CN116987809B - Bojioocyst algae reference gene and its amplification primer and application - Google Patents
Bojioocyst algae reference gene and its amplification primer and application Download PDFInfo
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Abstract
The invention discloses a reference gene of Bojioocyst algae, which is one or a combination of a plurality of UBCE genes, RPS27 genes and RPL7 genes, wherein the nucleotide sequence of the UBCE genes is shown as SEQ ID NO.1, the nucleotide sequence of the RPS27 genes is shown as SEQ ID NO.2, and the nucleotide sequence of the RPL7 genes is shown as SEQ ID NO. 3. Also discloses a real-time fluorescent quantitative PCR primer for amplifying the reference gene of the Bojioocyst algae, application of the reference gene as an RT-qPCR reference gene and application of the primer in the quantitative detection of the reference gene of the Bojioocyst algae. The screened reference genes are suitable for the Boji oocyst algae under different culture conditions, can improve the stability and reliability of quantitative expression analysis of the Boji oocyst algae related genes, and fills the blank of lacking the reference genes in the Boji oocyst algae related research field.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a oocyst algae reference gene, an amplification primer and application thereof.
Background
The oocyst algae (oocysts borgei) are classified as belonging to the phylum Chlorophyta, the class of Coccophyceae (Trebouxiophyceae), the order of Chlorophyceae (Chlorococcus), the family of Oocystaceae (Oocystaceae), the genus of oocysts (oocysts), not only have stronger environment adaptation capability, but also can maintain the balance of algae in the culture water body, and have an important role in regulating the balance of the shrimp pond ecosystem. The application of the Bojioocyst algae in the shrimp culture can inhibit the inundation of vibrio and blue algae in the culture water body, can effectively absorb substances such as ammonia nitrogen, nitrite nitrogen and the like in the culture water body, and prevent the eutrophication of the water body, thereby being beneficial to the development and growth of the shrimps, enhancing the stress resistance of the shrimps, improving the quality and the yield. The bojioocyst algae as a new generation microalgae preparation is widely applied to the water quality regulation of a shrimp culture pond, has the characteristics of green and environment-friendly, and still lacks related information output on the molecular level.
Real-time fluorescent quantitative PCR (real-time quantitative PCR, RT-qPCR) is a method for detecting the total amount of products after the PCR (polymerase chain reaction, PCR) cycle in a DNA amplification reaction with fluorescent chemicals. The RT-qPCR technology has become an important tool for understanding biological processes and molecular mechanisms of organisms because of higher quantitative accuracy, specificity, sensitivity and good repeatability and the advantage of supporting batch detection. In RT-qPCR analysis, it is necessary to normalize the expression of target gene mRNA using reference genes whose expression should be unaffected by experimental factors with minimal variation in expression between the physiological states of tissues and organisms in order to obtain accurate quantitative results. Common reference genes such as ACT (actin), 18S rRNA (18S ribosomal RNA), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) are widely used in the analysis of mRNA expression of plant target genes. However, some studies have shown that the expression levels of these genes vary depending on the species, the life stage of the organism and the experimental conditions, i.e. no particular single reference gene can be universally used for the analysis of different samples. Therefore, screening of the RT-qPCR reference gene of the target species is an indispensable step in the analysis and study of the relative expression amount of mRNA of the target gene.
At present, no related data and no report exist on research on fluorescence quantitative PCR reference gene screening of the oocyst algae. Therefore, the application aims at promoting the development of the industrial chain of the oocyst algae microecological preparation through the screening of the oocyst algae real-time fluorescence quantitative PCR reference genes under different conditions.
Disclosure of Invention
The invention aims to provide a oocyst algae reference gene.
The invention also aims at providing a primer for amplifying the oocyst algae reference gene.
The final object of the invention is to provide the application of the above-mentioned Bojioocyst algae reference gene as RT-qPCR reference gene and the application of the above-mentioned primer in quantitative detection of the Bojioocyst algae reference gene.
