CN107475416B - Primer for screening internal reference gene under low-temperature stress of wax apples and application of primer - Google Patents
Primer for screening internal reference gene under low-temperature stress of wax apples and application of primer Download PDFInfo
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Abstract
The invention provides a primer for screening an internal reference gene under low-temperature stress of wax apples and application thereof, wherein the primer comprises the following components: forward primer 5'-CCTGGCCTCTATGATCTTATCC-3', reverse primer 5'-CAGGCATCATGTGCTTTG-3'. The invention utilizes qRT-PCR to detect the expression levels of 7 candidate internal reference genes ACT-7, GAPDH, UBQ, alpha-TUB, CYP20-1, EF-2 and APRT-5 under the conditions of wax apple fruit development and low temperature stress, and gene stability evaluation is carried out through geNorm, NormFinder, BestKeeper and RefFinder software, and the result shows that the most stable internal reference gene CYP20-1 is expressed under the low temperature stress, so that the current situation that no internal reference gene exists in the existing quantitative PCR detection of wax apple is solved, and a foundation is laid for the subsequent gene expression work.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer for screening an internal reference gene under low-temperature stress of wax apples and application thereof.
Background
Wax apple [ ]Syzygium. samarangense(BI.) Merr. et Perry of Myrtaceae (Mytaceae) SyzygiumSyzygium) Evergreen trees, which are typical tropical fruit trees. The wax apple fruits are rich in nutrition, have the effects of stopping diarrhea, relieving pain, resisting bacteria, diminishing inflammation (2011 such as Tina) and the like, are bright in color, crisp and tasty, are popular with consumers, are rapidly developed in suitable planting areas in China, and are important tropical characteristic fruits. In production, unstable quality of wax apple fruits and low temperature and cold damage in winter are two prominent problems which hinder the development of the wax apple industry, and solution is needed urgently. For a long time, the quality and cold resistance of wax apple fruits are generally improved by adopting optimized cultivation measures and improved cultivation conditions, but the high quality and cold resistance genetic characteristics of varieties are the factors playing a role in determination. Therefore, the research on the quality of the wax apple fruits and the stress-resistant mechanism to the cold and freeze injury analyzes the whole metabolic network formed by the fruit quality and the gene expression regulation network responding to low temperature, and the related genes are purposefully discovered and utilized, thereby having important significance on the molecular breeding of the wax apple.
Gene expression and transcriptome analysis are important means for clarifying complex metabolic pathways and physiological responses of plants to adversity stress. The real-time fluorescence quantitative PCR has the characteristics of high sensitivity, good repeatability, strong specificity, high flux and the like, and is widely applied to the research of gene expression, transcriptome analysis and the like. Factors such as RNA quality, reverse transcription efficiency, selection of internal reference genes and the like influence the accuracy and reliability of qRT-PCR. Wherein, selecting stably expressed reference gene is an important precondition for ensuring accurate and reliable gene expression analysis result. The ideal internal reference gene can be stably expressed in all cells or under various physiological states, but a great deal of research shows that the traditional housekeeping genes have different stability under different tissue types or test conditions, so that the accuracy and the reliability of the target gene expression quantification can be ensured only by screening the proper internal reference gene or simultaneously using a plurality of internal reference genes according to different test materials and conditions. At present, no report about the screening of the wax apple reference genes is found, 7 common reference genes are selected, expression stability of the common reference genes under different test conditions is evaluated by utilizing a qRT-PCR technology, so that the reference genes stably expressed under low-temperature stress are screened, and a foundation is laid for subsequent gene expression work.
Disclosure of Invention
The invention aims to provide a primer for screening an internal reference gene under low-temperature stress of wax apples and application thereof, and solves the technical problems by the following technical scheme: the expression stability of 7 wax apple reference genes under the condition of low temperature stress is detected by utilizing a qRT-PCR technology, the gene stability is evaluated by 4 software of geNorm, NormFinder, BestKeeper and RefFinder, and the reference genes stably expressed under the condition of low temperature stress are screened out by integrating the analysis results of the software. Obtaining one of stably expressed reference genes: CYP20-1, and the primer pair thereof is shown as follows:
forward primer 5'-CCTGGCCTCTATGATCTTATCC-3'
Reverse primer 5'-CAGGCATCATGTGCTTTG-3'
The reference gene primer is applied to real-time fluorescence quantitative PCR under the low-temperature stress of wax apples.
The invention has the advantages that:
the invention provides an internal reference gene of wax apple, and simultaneously discloses a real-time fluorescent quantitative PCR primer designed on the basis of the Unigene sequence of the internal reference gene, thereby solving the problem that the internal reference gene does not exist in the existing quantitative PCR detection of wax apple; when the designed real-time fluorescent quantitative PCR primer is used for gene expression analysis under low-temperature stress of wax apples, the stability, reliability and repeatability of the wax apple gene expression analysis research can be improved; in addition, the designed real-time fluorescent quantitative PCR primer has strong specificity, so that the detection efficiency when the wax apple gene is detected by real-time fluorescent quantitative detection can be greatly improved, and the reliability of the detection result is improved.
