CN112980990B - miRNA fluorescent quantitative reference gene under stress of cryptomeria fortunei hormone and application of primer thereof - Google Patents
miRNA fluorescent quantitative reference gene under stress of cryptomeria fortunei hormone and application of primer thereof Download PDFInfo
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Abstract
The invention discloses a cryptomeria fortunei hormone hypochondriumAn application of forced miRNA fluorescence quantitative reference gene and primers thereof belongs to the field of plant molecular biology. According to the method, 13 candidate internal reference genes required by the implementation are finally selected according to reference of literature data and database search, and the results of gene stability evaluation and comprehensive analysis of RefFinder websites are obtained on the 13 internal references through four software of delta-CT, geNorm, normFinder and BestKeeper 3 Fluorescent quantitative reference genes cln-miR6725, novel1 and 18S under stress, and fluorescent quantitative reference genes U6 and novel16 under ABA stress. Provides powerful support for the quantitative expression of related functional genes of miRNA under the stress of different hormones of cryptomeria fortunei, thereby having important application value.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to a miRNA fluorescent quantitative reference gene under the stress of cryptomeria fortunei hormone and application of a primer thereof.
Background
Ramulus et folium taxi Cuspidatae (ramulus et folium taxi Cuspidatae) (ramulus et folium taxi Cuspidatae)Cryptomeria fortuneiHooibrenk ex Otto et Dietr), also known as Pinus longifola. The natural distribution of the trees is extremely narrow, and the trees are mainly distributed in China, japan and other places. The cedar tree has the characteristics of graceful tree shape, excellent material quality, strong adaptability, fast and durable growth, cold resistance, drought resistance, barren resistance, wind resistance, snow pressure resistance and the like. Abscisic acid (ABA) is a plant hormone with a hemiterpene structure, and is particularly important in regulating the adaptation of plants to stress. It can inhibit seed germination, promote dormancy, inhibit growth, promote leaf senescence and abscission, etc. GA 3 Has remarkable promoting effect on flower bud differentiation of Cryptomeria japonica, such as GA application of Cryptomeria japonica 3 Can obviously early bear fruit. However, it is now about the ABA stress and GA stress of cedar 3 The research reports of the difference of gene expression under the stress condition are few, and in the process, the stable and reliable reference gene is needed when the real-time fluorescent quantitative technology is used for analyzing and verifying the expression level of the gene, so that the screening of the stable-expression reference gene under the stress conditions of different hormones of cryptomeria fortunei is a key factor for the accuracy of the real-time fluorescent quantitative result.
Disclosure of Invention
Aiming at the problems in the prior art, one of the problems to be solved by the invention is to obtain the application of the miRNA fluorescent quantitative internal reference gene with stable expression of the cryptomeria fortunei under different hormone stress conditions, so that the requirement of real-time fluorescent quantitative detection on the transcription expression level of other cryptomeria fortunei genes can be met, and the stability, reliability and efficiency of cryptomeria fortunei gene expression analysis research are improved. The invention also aims to solve the problem of providing the application of the primer of the miRNA fluorescence quantitative reference gene under the stress of hormones.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
according to the invention, four software of delta-CT, geNorm, normFinder and BestKeeper are used for evaluating the gene stability of 13 internal references, and the internal reference genes stably expressed by the cedar under different hormone stress conditions are screened out through RefFinder website comprehensive analysis results.
Application of miRNA fluorescence quantitative reference gene under stress of cedar hormone, wherein the hormone stress is ABA stress or GA stress 3 And (4) stressing.
In the application of the cedar miRNA fluorescent quantitative internal reference gene under ABA stress, the internal reference gene is U6 and/or novel16 gene, and the nucleic acid sequence of the novel16 gene is shown as SEQ ID NO. 1.
The GA 3 In the application of the fluorescence quantitative internal reference gene of the cryptomeria fortunei miRNA under stress, the internal reference gene is cln-miR6725, novel1 and 18S genes, and the nucleotide sequence of the cln-miR6725 gene is shown as SEQ ID NO. 2; the nucleotide sequence of the novel1 gene is shown as SEQ ID NO. 3; the nucleotide sequence of the 18S gene is shown as SEQ ID NO. 4.
The application of the primer sequence of the U6 gene in the fluorescence quantification of the cryptomeria fortunei miRNA is that the primer sequence of the U6 gene is as follows:
u6 forward primer: 5 'ACAGAGAGAATTAGCATGGCC-3';
u6 reverse primer: 5'GACCAATTCTCGATTTGTGCG-3'.