The first object of the present invention can be achieved by the following technical means: the reference gene is one or a combination of a plurality of UBCE genes, RPS27 genes and RPL7 genes, wherein the nucleotide sequence of the UBCE genes is shown as SEQ ID NO.1, the nucleotide sequence of the RPS27 genes is shown as SEQ ID NO.2, and the nucleotide sequence of the RPL7 genes is shown as SEQ ID NO. 3.
More preferably, the reference gene is a combination of UBCE gene and RPS27 gene.
The invention screens out 9 candidate reference genes EF1 alpha, RPL7, RPS27, UBCE,18S,UBQ,TUB,GAPDH and RBCL based on the existing transcriptome data of the Bojioocyst algae, quantitatively analyzes the mRNA expression levels of the 9 candidate reference genes under different light intensity and salinity conditions and at different culture times by using a real-time fluorescence quantitative PCR (RT-qPCR) technology, evaluates the expression stability of the 9 candidate reference genes by software analysis, screens out the reference genes which can be stably expressed in the Bojioocyst algae under different light intensity and different salinity conditions, provides a molecular tool for researching the related functional genes of the Bojioocyst algae, and provides a tool support for solving some key problems in the Bojioocyst algae industrialization process.
The invention comprehensively analyzes the results of delta Ct, bestKeeper, normFinder and gemum by means of a ReFinder program, the program can assign an appropriate weight to each candidate reference gene according to the ranking of each software, and calculate the geometric mean of the weights, so that the candidate genes are comprehensively ordered, and the result shows that: the first three genes of stability under different light intensity conditions are UBCE, RPS27 and RPL7, and the last three genes are TUB, GAPDH and 18S; the genes of the first three of stability under different salinity conditions are RPS27, UBCE and RPL7, respectively, and the latter three are EF1 alpha, GAPDH and TUB, respectively. The top three genes with the stability under different light intensities and salinity are the same, which indicates that the expression of the three genes in the Bojioocyst algae is relatively stable, and the gene can be used as a candidate reference gene of the Bojioocyst algae under other processing conditions.
Further, the invention analyzes the paired variation coefficients V of the light intensity group and the salinity group by pairwise variation n/(n+1) The values are all smaller than 0.15, based on the least principle, the optimal internal reference factors are 2, and the optimal internal reference genes of the light intensity group and the salinity group are the combination of UBCE and RPS 27.
The second object of the present invention can be achieved by the following technical means: the primer for amplifying the oocyst algae reference gene comprises one or more of a primer pair for amplifying a UBCE gene, a primer pair for amplifying an RPS27 gene and a primer pair for amplifying an RPL7 gene, wherein the primer pair for amplifying the UBCE gene comprises UBCE-F, UBCE-R, the primer pair for amplifying the RPS27 gene comprises RPS27-F, RPS-R, and the primer pair for amplifying the RPL7 gene comprises RPL7-F, RPL-R.
Further, the nucleotide sequence of UBCE-F is shown as SEQ ID NO.11, the nucleotide sequence of UBCE-R is shown as SEQ ID NO.12, the nucleotide sequence of RPS27-F is shown as SEQ ID NO.13, the nucleotide sequence of RPS27-R is shown as SEQ ID NO.14, the nucleotide sequence of RPL7-F is shown as SEQ ID NO.15, and the nucleotide sequence of RPL7-R is shown as SEQ ID NO. 16.
The last object of the invention can be achieved by the following technical scheme: the application of the oocyst algae reference gene as an RT-qPCR reference gene.
The invention screens out internal reference genes which can be stably expressed in the Bojioocyst algae under different light intensity and salinity conditions, provides a molecular tool for researching the related functional genes of the Bojioocyst algae, and provides tool support for solving some key problems in the industrialization process of the Bojioocyst algae.
The invention further provides application of the primer in quantitative detection of the oocyst algae reference gene.
