CN117025810B - Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof - Google Patents

Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof Download PDF

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CN117025810B
CN117025810B CN202310809843.5A CN202310809843A CN117025810B CN 117025810 B CN117025810 B CN 117025810B CN 202310809843 A CN202310809843 A CN 202310809843A CN 117025810 B CN117025810 B CN 117025810B
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曾红
林能锋
吕艳红
柯翎
林晨韬
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Fujian Normal University
Institute of Biotechnology of Fujian Academy of Agricultural Science
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Abstract

The invention relates to an internal reference gene combination for expression analysis of a pseudokanbrix drop gene and application thereof. The reference gene combination comprises a C3228 gene and a C3765 gene; the C3228 gene sequence is a nucleotide sequence shown as SEQ ID NO.6, and the C3765 gene sequence is a nucleotide sequence shown as SEQ ID NO. 8. The technical scheme provides a stable internal reference gene combination for analysis of the difference genes of the nutrition period and the encapsulation period of the pseudokon water drop, finds 2 suitable internal reference genes for normalization of transcriptome data of the pseudokon water drop, and provides a reference for genetic research of the pseudokon water drop.

Description

Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof
Technical Field
The invention relates to the field of detection, in particular to an internal reference gene combination for analysis of expression of pseudokanbrix drop genes and application thereof.
Background
The ciliates of the shield fiber type are a kind of facultative parasitic protozoa which widely exist in various water environments, especially in eutrophic water bodies, can deteriorate the water environments and are parasitic on shellfish coat cavities, body surfaces of fishes, hearts, blood and other tissue organs, and have no obvious selectivity to hosts, so that the ciliates are extremely easy to directly or indirectly harm aquaculture animals. The shield ciliate disease is a common parasitic ciliate disease of indoor fingerling production and sea water cage cultured fishes, and causes great economic loss of various important economic sea fishes such as artificial cultured flounder, tetrodes, large yellow croakers and the like. Pseudokonjac (pseudokonjac budworms) are belonging to the genus pseudokonjac, phylum promiscuous, order of fouling, family pseudokonjac, genus pseudokonjac. It has been reported that the pseudokansui of water drop can be parasitic on the bodies of ill cultured animals such as prawn, flounder, etc., and the death rate is even more than 90%.
Some ciliates can dramatically increase cell metabolism secretion when the temperature is uncomfortable, food is insufficient and space is crowded, oxygen content, pH and the like also change, at this time, ciliates often form capsules to bridge adverse environments, and once the environments are proper, the cells forming the capsules can be de-encapsulated to restore normal activities. Earlier studies in the laboratory found that pseudoartemia of water droplets formed into capsules in the case of malnutrition or high population density and rapidly de-capsulated once food and space were sufficient, and thus were good materials for studying the mechanism of formation and de-capsulation of the scutella species. The formation of the parasitic ciliate capsule is also a most 'safe' method for avoiding the influence of severe environment and ensuring effective infection of a host, so that the differential analysis of the gene expression of the water drop pseudohealthy ciliate in the nutrition period and the encapsulation period can provide meaningful reference for the control of the scutellum diseases.
Real-time fluorescent quantitative PCR (quantitative-timePCR) is an important technical means for gene expression analysis, and is generally used for testing various application fields such as expression differences of different phases of specific genes and confirmation of cDNA or differential results in different samples under different experimental conditions. The selection of a proper reference gene is a precondition for ensuring the accuracy of qRT-PCR analysis results. The most suitable reference genes are genes which are constantly expressed and have no obvious difference in expression levels in different test conditions and different types of tissues and cells, and common reference genes include Actin (Actin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 18S ribosomal RNA (18S rRNA), UBQ (ubiquitin, polyubiquitin enzyme), EF 1a (Alpha transcriptional elongation factor gene), RPL (ribosomal protein), TUB (beta-tubulin ), and the like. Although these commonly used reference genes are highly conserved, the expression levels in different tissues and different physiological states are not stable, and thus, an appropriate reference gene is selected according to a specific species and specific experimental conditions. During the nutrition phase and encapsulation phase of pseudokanbrix, the physiological activities of the pseudokanbrix are obviously changed, while some common reference genes, such as TUB and GAPDH, are closely related to the physiological activities of exercise, respiration and the like, and the expression of the genes is presumed to be also strongly changed, so that the pseudokanbrix is not suitable for being used as the reference genes for researching the difference of the expression of the genes during the nutrition phase and the encapsulation phase. Therefore, two or more reference genes whose expression levels are stable should be selected according to the subject and the conditions of the study.