The application of the primer sequence of novel16 gene in the fluorescence quantification of the cedar miRNA is as follows:
novel16 forward primer: 5 'GCGTTTTTTTCCAATACTCTCTATACC-3';
novel16 reverse primer: provided by the Kit miRcute miRNA qPCR Detection Kit (SYBR Green).
The application of the primer sequence of the cln-miR6725 gene in the fluorescence quantification of the cryptomeria fortunei miRNA is that the primer sequence of the cln-miR6725 gene is as follows:
cln-miR6725 forward primer: 5' TGGCATCTGTCGAGGTCATCTTA-;
cln-miR6725 reverse primer: provided by the Kit miRcute miRNA qPCR Detection Kit (SYBR Green).
The application of the primer sequence of novel1 gene in the fluorescence quantification of cryptomeria fortunei miRNA is characterized in that the primer sequence of novel1 gene is as follows:
novel1 forward primer: 5 'CTTTCCGGATCCTCCCATGC-3';
novel1 reverse primer: provided by the Kit miRcute miRNA qPCR Detection Kit (SYBR Green).
The application of the 18S gene primer sequence in the fluorescence quantification of the Cryptomeria fortunei miRNA, wherein the 18S gene primer sequence is as follows:
18S forward primer: 5 'TCTGGTCCTGTTCCGTTGG + 3';
18S reverse primer: 5 'GCTTTCGCAGTGGTTCGTC-3'.
Has the advantages that: compared with the prior art, the invention has the advantages that:
according to the invention, through reference of literature data and database search, 10 mature miRNAs with abundant and stable expression quantity and 3 internal reference genes (5 Sr RNA,18Sr RNA and U6) of common miRNA are finally selected as candidate genes, the gene sequences of the internal reference genes are screened, the stability of the candidate genes is evaluated through 5 algorithms (delta-CT, geNorm, normFinder, bestKeeper and RefFinder), and the GA suitable for cryptomeria fortunei is obtained 3 Fluorescent quantitative internal reference genes cln-miR6725, novel1 and 18S under stress and fluorescent quantitative internal reference genes U6 and novel16 under ABA stress, and real-time fluorescent quantitative PCR primers of the internal reference genes are designed, the primers have strong specificity and high amplification efficiency, can greatly improve the detection efficiency when the real-time fluorescent quantitative detection of the cryptomeria fortunei genes is adopted, and improve the detection resultAnd (5) reliability.
Drawings
Fig. 1 is CQ values of the screened 13 miRNA fluorescence quantification reference genes, a: ABA stress; b: GA 3 Stress;
FIG. 2 is a graph analysis of results of the geNorm software ranking the expression stability values (M) of 13 reference genes, the lower the stability value, the more stable the gene is represented, A: ABA stress; b: GA 3 Stress;
figure 3 is a graph of the number of reference genes determined by geonorm to be optimal for accurate quantification, a: ABA stress; b: GA 3 Stress;
FIG. 4 shows the case where stable gene and unstable gene are used as reference genesaof-miR396aExpression level of gene, a: ABA stress; b: GA 3 And (4) stressing.
Detailed Description
The invention is further described with reference to specific examples.
The following examples used test materials: in 6 months of 2014, a good-growing and pest-free Cryptomeria fortunei forest farm (118 DEG 32'E, 25 DEG 23' N) was used as a mother tree, and semi-lignified branches with 2-3 lateral buds and growing in the same year were cut as cuttings (10-12 cm) (# 3 seedlings). Cutting the cutting slips, cutting the cutting slips at 45 deg.C, soaking in distilled water for 12h, soaking in 1% sodium hypochlorite for 10 min, washing with distilled water for 3 times, and washing with 0.1 g L −1 And soaking the GGR rooting powder for 4 hours. The treated cuttings are planted in black plastic bags, the soil matrix of the black plastic bags is 1. And returning to Nanjing forestry university in 2017 in 6 months, performing unified management, weeding periodically, and watering. In 10 months in 2020, 6 annual Cryptomeria fortunei #3 seedlings (3 in each group) with consistent growth vigor are selected and respectively treated with 200. Mu.M ABA and 200. Mu.M GA 3 Uniformly spraying 6-year-old cryptomeria fortunei seedlings with consistent growth vigor until the leaves are completely wet; and the seedlings were cultivated in a light incubator at 25 ℃ with a photoperiod (12-h light/12-h dark cycle) and humidity (60%). Three biological replicates were done. Young leaves were taken at 0, 2, 6, 12, 24 and 48h after stress, respectively. At the time of collecting the sample, it was rapidly frozen with liquid nitrogen, and thenStored at-80 ℃ for further analysis.
