CN103981272A - Application of GRMZM2G054655 gene as maize reference gene - Google Patents
Application of GRMZM2G054655 gene as maize reference gene Download PDFInfo
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Abstract
The invention provides a reference gene for maize genetic detection, relates to the field of molecular biology, and in particular relates to an application of maize GRMZM2G054655 gene as a reference gene for transgenic detection. The reference gene is GRMZM2G054655 maize specific intronless gene, the nucleotide sequence of the gene GRMZM2G054655 is shown in SEQ ID NO: 1, and the primers are designed to specifically amplify a DNA fragment of which the length is 302bp of GRMZM2G054655 genes to obtain the reference gene for qualitative and quantitative PCR reactions in maize transgenic detection process. According to the invention, the establishment of a common reference gene in maize transgenic PCR detection is proposed for the first time; an application of maize specific intronless gene GRMZM2G054655 as a reference genes in the maize transgenic detection is firstly proposed; the reference gene provided by the invention has the advantages of stability to different maize lines and specificity to different plant species, constant copy number and high detection sensitivity and has an important application value in maize transgenic qualitative and quantitative detection.
Description
Technical field
The present invention relates to biology field, particularly, relate to the application of GRMZM2G054655 gene as the reference gene of specific stable expression in different lines corn.
Background technology
Since the first transgenic Fructus Lycopersici esculenti " FLAVR SAVR " comes out, along with the development of biotechnology, genetically modified crops (Genetically Modified Plants, GMP) be more and more applied to agriculture field, promote agriculture production, boost agricultural yield, reduce costs, opened up new method for solving global food problem.China's transgenic technology development in recent years has rapidly tens kinds of genetically modified crops that carry out field test, and wherein 3 main productss are paddy rice, corn and wheat.Meanwhile, along with the extensive plantation in the world of global transgenic plant, a large amount of transgene agricultural products pour in China market.
Reference gene, is in certain kind of plant, to have specificity between kind, non-specific in planting, a genoid of low copy number feature.Between kind, to refer to selected gene be special for these species to specificity, and in other species, have very low homology.Can obtain preliminary data by the analysis of biological information of network data base, select candidate gene, then method is by experiment screened candidate gene, finally between selected suitable kind special gene as plant reference gene.In kind, the non-specific reference gene that refers to has very high homology and similarity in the different cultivars of same species.
The feature of plant reference gene and constant copy number non-specific because of specificity between its kind, in planting is being brought into play and important effect aspect the mensuration of copy number of foreign gene in qualitative, the quantitative PCR detection of transgenic plant and converted products thereof and transgenic plant production process.
The GRMZM2G054655 reference gene that the present invention chooses, detects by round pcr that to show that it has in B73, Zheng 22,14 kinds of different lines corns such as middle yellow 68 non-specific, and copy number is constant; In the 14 kind of plant species such as Arabidopis thaliana, paddy rice, Chinese sorghum, all there is specificity between kind; Detection sensitivity is high; For the qualitative and quantitative analysis of foreign gene in transgenic corns lays the foundation.
Summary of the invention
The object of this invention is to provide the application of GRMZM2G054655 gene as the reference gene of the qualitative and quantitative analysis stably express of foreign gene in transgenic corns, wherein, the nucleotide sequence of described GRMZM2G054655 gene fragment is as shown in the DNA sequence dna of SEQ ID NO:1 in sequence table.
According to the specific embodiment of the present invention, whether in different lines corn, have non-specificly as reference gene detecting above-mentioned GRMZM2G054655 gene, copy number is constant, in different plant species, has specificity.The detection sensitivity of GRMZM2G054655 gene qualitative PCR reaction is 0.5ng, and the detection sensitivity of quantitative PCR reaction system is 0.04ng.
Qualitative, the quantification PCR primer of amplification GRMZM2G054655 gene is to also within protection scope of the present invention.