At present, no report exists on screening of internal reference genes available in gene expression change research of the peltate caterpillars, and in order to accurately research the expression of differential genes in the nutrition period and the encapsulation period of the water drop pseudokansuis, it is necessary to screen the internal reference genes which are applicable to the water drop pseudokansuis and have no obvious difference in the expression quantity in the two physiological periods.
Disclosure of Invention
In view of the above problems, the present application provides an internal gene combination suitable for differential analysis of gene expression in the trophic and encapsulating phases of pseudokanbrix rosea.
The first aspect of the application provides an internal reference gene combination for the expression analysis of the pseudokanbrix water drop genes, wherein the internal reference gene combination comprises a C3228 gene and a C3765 gene; the C3228 gene sequence is a nucleotide sequence shown as SEQ ID NO.6, and the C3765 gene sequence is a nucleotide sequence shown as SEQ ID NO. 8.
Compared with the prior art, the technical scheme provides a stable internal gene combination for analysis of the differential genes of the water drop pseudohealthy fiber worm in the nutrition period and the encapsulation period, finds 2 suitable internal genes for the differential gene expression of the water drop pseudohealthy fiber worm in different periods and normalization of transcriptome data, and provides a reference for genetic research of the water drop pseudohealthy fiber worm.
Further, the PCR primer sequences of the C3228 gene are the nucleotide sequences shown as SEQ ID NO.23 and SEQ ID NO. 24.
Further, the PCR primer sequences of the C3765 gene are the nucleotide sequences shown as SEQ ID NO.27 and SEQ ID NO. 28.
A second aspect of the present application provides the use of the reference gene combination according to the first aspect of the present application in differential studies of gene expression in the trophic and encapsulating stages of pseudokanbrix roseus.
In a third aspect, the present application provides a PCR primer of C3228 gene, wherein the primer comprises a nucleotide sequence shown as SEQ ID NO.23 and a nucleotide sequence shown as SEQ ID NO. 24.
In a fourth aspect, the present application provides a PCR primer for a C3765 gene, the primer comprising the nucleotide sequence shown as SEQ ID NO.27 and the nucleotide sequence shown as SEQ ID NO. 28.
In a fifth aspect, the present application provides a PCR kit for pseudokanbrix drop genes, wherein the kit comprises the reference gene combination according to the first aspect of the present application.
In a sixth aspect, the present application provides a PCR kit for pseudokanbrix drop genes, wherein the kit comprises the PCR primer according to the third aspect of the present application or/and the PCR primer according to the fourth aspect of the present application.
Further, the primer of the C3228 gene comprises a nucleotide sequence shown as SEQ ID NO.23 and a nucleotide sequence shown as SEQ ID NO. 24.
Further, the primer of the C3228 gene comprises a nucleotide sequence shown as SEQ ID NO.27 and a nucleotide sequence shown as SEQ ID NO. 28.
The foregoing summary is merely an overview of the present application, and is provided to enable one of ordinary skill in the art to make more clear the present application and to be practiced in light of the present disclosure, as well as to enable one of ordinary skill in the art to make more readily understood by referring to the present application and its various aspects, features and advantages.
Drawings
The drawings are only for purposes of illustrating the principles, implementations, applications, features, and effects of the present application and are not to be construed as limiting the application.
