CN116904634A - Internal reference gene of Chinese capsicum in different fruit development periods, and primers and application thereof - Google Patents
Internal reference gene of Chinese capsicum in different fruit development periods, and primers and application thereof Download PDFInfo
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Abstract
The application belongs to the technical field of pepper gene research, and particularly relates to an internal reference gene of Chinese peppers in different fruit development periods, and a primer and application thereof. Aiming at the current situation of lack of internal reference genes in the development of pepper fruits, the method utilizes a real-time fluorescent quantitative PCR technology to analyze the gene expression quantity of different growth and development stages, fruit parts and tissues of the pepper fruits according to transcriptome data, screens candidate internal reference genes, carries out wide screening on the internal reference genes, comprehensively compares the stability of each internal reference gene by using a plurality of statistical analysis methods, designs corresponding real-time fluorescent quantitative PCR primers by taking a target gene sequence as a template, has high amplification efficiency, screens stable and suitable internal reference genes UBI-3, EIF and CT4 in the development process of Chinese peppers, develops and designs efficient amplification primers, and provides relatively effective internal reference genes for expression analysis, screening and timely air expression verification of the target genes in the development process of the pepper fruits.
Description
Technical Field
The application belongs to the technical field of pepper gene research, and particularly relates to an internal reference gene of Chinese peppers in different fruit development periods, and a primer and application thereof.
Background
The real-time fluorescent quantitative PCR technology is widely applied to analysis of gene expression quantity due to the advantages of accuracy, high efficiency, good repeatability and the like, but a proper and stable reference gene is a key factor for accurately calculating the relative expression quantity of a target gene. In ideal conditions, the reference gene should be stably expressed in the same species, different tissues and organs and at different growth stages, and the expression level is not affected by internal and external factors. In fact, the expression levels of any reference gene are relative, and the expression levels of the same reference gene in the same species are often affected by internal and external factors, so that appropriate and stable reference genes should be selected according to different experimental conditions, experimental environments and experimental purposes when related studies are performed.
The capsicum is one of the world vegetables and is also the vegetable crop with the most extensive planting area in China. The capsicum is not only an auxiliary food and a seasoning in a restaurant kitchen, but also a raw material of important products such as cosmetics, medicines, tear gas, ship bottom protective agents and the like, wherein the Chinese capsicum (Capsicum chinense Ja cquin) is one of five cultivation species which are widely planted, and the fruits of the capsicum have the advantages of rich capsaicin content, changeable fruit color, various flavors and the like. The fruit quality is important to the commodity value of the capsicum, and the space-time expression quantity analysis of genes of the fruit development, nutrition and flavor related quality traits is an important theoretical basis for improving the fruit quality of plants. However, the reference gene for the development of the Chinese pepper fruit is not available for reference at present, so that the research on molecular biology of a plurality of excellent properties of the Chinese pepper fruit is still under the current status.
Disclosure of Invention
Aiming at the problems, the application aims to solve the problem of lack of internal reference genes for the development of Chinese capsicum fruits and provide internal reference genes for different fruit development periods of Chinese capsicum (Capsicum chinense Jacquin) as well as primers and application thereof.
The technical content of the application is as follows:
the application provides an internal reference gene of Chinese capsicum (Capsicum chinense Jacquin) in different fruit development periods, which is characterized in that the internal reference gene is more than one of UBI-3, EIF and CT 4;
the nucleic acid sequence of the UBI-3 is shown in SEQ ID NO. 1;
the nucleic acid sequence of the EIF is shown as SEQ ID NO. 2;
the nucleic acid sequence of CT4 is shown as SEQ ID NO. 3;
the specific primers of the internal reference gene UBI-3 gene are shown as SEQ ID NO.6 and SEQ ID NO. 7;
specific primers of the reference gene EIF gene are shown as SEQ ID NO.12 and SEQ ID NO. 13;
the specific primers of the reference gene CT4 gene are shown as SEQ ID NO.24 and SEQ ID NO. 25.
The application also provides application of the internal reference gene or the specific primer thereof in preparing detection kits for different fruit development periods of Chinese capsicum.