Drawings
FIG. 1 electrophoresis of RNA detection, M: DL2000 DNA marker; 1-5: the purple red wax apple pulp is T1-T5; 6-10: the purple red wax apple pericarp is T1-T5; 11-14: purple red L1-L4; 15-18: black pearls L1-L4; 19-22: jadeite L1~ L4.
FIG. 2 shows the RT-PCR amplification results of 7 internal reference genes using purple red wax apple T5 stage pulp cDNA as a template, M: DL2000 DNA marker; 1: ACT-7; 2: GAPDH; 3: UBQ; 4: alpha-TUB; 5: CYP 20-1; 6: EF-2; 7: APRT-5.
FIG. 3 shows the melting curves of 7 reference genes qRT-PRC of wax apple.
FIG. 47 is a box plot of Ct values of the reference genes.
FIG. 5 comparative analysis of pairs of 7 reference genes of Syzygium samarangense.
Detailed Description
Example 1
1 materials and treatments
Tested wax apple [ ]Syzygium. samarangense(BI.) the Merr. et Perry fruits are from wax apple plantation in Dongshan county in Zhangzhou, Fujian province, and the varieties are 'purplish red' (Tub Ting Jiang), 'black pearl' (Heizhenzhu) and 'emerald' respectively. And collecting the wax apple fruits 10 d, 20 d, 30 d, 40 d and 50 d after full bloom in 2016 at different sampling periods of T1-T5. Fruit was harvested 5 times per tree in 4 directions for a total of 20 fruits, with 1 treatment per plant and 3 replicates. After cleaning fruits, separating peels and pulps by using a peeling knife, respectively and rapidly putting into liquid nitrogen for quick freezing, and then storing in a refrigerator at the ultralow temperature of minus 80 ℃ for later use.
The used seedlings of 'purplish red', 'black pearl' and 'emerald' wax apples were from the institute for subtropical plants in Xiamen. Selecting annual wax apple cutting seedlings with consistent growth vigor, placing the annual wax apple cutting seedlings in an GXA-0288 illumination incubator (Ningbo Jiangnan instrument factory), treating the annual wax apple cutting seedlings at a humidity of 60% and no illumination, and setting the test temperature to be 25 ℃, 4 ℃, 1 ℃ and-2 ℃ for 4 treatments, wherein the treatments at different temperatures are recorded as L1-L4. Each temperature was treated for 3 h, and the individuals were 1 replicate, 3 replicates. Then the tender leaves are taken, subjected to liquid nitrogen quick freezing and then placed in a refrigerator with ultralow temperature of minus 80 ℃ for storage and standby.
2 method
2.1 extraction of Total RNA and Synthesis of cDNA 1st Strand
Total RNA extraction of samples all uses a Baitake (BioTeke) general plant total RNA extraction kit, and the specific operation is carried out according to the product instruction. Total RNA integrity was checked by 1.5% agarose gel electrophoresis and concentration and purity of total RNA were determined using a Nanodrop2000C spectrophotometer (Thermo). Synthesis of 1st chain of cDNA Using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa), the detailed procedures were performed according to the product instructions.
2.2 selection of reference genes and primer design
In this study, actin gene was extracted from the previously constructed Nelumbo Nucifera transcriptome database: (ACT-7) 3-glyceraldehyde phosphate dehydrogenase gene (a)GAPDH) Polyubiquitin gene (c) <UBQ) Microtubulin gene (A)α-TUB) Cyclophilin gene (a)CYP20-1）A transcription elongation factor gene (EF-2) And adenine phosphoribosyl transferase gene (APRT-5) 7 genes with relatively stable expression are used as candidate reference genes (the gene sequences are respectively shown as SEQ ID NO. 15-22). Further selecting flavanone-3-hydroxylase gene(s) (ii)F3H) A validation study was performed. 8 pairs of primers (Table 1) were designed using Primer 3.0 according to the quantitative Primer design requirements, and all Primer sequences were synthesized by Shanghai Yingjun.
TABLE 1 reference genes andF3Hprimer sequences for genes
2.3 detection of target Gene by fluorescent quantitative PCR
Quantitative analysis was performed using 7500 read Time PCR System (Applied Biosystems) using 1st strand of cDNA from samples of extracted pericarp, pulp and leaf as template. The PCR reaction system is as follows: the total volume was 25. mu.L, 25 ng cDNA, 0.4. mu. mol.L-1Forward/reverse primer, 0.15 mmol.L-1 dNTP, 1U Taq DNA polymerase, 1.5 mmol.L-1 MgCl22.5. mu.L of each 10 XPCR buffer, and the amount of ultrapure water was adjusted to 25. mu.L. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, and extension at 72 ℃ for 30 s, for 40 cycles; finally, extension is carried out for 7 min at 72 ℃ and storage is carried out at 4 ℃.3 replicates per sample were set and a negative control was set.