Example 1
1. Screening of candidate reference genes and design of primers therefor
Through sequencing the cryptomeria fortunei miRNA transcriptome, 10 mature miRNAs with abundant and stable expression quantity and 3 internal reference genes of common miRNA are selected as candidate genes respectivelycln-miR6725、novel16、novel1、 novel14, pab-miR159a, pab-miR395b, U6, ppt-miR894, 5S, novel6, 18S, cln-miR162 and cas- miR166d。(U6 from Kou, S.J., wu, X.M., liu, Z, liu, Y.L., xu, Q, guo, W.W., 2012 Selection and differentiation of Suitable reference genes for miRNA expression by quantification RT-PCR in clinical systematic analysis and addition facilities Plant Cell Rep.31, 2151-2163.);5 Sr RNA、18Sr RNAdesigning primers of the internal gene by using Primer 5.0; the miRNA upstream primer is based on a mature miRNA sequence, U is replaced by T, bases are increased and decreased at two ends of the primer to achieve a proper Tm value at 65 ℃ and a GC content of 40-60%, and a reverse primer is provided by a Kit miRcute miRNA qPCR Detection Kit (SYBR Green). The candidate reference genes and primers thereof are shown in table 1 below:
TABLE 1 13 candidate reference genes and primer sequences
2. Extraction of total RNA of cryptomeria fortunei and preparation of cDNA
MiRNA was extracted using an anmiRNA isolation kit (Tiangen, beijing, china). 1.0% (w/v) agarose gel electrophoresis and spectrophotometer (NanoDrop 2000, thermo Scientific, wilmington, DE, USA) were used to check integrity, purity and concentration, respectively.
Detecting the synthesized sample, and performing reverse transcription by adopting a tailing method, namely: 1 ug of miRNA First Strand Synthesis of miRNA cDNA was performed according to the MiRcute miRNA First-Strand cDNA Synthesis Kit (KR 201, tiangen Biotech, china) instructions. The reaction was carried out at 42 ℃ for 60 minutes; after which the sample was subjected to an enzyme inactivation reaction at 95 ℃ for 3 minutes. The synthesized cDNA reaction solution was immediately stored at-20 ℃.
3. qPCR quantification
Preparing 20 mu L PCR reaction system according to the instruction of miRcute Plus miRNA qPCR Detection Kit (SYBRGreen), 2 Xmircute Plus miRNA Premix (SYBR)&ROX) 10. Mu.L, forward Primer (10. Mu.M) and Reverse Primer (10. Mu.M) each 0.4. Mu.L, 10-fold diluted miRNA first strand cDNA 2. Mu.L, 50 XROX Reference Dye 1.6. Mu.L, RNase-Free ddH 2 O5.6. Mu.L. Each sample was tested for amplified background by 3 technical replicates and no template negative control for each candidate reference gene. qRT-PCR was amplified using the ABI 7500 system (Applied Biosystems) with the following procedure: pre-denaturation at 95 ℃ for 15 min; then 40 times of circulation are carried out, denaturation at 94 ℃ is carried out for 20 s, and annealing and extension at 60 ℃ is carried out for 34 s; then, the resultant was melted at 60 to 95 ℃. And (3) obtaining a CT value through qRT-PCR, wherein the CT value is in inverse proportion to the expression quantity of the gene, the larger the CT value is, the lower the expression quantity of the gene is, and conversely, the smaller the CT value is, the higher the expression quantity of the representative gene is.
4. Stability evaluation
Respectively carrying out statistical analysis on the expression stability of the reference genes in different samples by adopting 4 different algorithms of delta-Ct, geonorm, normFinder and BestKeeper; and (4) screening out stable reference genes through the comprehensive analysis result of RefFinder website.
5. Results
delta-CT analysis: the mean value of the standard deviation was obtained by delta-CT analysis, wherein the higher the mean value of the standard deviation, the worse the gene stability, the results are shown in Table 2, under ABA stress,U6the mean standard deviation of (a) was 0.65, which is the most stable gene;cln-miR162the mean value of the standard deviation of (a) was 1.39, which is the gene with the worst stability. In GA 3 Under stress, the mean value of the standard deviation of cln-miR6725 is 0.89, and the cln-miR6725 is the most stable gene; the mean standard deviation of cas-miR166d is 2, and the gene with the worst stability is obtained.