In the drawings of the specification:
FIG. 1 is an agarose gel electrophoresis of total RNA. M: DL2000DNAMarker;1-3: total RNA samples in vegetative phase, 4-6: total RNA samples during encapsulation phase
FIG. 2 is a diagram showing the result of PCR product electrophoresis detection of 12 candidate reference genes. M: DL500DNAMarker;1-12: c1210 C 17610, c16121, c44763, c17860, c3765, c37508, c3228, c18657, c17293, c17299, c17076
FIG. 3 is a graph showing the dissolution profile of candidate reference genes.
FIG. 4 is a graph showing amplification of 8 candidate reference genes.
FIG. 5 is a graph showing the stability of expression of a candidate reference gene for gemm analysis.
FIG. 6 is a graph showing the number of reference genes best candidate for the gemm analysis.
FIG. 7 is a graph showing amplification standards of 2 reference genes.
Detailed Description
In order to describe the possible application scenarios, technical principles, practical embodiments, and the like of the present application in detail, the following description is made with reference to the specific embodiments and the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples and are not intended to limit the scope of protection of the present application.
The main instruments and reagents of this embodiment are as follows:
the main instrument is as follows: TG16-WS bench-top high-speed centrifuges (Hunan instrument), ABI2720PCR amplification instrument (applied biosystems); ultra-micro ultraviolet visible spectrophotometer DS-11 (DeNovix), gel electrophoresis apparatus (Beijing Liuyi instruments Co., ltd.); gel imaging System WD-9413D (Beijing Liuyi instruments Co., ltd.), refrigerated centrifuge (Eppendorf centrifuge 5417R); quantum studio3 real-time fluorescent quantitative PCR apparatus (applied biosystems).
Reagent: TAKARAMiniBESTUniversal RNAextraction kit (TAKARACODENo. 9767), primeScript TM RT reagent Kit with gDNAEraser(TAKARA code No.RR047A),TBPremixEx Taq TM II (Tli RNaseH Plus) (TAKARACODENo. RR5000A), DL2000Marker (TAKARACODENo. 3427A), DL500Marker (TAKARA CODENo. 3590A), premix Taq (TAKARACODENo. RR901A), DEPC treated water (Shanghai Korea); other reagents are homemade analytically pure.
1. Extraction of Total RNA and Synthesis of cDNA
1 cultivation and acquisition of nutritional insects
Inoculating 1mL of water drop pseudokonjac fibrinolytic culture solution into 50mL of filtered seawater, dropwise adding 500 μl of cultured E.coli DH5α bacterial solution overnight, culturing for 3-4d until the density is greater than 4×10 4 ind·mL －1 After the collection of the vegetative insects, the culture solution was filtered with a cell sieve of 70 μm and 40 μm respectively to a 15mL centrifuge tube, 4000 r.min -1 Centrifuging for 5min, discarding supernatant, adding 1mL sterile seawater, re-suspending the insect precipitate into 1.5mL tube, and standing at 8000 r.min -1 Centrifuging for 3min, and discarding supernatant to obtain nutritional insect for extracting total RNA of nutritional insect.
2 acquisition of capsules
10mL of seawater was added to the culture wells of the 6-well cell culture plate, 100. Mu.L of E.coli DH5α bacteria solution cultured overnight was added to each well, and 100. Mu.L of 3 d-cultured Desmodium vivax was inoculated. After 1 week of culture, the pseudokoning artemia of water drop formed a capsule adhered to the bottom of the hole, sucking off the culture seawater, adding 2mL of fresh sterile seawater, scraping the capsule with cells, concentrating in a 15mL centrifuge tube, 4000 r.min -1 Centrifuging for 5min, discarding supernatant, adding 1mL sterile seawater, re-suspending and precipitating into 1.5mL tube, and precipitating at 8000 r.min -1 Centrifuging for 3min, and removing supernatant to obtain capsule for extracting total RNA.