2.4 data analysis
After the fluorescent quantitative PCR analysis, the Ct value of the sample obtained automatically by the instrument is calculated 2-ΔCtThe stability of the reference genes and the quantity of the appropriately selected reference genes are compared by using geNorm and NormFinder software (Sinara and the like 2010), the Ct value of a sample is directly used, the stability of the candidate reference genes is analyzed by BestKeeper software (Deng and the like 2013), and finally RefFinder (Xie and the like 2011) software is used for comprehensive evaluation.
3 results of the experiment
3.1 RNA and primer quality detection
OD of RNA of each sample260/280And OD260/230All are 1.97-2.20; the 1.5% agarose gel electrophoresis detection shows that the 28S and 18S subunit bands are clear, the gray scale ratio is about 2.0, the 5S subunit band has low brightness and no other miscellaneous bands, and the requirement of fluorescent quantitative PCR (figure 1) is met.
In the development period of 5 pulps and peels of purplish red, black pearl and emerald, 4 melting curves in low-temperature stress treatment of 7 pairs of primers only have obvious single peaks, and electrophoresis detection also only has a single band (figure 2), which shows that the primers can specifically amplify corresponding products of various internal reference genes, no primer dimer exists, the PCR amplification curve of each sample to be detected has good repeatability, the template can specifically amplify, and the real-time fluorescence quantitative PCR result is accurate and reliable (figure 3).
3.2 analysis of Ct value of reference Gene
For the 7 piecesThe Ct value analysis of the internal reference genes shows that the expression level of each internal reference gene is different under different tissues and different low-temperature conditions of the wax apple, and the Ct value of each gene is different from 18.88 to 31.59. And (3) reflecting the expression abundance of the reference gene by the Ct value, wherein the lower the value, the higher the expression abundance, and the lower the expression abundance. In general, the expression abundance of each gene in the wax apple leaves is higher than that of fruits, and as shown in FIG. 4, the Ct value of each gene in the wax apple leaves is 18.88-29.87, and the Ct value in the pulp and the peel is 25.44-31.53. In addition, reference genesEF-2The expression abundance of the gene is higher than that of other genes,α-TUBis less abundant than other genes. The Ct value of 7 reference genes is changed between 0.96 and 4.52, whereinα-TUBThe Ct value of (A) is changed greatly in the wax apple leaves and is changed less in the pulp and the peel;CYP20-1the opposite is true. However, there was no regular pattern of expression changes for all 7 reference genes in these 3 treated samples.
3 evaluation of expression stability of reference Gene
3.1 GeNorm software analysis
According to the geonorm software, the average variation M value is used as an index for measuring the stability of the internal reference gene, and the larger the M value is, the lower the stability is; otherwise, the higher. The default rounding value for the geonorm software is M = 1.5. As can be seen from Table 2, the most stable reference genes differed among the 7 reference genes in the different treatment groups. In the process of pulp development, the most stable expression of the reference gene isACT-7(ii) a In the fruit peelα-TUBThe stability of (2) is best represented; under the stress of low temperature, the material is in a non-thermal state,CYP20-1is the most stable expression reference gene.
