CN112048511B - Reference gene for stable expression of club head grass under stress of ALS herbicide, screening method and application - Google Patents
Reference gene for stable expression of club head grass under stress of ALS herbicide, screening method and application Download PDFInfo
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Abstract
The application discloses an internal reference gene for stable expression of club head grass under the stress of ALS herbicide, a screening method and application thereof, wherein the gene is an internal reference gene UBQ, and a gene sequence is shown as SEQ ID NO. 1. The internal reference gene can meet the requirement of real-time fluorescence quantitative detection of the transcription expression level of the nostoc flagelliforme, and improves the stability, reliability and efficiency of the analysis and research of the gene expression of the nostoc flagelliforme.
Description
Technical Field
The application belongs to the technical field of plant molecular biology, and particularly relates to an internal reference gene for stable expression of club head grass under the stress of ALS herbicides, a screening method and application thereof.
Background
Radix seu herba Lespedezae CuneataePolypogon fugax) The grass is a common annual or perennial grass weed in summer crop fields, seedlings emerge in 9-10 months each year, flowers in 4-6 months of the next year, and the growth period of the grass is identical to that of a plurality of summer crops, so that the grass mostly occurs in summer crop fields such as wheat, rape and vegetables. Because the club head grass is a wet weed, the club head grass frequently occurs in areas with low-lying, moist and rich soil, so the club head grass is particularly serious in fields in a rice-wheat and rice-tanker mode, and the grain yield is seriously affected. At present, chemical herbicide is the main mode for preventing and controlling the clavate grass in agricultural production, but with the massive use of the herbicide, the problem of drug resistance of the clavate grass in fields is more and more serious, and the exploration of the action mechanism and the drug resistance mechanism of the herbicide on the clavate grass is helpful for preventing and controlling the clavate grass. Since the lolium clavatum is a hexaploid plant, the acquisition of whole genome information is difficult, and thus, the transcription level is developedThe regulation and control mechanism of physiological processes such as germination or flowering is a more feasible and effective method.
The real-time quantitative fluorescent PCR (RT-PCR) technology is a common method for researching the gene expression under a certain specific condition, has the characteristics of strong specificity, high sensitivity, repeatability and the like, and screens out an internal reference gene which can be stably and continuously expressed in a specific environmental condition or organ tissue, and is a precondition for determining the reliability of an RT-PCR result. Therefore, it is necessary to analyze and screen the optimally stably expressed reference gene of the sample to be tested by using a statistical method before performing the RT-PCR experiment.
At present, a blank research report on development and screening of the internal reference genes of the grass of bankoua is provided, and if the related molecular biological mechanism of the grass of bankoua is to be researched under the condition of herbicide stress, the development and screening of the internal reference genes are very important.
Disclosure of Invention
Aiming at the problems, the application aims to provide an internal reference gene for the stable expression of the nostoc flagelliforme, which can meet the requirement of detecting the transcription expression level of the nostoc flagelliforme by real-time fluorescence quantification, and improve the stability, reliability and efficiency of the analysis and research of the gene expression of the nostoc flagelliforme. Another object of the present application is to provide a method for screening the above-mentioned reference gene. It is still another object of the present application to provide an application of the above reference gene.
In order to achieve the above object, the technical scheme of the present application is as follows:
the internal reference gene of the stable expression of the club head grass under the stress of the ALS herbicide is the internal reference gene UBQ, the nucleotide sequence of the internal reference gene UBQ is shown as SEQ ID NO. 1.
As a further preferred aspect of the present application, the primer sequence of the internal reference gene UBQ is as follows:
forward primer F GCAAGAAGAAGACCTACACCAAG;
reverse primer R CCTTCTGGTTGTAGACGTAGGTG.
As a further preferred aspect of the application, the use of the internal reference gene UBQ for the fluorescent quantification of the grass of Leptoradix Erycibe under the stress of ALS herbicides.
As a further preferred aspect of the application, the use of the primer sequence of the internal reference gene UBQ for fluorescent quantification of the grass of Leptospermum under the stress of ALS herbicides.