Compared with the prior art, the invention has the following advantages:
(1) Aiming at the lack of reliable reference genes of the Bojioocyst algae in scientific research and industry, the invention screens out the relatively stable reference genes UBCE, RPS27 and RPL7 expressed by the Bojioocyst algae under different light intensity and different salinity culture time, and the detection primer has specificity, the data is true and reliable, provides a correction tool for researching molecular biology of the Bojioocyst algae in response to light intensity, salinity and other mechanisms, and simultaneously solves the current situation that the Bojioocyst algae does not have the reference genes;
(2) The invention firstly proposes the research of using the combination of UBCE and RPS27 as the reference genes of the oocyst algae for the expression of target gene mRNA, improves the accuracy and the reliability of data, and solves the problem that the research result is unstable because only a single reference gene is usually selected as a correction standard in the prior RT-qPCR;
(3) The reference gene and the combination thereof provided by the invention can be also suitable for researching the target gene of the Bojioocyst algae under other culture conditions such as different nitrogen concentrations, different carbon concentrations, different temperatures and the like, and simultaneously provide reference value for researching other microalgae of the oocyst algae;
(4) The screened reference genes are suitable for the Boji oocyst algae under different culture conditions, can improve the stability and reliability of quantitative expression analysis of the Boji oocyst algae related genes, and fills the blank of lacking the reference genes in the Boji oocyst algae related research field.
Drawings
The invention will be further described with reference to the accompanying drawings, in conjunction with examples.
FIG. 1 is an electrophoretic detection of PCR products of candidate reference genes in example 1;
FIG. 2 is a standard curve of nine candidate reference genes in example 1;
FIG. 3 is a melting curve of nine candidate reference genes in example 1;
FIG. 4 is a box plot of Ct value distribution of candidate reference genes in example 1, wherein A: a light intensity group; b: salinity group;
FIG. 5 shows the mean stable expression values of the geNorm analysis candidate reference genes in example 1, wherein A: a light intensity group; b: salinity group;
FIG. 6 is a graph showing the stable values of expression of candidate reference genes in the NormFinder assay of example 1, wherein A: a light intensity group; b: salinity group;
FIG. 7 is a graph showing the analysis of the number of geNorm optimal candidate reference genes in example 1, wherein A: a light intensity group; b: salinity group;
FIG. 8 shows the relative expression levels of GS obtained under different light intensity and salinity conditions using UBCE and RPS27 alone or in combination (UBCE+RPS27) as internal reference in example 1, wherein A: a light intensity group; b: salinity group.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The experimental methods used in the following implementation methods are conventional experimental methods unless otherwise specified.
The terms used in the following methods and examples, unless otherwise indicated, generally have meanings that are commonly understood by those of ordinary skill in the art.
Example 1
1. Instruments, reagents and materials
1.1 laboratory apparatus
JJ 124BC electronic balance, oven, high temperature sterilization pot, FPG 3 type three temperature zone illumination incubator, pipetting gun and gun head, 500mL and 1000mL conical flask, high speed refrigerated centrifuge, gene PCR amplifier, -80 ℃ low temperature refrigerator, vortex oscillator, ultra-micro biological detector, mortar, open algae cultivation rack equipped with illumination system, roger real-time fluorescent quantitative PCR instrument (LightCycler 96).
1.2 Experimental reagents
TIANGEN polysaccharide polyphenol plant total RNA extraction kit (DP 441), taKaRa reverse transcription kit (RR 037A), ROCHE fluorescence quantitative kit (FastStart Universal SYBR Green Master), liquid nitrogen, beta-mercaptoethanol, 100% ethanol, agarose and NaNO 3 、NaH 2 PO 4 、ZnSO 4 ·4H 2 O、MnCl 2 ·4H 2 O、CuSO 4 ·5H 2 O、ironic citrate、Na 2 MoO 4 ·H 2 O、CoCl 2 ·6H 2 O、Na 2 EDTA、V B12 、V B1 、Biotin。
1.3 Experimental materials
Oocyst algae (oolysis borgei) are provided by the university of ocean, guangdong, algae resource development and cultivation environment ecological restoration laboratory.