Brief description of the drawings
The specific qualitative analysis in different lines corn of Fig. 1 GRMZM2G054655 reference gene.M:DL2000Plus DNA Marker; 1:B73; 2: agricultural university 178; 3: Shen 3336; 4: Ji 35; 5:5213; 6:PI143; 7: Zheng 22; 8: Zheng 30; 9:7922; 10: lucky 4112; 11:8902; 12: middle yellow 68; 13: golden yellow 73; 14:5311; 15: blank.PCR reaction product electrophoresis showed can amplify size for 302bp, single-minded band in 14 different lines corns, and stripe size is consistent with expection.Thereby non-specific in having kind of GRMZM2G054655 gene is described.
The specific qualitative analysis in different plant species of Fig. 2 GRMZM2G054655 reference gene, M:DL2000Plus DNA Marker; 1: paddy rice; 2: Chinese sorghum; 3: barley; 4: wheat; 5: Arabidopis thaliana; 6: soybean; 7: mung bean; 8: tomato; 9: tobacco; 10: Sunflower Receptacle; 11: willow; 12: cotton; 13: rape 73; 14: millet; 15-17: corn B73; 18: blank.Only can increase in the Maize genome object band of 302bp, does not have object band in other species.Illustrate that GRMZM2G054655 gene has corn specificity.
The quantitative analysis of Fig. 3 GRMZM2G054655 reference gene copy number in different lines corn, ID1:B73; ID2: agricultural university 178; ID3: Shen 3336; ID4: Ji 35; ID5:5213; ID6:PI143; ID7: Zheng 22; ID8: Zheng 30; ID9:7922; ID10: lucky 4112; ID11:8902; ID12: middle yellow 68; ID13: golden yellow 73; ID14:5311.Result shows that GRMZM2G054655 gene copy number in different corn varieties is constant.
The sensitivity analysis of Fig. 4 GRMZM2G054655 qualitative detection.M:DL2000DNA Marker; 1-2: template concentrations 50ng/ μ L; 3-4: template concentrations 5ng/ μ L; 5-6: template concentrations 0.5ng/ μ L; 7-8: template concentrations 0.05ng/ μ L; 9-10: template concentrations 0.01ng/ μ L.Electrophoresis result shows that the sensitivity of qualitative PCR reaction detection is 0.5ng.
Embodiment
The existing gene order-checking of this research and utilization and PIGD, NCBI common data library information, first find the candidate gene of corn reference gene by BLAST and BLINK sequence alignment.Then carry out the screening of reference gene from planting the aspects such as specificity between interior consistence and stability, kind, copy number are stable,, quantitative PCR detection qualitative through the species specificity in non-corn species (paddy rice, Chinese sorghum, barley, Arabidopis thaliana etc.) and different corn variety, copy number is analyzed, and final choice detects reference gene using corn GRMZM2G054655 as corn gene.
Embodiment 1: corn specificity reference gene GRMZM2G054655 is specific qualitative analysis in non-specific and other plant species in different lines corn
1, extracting genome DNA: the water-bath of preparing 65 DEG C; Get 20g material and clay into power in liquid nitrogen, gained powder is moved in the 50ml centrifuge tube of precooling; Add 20ml to be preheated to 2 × CTAB Extraction buffer of 65 DEG C, fully mix, 65 DEG C of incubations 45 minutes, shake mixes gently during this time; Add the Proteinase K of 100ul100mg/ml, after mixing in 37 DEG C of incubations 30 minutes; After mixture is cooling, add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) mixing solutions, put upside down to mix and make to manage interior mixture and become emulsion; Centrifugal 10 minutes of 12000rpm, repeats for several times until that extract becomes is limpid; Get supernatant and add isopyknic Virahol, mix, place 1 hour for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is washed rear room temperature with 75% ethanol and is dried; With 100ul TE dissolution precipitation, add 1ul RNA enzyme solution (5mg/ml), 37 DEG C of incubations 30 minutes; Add isopyknic phenol: the extracting of chloroform (1:1) mixing solutions, centrifugal 5 minutes of 12000rpm: get supernatant, add the NaAc solution of 3M and the dehydrated alcohol of 2 times of volumes of 1/10 volume, place 30 minutes for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is dried after washing with 75% ethanol, and-20 DEG C save backup.