TABLE 2 Steady values (M) and ranking for analysis of GeNorm data
The geNorm software can obtain the paired difference value V of the candidate reference genesn/n+1To determine the appropriate reference base factor with a default threshold ofVIs less than 1.5. As shown in FIG. 5, V occurs at different development stages of wax apple pulp4/5=0.13, indicating the optimum internal referenceThe base factor being 4, i.e.ACT-7、α-TUB、GAPDHAndUBQ(ii) a And the syzygium samarangense pericarp is in different development stages V2/3=0.135, indicatingα-TUBAndACT-7the most suitable reference gene is the gene; under low temperature stress conditions V2/3=0.141, indicating that the optimum internal reference factor is also 2, beingCYP20-1AndACT-7。
3.2 NormFinder software analysis
The NormFinder software carries out gene evaluation by calculating the stability value of the candidate reference gene, and the stability value is in negative correlation with the stability of the reference gene. Therefore, as shown in Table 3,ACT-7stable expression in different development stages of wax apple pulp, and is the most suitable internal reference gene, and in the fruit peelα-TUB(ii) a Under low temperature stressCYP20-1Is the most stable reference gene, and the second isACT-7。
TABLE 3 Stable values and ranking for NormFinder data analysis
3.3 comprehensive evaluation of expression stability of 7 reference genes
According to the comprehensive evaluation of RefFinder software, the stability of the candidate reference gene in different development stages of the pulp is from high to low in the development process of the wax apple fruitACT-7＞GAPDH＞UBQ＞α-TUB＞CYP20-1＞APRT-5＞EF-2Optionally, the results are analyzed by the GeNorm softwareACT-7、GAPDH、UBQAndα-TUB4 most suitable reference genes are obtained; different development stages of pericarpα-TUB＞UBQ＞ACT-7＞EF-2＞GAPDH＞CYP20-1＞APRT-5Since geNorm recommends 2 optimal reference genes, it can be usedα-TUBAndUBQis an internal reference gene; under low temperature stressCYP20-1＞UBQ＞ACT-7＞APRT-5＞EF-2＞GAPDH＞α-TUB2 most suitable internal parameters can be selected asCYP20-1AndUBQ。
the above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> research institute of fruit trees of academy of agricultural sciences of Fujian province
<120> primer for screening internal reference genes under wax apple low-temperature stress and application thereof
<130> 23
<160> 23
<170> PatentIn version 3.3
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<400> 19
gcggcaagta gcctataaaa aaaaataccc acacgatatc acgctctaat atttcgcagc 60
tggttgcaaa agaaaactca aaccctaggt gctctccgtt cgacctctcg tggttcttct 120
ctcttcgcct caagatgcaa atctttgtga aaacccttac tggcaagaca atcaccctcg 180
aggtggaaag ctccgacaca atcgataatg tgaaagcaaa aatccaggac aaggaaggga 240
tccctccgga ccagcagagg cttatctttg ctggcaagca gctcgaggac ggccgaacct 300
tggccgatta caacattcag aaggagtcca ccctccactt ggtgctccgc ctcaggggag 360
gcatgcaaat ctttgtgaag accctcactg ggaagacaat taccctagaa gttgagagct 420
ctgacaccat tgacaatgtc aaggccaaga tccaagacaa ggagggtatc cccccggacc 480
aacaaaggct catctttgct ggcaaacagc ttgaggatgg gcgtactttg gctgattaca 540
acatccagaa ggaatcaacc ctgcacctgg ttcttcgcct taggggtggc atgcagatct 600
ttgtgaagac cctcaccggc aagaccatta ctcttgaggt tgagagttct gataccattg 660
acaatgtgaa ggccaaaatc caggacaaag aagggatccc cccagaccag cagcggttga 720
tctttgctgg caagcaactt gaggatggcc ggacactagc ggactacaac attcaaaagg 780
aatccaccct tcacttggtc ctccgtttga gaggaggaat gcaaatcttt gtcaagaccc 840
ttactgggaa aacaatcact ttggaagttg agagctccga caccatcgac aatgtcaaag 900
ccaagattca ggacaaggaa ggtattcctc cggaccaaca gcgattgatc tttgctggta 960