2. Experimental method
2.1 Pre-cultivation of algal species
Oocyst algae were inoculated into modified f/2 medium (containing 74.8mg/L NaNO) 3 ，4.4mg/L NaH 2 PO 4 ，23μg/LZnSO 4 ·4H 2 O，178μg/L MnCl 2 ·4H 2 O，10μg/L CuSO 4 ·5H 2 O，3.9mg/L C 6 H 5 FeO 7 ，7.3μg/LNa 2 MoO 4 ·H 2 O，12μg/L CoCl 2 ·6H 2 O，4.35mg/L Na 2 EDTA，0.5μg/L V B12 ，100μg/L V B1 and 0.5. Mu.g/L Biotin), at 25℃with 1500lx light intensity, 12h light/12h dark photoperiod. After 7d, algae seeds are collected, artificial seawater is replaced and starved for 48 hours, and control culture and different treatments are carried out.
2.2 cultivation and concentration of algal species
800mL of culture medium and 30mL of precultured algae are added into a 1000mL conical flask, the light intensity gradients are set to be 500lx, 1500lx, 3500lx and 5000lx, the salinity gradients are set to be 10, 20, 30, 40 and 50, 3 biological parallels are set for each gradient, and the rest culture conditions are consistent with preculture. Samples were collected by centrifugation at 5000r/min for 10min at 0h,3h,6h,12h,24h, respectively, washed 2 times with 1 XPBS, and then snap frozen in liquid nitrogen for subsequent RNA extraction at-80 ℃.
2.3 extraction of Total RNA and Synthesis of cDNA
Extracting total RNA of the oocyst algae sample according to the instruction of the total RNA extraction kit of the polysaccharide-polyphenol plant of the root of the Chinese day. By detecting the ratio (A) of the absorption peak at 260nm to the absorption peak at 260:280 nm 260/280 ) To evaluate the concentration and purity of each RNA sample, and the integrity was checked by 1.5% agarose gel electrophoresis. cDNA synthesis was performed according to TaKaRa reverse transcription kit instructions and stored in a-20deg.C refrigerator for use.
2.4 primer design and detection of candidate reference genes
Screening 9 candidate internal reference genes according to the oocyst algae transcriptome data (table 1): ubiquitin-binding enzyme E gene (ubiquitin-conjugating enzyme E, UBCE, shown as SEQ ID NO. 1), ribosomal small subunit protein gene (ribosomal protein S, RPS27, shown as SEQ ID NO. 2), ribosomal large subunit protein gene (ribosomal protein L, RPL7, shown as SEQ ID NO. 3), eukaryotic elongation factor gene (elongation factor- α, EF1 α, shown as SEQ ID NO. 4), ribulose-1, 5-bisphosphate carboxylase gene (ribulose 1,5-bisphosphate carboxylase, RBCL, shown as SEQ ID NO. 5), glyceraldehyde-3-phosphate dehydrogenase gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, shown as SEQ ID NO. 6), ubiquitin gene (ubiquitin, UBQ, shown as SEQ ID NO. 7), 18S ribosomal RNA (18S ribosomal RNA,18S, shown as SEQ ID NO. 8), β -tubulin gene (β -tublin, B, shown as SEQ ID NO. 9). The quantitative primers were designed using the software Primer premier 5.0 and submitted to the company su Jin Weizhi to complete the synthesis.
cDNA mixture (diluted 10 times) under different treatments and different culture times is used as template, ddH 2 O is used as a control, and the quantitative primer is detected.
PCR reaction system: ddH 2 O11.67. Mu.L, 10 XBuffer 1.5. Mu.L, 2.5mmol/L dNTPs 0.6. Mu.L, 10. Mu. Mol/L upstream and downstream primers each 0.2. Mu.L, rTaq 0.08. Mu.L, and template 0.75. Mu.L. Reaction conditions: 95 ℃ for 5min;95 ℃ for 30s,60 ℃ for 20s,72 ℃ for 30s,33 cycles; 72 ℃ for 10min; preserving at 4deg.C.
After the specificity of the PCR product was confirmed by 1.5% agarose gel electrophoresis, the clone product was submitted to the company Jin Weizhi, suzhou for sequencing. The sequencing results were analyzed using DNASTAR-laser v6 software and aligned with the gene sequences obtained from the oocyst transcriptome.