The application also provides a screening method of reference genes of different fruit development periods of the Chinese capsicum, which comprises the following steps:
1) Pretreatment of fruits: obtaining Chinese pepper fruits in different growing and developing periods, and grinding the Chinese pepper fruits into powder;
2) Extracting RNA: extracting RNA of pepper fruit powder in different growing and developing periods;
3) Synthesizing a first strand of cDNA;
4) Screening candidate reference genes and designing primers;
the candidate internal reference genes comprise GAPDH, UBI-3, UBI-1, ACT, EIF, beta-TUB, alpha-TUB, CT1, CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CT11 and CT12;
the nucleic acid sequence of the candidate reference gene can be obtained according to NCBI gene library, and the specific primer sequences are respectively shown in SEQ ID NO. 4-SEQ ID NO. 41.
5) Amplifying cDNA by PCR;
6) PCR amplifying candidate reference genes, and calculating primer amplification efficiency and slope R2 by using software;
7) Screening of reference genes: the stability of candidate reference genes is analyzed by using four software of NormFinder, delta Ct, bestKeeper and gem, comprehensive sequencing and evaluation are carried out according to the obtained results, and the optimum reference genes UBI-3, EIF and CT4 for the development of the Chinese pepper fruits according to claim 1 are screened.
The beneficial effects of the application are as follows:
according to the method for screening internal reference genes of Chinese peppers (Capsicum chinense Jacquin) in different fruit development, according to the current situation of lack of internal reference genes of the current pepper fruit development, the real-time fluorescent quantitative PCR technology is utilized to analyze gene expression quantities of different growth development stages, fruit parts and tissues of the pepper fruit according to transcriptome data, 19 candidate internal reference genes are screened out, the internal reference genes are widely screened out, various statistical analysis methods are used, stability of the internal reference genes are comprehensively compared, a target gene sequence is used as a template to design corresponding real-time fluorescent quantitative PCR primers, and through verification, the designed primers have good specificity and high amplification efficiency, stable and suitable internal reference genes UBI-3, EIF and CT4 in the pepper fruit development process are screened out, and the real-time fluorescent quantitative PCR efficient amplification primers in the pepper fruit related gene research field are developed and designed, so that relatively effective internal reference genes are provided for calibration in the pepper fruit development process, and the problem of lack of suitable stable internal reference genes in the pepper fruit development research field is solved;
aiming at the output of capsicum fruits as important commodity, sampling points are designed at different development stages of capsicum, the screened reference gene can be used for calibrating target genes, so that time loss caused by the selection of the reference gene is greatly saved, the accuracy and reliability of the result are ensured, and the screened reference gene can also provide reference for research on the development of fruits of other solanaceae crops.
Drawings
FIG. 1 is a diagram of electrophoresis gel obtained by performing ordinary PCR amplification on 19 primers screened by the application by taking pepper fruit cDNA as a template;
FIG. 2 is a graph showing the Cq value distribution of the real-time fluorescent quantitative PCR of 16 candidate internal reference genes of Chinese capsicum;
FIG. 3 is a graph showing the dissolution profile of 16 candidate reference genes selected according to the present application.
Detailed Description
The application is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the application are materials and reagents of the conventional market unless specified otherwise.