TABLE 2 Delta-CT analysis results
Analysis by using the GeNorm software: when using geNorm, the original Ct value is converted to 2 -ΔCt (delta Ct = original Ct value-lowest Ct value in this group) was then used for expression stability analysis of the reference genes, and the expression stability M value of each candidate reference gene was calculated by the geonorm software, with a larger M value indicating a lower stability, where M =1.5 is the upper limit (fig. 1). Furthermore, geNorm may be 2 -ΔCt For the raw data, the paired difference values V of two continuous normalization factors are calculated n /V n+1 To determine the appropriate internal reference factor, V2/V3, due to pairwise variation of examples under ABA stress<0.15 (FIG. 2A), therefore, only 2 internal reference genes were required under Salicis Babylonica ABA treatment: (A)U6 And novel16) Analysis of relative expression amount of genes was conducted in GA 3 Under stress, due to pairwise variation of examples V 3 /V 4 <0.15 (FIG. 2B), therefore in cedar GA 3 Only 3 reference genes (cln-miR 6725, novel1 and 18S) are needed for relative expression analysis of the genes under stress (FIG. 3).
Analysis by NormFinder software: using NormFinder, convert 2 the original Ct value -ΔCt (delta Ct = original Ct value-lowest Ct value in this group) was then used for expression stability analysis of the reference gene. The stability values of the candidate reference genes are calculated by NormFinder software, and the stability is better when the stability value is lower, namely the gene with the minimum stability value is the most stable gene. The results are shown in Table 3.
TABLE 3 analysis results of the NormFinder software
Bestkoeper software analysis: for bestkoeper, the amplification efficiency calculated from the raw Ct values by the LinRegPCR program and the raw Ct values were used to calculate the Coefficient of Variation (CV) and Standard Deviation (SD) of candidate reference gene expression, with the smaller the SD value, the more stable the reference gene. If SD value >)1.0, the reference gene is considered unstable and should be avoided for gene expression normalization. The results are shown in Table 4. Under the stress of the ABA of the cryptomeria fortunei, the stability of the gene isU6 > ppt-miR894 > novel1 > 5 S > cln-miR6725 > novel16 > pab-miR159a > pab-miR395b > novel14 > novel6 > 18S > cas-miR166d > cln-miR162 The most stable expression isU6. In cryptomeria fortunei GA 3 Under stress, the stability of the gene isU6 > cln-miR6725 > 5S > novel14 > novel16 > 18S > cln-miR162 > novel1 > novel6 > ppt-miR894 > pab-miR159a > pab-miR395b > cas-miR166dThe most stably expressed gene isU6。
TABLE 4 BestKeeper software analysis results
RefFinder website analysis: to verify the accuracy of the reference gene screening, candidate reference genes were ranked together by the geometric mean of the delta-Ct, geonorm, normFinder and bestkoever ranking results under each treatment. The smaller the overall ranking, the better the stability of gene expression. The results are shown in Table 5, and the stability of the gene is shown in the condition that the Abelia of the cedar is stressedU6 > novel16 > novel1 > cln-miR6725 > ppt-miR894 > pab-miR159a > pab- miR395b > 5S > novel6 > novel14 > 18 S > cas-miR166d > cln-miR162Wherein the geneU6The geometric mean value of (a) is 1.41, which is the most stable gene; genecln-miR162 The geometric mean of (2) is 13, which is the most unstable gene.
In Cryptomeria fortunei GA 3 The stability of the gene under stress iscln-miR6725 > novel1 > 18S > U6 > 5S > pab-miR159a > novel16 > novel6 > novel14 > cln-miR162 > ppt-miR894 > pab- miR395b > cas-miR166dWherein the geometric mean value of the gene cln-miR6725 is 1.19, and the gene cln-miR6725 is the most stable gene; the geometric mean value of the gene cas-miR166d is 13, and the gene is the most unstable gene.
TABLE 5 RefFinder Web site analysis results
(3) Verification of stability of reference gene
And selecting the MIR396 family with high expression quantity and obvious difference as the target miRNA according to the sequencing result of the selection flux. Use of the aoof-miR 396a as a target Gene to confirm the suitability of the candidate Gene evaluated in the present invention, 2 -△△Ct The method calculates the expression of the target gene. Under ABA stress, stable genes are foundU6 Andnovel16when the internal reference is made, the change trends of the expression quantity of the target gene are basically consistent, and the expression quantity is low. But using unstable genescln-miR162 When the internal reference is made, the expression level is higher in 2h and 24h, and the expression condition of the target gene is different compared with that of the stably screened stable gene. In GA 3 Under stress, stable genes cln-miR6725, novel1 and 18S and unstable genes cas-miR166d are used as standards for calculation, and when the unstable genes cas-miR166d are used as reference genes, the expression levels of the aof-miR396a genes are generally low at 2h, 6 h and 24 h. Therefore, selection of an appropriate gene has a large influence on the expression amount of the target gene (FIG. 4).