As a further preferred aspect of the present application, the use of the reference gene UBQ for detecting the expression level of an ALS-based herbicide resistance gene is provided.
The application provides a screening method of internal reference genes for stable expression of club head grass under the stress of ALS herbicides, which comprises the steps of sample processing, RNA extraction, cDNA preparation, candidate internal reference gene design and analysis, wherein the candidate internal reference gene design and analysis comprises the following steps:
(1) Screening 5 candidate reference genes, namely UBQ, EF1, 18srRNA, 25srRNA and 26srRNA, and designing amplification primers of the candidate reference genes;
(2) Using the cDNA as an amplification template, and measuring the specificity of the amplification primer by utilizing PCR;
(3) Performing stability analysis on the candidate internal reference genes by using Ct values obtained after fluorescent quantitative PCR reaction;
(4) And calculating and analyzing the expression quantity stability of the reference gene in different organ tissues under the treatment of ALS herbicides.
As a further preferred aspect of the present application, the fluorescent quantitative PCR reaction system in the step (3) of designing and analyzing the candidate internal reference gene comprises: 10 μl of iTaq ™ uniactive SYBR [ beta ] (Green Supermix (2X), 2 μl of cDNA template, 1 μl of upstream or downstream primer (10 μM) each, 6 μl of sterile ultra pure water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s,40 cycles.
As a further preferred aspect of the present application, the sample processing is performed by: an AR clavicle population exhibiting a 3-4 fold level of resistance to ALS herbicides and an AS clavicle population susceptible to ALS herbicides.
As a further preferred aspect of the present application, the sample processing includes: a. seeds in the AR club head grass population and the AS club head grass population are respectively selected for grouping sowing, and ALS herbicide with the concentration of 4.0-5.0 g.a.i/ha is sprayed after the seeds grow to 3-4 leaf periods; b. four sampling times were taken with ALS herbicides sprayed for 0h, 24h, 48h, 72h, and leaves were removed and frozen in liquid nitrogen for RNA extraction.
According to the application, three pieces of software, namely Bestkeeper, normFinder and gemum, are used for evaluating the stability of 5 reference genes, comprehensive analysis is carried out on comprehensive software analysis results, reference genes which are stably expressed by the club head grass under experimental conditions are screened out, the reference genes which are the most stably expressed by the club head grass population (AS and AR) under herbicide treatment can be obtained, and the method can be used for analyzing the research on the expression level of functional genes of the club head grass under ALS herbicide treatment conditions.
In summary, the application has the following beneficial effects:
compared with the prior art, the application screens out the internal reference genes of the club head grass, which are stable to express under the stress of ALS herbicides, from the 5 candidate internal reference genes, has true and reliable data, provides a correction tool for the molecular biology research of response mechanisms of the club head grass, such as ALS herbicides, and the like, and fills the blank of lacking proper internal reference genes in the club head grass research field.
The screened internal reference genes are suitable for the expression analysis of key genes of the lolium head grass under the stress of ALS herbicides, and can improve the accuracy of data.
The application also provides the application of the internal reference gene UBQ in the practical test of the resistance and the difference experiment of the toxicity and metabolism capacity of sensitive clavate grass on herbicide, and proves that the screened internal reference gene UBQ has the value of researching the related molecular biological mechanism under the condition of the clavate grass stress.
Drawings
FIG. 1 is a fragment electrophoresis of candidate internal reference genes using a cDNA template of the grass of Leptoradix et rhizoma Rhei in the present application.
FIG. 2 is a bar graph showing the expression levels of CYP81B1 genes of resistant and sensitive lolium clavatum AR and AS under the condition of metsulfuron-methyl treatment and untreated conditions in the present application.
Detailed Description
Example 1
1. Material selection and treatment:
two populations of lolium clavatum (AR and AS) were selected, and early studies showed that lolium clavatum AR exhibited 3-4 fold levels of resistance to metsulfuron, whereas AS was susceptible to metsulfuron, a widely used ALS herbicide.
