CN110079627B

CN110079627B - Reference gene for gene expression analysis of primula forbesii in different flowering phases and primer thereof

Info

Publication number: CN110079627B
Application number: CN201910339338.2A
Authority: CN
Inventors: 贾茵; 黄远祥; 李西; 周鹏; 刘思岑; 江明艳
Original assignee: Sichuan Tianyi Youjing Environment Technology Co ltd; Sichuan Agricultural University
Current assignee: Sichuan Tianyi Youjing Environment Technology Co ltd; Sichuan Agricultural University
Priority date: 2019-04-25
Filing date: 2019-04-25
Publication date: 2022-07-22
Anticipated expiration: 2039-04-25
Also published as: CN110079627A

Abstract

The invention discloses internal reference genes for fluorescent quantitative PCR (polymerase chain reaction) of primula forbesii in different flowering phases, which are GAPDH, HIS, 28S and RNA pol II, wherein the sequence of the GAPDH is shown as Seq ID No.1, the sequence of the HIS is shown as Seq ID No.2, the sequence of the 28S is shown as Seq ID No.3, and the sequence of the RNA pol II is shown as Seq ID No. 4. The method takes the floral organs and different tissues of primula forbesii in different florescence as test materials, selects 10 reference genes from the test materials based on the sequencing data of a transcriptome in the previous stage, and analyzes and evaluates the obtained results by respectively utilizing the software of geonorm, NormFinder and Bestkeeper. The combined use of GAPDH, HIS, RNA pol II and 28S as internal controls under different flowering conditions may lead to more reliable results.

Description

Internal reference gene for gene expression analysis of primula forbesii in different flowering phases and primer thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to an internal reference gene for analyzing gene expression of primula forbesii in different flowering phases and a primer thereof.

Background

In the real-time fluorescent quantitative PCR (quantitative real-time PCR, qRT-PCR), fluorescent substances are added during the PCR amplification process so as to detect the PCR process in real time. The quantitative basis is that in the exponential period of PCR amplification, the Ct value of the template and the initial copy number of the template have a linear relationship, the technology overcomes the defects of the conventional PCR, and the technology is rapidly developed due to the advantages of simple operation, high sensitivity, good repeatability and the like, and is applied to various fields of life science research.

The gene expression analysis can be performed by using the qRT-PCR technology, however, in the analysis process, the quality and yield of RNA, the difference of reverse transcription efficiency and other hindering factors can influence the accuracy of the target gene expression result. Therefore, it is necessary to select an appropriate reference gene for calibration and normalization in order to reduce the difference between test samples.

The ideal reference gene should be stably expressed in all cells and physiological states, and can be completely used for different types of samples, but in the actual process, the expression of any reference gene is always stable along with the change of test conditions, i.e. the suitable reference gene under one test condition is not necessarily suitable for another test condition, or the reference genes between different species may be different. Using a gene as an internal reference without verification, small differences in gene expression may be difficult to find at a low level, and the exact opposite may occur at a high level. The use of an unstably expressed reference gene may have a great influence on the analysis of the expression level of a target gene, and therefore, it is important to select an stably expressed reference gene under specific experimental conditions.

Researchers often use housekeeping genes as internal controls because these genes are widely involved in the basic biochemical reactions of organisms, which allows for their stable expression in certain organ tissues. At present, the commonly used traditional internal reference is actin gene, glyceraldehyde-3-phosphate dehydrogenase gene, histone gene, tubulin gene, ubiquitin-conjugating enzyme gene, elongation factor and the like. So far, the field of reference gene screening of primula plants is still a blank.

Disclosure of Invention

In order to solve the problems, the invention provides an internal reference gene with higher stability for gene expression analysis of primula forbesii in different flowering phases.

The internal reference genes for the fluorescent quantitative PCR of primula forbesii in different flowering phases are GAPDH, HIS, 28S and RNA pol II, wherein the sequence of the GAPDH is shown as Seq ID No.1, the sequence of the HIS is shown as Seq ID No.2, the sequence of the 28S is shown as Seq ID No.3, and the sequence of the RNA pol II is shown as Seq ID No. 4.

The invention also provides a reagent for analyzing the gene expression of primula forbesii in different flowering phases, which comprises the following components:

1) a substance that detects the expression level of GAPDH;

2) (ii) a substance that detects the expression level of HIS;

3) detecting said 28S expression level; and

4) (II) a substance that detects the expression level of said RNA pol II.

In a preferred embodiment of the present invention, said means for detecting the expression level of GAPDH is a primer for amplifying said gene or a fragment thereof; the substance for detecting the expression level of the HIS is a primer for amplifying the gene or the segment thereof; the substance for detecting the 28S expression level is a primer for amplifying the gene or the segment thereof; the substance for detecting the expression level of the RNA pol II is a primer for amplifying the gene or the segment thereof.