Extraction method of 3 total RNA
Total RNA extraction was performed with reference to the MiniBESTUniversal RNAextraction kit (TAKARACODENo. 9767) kit. The operation steps are as follows:
1. washing appropriate amount of nutritional insect with 1×PBS, centrifuging at 8,000Xg at 4deg.C for 2min, and discarding supernatant; adding a proper amount of cracking Buffer RL to repeatedly blow and suck until no obvious precipitate exists in the cracking liquid;
2. standing the lysate at room temperature for 2min;
3. gDNAEraser Spin Column was mounted on 2mL Collection Tube;
4. transferring the lysate to gDNAEraser Spin Column;
5.12000rpm, and centrifuged for 1min.
6. Discarding gDNAEraser Spin Column, and retaining the filtrate in 2 mLTube;
7. adding 70% ethanol with the same volume as the liquid into the filtrate, and uniformly mixing the solution;
8. immediately transferring the whole mixed solution to an RNA Spin Column;
9.12,000rpm, centrifuged for 1min, and the filtrate was discarded. Placing the RNA Spin Column back into 2mL Collection Tube;
10. 500. Mu.L of Buffer RWA was added to RNA Spin Column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded;
11. 600. Mu.L of Buffer RWB was added to RNA Spin Column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded;
DNase I digestion
A50 mu LDNase I reaction solution is added to the center of the RNA Spin Column membrane, and the mixture is allowed to stand at room temperature for 15min.
b 350. Mu.L of BufferRWB was added to the center of the RNA Spin Column membrane, centrifuged at 12,000rpm for 30s, and the filtrate was discarded.
13. Repeating the operation step 11;
14. the RNA Spin Column was re-mounted on 2mL Collection Tube and centrifuged at 12,000rpm for 2min;
15. placing the RNA Spin Column on 1.5mL RNase Free Collection Tube, adding 50-200 mu L of 0.1% DEPC treated water at the center of the RNA Spin Column membrane, and standing for 5min at room temperature;
centrifuging at 16.12000rpm for 2min to elute RNA;
17. the integrity and quality of the RNA was checked by 1.2% agarose gel electrophoresis, and the concentration and purity of the extracted RNA was checked by using an ultra-micro ultraviolet spectrophotometer.
OD of total RNA of sample is detected by ultra-micro ultraviolet visible spectrophotometer DS-11 260/280 The value is between 1.8 and 2.2. Detection of the water drop pseudoartemia artemia by agarose gel electrophoresis and total RNA encapsulated samples the total RNA samples are shown in fig. 1, wherein the 28S and 18S band brightness ratio is close to 2:1.
synthesis of 4cDNA
Reverse transcription reaction referring to PrimeScript TM RTreagent kit (TAKARACODENo. RR037A) kit instructions. Reaction system 20 μl: 1. Mu.g of total RNA, 1. Mu.L of each of the upstream and downstream primers, primeScript enzyme mix I1. Mu.L, RTPrimerMix 1. Mu.L, 5 XPrimeScript buffer 24. Mu.L, and RNAfreeddH2O to 20. Mu.L. Firstly, the cDNA is obtained by extending for 15min at 37 ℃ and then placing at 85 ℃ for 5s, and the cDNA is packaged and stored in an ultralow temperature refrigerator at-80 ℃.
2. Screening of candidate reference genes and primer design of qRT-PCR
According to the sequencing result of transcriptome in the nutrition phase and encapsulation phase of pseudokanbrix, 12 genes with higher expression level and similar expression level in two physiological phases are screened out, and the names and sequences of the genes are shown in the table 1 (SEQ ID NO. 1-12)
Based on the sequencing results, qRT-PCR primers were designed using the on-line Primer design tool Primer3 (https:// Primer3.Ut. Ee /), the Primer sequences (SEQ ID NOS.13-36) are shown in Table 1.
TABLE 1 candidate internal reference gene qRT-PCR primers
3. PCR amplification
Performing PCR amplification on 12 candidate internal reference genes by using mixed cDNA of the water drop pseudokanbrix in the nutrition period and the encapsulation period as a template, wherein the reaction system is 20 mu L: 12.5. Mu.L of 2 XTaqmix, upstream and downstream primers (10. Mu. Mol. L -1 ) mu.L of each, 1. Mu.L of cDNA template, and ddH were added 2 O to 20. Mu.L. The PCR reaction procedure was 95℃melting for 4min; 30s at 95℃and 30s for annealing (annealing temperature see Table 1), 30s for 72℃extension, 30 cycles; and then the extension is carried out for 7min at 72 ℃. The PCR products were subjected to agarose gel (2.0%) electrophoresis.