2, the primer of corn specificity reference gene GRMZM2G054655 is synthetic:
The qualitative primers F 1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:2, for:
5′-TCCGACAAGAAAGATGAG-3′；
The qualitative primer R1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:3, for::
5′-TGACGCAAGTGTAAGGTG-3′；
Synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
3, pcr amplification reaction
PCR detection system and program are:
PCR response procedures is: 94 DEG C of sex change 10 minutes; 94 DEG C of sex change 1 minute, 68 DEG C of annealing 45 seconds, 72 DEG C are extended 1 minute, 35 circulations; 72 DEG C are extended 10 minutes, 10 DEG C of preservations.
4, PCR result detects and analyzes
1.0% agarose gel electrophoresis detects, and in the kind of corn specificity reference gene GRMZM2G054655 in 14 kinds of different lines corns, non-specific situation is as Fig. 1; The middle specificity situation of corn specificity reference gene GRMZM2G054655 in 14 kinds of different plant varieties, as Fig. 2, illustrates that this gene can be used as the reference gene that corn gene qualitative PCR detects.
Embodiment 2: corn specificity reference gene GRMZM2G054655 is specific quantitative analysis in non-specific and other plant species in different lines corn
1, extract the method extraction DNA of plants of DNA according to embodiment 1, as genomic dna.
2, the specific regions of choosing GRMZM2G054655 gene carries out design of primers, and primer uses Primer Express3.0 (ABI) software to design.Corn specificity reference gene GRMZM2G054655 fluorescent quantitation primer is synthetic:
The quantitative primers F 2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:4, for::
5′-GCTACGCCCTTCCCCATT-3′；
The quantitative primer R2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:5, for::
5′-GGCTTGCATGGATCCAAAAA-3′；
Synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
3, quantitative pcr amplification reaction
Quantitative PCR reaction system:
PCR reaction parameter is as follows: 95 DEG C of denaturation 10min; 95 DEG C of 15sec, 60 DEG C of 1min, totally 40 circulations.After reaction finishes, product is heated, obtain the solubility curve of product.Adopt 2
– Δ Δ CT[Δ CT=CT
target gene– CT
reference gene. Δ Δ CT=Δ CT
after processing– Δ CT
contrast] method to obtain signal and data process.Each gene does 3 secondary pollutants and learns repetition and 3 experiment repetitions.
4, quantitative PCR result detects and analyzes
Corn specificity reference gene GRMZM2G054655 can obtain the fluorescent signal of similar intensity in 14 kinds of different lines corns, this result shows in 14 kinds of different lines of corn, all to contain corn specificity reference gene GRMZM2G054655, thereby it is non-specific to illustrate that this gene has in kind; Corn specificity reference gene GRMZM2G054655 copy number proterties condition in 14 kinds of different lines corns, as Fig. 3, illustrates that this gene has specificity between kind of an internal stability, kind, copy number is constant, can be used as the reference gene of corn gene quantitative PCR detection.