TABLE 1 primer sequences, PCR efficiencies and correlation coefficients for candidate reference genes
2.5 RT-PCR amplification of candidate genes
Respectively diluting cDNA samples at different times under different treatments by 10 times, respectively taking equal volumes, mixing, continuously diluting for 4 times by 5 times to obtain 5 cDNAs with different gradients, using the cDNAs as templates, amplifying candidate genes in a LightCycler96 real-time fluorescence quantitative instrument by using a Roche quantitative kit FastStart Essential DNA Green Master, and drawing a standard curve.
The reaction system: 2 XSYBR Premix 7.5. Mu.L, water (PCR Grade) 1.7.7. Mu.L, 0.4. Mu.L each of the upstream and downstream primers (10. Mu. Mol/L) and 5. Mu.L of cDNA.
The reaction procedure: after 30s at 95 ℃, 40 cycles of 10s at 95 ℃,60 s at 60 ℃ and collecting fluorescent signals; melting curves 95℃10s,65℃1min,95℃1s.
According to the standard curve result, after all cDNA samples are diluted by 20 times, RT-qPCR amplification is carried out on candidate internal reference genes, independent RNA samples from three biological repetition at different treatment times are used for experiments, and average Ct values are calculated; the reaction system and the conditions are the same as the above.
2.6 analysis of stability and quantity optimization of reference genes
To select the best reference gene or combination of reference genes, four are usedDifferent methods separate statistical analyses were performed for the stability of expression of each candidate gene: delta Ct, gem, normFinder and BestKeeper, and finally, were analyzed in an integrated manner using an on-line tool ReFinder (http:// bloom. Cn/RefFinder /). In addition, paired differential analysis of candidate reference genes by using gemm as normalization factor (V n/(n+1) ) The optimal number of reference genes was obtained.
2.7 verification of stability of reference Gene
Quantitative primers (Table 1) of GS (glutamine synthase, glutamyl ammonia synthetase, shown as SEQ ID NO. 10) were designed based on the transcriptional genome data of Bojioocyst algae, and GS expression was verified under the same RT-qPCR conditions as described above using the most stable and least stable reference genes screened, respectively, for Bojioocyst algae cultured at different light intensities and salinity.
2.8 data analysis
From Microsoft Excel 2010 according to formula e= (10 [-1/slope] -1) x 100% slope, and the correlation coefficient (R) was calculated from Ct value 2 ) And amplification efficiency (E). The correlation coefficient should be greater than 0.99 and the amplification efficiency should be between 90% and 110%. The results were plotted using GraphPad Prism 9.
3. Results and analysis
3.1 quantitative primer mass detection
After PCR amplification of candidate reference genes, all primers can amplify a single PCR product (reference numeral 1 in figure 1) in a cDNA template, the fragment length is about 100-200bp, the strip size is consistent with theory, the specificity of the primers is perfect and no dimer amplification is carried out, and the negative control (reference numeral 2 in figure 1) has no PCR product, thus indicating that the reaction is pollution-free.
The cloning result sequencing result shows that the size of the PCR product is consistent with the electrophoresis result, and the PCR product is consistent with the target gene sequence of the Bogeric oocyst by BLAST comparison.
The analysis results of the primer standard curve (FIG. 2) show that the correlation coefficient (R 2 ) Meets the requirement of stabilizing reference genes, and the dissolution curve of all primers is obvious and the peak value is single (figure 3), which shows that the primers used in RT-qPCR can be specifically combined with cDNA template and amplify target genes.
The value of the amplification efficiency (E) was calculated to be 95% < E <106% (Table 1), and the data were in agreement with theory. In summary, the primers can be used for subsequent detection and analysis.
3.2Ct value analysis results
In the fluorescent quantitative PCR reaction, the smaller the Ct value of the gene is, the higher the expression level of the gene in a sample is, and if the smaller the variation interval of the Ct value is, the better the stability of the gene is, so that the stability of the gene expression can be primarily analyzed by comparing the Ct value.
The distribution box graphs of Ct values of 9 candidate reference genes under different conditions are shown in FIG. 4.
Under different light intensities (A diagram in FIG. 4), the Ct values of 9 candidate reference genes range from 14 to 30, wherein the UBCE has the smallest range, and the UBCE has a difference of 2.05 cycles, so that the 9 candidate reference genes are the most stable genes.