In a more preferred embodiment of the present invention, said substance for detecting the expression level of GAPDH is a single-stranded DNA having the sequence shown in Seq ID No.17 and a single-stranded DNA having the sequence shown in Seq ID No. 18; the substance for detecting the HIS expression level is single-stranded DNA with a sequence shown as Seq ID No.19 and single-stranded DNA with a sequence shown as Seq ID No. 20; the substance for detecting the 28S expression level is single-stranded DNA with a sequence shown as Seq ID No.11 and single-stranded DNA with a sequence shown as Seq ID No. 12; the substance for detecting the expression level of the RNA pol II is single-stranded DNA with a sequence shown as Seq ID No.21 and single-stranded DNA with a sequence shown as Seq ID No. 22.

The invention also provides a kit containing the reagent and used for analyzing the gene expression of primula forbesii in different flowering phases.

In one embodiment of the invention, the kit may also contain other components necessary for analysis of gene expression, such as other reagents necessary for PCR.

In one embodiment of the present invention, the kit may further comprise a substance for detecting the expression level of the target gene expressed at different florescence, such as primers required for target PCR, and preferably further comprises other reagents required for PCR.

The target genes expressed in different florescence can be determined by those skilled in the art, including but not limited to controlling flowering time, controlling flower color, controlling volatile substances in the flower, etc.

The invention also provides application of the reagent in gene expression analysis of primula forbesii in different florescence.

The invention takes the floral organs and different tissues of primula forbesii in different florescence as test materials, selects 10 reference genes from the test materials based on the sequencing data of the transcriptome in the early stage, and analyzes and evaluates the obtained results by respectively using the software of geonorm, NormFinder and Bestkeeper. Under different flowering phase conditions, GAPDH, HIS, RNA pol II and 28S all showed similar trends when a single internal reference gene was used, with only slight differences in expression; when GAPDH, HIS, RNA pol II and 28S were used in combination, the results tended to be more stable, indicating that GAPDH, HIS, RNA pol II and 28S used in combination as an internal reference gave more reliable results than a single-gene internal reference.

Drawings

FIG. 1 shows the primer amplified fragments of candidate reference genes.

FIG. 2 is a graph showing the melting curves of 10 candidate reference genes.

FIG. 3 shows the Ct values of qRT-PCR in all samples. The lines in the box represent the median, the upper and lower ends of the box represent 25% and 75%, respectively, and the upper and lower margin lines represent the maximum and minimum values, respectively.

FIG. 4 shows the results of analysis by the geNorm software (upper: all samples; lower: different flowering stages).

FIG. 5 shows the number analysis of the geoNorm best reference gene.

FIG. 6 shows Venn diagram analysis of the best reference genes.

FIG. 7 shows the effect of different combinations of reference genes on the expression level of PfLIS/NES at different flowering stages.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

Primula forbesii used in the test was collected from 2016 months 2 to 4 months. Taking the flowers in different flowering periods. The different flowering stages include 4 stages: bud phase, initial flowering phase, full flowering phase and final flowering phase. The description criteria for each stage are as follows: in the bud stage: the flower is in an unopened state, and the calyx tightly wraps the petals; at the initial flowering stage: the flower is in a semi-open state, the calyx is expanded, and the petals are extended; full bloom stage: the flower is in a full bloom state, and the calyx and the petals are fully unfolded; and (3) at the end flowering stage: the flower is in the open state, and the calyx and the petals appear to wilting and turn backwards. The different organizations include: root, scape, leaf and flower (full bloom). After harvesting, the roots were carefully washed out of the soil with clear water and blotted dry with filter paper. All samples were frozen immediately after collection with liquid nitrogen and stored in an environment at-80 ℃.

10 gene sequences with high homology to Arabidopsis were selected from the transcriptome sequencing database as candidate reference genes (Table 1), including 6 conventional reference genes (28S, ACT, TUA, TUB, HIS, GAPDH) and 4 novel reference genes (RPL18a, STPP, CSD, RNA pol II). Primer design was performed using Primer Premier 5.0.

TABLE 1 information on candidate reference genes

Note: BLAST (https:// blast.ncbi.nlm.nih.gov/blast.cgi)

The specificity of the designed 10 pairs of qRT-PCR primers was verified by melting curve analysis and general PCR.