Embodiment 3: the sensitivity analysis of corn specificity reference gene GRMZM2G054655 qualitative and quantitative analysis
1, qualitative detection sensitivity
Extract the DNA of the method extraction corn B73 of DNA according to embodiment 1, taking the DNA of corn B73 as template DNA, be 50ng/ μ L by DNA concentration dilution, 5ng/ μ L, 0.5ng/ μ L, 0.05ng/ μ L, 0.01ng/ μ L, gets each concentration 1ul, tests according to the method for embodiment 1, electrophoresis result, as Fig. 4, shows that the sensitivity of corn specificity reference gene GRMZM2G054655 qualitative detection is 0.5ng
2, detection by quantitative sensitivity
Extract the DNA of the method extraction corn B73 of DNA according to embodiment 1, taking the DNA of corn B73 as template DNA, be 50ng/ μ L by DNA concentration dilution, 5ng/ μ L, 0.5ng/ μ L, 0.05ng/ μ L, 4ul is got in the each quantitative PCR reaction of 0.01ng/ μ L, method according to embodiment 2 is tested, detection by quantitative result shows that each concentration DNA profiling has all obtained the fluorescent signal of respective strengths, and the CT value of amplification curve increases gradually, consistent with the variation of template DNA concentration, show that corn specificity reference gene GRMZM2G054655 detection sensitivity is 0.04ng.
Claims (10)
1.GRMZM2G054655 gene is as the application of corn reference gene, and the sequence of described GRMZM2G054655 gene is as shown in sequence table SEQ ID NO:1.
2. GRMZM2G054655 gene as claimed in claim 1, as the application of corn reference gene, is characterized by: non-specific in having in 14 kinds of corn varieties kind, copy number is constant; In 14 kinds of different plant species, there is specificity between kind.
3. GRMZM2G054655 gene as claimed in claim 1 application in corn gene qualitative detection as corn reference gene.
4. GRMZM2G054655 gene as claimed in claim 3 application in corn gene qualitative detection as corn reference gene, is characterized by: non-specific in having in 14 kinds of corn varieties kind, copy number is constant; In 14 kinds of different plant species, there is specificity between kind; Qualitative detection sensitivity reaches 0.5ng.
5. GRMZM2G054655 gene as claimed in claim 1 application in corn gene detection by quantitative as corn reference gene.
6. GRMZM2G054655 gene as claimed in claim 5 application in corn gene detection by quantitative as corn reference gene, is characterized by: non-specific in having in 14 kinds of corn varieties kind, copy number is constant; In 14 kinds of different plant species, there is specificity between kind; Detection by quantitative sensitivity reaches 0.04ng.
7. a method of qualitative analysis corn reference gene GRMZM2G054655, is characterized by and comprise the following steps:
(1) extracting genome DNA: the water-bath of preparing 65 DEG C; Get 20g material and clay into power in liquid nitrogen, gained powder is moved in the 50ml centrifuge tube of precooling; Add 20ml to be preheated to 2 × CTAB Extraction buffer of 65 DEG C, fully mix, 65 DEG C of incubations 45 minutes, shake mixes gently during this time; Add the Proteinase K of 100ul100mg/ml, after mixing in 37 DEG C of incubations 30 minutes; After mixture is cooling, add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) mixing solutions, put upside down to mix and make to manage interior mixture and become emulsion; Centrifugal 10 minutes of 12000rpm, repeats for several times until that extract becomes is limpid; Get supernatant and add isopyknic Virahol, mix, place 1 hour for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is washed rear room temperature with 75% ethanol and is dried; With 100ul TE dissolution precipitation, add 1ul RNA enzyme solution (5mg/ml), 37 DEG C of incubations 30 minutes; Add isopyknic phenol: the extracting of chloroform (1:1) mixing solutions, centrifugal 5 minutes of 12000rpm: get supernatant, add the NaAc solution of 3M and the dehydrated alcohol of 2 times of volumes of 1/10 volume, place 30 minutes for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is dried acquisition genomic dna after washing with 75% ethanol, and-20 DEG C save backup;
(2) primer of corn specificity reference gene GRMZM2G054655 is synthetic:
The qualitative primers F 1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:2, for:
5′-TCCGACAAGAAAGATGAG-3′；
The qualitative primer R1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:3, for::
5′-TGACGCAAGTGTAAGGTG-3′；
(3) pcr amplification reaction
PCR detection system and program are:
PCR response procedures is: 94 DEG C of sex change 10 minutes; 94 DEG C of sex change 1 minute, 68 DEG C of annealing 45 seconds, 72 DEG C are extended 1 minute, 35 circulations; 72 DEG C are extended 10 minutes, 10 DEG C of preservations; After reaction finishes, PCR product is carried out to 1.0% agarose gel electrophoresis determination and analysis.