Under different salinity conditions (panel B in FIG. 4), ct values of 9 candidate reference genes varied in a range of 13-30, with the smallest variation being UBQ, differing by 1.53 cycles, but the expression levels were the lowest among all genes, requiring further analysis of their applicability as reference genes.
Under both conditions, 18S was the largest in the range of variation and the expression level was high, which was not suitable as a reference gene.
3.3 analysis of stability of candidate internal reference genes
3.3.1BestKeeper analysis results
The correlation coefficient (r), standard Deviation (SD) and variation Coefficient (CV) of the light intensity group and salinity group candidate internal reference genes are respectively judged by a BestKeeper program, and the judgment principle is that the larger the correlation coefficient is, the smaller the standard deviation and variation coefficient is, the better the gene stability is, and the worse the stability is, meanwhile, when SD >1, the internal reference gene expression is unstable.
As can be seen from table 2:
in the light intensity group, SD (1.46) >1 of 18S is expressed unsteady genes, and the three reference genes in comprehensive ranking under different light intensities according to the judgment principle are UBCE, RPL7 and UBQ respectively.
In the salinity group TUB is an unstable gene, its SD is 1.17, and the three reference genes in the comprehensive rank are UBQ, 18S and UBCE, respectively.
TABLE 2 BestKeeper analysis of candidate internal reference genes
3.3.2 results of GeNorm analysis
The data obtained by the calculation of the gemum program is a gene expression stable value (M) corresponding to the gene, 1.5 is taken as a zero boundary point, the M value is smaller than 1.5, the gene expression is relatively stable, and the stability of the gene with smaller M value is better.
As shown in FIG. 5, the M values of 9 candidate genes under different light intensity and salinity conditions are less than 1.5, which indicates that the expression of candidate genes under both conditions is relatively stable, the M values of UBCE and RPL7 genes in the light intensity group are equal and minimum, and are 0.253, the more stable of RPS27, the M value of which is 0.276 (FIG. 5A), the M values of RPS27 and RPL7 in the salinity group are equal and the minimum value is 0.299, and the more stable of UBCE, the M value of which is 0.376 (FIG. 5B).
3.3.3NormFinder analysis results
The calculation principle of the NormFinder program is similar to that of the GeNorm program, and the optimal reference gene is screened according to the expression stability value of the reference gene, and the reference gene with the minimum expression stability value is the optimal reference gene.
The normFinder analysis results indicated (FIG. 6):
the stable expression value of the RPS27 gene in the oocyst algae is the smallest (0.027) under different light intensities (A diagram in FIG. 6), the gene stability is the highest, and the expression stability of the UBQ and UBCE,18s genes is the worst (1.771); the stability of the candidate internal reference genes is RPS27> UBQ > UBCE > RPL7> RBCL > EF1 alpha > TUB > GAPDH >18S in sequence from high to low.
At different salinity (panel B in fig. 6), the stable expression value of RPS27 gene in oocyst algae is minimal (0.268), the gene stability is highest, next to UBCE and RPL7, the stability of the TUB gene is worst (1.241); the stability of the candidate internal reference genes is RPS27> UBCE > RPL7> UBQ > RBCL >18S > GAPDH > EF1α > TUB from high to low in sequence.
3.3.4 comprehensive analysis results of RefFinder reference Gene stability
The results of the analysis of Δct, bestKeeper, normFinder and gemm were analyzed together by the ReFinder program, which assigns an appropriate weight to each candidate reference gene according to the ranking of each software, and calculates the geometric mean of the weights, thereby comprehensively ranking the candidate genes, the results of which are shown in table 3.
The genes of the first three of stability under different light intensities are UBCE, RPS27 and RPL7, respectively, and the latter three are TUB, GAPDH and 18S, respectively.
The front three of stability at different salinity are RPS27, UBCE and RPL7, respectively, and the rear three are EF1 a, GAPDH and TUB, respectively.