The test steps are as follows: sowing seeds into plastic pot with diameter of 7.5cm, 20 seeds per pot, planting 12 pots, spraying mesosulfuron with concentration of 4.5g a.i./ha after growing to 3-4 leaf period, taking leaves after spraying mesosulfuron for 0h, 24h, 48h and 72h, freezing in liquid nitrogen for RNA extraction, selecting more than three plant leaves as one sample per sampling, and repeating 3 groups of each experimental material, thus obtaining 6 samples per sampling time, and obtaining 24 samples to be tested for analyzing the expression stability of candidate reference genes in different herbicide treatment time periods in total for 4 sampling times.
Extraction of RNA and preparation of cRNA
Total RNA extraction of the samples was performed using plant total RNA extraction kit (Tiangen, china), and the concentration and purity of the RNA samples were determined by OD (ultra drop, U.S.) using an ultra-micro spectrophotometer 260 /OD 280 And OD (optical density) 260 /OD 230 The ratio is determined. A1. Mu.g sample of RNA was reverse transcribed using a PrimeScript RT Reagent Kit with gDNA Eraser kit and the final product was diluted 400-fold for use as a template for RT-PCR.
3. Designing candidate internal reference gene primers:
the inventors performed primer design based on homologous genes in the physalis alkekengi (Alopecurus myosuroides) of the kindred grass weeds, and the primer sequence information and the amplification product sizes are shown in table 1. All primers were designed using Primer Premier 5.0 software and primers meeting the following conditions were selected: the annealing temperature is 50-65 ℃, the primer length is 18-24 bp, the GC content is 50-60%, and the PCR product size is 100-225bp.
TABLE 1 information about candidate reference genes
4. Specificity analysis of candidate reference genes:
the specificity of the primer is determined by using conventional PCR, and the PCR product is observed through 2% gel electrophoresis, as shown in FIG. 1, the amplified product has single band and the size accords with the expectation, which shows that the specificity of the primer is good.
5. Real-time quantitative fluorescent PCR (RT-PCR) reaction system and reaction conditions:
the RT-PCR reaction (20. Mu.l) contained: 10 μl of iTaq ™ uniactive SYBR [ beta ] (Green Supermix (2X), 2 μl of cDNA template, 1 μl of upstream or downstream primer (10 μM) each, 6 μl of sterile ultra pure water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s,40 cycles.
The RT-PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s,40 cycles. And (3) performing fluorescent quantitative PCR amplification on each candidate reference gene to obtain a Ct value, and calculating the stability of the reference gene.
6. Analysis of amplification efficiency of candidate internal reference Gene
Reverse transcribed cDNA was diluted into 5 concentration gradients (dilution factors of 1000, 100, 10, 1, 0.1) as templates for standard curve establishment, fluorescent quantitative PCR was performed on each pair of primers, and the amplification efficiency (E) and correlation coefficient (R2) of each pair of primers to be tested were calculated as shown in Table 2:
TABLE 2 amplification efficiency of candidate internal reference genes
The results showed that the amplification efficiency (97% -109%) and its correlation coefficient (0.94-0.99) of all genes were within acceptable ranges and could be used for the following stability analysis.
7. Gene sequence analysis of candidate internal reference genes
And amplifying the gene fragments of UBQ, EF1, 18srRNA, 25srRNA and 26srRNA by using a conventional PCR method and using the club head grass cDNA as a template and using each pair of primers, and sequencing and analyzing, wherein the sequence information is shown as SEQ ID NO. 1-SEQ ID NO. 5.
