014.
Compared with prior art beneficial effects of the present invention: this patent uses Real-Time Fluorescent Quantitative PCR Technique, with geNorm, NormFinder, on the basis of a series of reference genes in rhodiola root are evaluated by Bestkeeper software, final selection determines that foundation carries out dual-gene combination correction with bis-reference genes of GAPDH and PCS and detects three rhodioside synthesis key gene TyDC, TAT, the expression activity of PAL, this is to rhodiola plantlet in vitro, cell suspension cultures, the quality control that hairy root cultivation etc. produce, the aspects such as its lytic activity detection, have quickly, accurately, practical, the advantage such as stable, it it is a kind of new technology with applications well prospect.
Accompanying drawing explanation
Fig. 1. double reference gene GAPDH and PCS are to TyDC activity of gene expression correction detection Activity Results figure
Fig. 2. double reference gene GAPDH and PCS are to TAT activity of gene expression correction detection Activity Results figure
Fig. 3. double reference gene GAPDH and PCS are to PAL activity of gene expression correction detection Activity Results figure
Detailed description of the invention
The fungi used in embodiment be deposit number be the fungi Phialocephala fortinii fungi of CGMCC No.9347.
Embodiment 1
1) method for tissue culture is used to obtain rhodiola plantlet in vitro.Inoculated fungi CGMCC No.9347, hot-house culture (intensity of illumination 30 μm ol m-2 s-1；Light cultivates 14h, temperature 28 DEG C；Light culture 10h, temperature 22 DEG C；Humidity 70%～75%), periodically (4d, 6d, 8d, 10d, 12d, 14d, 16d) gathers rhodiola root plantlet in vitro；Gather the plantlet in vitro do not inoculated as comparison (ck).
2) Total RNAs extraction: use conventional method to extract the total serum IgE of rhodiola plantlet in vitro, and synthesize cDNA the 1st chain.
3) primer provided with table 1, with cDNA the 1st chain as template, 5 genes of quantitative pcr amplification.Amplified reaction program is: 94 DEG C of denaturations 30s, 95 DEG C of sex change 5s, and 56 DEG C of annealing 15s, 72 DEG C extend 10s, 45 circulations.
4) expression activity of 2 reference gene GAPDH, PCS combination correction detection rhodioside synthesis key gene TyDC is used.
Fig. 1 shows, when endogenetic fungus and rhodiola plantlet in vitro interaction 4d and 6d, the relative expression quantity of TyDC gene and the difference of control group notable (*P>0.05)；When interaction 8d～12d, its relative expression quantity significantly raise (*P<0.05)；During 12d, the expression of process group is maximum, for 2.9 times of control group, then begins to decline, and the relative expression of 14d and 16d, TyDC gene is reduced to about 0.7 times of comparison.This shows the fungi of the numbered CGMCC No.9347 effect difference in the different time periods, to the regulation and control of rhodioside isoreactivity Composition accumulation in host's rhodiola.This demonstrates double reference gene good result to key enzyme TyDC activity of gene expression.
Embodiment 2
1) method for tissue culture is used to obtain rhodiola plantlet in vitro.Inoculated fungi CGMCC No.9347, hot-house culture (intensity of illumination 30 μm ol m-2 s-1；Light cultivates 14h, temperature 28 DEG C；Light culture 10h, temperature 22 DEG C；Humidity 70%～75%), periodically (4d, 6d, 8d, 10d, 12d, 14d, 16d) gathers rhodiola root plantlet in vitro；Gather the plantlet in vitro do not inoculated as comparison (ck).
2) Total RNAs extraction: use conventional method to extract the total serum IgE of rhodiola plantlet in vitro, and synthesize cDNA the 1st chain.
3) primer provided with table 1, with cDNA the 1st chain as template, 5 genes of quantitative pcr amplification.Amplified reaction program is: 94 DEG C of denaturations 30s, 95 DEG C of sex change 5s, and 56 DEG C of annealing 15s, 72 DEG C extend 10s, 45 circulations.
4) expression activity of GAPDH, PCS double reference gene combination correction detection rhodioside key gene TAT is used.
Fig. 2 shows, endogenetic fungus does not significantly increase the expression of rhodiola root TAT gene.Interaction 4d, when 14d, 16d, the relative expression quantity of TAT be markedly inferior to comparison (*P<0.05)；Interaction 6d～12d, the relative expression quantity of TAT does not has significant difference with compareing, and shows that tyrosine ammonia lyase compound direction in rhodioside route of synthesis in rhodiola is not affected by fungi.This demonstrates double reference gene good detection effect to key enzyme TAT activity of gene expression.
Embodiment 3
1) method for tissue culture is used to obtain rhodiola plantlet in vitro.Inoculated fungi CGMCC No.9347, hot-house culture (intensity of illumination 30 μm ol m-2 s-1；Light cultivates 14h, temperature 28 DEG C；Light culture 10h, temperature 22 DEG C；Humidity 70%～75%), periodically (4d, 6d, 8d, 10d, 12d, 14d, 16d) gathers rhodiola root plantlet in vitro；Gather the plantlet in vitro do not inoculated as comparison (ck).
2) Total RNAs extraction: use conventional method to extract the total serum IgE of rhodiola plantlet in vitro, and synthesize cDNA the 1st chain.