The general PCR reaction system is as follows:

the reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 95 ℃ for 30s, 48-58 ℃ for 30s, and 72 ℃ for 30 s; 5min at 72 ℃. After completion of the reaction, each PCR product was detected by 1% agarose gel electrophoresis. The results are shown in FIG. 1. As can be seen from FIG. 1, the ordinary PCR of each candidate reference gene has a PCR product with the same size as expected, the band is obvious and single, and no non-specific amplification exists, which indicates that the primer has good specificity and can be used for subsequent tests.

cDNA as template, using ddH₂O sequentially dilutes 5 gradients, each gradient dilutes 10 times, namely the concentration of each template is 1 and 10 respectively^-1、10^-2、10^-3、10^-4.3 replicates were set up for each reaction. qRT-PCR was performed using Analytik Jena qTOWER 2.2 fluorescence quantifier (AG, Germany) with the following reaction system and reaction conditions: 3min at 95 ℃; 5s at 95 ℃, 30s at 80 ℃ and 44 cycles; melting curve analysis at 60-95 deg.C. The experiment was set up for 3 technical replicates and relevant data was automatically read by the instrument. Candidate primers for reference genes and related information are shown in table 2.

qRT-PCR reaction System:

the results are shown in Table 2 and FIG. 2. The correlation coefficient r of the standard curve of each candidate reference gene can be known²>0.988, the amplification efficiency is between 90% and 110%, and the melting curves only generate a single melting peak without non-specific amplification interference. The test results show that the linear analysis and amplification efficiency of each candidate internal reference gene meet the requirements.

TABLE 2 candidate reference gene primers and related information

In 7 samples of primula forbesii, Ct values of 10 candidate reference genes are between 17 and 26. The larger the Ct value is, the lower the gene expression amount is; and vice versa. As can be seen in FIG. 3, the minimum Ct value for ACT (17.63) and the maximum Ct value for CSD (26.58) indicate that ACT is expressed in high abundance and CSD is expressed in low abundance. In addition, the expression changes of each candidate reference gene in different samples were distinct, with greater fluctuations in ACT, next to TUA, and smaller changes in expression of TUB and RNA pol II.

Example 2 analysis of reference Gene stability

In order to screen out the best reference gene, the stability of each candidate reference gene was assessed by analysis of geonorm (version 3.5), NormFinder (version 0.953) and Bestkeeper (version 1.0). Wherein, Bestkeeper uses original CT value to calculate, and the data of GeNorm and NormFinder need to be input according to the formula Q ═ E^-△CtCalculated, wherein E is gene amplification efficiency, and delta Ct ═ Ct_{Sample (I)}-Ct_min。

The results of qRT-PCR data were analyzed by three different software, geoNorm, NormFinder and BestKeeper, to determine the best reference genes for candidate reference genes in two different groups (all samples, different flowering phases).

GeNorm analysis of candidate reference Gene stability

The geonorm software is able to calculate the expression stability value M of each candidate reference gene, i.e.: and according to the average standard deviation calculated by logarithmic transformation after pairwise ratios of the expression levels of the single reference gene and other reference genes, the lower M value indicates that the expression of the candidate reference gene is more stable, and vice versa. When M is more than 1.5, the gene is very unstable and is not suitable for being used as an internal reference gene.

As can be seen from fig. 4, most of the M values are less than 1.5. HIS and 28S expression was most stable in all sample groups; HIS and GAPDH are the most stable genes in different florescence groups.

In addition, the software can predict the number of the best reference genes by calculating the pair variation (V) value of normalization factor (normalization factor) after introducing 1 new reference gene, wherein the default V value is 0.15, and if V is V, V is 0.15_n/V_n+1>0.15, then the n +1 th gene needs to be introduced, otherwise no new one needs to be introducedAn internal reference gene. FIG. 5 shows the optimal number of reference genes under different conditions of primula forbesii. In general, when V>At 0.15, the (n + 1) th gene should be introduced, but this value is too strict under certain conditions, since it depends not only on the number of genes but also on the sample itself. In this test, the V values for all three groups were greater than 0.15. In all sample groups, when the 5 th gene was introduced, the V value was the lowest, and then the V value did not significantly decrease with the increase in the number of genes, so the optimal number of reference genes for the group was 4; similarly, in the different florescence groups, when the 5 th gene was introduced, the V value was as low as 0.155, so 4 genes were the optimal number of reference genes in this group.

NormFinder analysis of candidate reference gene stability

NormFinder generates stable values of gene expression based on variance analysis, and then takes the gene with the minimum stable value as the most stable gene according to the magnitude sequence of the stable values.

As can be seen from table 3, GAPDH is stably expressed in all sample groups, whereas ACT is very unstable; GAPDH expression was stable and TUA ranked last in different florescence groups.