8. Method for analyzing expression stability of reference gene
Stability of the expression of the reference gene was accomplished by three statistical software (Bestkeeper, normFinder and geNorm). Bestkeeper directly calculates a standard error (SD) by using a Ct value to determine the stability of the expression quantity, and when the SD value is greater than 1, the expression of the reference gene is proved to be unstable; when the SD values are all smaller than 1, the sequence of the expression stability of the reference genes is determined by the size of the SD values, and the smaller the SD values are, the more stable. NormFinder and gemm are calculated using the relative expression levels:
NormFinder calculates an expression Stability Value (SV) using the relative expression amount, and the lower SV means the more stable the gene expression amount. The average degree of variation (M) of each reference gene was calculated for gemm, and reference genes with M values less than 1.5 were considered to be stably expressed, with stability being higher as the M value was smaller. And finally, comprehensively analyzing according to the calculation results of the three software to determine the most suitable internal reference gene under specific experimental conditions.
9. Analysis of expression stability of candidate reference genes under herbicide treatment
To select the most stably expressed reference genes under herbicide treatment, we used three statistical software to comprehensively analyze the expression stability of the 5 candidate reference genes after herbicide mesosulfuron-methyl treatment for 0h, 24h, 48h, and 72h for two lolium populations, as shown in table 3:
TABLE 3 scoring and ranking of candidate reference genes Bestkeeper, normfinder and gemm under herbicide treatment conditions
The Bestkeeper calculation shows that the SD value of 26srRNA and EF1 in the two club head grass populations at all treatment times is greater than 1, the expression is unstable, the SD values of 18srRNA, 25srRNA and UBQ are all less than 1, and the order of the expression stability from high to low is 18srRNA > 25srRNA > UBQ.
The gemm calculation result shows that, except that the M value of 26srRNA exceeds the standard threshold value of 1.5, the M values of the other 4 candidate reference genes are less than 1.5 for unstable expression of the reference genes, and the stability ranking is EF1 > 25srRNA > UBQ > 18srRNA.
Normfinder can divide all samples into subsets with a single variable to study the effect of plant population or herbicide treatment time on gene expression stability. EF1, 25 srrrna and UBQ are the most stably expressed genes when all samples were taken as subjects; when herbicide treatment time was the subject, we divided all samples into 4 subsets (0 HAT, 24HAT, 48HAT and 72 HAT) by treatment time, which indicated EF1, 26srRNA and ubq. When the effect of plant populations was the subject of study, we divided all samples into 2 subsets by population (AS and AR), and the results indicated that EF1, 18 srrrna and UBQ were the most stably expressed genes. Comprehensive Normfinder calculation results, EF1 and UBQ are the most stably expressed internal reference genes.
The calculation results combined with the three analysis software can prove that UBQ is the reference gene which is most stably expressed by the group of the clavate grass (AS and AR) under the herbicide treatment, and can be used for researching the expression level of the functional gene of the clavate grass under the herbicide treatment condition.
Example 2
To detect differences in resistance and the metabolic capacity of sensitive clavicle to detoxify herbicides, this example was further tested on the basis of the study of example 1: in this example, the inventors selected a gene of the cytochrome P450 family (CYP 81B 1) for measurement, and CYP81B1 has been confirmed to be involved in the regulation of weed metabolism herbicides, and differences in resistance and sensitivity to the herbicide metabolism ability of the lolium clavatum could be reflected by measuring the expression level of the gene under herbicide treatment conditions. The specific method comprises the following steps: seeds of a club head grass resistant population (AR) and a club head grass sensitive population (AS) are sown in plastic pots with the diameter of 7.5cm, 20 seeds of each pot are planted in 12 pots, the club head grass resistant population (AR) and the sensitive population (AS) are sprayed with mesosulfuron with the concentration of 4.5g a.i./ha after growing to 3-4 leaf periods, the leaves are taken after 24 hours of drug application and are quickly frozen and preserved in liquid nitrogen, meanwhile, the leaves which are not applied are taken AS a reference, more than three plant leaves are taken AS one sample for each sampling, 3 groups of experimental materials are repeated, thus 6 samples are obtained for each population, 12 samples to be tested are obtained, and the samples are named AS AS1-CK, AS2-CK, AS3-CK, AS1-CK, AS2-T, AS3-CK, AR2-CK, AR3-CK, AR1-T, AR2-T and AR3-T respectively. RNA extraction and cDNA preparation steps were as described in example 1.