Examples
Real-time fluorescent quantitative PCR method for screening pepper fruit development internal reference genes
1) Material cultivation and pretreatment: soaking pepper seeds in warm water for 12 hours, then placing the pepper seeds in a 28 ℃ incubator for germination, and sowing the pepper seeds in a seedling tray filled with seedling raising matrixes after the seeds are exposed. Transplanting the pepper seedlings to the open field for cultivation when the pepper seedlings grow to 5-6 leaves, and carrying out the management in the future according to the conventional field cultivation;
the method comprises the steps of selecting Chinese pepper fruits after fruit setting, respectively listing, sampling the whole pepper fruits at 6 time points of listing time of 0 day, 7 days, 14 days, 21 days, 28 days and 35 days, and performing three times of biological repeated sampling at each time point, namely selecting 6 fruit samples of the Chinese pepper in different growing and developing periods;
the collected 6 fruit samples with different growth and development periods are rapidly put into liquid nitrogen for freezing and then brought back to a laboratory to be ground into powder under the liquid nitrogen, and the powder is stored in a refrigerator with the temperature of-80 ℃ for subsequent experiments;
2) Extracting RNA: taking 6 Chinese pepper fruit powders with different growing and developing periods, and extracting total RNA of the Chinese pepper fruits by using a Trizol Reagent (Beijing full-scale gold biotechnology Co., ltd.) kit, wherein the extraction conditions are consistent with the kit instruction;
the integrity of the extracted RNA was checked by gel electrophoresis, 1% agarose gel, TBE buffer, electrophoresis at 120V for 15min, and the integrity of the RNA bands was observed under a gel imaging system;
detecting the concentration and purity of RNA by using Nanodrop-2000c, wherein the OD value 260/280 of pure RNA is between 1.8 and 2.2, and then quantifying the concentration of RNA to 100 ng/. Mu.L;
3) First strand cDNA Synthesis: accurately sucking 1 μg of RNA sample, and performing reverse transcription by using PrimeS script O RRT reagent Kit with g DNA Eraser (Perfect Real Time) reverse transcription kit to obtain cDNA first strand;
4) Collecting transcriptome data related to pepper fruits, and preliminarily selecting 19 pepper candidate reference genes, namely GAPDH, UBI-3, UBI-1, ACT, EIF, beta-TUB, alpha-TUB, CT1, CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CT11 and CT12, wherein corresponding nucleic acid sequences can be obtained according to NCBI gene libraries, and the numbering is shown in table 1 and can be used as the candidate reference genes;
wherein the primers for GAPDH, UBI-3, UBI-1, ACT, EIF, beta-TUB, alpha-TUB are obtained according to the prior art, and other primers are designed using software Primier5.0 with candidate reference gene sequences as templates, followed by designing specific primers using Primer-BLAST in NCBI database;
GAPDH、UBI-3、
5) cDNA obtained by reverse transcription of pepper fruit RNA was amplified by PCR using the designed primers. The common PCR polymerase used for detecting the specificity of the primer of the candidate reference gene is purchased from Beijing Kang Runcheng Biotechnology Co., ltd (GenStar), the reaction system is a 10 mu L system, the polymerase is 5 mu L, the sterile water is 4 mu L, the upstream and downstream primers are 0.25 mu L respectively, and the cDNA template is 0.5 mu L; the amplification procedure was 94℃for 5min,94℃for 30s,55℃for 30s,72℃for 1min,35 cycles, followed by final extension at 72℃for 10min, 1% agarose gel electrophoresis detection of primer specificity after amplification was completed, electrophoresis was performed for 20min at 120V voltage in TBE buffer, and the specific band length was observed under gel imaging system to confirm whether amplification was the target band. As shown in FIG. 1, the agarose gel electrophoresis results show that CT1 is not amplified, CT2 and CT3 are non-specifically amplified, so that the two candidate genes are eliminated, while CT9 and CT10 are non-specifically amplified, but the length of a non-specific fragment is close to 500bp, and the non-specific fragment cannot influence the fluorescent quantification, so that the next experiment can be kept.
6) qRT-PCR: the cDNA obtained by reverse transcription is used as a template to carry out real-time fluorescence quantitative PCR amplification on the candidate reference genes to obtain corresponding Cq values, and the result is shown in FIG. 2.
The results show that the Ct values of all candidate reference genes are between 19.18 (UBI-3) and 30.78 (CT 6), and the expression quantity of the candidate genes is high, so that the requirements of the reference genes are met. The Ct values of the five genes of alpha-TU B, EIF, GAPDH, UBI-3 and UBI-1 are obviously lower than those of other genes, which indicates that the genes are high in expression abundance in the development stage of pepper fruits, and the Ct value of CT6 is obviously higher than those of other genes, which indicates that the genes are the lowest in expression level in all candidate internal reference genes.