3) primer provided with table 1, with cDNA the 1st chain as template, 5 genes of quantitative pcr amplification.Amplified reaction program is: 94 DEG C of denaturations 30s, 95 DEG C of sex change 5s, and 56 DEG C of annealing 15s, 72 DEG C extend 10s, 45 circulations.
4) expression activity of GAPDH, PCS three rhodioside synthesis key gene PAL of double reference gene combination correction detection rhodioside is used.
Fig. 3 shows, compared to comparison, the expression of rhodiola PAL gene experienced by and first raises, reaches peak value, then downward trend, have conspicuousness result of variations (*P<0.05).Wherein, when interaction 6d～14d, endogenetic fungus can significantly improve the expression of host's rhodiola root PAL gene, and during interaction 16d, process group is reduced to 0.52 times of control group.This demonstrates double reference gene good detection effect to key enzyme PAL activity of gene expression.
Claims (4)
1. a double reference gene primer pair, is that nucleotides sequence is classified as SEQ ID NO:1 and the glyceraldehyde of SEQ ID NO:2
Phosphate dehydrogenase gene primer to and nucleotides sequence be classified as SEQ ID NO:3 and SEQ ID NO:4 phytochelatin synthesis
Enzyme gene primer pair.
2. rhodioside synthesis key gene primer pair, is that nucleotides sequence is classified as SEQ ID NO:5 and SEQ ID NO:
The tyrosine decarboxylase primer of 6 is classified as the TAT of SEQ ID NO:7 and SEQ ID NO:8 and draws, nucleotides sequence
Thing to or nucleotides sequence be classified as SEQ ID NO:9 and the PAL primer pair of SEQ ID NO:10.
3. the method using double reference gene detection rhodioside synthase gene expression activity, comprises the steps:
1) use method for tissue culture to obtain rhodiola plantlet in vitro, inoculated fungi elicitor, hot-house culture, gather rhodiola root
Plantlet in vitro；The plantlet in vitro do not inoculated is as comparison；
2) use conventional method to extract the total serum IgE of rhodiola plantlet in vitro, and synthesize cDNA the 1st chain；
3) with the double reference gene primers described in claim 1 to and claim 2 described in key gene primer to as primer,
With cDNA the 1st chain as template, quantitative pcr amplification GAPDH gene, PCS gene, TyDC gene, TAT base respectively
Cause and PAL gene；Amplified reaction program is: 94 DEG C of denaturations 30s, 95 DEG C of sex change 5s, 56 DEG C of annealing 15s, 72 DEG C of extensions
10s, 45 circulations；
4) geNorm, Normfider, Bestkeeper software analysis GAPDH gene, the stability of PCS gene expression are used
With CT value etc.；
5) excel software is utilized to calculate each sample TyDC respectively, the relative expression quantity of TAT, PAL gene, and with GAPDH
With PCS gene expression as double internal references, carry out experimental error rectification；Utilize 2-ΔΔCT[Δ Δ CT=be (to be measured group of key gene
The ct value of ct value-to be measured group of reference gene)-(the ct value of the ct value-control group reference gene of control group key gene)]
Method calculate symbiosis seedling TyDC, TAT, PAL activity of gene expression.
4. the method using double reference gene detection rhodioside synthase gene expression activity as claimed in claim 3, described
Step 1) in fungi be the fungi Phialocephala fortinii that preservation is numbered CGMCC No.9347.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653339A (en) * | 2017-11-21 | 2018-02-02 | 刘志伟 | For identifying the special primer and authentication method of Tibet Langkazi area rhodiola |
| CN108085409A (en) * | 2017-12-28 | 2018-05-29 | 福建农林大学 | The application of the screening technique of China fir reference gene and screening-gene as reference gene in different tissues |
| CN109486806A (en) * | 2018-12-27 | 2019-03-19 | 三明学院 | A kind of phenylalanine lyase and its encoding gene, recombinant vector, recombination engineering and application of anoectochilus formosanus |
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| CN104212725A (en) * | 2014-09-15 | 2014-12-17 | 山西大学 | Method for improving content of salidroside and tyrosol in rhodiola rosea tissue culture seedling |
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| CN104212725A (en) * | 2014-09-15 | 2014-12-17 | 山西大学 | Method for improving content of salidroside and tyrosol in rhodiola rosea tissue culture seedling |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653339A (en) * | 2017-11-21 | 2018-02-02 | 刘志伟 | For identifying the special primer and authentication method of Tibet Langkazi area rhodiola |
| CN107653339B (en) * | 2017-11-21 | 2021-06-22 | 刘志伟 | Specific primer and identification method for identifying rhodiola crenulata in Tibet wave checkpost region |
| CN108085409A (en) * | 2017-12-28 | 2018-05-29 | 福建农林大学 | The application of the screening technique of China fir reference gene and screening-gene as reference gene in different tissues |
| CN108085409B (en) * | 2017-12-28 | 2020-09-08 | 福建农林大学 | Screening method of fir reference gene in different tissues and application of screening gene as reference gene |
| CN109486806A (en) * | 2018-12-27 | 2019-03-19 | 三明学院 | A kind of phenylalanine lyase and its encoding gene, recombinant vector, recombination engineering and application of anoectochilus formosanus |
| CN109486806B (en) * | 2018-12-27 | 2022-03-01 | 三明学院 | Phenylalanine ammonia lyase of anoectochilus formosanus, and coding gene, recombinant vector, recombinant engineering bacterium and application thereof |
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