Primers were designed based on published gene sequences of CYP81B1, the primer sequences are shown in Table 1, and RT-PCR amplification reactions of UBQ gene and CYP81B1 gene were performed for each sample, and the reaction system and reaction conditions of RT-PCR were the same. And (3) obtaining Ct values of the UBQ gene and the CYP81B1 gene in each sample by RT-PCR, and further calculating the relative expression quantity of the genes. CYP81B1 is used as a target gene, UBQ is used as an internal reference gene, a non-applied sample (CK) is used as a calibration sample, an applied sample (T) is used as a test sample, and the expression ratio is calculated according to the following formula:
the results are shown in FIG. 2: compared with the 0h sample, the CYP81B1 gene of the resistant population AR has significantly increased expression level at 24h, while the CYP81B1 gene of the sensitive population AS has no significantly changed expression level at 24 h. This result shows that the expression level of CYP81B1 gene is increased in the resistant lolium clavatum population under the treatment of metsulfuron, so that the metabolic capacity of the resistant lolium clavatum on metsulfuron is enhanced, and the drug resistance is further generated.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the application. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the application or exceeding the scope of the application as defined in the accompanying claims.
Sequence listing
<110> China institute of Rice
<120> internal reference gene for stable expression of different tissues of nostoc flagelliforme under stress of ALS herbicide, screening method and application
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tatgaagaaa ttgtgaagga agtctcttca tacctgaaga aggtcggcta caaccctgag 480
aaggtccctt ttgtccccat ctctggcttt gagggtgaca acatgattga aaggtccacc 540
aaccttgact ggtacaaggg ccccaccctt cttgaggctc ttgaccagat caacgagccc 600
aagaggccct cagacaagcc cctccgcctt ccccttcagg atgtctacaa gattggaggc 660
attggaactg tgccagtggg gcgtgttgag actggagtga tcaagcctgg tatgcttgtc 720
acctttggcc caactgggct gaccactgag gtgaagtctg tggagatgca ccacgagtct 780
atgcaggagg ctctgccagg tgacaatgtt ggcttcaacg tcaagaatgt tgctgtcaag 840
gatatcaagc gtgggtatgt tgcctccaac gccaaggacg accctgccaa ggaggctgcc 900
aactttgttg cccaggtcat catcatgaac caccctggcc agatcggcaa cggctacgcc 960
ccagtgctgg actgccacac ttcccacata gccgtcaagt tctctgagat ccagaccaag 1020
gtcgacaggc gttccggcaa ggagattgag gccttcccca agttcctcaa gaacggtgat 1080
gccggtttcg tgaagatgat tcccaccaag cccatggtgg tggagacctt cgcccagtac 1140
cctccccttg gacgctttgc tgtgcgcgac atgaggcaga ccgttgctgt tggtgtcatc 1200
aagagcgtgg agaagaagga cccaaccggc gccaaggtga ccaaggctgc cgccaagaag 1260
aaatgagatg gcggtatttt gttggtagtc atgttagtgg attttaaaac ttgcaccgtt 1320
ttggttatgt gcgtggtgca gccttcgtgc tgtttaccat tttatcttcg gttgtgttgg 1380
tgaaacaatg tcatgctatg ttcgattaaa gttgcaactc ttcagtacgt cagaaatttc 1440
gagtcattat atgcttttag tttactgtct tcactcttaa atatctttga ctgatattta 1500
cgatgggaag ttgttttagt tgctgttgcc taatatttaa acttcactct ttgctcgacg 1560
actgatattt accagcgtgc ctcttctggg tactttcaga ggttttcagt cgattgaagt 1620