TABLE 3 analysis results of the NormFinder software

BestKeeper analysis of candidate reference Gene stability

The BestKeeper program is a program for analyzing the expression level of an internal reference gene and a target gene, which is written by Michael et al. According to the requirement of software, the original Ct value is directly input, and the program can generate a correlation coefficient (r) of pairing between each gene and a variation coefficient of each gene in different samples, wherein the higher the value of the correlation coefficient r is, the more stable the candidate reference gene is. As can be seen from table 4, GAPDH is first in both groups, indicating the highest stability, and furthermore HIS and 28S rank first and ACT and TUA rank last in the different florescence groups.

TABLE 4 BestKeeper software analysis results

Example 3 verification of reference Gene

By referencing the number of best reference genes predicted by geonorm, and combining with analysis of wien chart (fig. 6), experiments finally determined different combinations of best reference gene validation, wherein the best reference genes for different flowering phases include: HIS, RNA pol II, GAPDH, 28S, GAPDH + HIS, GAPDH + RNA pol II, 28S + RNA pol II, GAPDH +28S + HIS, and GAPDH + HIS +28S + RNA pol II, and the most labile reference: TUA.

The PfLIS/NES gene was used to verify the accuracy of the best reference gene results in primula forbesii at different flowering stages and under different tissue conditions. Primers for fluorescent quantitative RT-PCR of PfLIS/NES gene are shown in SEQ ID No.31 and 32.

In primula forbesii flower opening process, when PfLIS/NES is subjected to expression quantity analysis by using stably expressed candidate reference genes, results of different combinations show highly consistent trends and reach a peak value in the full-bloom stage. However, when the unstable gene TUA was used, the results were very inaccurate, and the expression level of PfLIS/NES was much lower than the normal expression level (FIG. 7), which is consistent with the results predicted by the software. Furthermore, GAPDH, RNA pol II and 28S all showed similar trends when a single reference gene was used, with only slight differences in expression; when multiple reference genes were used, the results tended to be more stable, indicating that more reliable results were obtained using multiple genes as references than a single gene reference.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Sichuan Process Environment science and technology Limited

Sichuan Agricultural University

<120> reference gene for gene expression analysis of primula forbesii in different flowering phases and primer thereof

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attgacgaaa agatactatg tatgcacggt gggctttctc ccgatctcaa taatttggat 540

caaataagga atttacagcg tccaacggat gttcccgagt ctggtttatt gtgtgattta 600

ctgtggtcag atccaagcaa agacgttaaa ggttggggca tgaatgatcg gggggtttca 660

tatacgtttg gctctgataa agtgaccgag tttctccaaa agctcgacct tgacctagtt 720

tgccgagccc accaggttgt ggaagatggg tatgagtttt ttgctgatag acaacttgta 780

accatatttt ctgctcctaa ctactgtgga gaatttgata atgcgggtgc gatgatgagt 840

gtt 843

<210> 10

<211> 570

<212> DNA

<213> Primula forbesii

<400> 10

gtttaccagc caatagaatc tctgtatctg ctgcttttaa caaacaaaca gagcaacata 60

cttgaagatc tggatactct gaggctgctt tccaaattag ttcgtgaatt tgctttttct 120

cttgatgaag aaggcattgg caaaacagcc tttgagctta tttttgcatt cgatgaagtc 180

atttctcttg ggcacaagga aaatgtcact gctgcacagg ttaaacaata ctgcgagatg 240

gagagtcagg aggagaaggt gcataagtta gtaatgcaga gtaagatcaa tgaaactaag 300

gatcacatgc agcgcaaagc tattgaaatt gacaaaacta agatagaaaa gagaggaagt 360

gataagggag gattcatgag ctctggtaga gttgattctg ggtttggtag cgagatgaga 420

acctctagca gtggaaccgg ctttggaagt ggttctggat ttggattaac ccctgatatc 480

gattcattct ctagcaagca caaaggtcct ccacctgcat ctgctaacgc acctcctagg 540

ggtctcggta tgaagcttgg taaaacgcag 570

<210> 11

<211> 20

<212> DNA

<213> Primula forbesii

<400> 11

ttccagatgt tccagtagat 20

<210> 12

<211> 18

<212> DNA

<213> Primula forbesii)

<400> 12

aaggtagtag tggcagac 18

<210> 13

<211> 18

<212> DNA

<213> Primula forbesii)

<400> 13

ttcaacactc ctgctatg 18

<210> 14

<211> 19

<212> DNA

<213> Primula forbesii

<400> 14

ccagaacaat accagtagt 19

<210> 15

<211> 18

<212> DNA

<213> Primula forbesii)