The real-time fluorescent quantitative PCR kit is SYBRRPremix Ex Taq TM II (Tli RN aseH Plus) (Takara) the amplification system was a 25. Mu.L system, and the amplification conditions were: 95 ℃ for 30s,95 ℃ for 5s,60 ℃ for 30s,40 cycles; the melting curve drawing program is that plates are sequentially read at 65 ℃ to 95 ℃ every 0.5 ℃, and each sample is subjected to three technical repetitions, so that the specificity of the primer can be judged according to melting peaks. The dissolution curve graphs of all candidate reference genes are shown in figure 3, and the dissolution curve results show that all the dissolution curves are single and have no impurity peak, so that the primer specificity is proved to be good, and the requirement of the specificity is met;
7) Primer amplification efficiency and R 2 And (3) calculating: deriving real-time fluorescent quantitative PCR data results, and calculating primer amplification efficiency and slope R using software LinRegPCR 2 The primers, amplification efficiencies and slopes of the obtained candidate reference genes are as follows:
TABLE 1 Pepper fruit development candidate internal reference Gene and amplification primer
The results of the table 1 combined with the results of the fig. 1 and the fig. 2 show that the designed primer has good specificity and high amplification efficiency, which shows that the screened reference gene has stable performance in the pepper development process, and the designed real-time fluorescence quantitative PCR high-efficiency amplification primer of the related gene provides relatively effective reference gene for calibration for target gene expression analysis, screening and timely empty expression verification in the pepper fruit development process.
8) Candidate reference gene stability was analyzed using four software of normFinder, Δ Ct, bestKeeper and georm;
9) Comprehensively sequencing and evaluating the results obtained by the 4 software by using an online tool RefFinder, and screening out the optimal internal reference gene for pepper fruit development;
the method for calculating the relative expression level of the gene uses 2 -ΔΔCt The specific calculation method comprises the following steps: delta ct=ct (target gene) -Ct (reference gene), delta ct=delta Ct (treatment) -delta Ct (control), 2 -ΔΔCt Relative expression amount.
The results are shown below:
TABLE 2 stability and ordering of candidate genes in Chinese Capsicum
According to the results of Table 2, the candidate reference genes screened by the application have good stability, wherein the genes EIF, CT4 and UBI-3 can be used as reference genes for screening fruit development of Chinese peppers in different periods.
Claims (7)
1. The internal reference gene of the Chinese capsicum in different fruit development periods is characterized in that the internal reference gene is more than one of UBI-3, EIF and CT4.
2. The internal reference gene for different fruit development periods of Chinese capsicum according to claim 1, wherein the nucleic acid sequence of UBI-3 is shown in SEQ ID No. 1;
the nucleic acid sequence of the EIF is shown as SEQ ID NO. 2;
the nucleic acid sequence of CT4 is shown as SEQ ID NO. 3.
3. The application of the reference gene in claim 1 in preparing detection kit for different fruit development periods of Chinese capsicum.
4. The specific primer of the internal reference gene of the Chinese capsicum in different fruit development periods of claim 1, wherein the specific primer of the internal reference gene UBI-3 gene is shown as SEQ ID NO.6 and SEQ ID NO. 7;
specific primers of the reference gene EIF gene are shown as SEQ ID NO.12 and SEQ ID NO. 13;
the specific primers of the reference gene CT4 gene are shown as SEQ ID NO.24 and SEQ ID NO. 25.
5. The use of the specific primer as defined in claim 4 for preparing detection kit for different fruit development periods of Chinese capsicum.
6. The screening method of the reference genes of the Chinese capsicum in different fruit development periods is characterized by comprising the following steps:
1) Pretreatment of fruits: obtaining Chinese pepper fruits in different growing and developing periods, and grinding the Chinese pepper fruits into powder;
2) Extracting RNA: extracting RNA of pepper fruit powder in different growing and developing periods;
3) Synthesizing a first strand of cDNA;
4) Screening candidate reference genes and designing primers;
the candidate internal reference genes comprise GAPDH, UBI-3, UBI-1, ACT, EIF, beta-TUB, alpha-TUB, CT1, CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CT11 and CT12;
5) Amplifying cDNA by PCR;
6) PCR amplification of candidate reference genes, calculation of primer amplification efficiency and slope R using software 2 ；
7) Screening of reference genes: the stability of candidate reference genes is analyzed by using four software of NormFinder, delta Ct, bestKeeper and geNorm, comprehensive sequencing and evaluation are carried out according to the obtained results, and the optimum reference genes UBI-3, EIF and CT4 for the development of the Chinese pepper fruits according to claim 1 are screened.
7. The method for screening reference genes of different fruit development periods of Chinese capsicum according to claim 5, wherein the primer sequence of the candidate reference genes in step 4) is shown as SEQ ID NO. 4-SEQ ID NO. 41.
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