ttctgtatca atgcgagtat gctactctga aacctgcatg tttccttc 1668
<210> 3
<211> 1018
<212> DNA
<213> 18srRNA
<400> 3
aacgatgccg accaaggatc ggcggatgtt gcttatagga ctccgccggc accttatgag 60
aaatcaaagt ctttgggttc cggggggagt atggtcgcaa ggctgaaact taaaggaatt 120
gacggaaggg caccaccagg cgtggagcct gcggcttaat ttgactcaac acggggaaac 180
ttaccaggtc cagacatagc aaggattgac agactgagag ctctttcttg attctatggg 240
tggtggtgca tggccgttct tagttggtgg agcgatttgt ctggttaatt ccgttaacga 300
acgagacctc agcctgctaa ctagctatgc ggagccatcc ctccgcagct agcttcttag 360
agggactatc gccgtttagg cgacggaagt ttgaggcaat aacaggtctg tgatgccctt 420
agatgttctg ggccgcacgc gcgctacact gatgtattca acgagtatat agccttggcc 480
gacaggcccg ggtaatcttg ggaaatttca tcgtgatggg gatagatcat tgcaattgtt 540
ggtcttaaac gaggaatgcc tagtaagcgc gagtcatcag ctcgcgttga ctacgtccct 600
gccctttgta cacaccgccc gtcgctccta ccgattgaat ggtccggtga agtgttcgga 660
tcgcggcgac gggggcggtt cgccgccccc gacgtcgcga gaagtccatt gaaccttatc 720
atttagagga aggagaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt 780
gtcgtgaccc tgaccaaaac agaccgcgca cgcgtcatcc agcctgccgg cggcgacacc 840
gttcgtcgct cggccaagtc ctcgattcct actctcctcg gagcggggga tcggggtaaa 900
agaacccacg acgcctaagg cgtcaaggaa cactgtgcct agctcgagga cgcggacggc 960
ttgctggccg cccctcgtgc tgcaatgcta tttaatccac acgactctcg gcaacgga 1018
<210> 4
<211> 975
<212> DNA
<213> 25srRNA
<400> 4
gaaaccacag ccaagggaac gggctgggcg gaatcagcgg ggaaagaaga ccctgttgag 60
cttgactcta gtccgacttt gtgaaatgac ttgagaggtg taggataagt gggagccttt 120
acgggcgcaa gtgaaatacc actactttta acgttatttt acttattccg tgggtcggaa 180
gcggggcaag tcccctcctt ttggctccaa ggcccggtct taccgggccg atccgggcgg 240
aagacattgt caggtgggga gtttggctgg ggcggcacat ctgttaaaag ataacgcagg 300
tgtcctaaga tgagctcaac gagaacagaa atctcgtgtg gaacaaaagg gtaaaagctc 360
gtttgattct gatttccagt acgaatacga accgtgaaag cgtggcctat cgatccttta 420
gatcttcgga gtttgaagct agaggtgtca gaaaagttac cacagggata actggcttgt 480
ggcagccaag cgttcatagc gacgttgctt tttgatcctt cgatgtcggc tcttcctatc 540
attgtgaagc agaattcacc aagtgttgga ttgttcaccc accaataggg aacgtgagct 600
gggtttagac cgtcgtgaga caggttagtt ttaccctact gatgacagtg tcgcgatagt 660
aattcaacct agtacgagag gaaccgttga ttcacacaat tggtcatcgc gcttggttga 720
aaagccagtg gcgcgaagct accgtgtgcc ggattatgac tgaacgcctc taagtcagaa 780
tccaagctag caagcgacgc ctgcgcccgc cgcccgcccc gacccacgtt aggggcgctt 840
gcgcccccaa gggcccgtgc cattggctaa gccggtccgg ccgaagtgtc gtggccggcc 900
gcctcgaagc tcccttccca acgggcggtg ggctgaatcc tttgcagacg acttaatacg 960
cgacgggcat gtatg 975
<210> 5
<211> 1066
<212> DNA
<213> 26srRNA
<400> 5
cagtgagcta ttacgctttc ttcaaggatg gctgcttcca agcccacctc ctggttgtca 60
tcgctcgatc acttcctttt ccactaagtg attgcttagg gaccttagcg tacgatctgg 120
gctgtttccc tctcgacttt ggatcttagc acccaatcag tctgtctgta ctaaggatga 180
cggcctgtat tcggagtttc cctggggttg gtaaagcgaa atgggggcca ccctagccca 240
ttgagtgctc tacctcgggc catcgacatc atacgctcta ctgaaataga tttcgcggaa 300
aaccagctat atccgatctt ggttggcctt tcacccctag ccacaagtca tccccgtatt 360
ttgccacata cgtgggttcg gtcctccaag gcctgttaga gctctcttca acctgctcat 420
ggctagatcg atcggtttcg ggtcaaatag gaagaacgta aatcttccac ctctggaaag 480
cgcctacacc taatggctta agccgctgtt cccatttcct cgctgaccca tcatgcgaaa 540