agcaattgga agatggacga actcttgcgg actataacat ccaaaaggag tcgacccttc 1020
atctggtgtt gaggctcagg ggaggaacta tgatcaaggt taagaccctt actgggaagg 1080
aaattgaaat agatattgag ccgaccgata ccatcgaccg tatcaaggaa agggtcgaag 1140
agaaagaagg aattccacct gtccagcaaa ggcttatata tgccggtaag cagcttgcag 1200
atgataaaac agctcgagac tacaacattg agggtggctc tgtcctccat cttgtgcttg 1260
cgctgagagg tggcagattt taattgaatc ttaccatacc ggggttgtgt tcactgttgg 1320
cacttcattg aactaaagaa gcttgcttaa tccttttgca gtgtggatag acttgagatg 1380
ttatgagcaa ctctatgaac atgataattg cctttttgct ctattcctgt tgttttttct 1440
taaatccgct tttgcatcag agcttggatc accaatattt gccttcttaa attatagcgc 1500
acaagcatgc tgattgccac tggttttgct caagcagtgc agcgtattta gagcgttgag 1560
tctgtgcatg gattcatctt aggattgtat cccgcaagat atatatgttc acaacttcgg 1620
tatgacatct cacaaacttt 1640
<210> 20
<211> 1905
<212> DNA
<213> α-TUB
<400> 20
gtttggggat aaaaagtttg gattcaacat ttagccaatg ataggccgaa tgctgaacct 60
ttgtggccca cgatcagttt tgacaatgtc tctgtctcac atgtggcaga gctacgggta 120
aacagcaaaa tcacgactcc tttaagccta tgtcgtaacc cctccaagtc cactattgca 180
aaagctactc aaatctcaaa agtgaaaata gtacccgaat gggagggatg cagaaagaag 240
aagtaagctg cggaatacta agatactgat ttgatagcaa ttcacaatga aaaaccatca 300
tgccacgaaa atacccacac acgcgcactc aattttgaca ggacaaacat aaaaccgatg 360
catattgaag caaacaagcc agtgaattca caaccatatc agtactcatc tccttcatca 420
ccttcatcgc cctcagcaga ctcggcaccg acttcttcgt agtccttctc cagggcagca 480
aggtcctcac gagcctcaga gaactctccc tcctccatac cctcaccgac ataccaatgc 540
acaaaagcac gcttggcata catgaggtca aatttgtggt cgatgcgaga gaacacctcg 600
gcaacactgg tggagttgga gatcatgcag acagccctct gcaccttggc aaggtcacct 660
cccggaacaa cagtaggtgg ttggtagtta ataccgcact tgaatccggt tgggcaccag 720
tcaacaaatt ggatggtgcg tttggtcttg atggtggcca cagctgcatt cacatccttt 780
ggaacgacat ctcctcggta catgagacag caagccatgt acttgccatg gcgaggatca 840
cacttggcca tcatagatga tggctcgaat gcactgttgg tgatctcagc caccgagagc 900
tgctcatgat atgctttctc agctgagata actggagcat acgaagaaag cataaagtgg 960
atcctggggt aagggaccag gttggtctgg aactcggtga catccacatt caaggctcca 1020
tcaaacctca gagaggcagt caaggatgag atgacctgag agaccaagcg gttcagatta 1080
gtgtatgtgg gacgctcaat atcgagggat cttcggcaga tgtcatagat ggcttcatta 1140
tcaagaagca cggcaacatc agtgtgctca aggagggagt gggtagaaag gacgctgttg 1200
tagggttcca caacagaggt agaaacctga ggagagggat aaacggtgaa actgagcttt 1260
gattttttgc catagtcaac tgagagtcgc tccaagagaa gtgatccgag accagaacca 1320
gtgccacctc caacagaatt aaagacaagg aagccctgga gaccagtgca gttgtctgca 1380
agcttgcgga ttctgtccaa gcagagatcc acgatctcct ttccaatcgt gtaatgacca 1440
cgggcgaagt tgttggcggc atcttctttg ccactgatga gctgctcagg gtggaagagc 1500
tggcggtaag cgccagtcct cacttcatcg ataacggtgg gctccagatc cacaaacaca 1560
gcacgaggga cgtgcttgcc agcaccggtc tcactgaaga aggtgttgaa agcatcatcg 1620
cctccgccaa ctgtcttgtc acccggcaac tggccatcag gctgaatgcc gtgctcgaga 1680
cagtagagct cccagcaggc gttgccgacc tgaatgccgg cctggccgat gtggatcgag 1740
atgcactccc tcatctccgc cggaggaagg gatcgagaga atgggcggcg ggaatcggag 1800
gacgaaggag gggatggggg ggggaagcac gggaggtcga aggcgtttat gaagacgcct 1860
tcttcggagg aaatctctct tctcgctctg ccggaaagat gaaac 1905
<210> 21
<211> 2148
<212> DNA
<213> CYP20-1
<400> 21
acattacggg gttttcttca attcgggatt ttatccgtga cagtaattcg tttgacggga 60
tcgtgaccca ttggaaacga ggtcaacaca aatcttaaag agcgccgaat ccacagcaac 120
agccgcaaca ttcagtttgc gaaaacagag gaacttttct caatttacaa atcaagtaat 180
ggtcacagaa ctatacaaaa ccactctcga tgtactggaa cattttaggt ttaccagaag 240
tcttttgttt tgtctttcat tttcagagag attgtttccc gttcgaatat tgctttcact 300
cgttgcaaat atcatgctgt cggcatcagt ttttacccgt tcattcggct ggaatttatt 360
aataaaatga aattccccaa aaatgactct tctcatttgg acaatggatt gaagattttt 420