Sequence listing
<110> Nanjing university of forestry
<120> miRNA fluorescent quantitative reference gene under stress of cryptomeria fortunei hormone and application of primer thereof
<130> 100
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> Cryptomeria fortunei Hooibrenk ex Otto et Dietr
<400> 1
uuuuuccaau accuccuaua cc 22
<210> 2
<211> 22
<212> RNA
<213> Cryptomeria fortunei Hooibrenk ex Otto et Dietr
<400> 2
uggcaucugu cgaggucauc ua 22
<210> 3
<211> 22
<212> RNA
<213> Cryptomeria fortunei Hooibrenk ex Otto et Dietr
<400> 3
ucuuuccgga uccucccaug cc 22
<210> 4
<211> 516
<212> DNA
<213> Cryptomeria fortunei Hooibrenk ex Otto et Dietr
<400> 4
ggtgtgcact ggccctcacg tcccttctgc cggcggcgtg ttcctggcct taattggctg 60
ggtcgcggtt ccggcgccgt tactttgaaa aaattagagt gctcaaagca agcctacgct 120
ctgaatacat tagcatggaa taacgcgata ggagtctggt cctgttccgt tggccttcgg 180
gaccggagta atgattaata gggactgtcg ggggcattcg tatttcattg tcagaggtga 240
aattcttgga tttatggaag acgaaccact gcgaaagcat ttgccaagga tgttttcatt 300
aatcaagaac gaaagttggg ggctcgaaga cgatcagata ccgtcctagt ctcaaccata 360
aacgatgccg accagggatc ggcggatgtt gctctaagga ctccgccagc accttctgag 420
aaatcagagt gtttgggttc cggggggagt atggtcgcaa ggctgaaact taaaggaatt 480
gacggaaggg caccaccagg agtggagcct gcggct 516
Claims (6)
1. The application of the miRNA fluorescent quantitative internal reference gene under the stress of cedar hormones is characterized in that the internal reference gene under the stress of ABA is U6 and/or novel16 gene, and the nucleic acid sequence of the novel16 gene is shown as SEQ ID NO. 1; in GA 3 Under stressThe internal reference genes are cln-miR6725, novel1 and 18S genes, the nucleotide sequence of the cln-miR6725 gene is shown as SEQ ID NO. 2, the nucleotide sequence of the novel1 gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the 18S gene is shown as SEQ ID NO. 4.
2. The application of the primer sequence of the U6 gene in miRNA fluorescence quantification under the stress of Cryptomeria fortunei ABA in claim 1 is characterized in that the primer sequence of the U6 gene is as follows:
u6 forward primer: 5 'ACAGAGAGAATTAGCATGGCC-3';
u6 reverse primer: 5'GACCAATTCTCGATTTGTGCG-3'.
3. The application of the primer sequence of the novel16 gene in miRNA fluorescence quantification under the stress of Cryptomeria fortunei ABA in claim 1 is characterized in that the primer sequence of the novel16 gene is as follows:
novel16 forward primer: 5 'GCGTTTTTCCAATACTCTCTATACC-3';
novel16 reverse primer: the Kit is used for detecting the miRNA qPCR Detection Kit.
4. The primer sequence of cln-miR6725 gene in claim 1 in Cryptomeria fortunei GA 3 The application of miRNA fluorescence quantification under stress is characterized in that the primer sequence of cln-miR6725 gene is as follows:
cln-miR6725 forward primer: 5' TGGCATCTGTCGAGGTCATCTTA-;
cln-miR6725 reverse primer: provided by a Kit miRcute miRNA qPCR Detection Kit.
5. The primer sequence of novel1 gene in claim 1 is in cryptomeria fortunei GA 3 The application of miRNA fluorescence quantification under stress is characterized in that primer sequences of novel1 genes are as follows:
novel1 forward primer: 5 'CTTTCCGGATCCTCCCATGC-3';
novel1 reverse primer: provided by a Kit miRcute miRNA qPCR Detection Kit.
6. The primer sequence of 18S gene in claim 1 in cedar GA 3 The application of miRNA fluorescence quantification under stress is characterized in that the 18S gene has the following primer sequences:
18S forward primer: 5 'TCTGGTCCTGTTCCGTTTGG-3';
18S reverse primer: 5 'GCTTTCGCAGTGGTTCGTC-3'.
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