ggtacgccgt tagagtgagt gcgcccgcgc tccgctcctt cgactgattg ttcgcatcgg 600
atctcaggtt ctctattgca ctccccgata ggggttcttt tcacctttcc ctcacggtac 660
ttgtacgcta tcggtcattg aggaatactt aggcttagag ggtggtcccc ctttctcgcg 720
taaaagcgat catcattcga acacgccgcg ttttactggg aaggatcgaa ccatgggaac 780
gaatgaacag ggctatcacc ttctttggcc ggatcttcca atctttttag aaaaccagtt 840
cactgcgcgc cccaccccgt catcagtcct gtgttgccta tgcttgcgcc ccaaaacggt 900
tgctgacttt gtacaactcc gtagggcgcg ctacggcaac acagcctacg cttgctgctt 960
gctggttttt ccatcatcca atccacaaca aatcgaatga aaccgcttcg gatttctttt 1020
gatgaaaacc cttgttccgc tctcgctcgc cgctactaac ggggtc 1066
Claims (4)
1. The screening method of the internal reference gene for stable expression of the club head grass under the stress of the ALS herbicide is characterized by comprising the steps of sample treatment, RNA extraction, cDNA preparation, candidate internal reference gene design and analysis, wherein the candidate internal reference gene design and analysis comprises the following steps:
(1) Screening 5 candidate reference genes, namely UBQ, EF1, 18srRNA, 25srRNA and 26srRNA, and designing amplification primers of the candidate reference genes;
(2) Using the cDNA as an amplification template, and measuring the specificity of the amplification primer by utilizing PCR;
(3) Performing stability analysis on the candidate internal reference genes by using Ct values obtained after fluorescent quantitative PCR reaction;
(4) And (5) respectively calculating and analyzing the expression stability of the reference genes under the treatment of ALS herbicides by using Bestkeeper, normFinder, geNorm.
2. The method for screening reference genes for stable expression of club head grass under the stress of ALS herbicides according to claim 1, wherein the fluorescent quantitative PCR reaction system in the step (3) of designing and analyzing the candidate reference genes comprises: 10. Mu. l iTaq universal SYBR Green Supermix (2X), 2. Mu.l cDNA template, 1. Mu.l each of the upstream or downstream primers 10. Mu.M, 6. Mu.l sterile ultra pure water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s,40 cycles.
3. The method for screening internal genes for stable expression of club head grass under the stress of ALS herbicides according to claim 1, wherein the sample collection objects in the sample treatment are: an AR clavicle population exhibiting a 3-4 fold level of resistance to ALS herbicides and an AS clavicle population susceptible to ALS herbicides.
4. The method for screening an internal reference gene for stable expression of club head grass under the stress of ALS herbicides according to claim 3, wherein the sample treatment comprises: a. seeds in the AR club head grass population and the AS club head grass population are respectively selected for grouping sowing, ALS herbicide with the concentration of 4.0-5.0 g.a.i/ha is sprayed after the seeds grow to 3-4 leaf periods; b. four sampling times were taken with ALS herbicides sprayed for 0h, 24h, 48h, 72h, and leaves were removed and frozen in liquid nitrogen for RNA extraction.
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