The electrophoresis results are shown in FIG. 2,2.0% agarose gel electrophoresis, which shows that candidate reference gene amplification produces a single band, that the size of the target band is as expected, and that no non-specific amplification or primer dimer is present, indicating that the screened primers are well specific.
4. Real-time fluorescent quantitative PCR
Using TBPremix Ex Taq TM II (Tli RNaseH Plus) (TAKARA code No. RR820A) kit the qRT-PCR amplification reaction was performed in a Quantum studio3 real-time fluorescent quantitative PCR instrument (applied biosystems). Reaction system 20 μl: 10. Mu.L 2X TB GreenPremixExTaq II, upstream and downstream primers (10. Mu. Mol. L) -1 ) Each 0.8. Mu.L of cDNA template 1. Mu.L, 0.4. Mu.L of 50X ROX ReferenceDye II, add ddH 2 O to 20. Mu.L. PCR reaction procedure: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 34s; the reaction was carried out 40 times and the dissolution profile analysis procedure was 95℃15s,60℃1min,95℃15s. No template and no primer were set as two controls and 3 technical replicates of qPCR assays were performed per sample.
qPCR obtained the dissolution curve of candidate reference genes, and the results are shown in FIG. 3, c1210, c17076, c17293 and c17299 are shown to have obvious double peaks, the four candidate reference genes are discarded, and the rest candidate reference genes are subjected to stability analysis.
And analyzing the expression abundance of the candidate genes by using an average CT value, analyzing the stability of the candidate reference genes by using three software of geNorm, normFinder and Bestkeeper, and screening the comprehensive results to obtain the optimal reference genes and determining the number of the reference genes.
1. CT values and amplification curves:
the CT value distribution of 8 candidate genes with better specificity in the sample is analyzed, and the results are shown in Table 2: the average CT value of the 8 candidate genes is 11.631 (c 17860) to 22.405 (c 3228), and the distribution range of the expression abundance is wider. Among these 8 candidate reference genes, c3765, c18657, c3228, c37508 showed little change in gene expression level in different samples, and c44763 showed large difference in gene expression level in different samples.
FIG. 4 shows amplification curves of candidate genes. Therefore, from the point of uniformity of the expression level of the pseudokanbrix in different periods, the c3765, c18657, c3228 and c37508 genes are suitable as internal reference groups for analyzing the differential gene expression in the vegetative period and the encapsulation period.
TABLE 2 CT value Table of candidate reference genes
2. GeNorm software analysis
From the results of FIG. 5, it was found that the M values of the 8 candidate reference genes were less than 1.5, with the smallest being c3765 and the largest being c44763, indicating that these genes could be used as candidate reference genes, with c3765< c3228< c16121< c37508< c18657< c 17114 < c17860< c44763 in order from low to high. It was shown that c3765 and c3228 are the most stable genes expressed therein, while the gene expression stability of c44763 was slightly inferior.
From the results of FIG. 6, the gemum program analyzed the paired differences of the reference genes, vn/n+1, which showed that all of the values of Vn/n+1 were less than 0.15, and V 2/3 The expression analysis of the differential genes in the nutrition and encapsulation phases of water drop pseudokan was carried out by using the reference gene c3765 and c3228, which had the best stability, because the 3 rd reference gene was not required to be added for correction and the number of suitable reference gene combinations was 2.
3. NormFinder software analysis
The analytical results are shown in Table 3, and the candidate gene expression stability values are arranged from low to high: c3228 =c 3765< c37508< c18657< c16121< c17860< c 17610 < c44763. Among them, the Stability value of c3228 and c3765 was the lowest (0.040), indicating that they were the most stable and most suitable as reference genes.