aattaataca aaggagaaat acatcatttg ctttccagtt ttcagaaaaa aaaatttccg 480
ttttctagat ttcattcggc tatatttaat ctaacttcca attcagactc agagcagttc 540
tctctattcc tggcctctat gatcttatcc aagctctttc aagcatgccc caatatccaa 600
tgaacaagat tgcctcgcgt tttcttcacc atataaaaac ttcatactca tcaaagcagt 660
acatgatgcc tgagccctac attcatgtgc aatctggagc gaagtgctca tagagccaat 720
tctccgctgt ctgaaatgac gactgtactt ttgggttggc cgctctgtct cccttcagcc 780
tcaatcttgc ggacaacatc cattccagac agcacctttc caaacacaac atggtggcca 840
tccaacctac aagagattga aaggagttca aatccttgag ttagtttacg atcaacctaa 900
caactcaaaa gattaccaaa agaactcaga ttaactagat tatgacaata atccatgata 960
aagttgccta gatgttttta ggcatacgca cattttttcc ccacaaggac ttgagatgaa 1020
gctaacaata gcctcaaact cattcaaatc atggtaacat ctccaacagt aacagcaatg 1080
agctgaatgt cccacgaatt aggtgacaat gttgcttatc aaagaaatga gtaaaggaag 1140
atagtcgatt accaactagt tgtcacagtg gtaatgaaaa attgtgaccc attcgtgtct 1200
ggcccagcat ttgccatcga aaggcgccct gcatctgtat gcttcaattt gaagttttca 1260
tcagaaaatt tgttcccgta gatcgactct ccccctctcc cgtctccaag agtgaagtca 1320
gcaccgtgga tcataaagct aggaatgatc ctgtggaatc ggctcccttt gtaatgcagg 1380
ggcttcccac tctttccgat ccctttctcc cctgcaataa gactgataat aatctaggaa 1440
agacaccctg acatgtttag gcaggaagta acaatttagg gatacctctc tcttctctac 1500
acatgaatga ttctttctta ttattagttt gtagagaaat cattcagcaa aaagccgatt 1560
gctcttcctt caacttggtc atgagtgttc aggggtttaa aggaaattca aaaaggaagc 1620
agatgtcact tggctacccg tgcaaagagc tcggaagtta tcaactgttt tggggacggt 1680
tttgccaaag agacccatga caattcgacc cactggtttt cctccaatct caacatcgaa 1740
ataaaccttg tgagtcacct cttccaaatc gtctggctcc ttctttaccg caccaacttt 1800
gcgatgaaca agtttcggat cagacgaagc tccggcgtcg ctcaatcggt tctggatgag 1860
agcaagggtt ccgaacaaga ccaagatcca cgccatcgtg atcgtgaccc atctcgtcct 1920
cgtcgccatg gccatttcca gatttcgctc ccctcctcct cctcctccgc gctcgaattt 1980
cccgcctttt ccccgggaat tcgtcgaaac tcgcggcgaa ggagagcgcg ggcttcgtgg 2040
gtgttcgtgg gtcaaccctc tcgtctctgc gcttgacgat ggtcaacggt cggccatgag 2100
cgaagctcga gctcttcgat caggctttgc tttcctcttc cttttttc 2148
<210> 22
<211> 4502
<212> DNA
<213> EF-2
<400> 22
gcacccccca catcctcctt catggagaac aaaagaaaac ttattcataa taataaacct 60
ctagaagcat aaaaactctg gtgggagagt caacaattaa gttgcgatga tctggtacat 120
tatggcacca tgaactttaa tcagctccaa caaacaaaac cacctatgag aaaataaaaa 180
ttgcacctct caagagacca ggacaatgtt caaaaaactc aacatgacga cgacaagcag 240
aacaattaaa ctgaaaagtt tgtgactact actacacaag ggacagcatc cagggaaagg 300
agaactagtg tgctgagcca tgaatatcac atcctcaatc catcctgcaa ttacagcttg 360
tcctcgaact cagacagcgg ggtcatctgc tccttcaagc ccttcctctt gcggatatca 420
gcaacaagct gagatgcctg agacccagac tccagaggat cggacgacat catatcccag 480
tggtcgaaga cacactgagg gaacgcctgc ccagaggttg cagctctcaa tgtactggag 540
aaaccgaagg actcgataac aggaaggtag gccttgatgt tgtagagagg ggttccaggc 600
ctctgcatct cctcaaacac atgcccacgc ttctggttaa ggacactgta aatacctcca 660
agggcctgct ccggagcttg gatctcgaca aggtagacag gctcaagaag cctgggcttg 720
gctgtcaact gcgaagcata gataaccctc ctagcagtcg gaatgacctg gccaccacct 780
ctgtgaatgg cgtcggcatg cagaaccaca tcacagacct caaagcaaat accacgcata 840
ttctcctcag ccaaagcacc ttcttttgat gcccactgga aaccagcaac aacagaatcc 900
ttgatttcat tgaggtactg aacaccctta cacatatcaa caaccatgtt aggtccagta 960
gtctcaggac cgaagcacca aatcttcttc gcaagatcct tatcccagcc aaactcttca 1020
gacaagatct ttgagcgagc cttgggatca tccctgggac caatacggcc atcatcaatg 1080
gcctcagcaa gcccctcctc caaaggtcga gcctccatgt acaacctgtt gtgcttattg 1140
ggtgatttgc tcatgacagt acggcacgat ttctccaaca cagtttcacg gaaagacaca 1200
actggatctg acttcacaat ctcagctccg cccatgaagt catcctgcaa atccttaagg 1260
caaatttcca aatgaagttc tccagcacca gcaatgatgt gttcgccaga ttcctcaatg 1320