The top three genes ranked under different light intensities and salinity are the same, which indicates that the expression of the three genes in the Bojioocyst algae is relatively stable, and the gene can be used as a candidate reference gene of the Bojioocyst algae under other processing conditions.
TABLE 3 ReFinder-based ranking of stability of oocyst algae reference genes
3.4 optimization of the number of candidate reference genes
In the conventional RT-qPCR test, only a single reference gene is usually selected as a calibration standard, but it is usually recommended to use two or more reference genes, since even the most stable genes may vary depending on conditions and organisms. In order to obtain the optimal gene number of the oocyst algae under different light intensity and salinity conditions, the GeNorm software is used for obtaining the gene number by the coefficient of variation V n/(n+1) Evaluating the number of the optimal reference genes accurately normalized when V n/(n+1) When the ratio is less than 0.15, the reference factor is "n", when V n/(n+1) If the number is greater than 0.15, the screening is continued for "n+1", and the result is shown in FIG. 7.
The paired variation coefficient values of the light intensity group and the salinity group are smaller than 0.15, and based on the least principle, 2 optimal reference genes are adopted, namely, the optimal reference genes of the light intensity group and the salinity group are the combination of UBCE and RPS 27.
3.5 verification of stability of reference Gene
To further verify the stability of the reference genes of the above-screened Bojioocyst algae, 2 candidate genes (UBCE, RPS 27) with the best stability and the least stable candidate gene (18S or TUB) were selected as reference genes based on the results of Table 3, and the expression patterns of the GS genes in Bojioocyst algae under different light intensity and salinity culture were compared and analyzed, and the results are shown in FIG. 8.
The expression quantity, the expression trend and the significance result of GS obtained by taking UBCE and RPS27 alone or in combination (UBCE+RPS27) as internal references are similar under different light intensity (A diagram in figure 8) or different salinity conditions (B diagram in figure 8), but the double internal references effect is still superior to that of a single internal reference; in the case of 18S (FIG. 8, panel A) or TUB (FIG. 8, panel B) as the reference gene, the expression level, expression trend and the significance result of GS were quite different. In summary, it is important to select stable reference genes when the quantitative expression analysis of mRNA of target genes is performed.
In addition, the reference gene and the combination thereof provided by the invention can be also suitable for researching the target gene of the Bojioocyst algae under other culture conditions such as different nitrogen concentrations, different carbon concentrations, different temperatures and the like.
Wherein:
optimal internal reference at different carbon concentrations: UBCE, RPS27, UBQ;
optimal internal reference at different nitrogen concentrations: RPL7, RPS27, UBCE;
optimal internal parameters at different temperatures: EF1 alpha, RPS27, UBCE;
it is further described that the reference gene is one or a combination of several of UBCE gene, RPS27 gene and RPL7 gene, and can be used as the reference gene of oocyst algae.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (2)
1. The application of the oocyst algae reference gene serving as the RT-qPCR reference gene in analyzing the quantitative expression of the oocyst algae related gene; the reference genes are UBCE genes and RPS27 genes; the nucleotide sequence of the UBCE gene is shown as SEQ ID NO.1, and the nucleotide sequence of the RPS27 gene is shown as SEQ ID NO. 2.
2. The application of the primer for amplifying the reference gene of the bojioocyst algae in the quantitative detection of the reference gene of the bojioocyst algae; the oocyst algae reference genes are UBCE genes and RPS27 genes; the nucleotide sequence of the UBCE gene is shown as SEQ ID NO.1, and the nucleotide sequence of the RPS27 gene is shown as SEQ ID NO. 2; the primer comprises a primer pair for amplifying UBCE genes and a primer pair for amplifying RPS27 genes; the primer pair for amplifying the UBCE gene is UBCE-F and UBCE-R, and the primer pair for amplifying the RPS27 gene is RPS27-F and RPS27-R; the nucleotide sequence of UBCE-F is shown as SEQ ID NO.11, the nucleotide sequence of UBCE-R is shown as SEQ ID NO.12, the nucleotide sequence of RPS27-F is shown as SEQ ID NO.13, and the nucleotide sequence of RPS27-R is shown as SEQ ID NO. 14.
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