TABLE 3 SV value of candidate reference genes
4. Bestkeeper software analysis
The analysis results are shown in Table 4. The stability of these 7 genes was ranked from high to low by combining standard deviation and coefficient of variation: c3765 > C3228 > C37508 > C18657 > C16121 > C17610 > C44763, wherein C3765 and C3228 are also reference genes screened by the geNorm software and the NormFinder software in the present study to be most suitable for differential gene expression studies of the nutritional phase and the encapsulation phase of pseudoKangbrix in water drops.
TABLE 4BestKeeper analysis of expression stability of 8 candidate internal reference genes
5. Primer amplification efficiency
The cDNA stock was diluted to five concentration gradients (1/4, 1/16, 1/64, 1/256, 1/1024) as templates for establishing standard curves, each concentration was repeated 3 times in parallel with ddH 2 O replaces cDNA as a negative control. The reaction system and the reaction procedure were the same as those of the fourth (real-time fluorescent quantitative PCR). Calculation of amplification efficiency (E) and Linear correlation coefficient (R) using Quantum studio3 real-time fluorescent quantitative PCR self-contained application 2 )。
The amplification standard curve obtained by the Quantum studio3 real-time fluorescent quantitative PCR instrument is shown in FIG. 7, and the good fitting degree of the standard curves of c3765 and c3228 can be seen. R of c3765 2 0.995, amplification efficiency of 119.903%, R of c3228 2 The amplification efficiency is 109.489% and meets the requirement of qRT-PCR on the amplification efficiency of the primer, so that C3228 and C3765 are suitable to be used as reference genes in the nutrition period and the encapsulation period of the pseudokanbrix water drop.
The analysis results of the above geNorm, normFinder software and Bestkeeper software are basically consistent, in addition, the selection of a single reference gene can cause larger experimental error, and the pairing mutation number V is obtained by using the gem software n/n+1 Analysis was performed to determine that the optimum number of desired reference genes was 2, so that the optimum reference gene combinations in the nutrition period and the encapsulation period of water drop pseudokanbrix were C3228 and C3765.
The application screens 8 candidate genes suitable for differential gene expression analysis of the nutrition period and the encapsulation period of the pseudokanbrix water drop, namely C3765, C3228, C37508, C18657, C17860, C16121, C17610 and C44763. The optimal number of the required reference genes is comprehensively judged to be 2 through analysis of three software, namely geNorm, normFinder and Bestkeeper. And C3228 and C3765 are reference gene combinations which are screened by the research and are most suitable for analysis of differential gene expression in the nutrition phase and the encapsulation phase of the pseudokanbrix water drop.
The application provides a selection basis for analyzing differential gene expression analysis of the nutrition period and the encapsulation period of the water drop pseudociliates, and can provide references for screening of the reference genes of other peltate ciliates and related gene expression analysis.
Finally, it should be noted that, although the foregoing embodiments have been described in the text and the accompanying drawings of the present application, the scope of the patent protection of the present application is not limited thereby. All technical schemes generated by replacing or modifying equivalent structures or equivalent flows based on the essential idea of the application and by utilizing the contents recorded in the text and the drawings of the application, and the technical schemes of the embodiments are directly or indirectly implemented in other related technical fields, and the like, are included in the patent protection scope of the application.

Claims (3)

1. Use of an internal reference gene combination in research on gene expression differences in the nutritional phase and the encapsulation phase of a pseudokansui in water droplets, characterized in that the internal reference gene combination comprises a C3228 gene and a C3765 gene; the C3228 gene sequence is a nucleotide sequence shown as SEQ ID NO.6, and the C3765 gene sequence is a nucleotide sequence shown as SEQ ID NO. 8.
2. The use according to claim 1, wherein the PCR primer sequence of the C3228 gene is the nucleotide sequence shown in SEQ ID No.23 and SEQ ID No. 24.
3. The use according to claim 1, wherein the PCR primer sequence of the C3765 gene is the nucleotide sequence shown in SEQ ID No.27 and SEQ ID No. 28.
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