ctacatacca ccatagggtc tgacttggcc agacgtttaa gtccttcaac gagcttggga 1380
aggtcagaag ccaccttgca ctgaacagca acacgcacaa ctggagagac ggagaatttc 1440
atggcacgga ttgggtgggc atcgacttct ttctcatttg tcaaagttgc attcttcgtg 1500
atatactgat caagaccaac cagagccaca gtgttaccac acggcacatc ctcaacagtc 1560
tcctgcctct ttcccatcca aataacagtt ctctggacac tcttcacata taaatccttc 1620
ttctcacctg ggacgtagtt gggacccatg atcctgacct tcagaccagt agagaccttg 1680
ccagcgaaaa cacgaccaaa agcaaagaat ctacccttat cagaagcagg aatcatcttg 1740
gagacataga gcatgagagg tccctcagga tcacagttcc taatggcagt ggcatattga 1800
tcgtcaaggg ggccctcata caagttctca acacgatact tctgagcctt agctggagat 1860
ggaaggtgga atatcatcat ttccaacaaa gcagtacttg ccggaagcca agtctgcatc 1920
acacgcttca tcaatgcctt acccatcaag tccttctcct cagacttcat ggtgacccca 1980
agcttctgta gcatgggcca cagcttatct ttctggtcat tcatgcatgt attgatgatc 2040
tgcttgattg gctcatagca gaactggaca aaaccacgct tacatgttgc agaaccggtg 2100
ttcttgcttg tccatttctt ggtggcagga tcgaagaaat tctcacccca caaacgttcc 2160
atcatcttcg actcgtcaac accaaacttg gatgcataca tcttggcgaa attggtgaga 2220
gtaaaggccc aaccatgcag accagcagaa aaagcaacag ttcctttctc tgggtacacc 2280
tggacatcac cgagaagggg atcctcatat gtggccatga tgacattagc attctcaata 2340
actctttgga atgtttgata tgcctcctca ccatcaacct gcagctcgag gaagcatctg 2400
tccatcttgt tgacagtcaa gacaggcctg atcctctcac ccaaagcctg acgaagaaca 2460
gtttctgtct ggacacagac accctcaaca cagtccacaa ccactaacgc accatcagta 2520
atacggaggg cagctgtaac ctcagaagag aagtccacgt gcccaggtga gtcgatcaag 2580
ttgatcagat actcattgcc atttctctct cccttgtagg tctttaagga ttcgtcagac 2640
atttcatagt atagagaaat tccagtagac ttgatcgtga taccacgctc tgcctcatct 2700
gcacgagtat ctgtcatacg aacgtctcca gcaacttcct gagcaataat accagcagca 2760
gccacaaggg aatcggtgag ggtggatttc ccgtgatcaa catgggcgat gacagacata 2820
ttccgaatgt tgtgcttgta gtccatgatt cttcggagct catcagccgt gaacttcacc 2880
atcttgtcgg gttcttaaaa ggcactcctc cacaaaatta gaagaaatct gaggcgagga 2940
gaggcgagag gagaggagag gagagagaga gcgggaacac cacgccgagt gtcgttccgc 3000
tccttttgct ctccgaccaa ggcccgagct tcttagggtt gtgcgctcgc tcctcgcctt 3060
ggccttcgcc ttcgagatct gcgcggtttc tgctctccgg agggaggttt tgctcgatga 3120
gtttgactga tacagaaatg gctcctccta ttgaaacccc acagaaagtt tatcaaaacg 3180
atcatccatc ccatccacct cttaatgaaa ggattctctc atcaatgact aggaggtctg 3240
ttgctgcgca cccttggcat gaccttgaga taggacctgg agctcctaca gtcttcaact 3300
gtgtgattga aattagtaaa gggagcaagg tcaagtatga acttgacaag aaaactggac 3360
tgatcaaggt cgaccgtgtg ctgtattctt cagttgtgta cccccacaac tatggtttca 3420
tccctcgaac tctctgcgag gataatgacc ctatggatgt cttgatcatc atgcaggaac 3480
ctgttcttcc tggatgcttt cttagggcta aagctattgg gctcatgcct atgattgatc 3540
agggcgagaa agatgacaag ataattgctg tttgcgctga tgatcctgaa taccgtcatt 3600
acaatgatat taaggagctc ccaccccatc gtttggctga aattcgtcgt ttctttgaag 3660
attacaagaa aaatgagaac aaggaagttg cagttaatga tttcctgcct gcctctgctg 3720
cctttgatgc aattcagcac tccatgaacc tgtacgcaga ctacattgtg gagagcttga 3780
ggagatagat agcccattcc agcattggtg atgctgatct cccgaagtga cttgaataca 3840
cagataacga cgatatatac agtatctata tttactacag gaaagtgggt attttgctac 3900
atgtacgagc attgtttcat ggttctgttt atattatgat ggtggattgg cttttatatc 3960
cttttccttc ttaaaaggaa agcaaacggc ttggcttctg tattcttttt tctaatttta 4020
tggaactagt atctcaacct agtcctcttg atgttttggt gtcgcaacct aaaaaaaata 4080
tcatcgagat tcgatctcag gctaatggat tactagatta aataaattaa ctaatctagc 4140
tcgagttctc tcaaactcta ccaaatctca acttaggtcc aattgtgagc atgtaaatat 4200
tttcaatggg agtcgctact aatcatttaa ggtaaattga ttagaaacct aaataaaagg 4260
cagaaggaca ctattggtgc cataacttgt gtacggcgat cactttggtg ccataacttt 4320
ttttcggatc actttggtgc taaaatctga aaaaatagat cactttagtg actccggcca 4380