8. a method of quantitative analysis corn reference gene GRMZM2G054655, is characterized by and comprise the following steps:
(1) extracting genome DNA: the water-bath of preparing 65 DEG C; Get 20g material and clay into power in liquid nitrogen, gained powder is moved in the 50ml centrifuge tube of precooling; Add 20ml to be preheated to 2 × CTAB Extraction buffer of 65 DEG C, fully mix, 65 DEG C of incubations 45 minutes, shake mixes gently during this time; Add the Proteinase K of 100ul100mg/ml, after mixing in 37 DEG C of incubations 30 minutes; After mixture is cooling, add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) mixing solutions, put upside down to mix and make to manage interior mixture and become emulsion; Centrifugal 10 minutes of 12000rpm, repeats for several times until that extract becomes is limpid; Get supernatant and add isopyknic Virahol, mix, place 1 hour for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is washed rear room temperature with 75% ethanol and is dried; With 100ul TE dissolution precipitation, add 1ul RNA enzyme solution (5mg/ml), 37 DEG C of incubations 30 minutes; Add isopyknic phenol: the extracting of chloroform (1:1) mixing solutions, centrifugal 5 minutes of 12000rpm: get supernatant, add the NaAc solution of 3M and the dehydrated alcohol of 2 times of volumes of 1/10 volume, place 30 minutes for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Remove supernatant, precipitation is dried acquisition genomic dna after washing with 75% ethanol, and-20 DEG C save backup;
(2) specific regions of choosing GRMZM2G054655 gene carries out design of primers, and primer uses Primer Express3.0 software to design; Corn specificity reference gene GRMZM2G054655 fluorescent quantitation primer is synthetic:
The quantitative primers F 2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:4, for::
5′-GCTACGCCCTTCCCCATT-3′；
The quantitative primer R2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:5, for::
5′-GGCTTGCATGGATCCAAAAA-3′；
(3) quantitative pcr amplification reaction
Quantitative PCR reaction system:
PCR reaction parameter is as follows: 95 DEG C of denaturation 10min; 95 DEG C of 15sec, 60 DEG C of 1min, totally 40 circulations; After reaction finishes, product is heated, obtain the solubility curve of product; Adopt 2
– Δ Δ CT[Δ CT=CT
target gene– CT
reference gene. Δ Δ CT=Δ CT
after processing– Δ CT
contrast] method to obtain signal and data process and analyze; Each gene does 3 secondary pollutants and learns repetition and 3 experiment repetitions.
9. the qualitative primer of the GRMZM2G054655 using in the method for a kind of qualitative analysis corn reference gene GRMZM2G054655 as claimed in claim 7, is characterized by:
The qualitative primers F 1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:2, for:
5′-TCCGACAAGAAAGATGAG-3′；
The qualitative primer R1 of GRMZM2G054655 as shown in sequence table SEQ ID NO:3, for:
5′-TGACGCAAGTGTAAGGTG-3′。
10. the quantitative primer of the GRMZM2G054655 using in the method for a kind of quantitative analysis corn reference gene GRMZM2G054655 as claimed in claim 8, is characterized by:
The quantitative primers F 2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:4, for:
5′-GCTACGCCCTTCCCCATT-3′；
The quantitative primer R2 of GRMZM2G054655 as shown in sequence table SEQ ID NO:5, for:
5′-GGCTTGCATGGATCCAAAAA-3′。
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| SCHNABLE: "《The B73 maize genome:complexity,diversity and dynamics》", 《SCIENCE》 * |
| 袁伟: "《植物实时荧光定量PCR内参基因的特点及选择》", 《植物学报》 * |
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