<400> 15

tctctgtatc tgctgctt 18

<210> 16

<211> 18

<212> DNA

<213> Primula forbesii

<400> 16

ccaatgcctt cttcatca 18

<210> 17

<211> 20

<212> DNA

<213> Primula forbesii

<400> 17

aacgagaagg aatacacatc 20

<210> 18

<211> 19

<212> DNA

<213> Primula forbesii)

<400> 18

ctaatggagc aagacagtt 19

<210> 19

<211> 18

<212> DNA

<213> Primula forbesii

<400> 19

taccagaaga gcactgag 18

<210> 20

<211> 18

<212> DNA

<213> Primula forbesii

<400> 20

cggtcttgaa atcctgag 18

<210> 21

<211> 18

<212> DNA

<213> Primula forbesii)

<400> 21

aagaaccttg ctctgatg 18

<210> 22

<211> 20

<212> DNA

<213> Primula forbesii)

<400> 22

ttccatactc cattcctcta 20

<210> 23

<211> 24

<212> DNA

<213> Primula forbesii

<400> 23

gaagaactac ggtatgtgga taag 24

<210> 24

<211> 24

<212> DNA

<213> Primula forbesii

<400> 24

ttgtaatgtc acgatattcc ttgt 24

<210> 25

<211> 20

<212> DNA

<213> Primula forbesii)

<400> 25

ctaatttgtt ggagattgag 20

<210> 26

<211> 20

<212> DNA

<213> Primula forbesii)

<400> 26

aataagtagt tggattgagg 20

<210> 27

<211> 18

<212> DNA

<213> Primula forbesii

<400> 27

gcgtgttgtc ttatgtat 18

<210> 28

<211> 19

<212> DNA

<213> Primula forbesii)

<400> 28

aatagtcctc ttcgtctta 19

<210> 29

<211> 18

<212> DNA

<213> Primula forbesii)

<400> 29

accctttccg ttcatcag 18

<210> 30

<211> 23

<212> DNA

<213> Primula forbesii

<400> 30

aatatcgtat agtgcctcgt tat 23

<210> 31

<211> 20

<212> DNA

<213> Primula forbesii)

<400> 31

gggtggcgga attgacaagg 20

<210> 32

<211> 20

<212> DNA

<213> Primula forbesii)

<400> 32

acggagaatc gtggctgtgt 20

Claims

1. Internal reference genes for fluorescent quantitative PCR of primula forbesii in different flowering phases are GAPDH, HIS, 28S and RNA pol II, wherein the sequence of the GAPDH is shown as Seq ID No.1, the sequence of the HIS is shown as Seq ID No.2, the sequence of the 28S is shown as Seq ID No.3, and the sequence of the RNA pol II is shown as Seq ID No. 4.

2. Reagent for the analysis of the gene expression of primula forbesii in different flowering phases, comprising:

1) a substance that detects the expression level of GAPDH according to claim 1;

2) a substance for detecting the expression level of HIS according to claim 1;

3) detecting a substance of 28S expression level according to claim 1; and

4) an agent that detects the expression level of RNA pol II according to claim 1.

3. The reagent according to claim 2, wherein the agent for detecting the expression level of GAPDH according to claim 1 is a primer for amplifying the gene or a fragment thereof; the substance for detecting the expression level of HIS according to claim 1, which is a primer for amplifying the gene or a fragment thereof; the substance for detecting the 28S expression level according to claim 1 is a primer for amplifying the gene or a fragment thereof; the agent for detecting the expression level of RNA pol II according to claim 1 is a primer for amplifying the gene or a fragment thereof.

4. The reagent according to claim 3, wherein the substance for detecting the expression level of GAPDH according to claim 1 is a single-stranded DNA having a sequence shown by Seq ID No.17 and a single-stranded DNA having a sequence shown by Seq ID No. 18; the substance for detecting the expression level of HIS according to claim 1 is single-stranded DNA with the sequence shown as Seq ID No.19 and single-stranded DNA with the sequence shown as Seq ID No. 20; the substance for detecting the 28S expression level of claim 1 is single-stranded DNA with a sequence shown as Seq ID No.11 and single-stranded DNA with a sequence shown as Seq ID No. 12; the substance for detecting the expression level of RNA pol II according to claim 1 is single-stranded DNA with a sequence shown as Seq ID No.21 and single-stranded DNA with a sequence shown as Seq ID No. 22.

5. A kit for analyzing gene expression of primula forbesii at different flowering stages, comprising the reagent according to any one of claims 2 to 4.

6. Use of an agent according to any one of claims 2 to 4 for the analysis of gene expression in primula forbesii at different flowering stages.