aaattcctgc caaaaagctg acttggcatt tattttttta aaaaaataat ttaaattata 4440
cacgtggaaa ttatgtgact tttttttgaa tttttgaatt ttttttttct ttttttttct 4500
tt 4502
<210> 23
<211> 1146
<212> DNA
<213> APRT-5
<400> 23
ggcgaggttt gaggggacgc ctttgccagt gctgacaccc ctctctctgt ctctcttctc 60
aggccagtgc tgacgcacgc acactttctc tctctatctc ttcccccctc tctctctcca 120
tccgctcgca cagagagagt gaaacagaga gcgaaggaga agaagacgat gctggcggca 180
gaggatggaa gagacccgag attgaaggcc atctcagaca ccataagggt cgttcctcac 240
ttcccaaaac caggcataat gttccacgac ataaccaccc tgttgctcga tcacaaggcc 300
ttcaaagaca cggttgacct gtttgtggag cggtatagag acatgggcat ctccattgtt 360
gctggcattg aagccagggg attcatattt gggccatcta ttgctttggg tattggtgcc 420
aaatttgtcc ctctccggaa accagggaag ctgccaggaa gagtcatatc tgaatcgtat 480
gaactcgaat atggcactga tcaactcgag atgcatgtcg gagctgttca gccaggggaa 540
cgtactataa tcatagacga tttggtagcc actggcggga ccctttccgc tgcaataaga 600
cttctggaac gttttcaggc tgaagtggtt gagtgcgctt gtgtcattgg ttcgcctgtt 660
gtccaggtaa tcctcaaata aagcagtttg gtaatatctt atccatgtgt tcaattttgc 720
gactcattga tggattttgc tgctcacatt gtccagtttt catgcatgca tttctcttct 780
cgctcatctt tttctatcta ctgtagaaat ggaattgtat gatcattcgt gttgactttc 840
taaccatgca tatcttccct aactgaacgt tgaagtgcac aactttttct caatagcagg 900
cttttattgt cgtaccagtg tcgtttcaag tatatattgt gttattttgt ataagatcaa 960
gttataactg ctgatcagct ctgctgtgca gaagtttcgt tcatgaggtt tatggggtcc 1020
tctgttggtt aatggtgtct gctctctctg ttgacttgaa aaatgctttt aagctcagct 1080
gtaacggata aagtagtcac aaacaagcgg tagcttgagt ttgttcgact gatcagataa 1140
actact 1146
Claims (2)
1. The primer for screening the internal reference gene of wax apple under low-temperature stress is characterized by comprising the following steps: the primer is as follows:
a forward primer 5'-CCTGGCCTCTATGATCTTATCC-3';
a reverse primer 5'-CAGGCATCATGTGCTTTG-3';
and (3) screening the primers to obtain an internal reference gene which is a cyclophilin gene.
2. The primer of claim 1 is applied to real-time fluorescent quantitative PCR detection of the wax apple related gene under low temperature stress.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710859453.3A CN107475416B (en) | 2017-09-21 | 2017-09-21 | Primer for screening internal reference gene under low-temperature stress of wax apples and application of primer |
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| CN107475416B true CN107475416B (en) | 2021-04-06 |
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| CN108570473B (en) * | 2018-04-11 | 2021-07-09 | 郑州师范学院 | Phalaenopsis cyclophilin CYP gene and application thereof |
| CN109182583B (en) * | 2018-10-19 | 2021-07-09 | 中国农业科学院蔬菜花卉研究所 | Internal reference gene for eggplant gene expression analysis and related primers thereof |
| CN109266775B (en) * | 2018-10-19 | 2021-07-13 | 中国农业科学院蔬菜花卉研究所 | Reagent for eggplant gene expression analysis and internal reference gene used by reagent |
| CN109722434A (en) * | 2019-03-15 | 2019-05-07 | 中国农业科学院特产研究所 | Ginseng reference gene and its PCR primer, reagent, kit and application for ginseng gene expression analysis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132417A (en) * | 2015-09-07 | 2015-12-09 | 安徽农业大学 | Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene |
| CN105274239A (en) * | 2015-11-19 | 2016-01-27 | 浙江省农业科学院 | Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132417A (en) * | 2015-09-07 | 2015-12-09 | 安徽农业大学 | Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene |
| CN105274239A (en) * | 2015-11-19 | 2016-01-27 | 浙江省农业科学院 | Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes |
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