CN109266775B - Reagent for eggplant gene expression analysis and internal reference gene used by reagent - Google Patents

Abstract

The invention discloses a reagent for eggplant gene expression analysis and a reference gene used by the reagent. The invention provides an application of any one of M1-M3 as an internal reference gene in eggplant gene expression analysis, which comprises the following steps: m1, a set of two genes consisting of D1 and D4; m2, D1; m3, D4; M1-M3, wherein D4 is a gene encoding a protein having an amino acid sequence of SEQ ID No. 4; the D1 is a gene encoding a protein of which the amino acid sequence is SEQ ID No. 6. The invention not only provides reliable reference genes for the standardized quantification of the gene expression levels of the eggplant in different fruit development periods under the condition of low temperature and/or normal temperature. The invention further promotes the research of the low-temperature resistance genes of the eggplant in different fruit development periods.

Description

Reagent for eggplant gene expression analysis and internal reference gene used by reagent

Technical Field

The invention relates to a reagent for eggplant gene expression analysis in the field of biology and a reference gene used by the reagent.

Background

Eggplant (Solanum melongena L.) is one of the most popular vegetable crops in the world. Like most warm vegetables, eggplant does not easily set fruit at low temperature, thereby seriously affecting the yield and quality of eggplant (Nothmann J, Koller D.Effect of growing regulators on fruit and seed degradation in eggplants L.). J Hort Sci.1975; 50: 23-7). In order to avoid the serious loss caused by low temperature, a method for cultivating a low-temperature-resistant eggplant variety is often adopted in production. However, the major genes related to low temperature resistance in eggplants are not yet discovered, and the molecular mechanism of the low temperature resistance of the eggplants is still to be further researched.

When functional gene screening and related molecular mechanism research are carried out, gene transcription expression quantity detection is an important means. At present, techniques such as DNA microarray technology, Northern blot analysis, ribonuclease protection, and real-time fluorescent quantitative PCR are widely used for the study of gene expression levels. Among these techniques, the fluorescent quantitative PCR technique is currently the most commonly used tool for detecting gene expression level by virtue of its advantages of high sensitivity, high accuracy, high specificity, high throughput, and low cost (Wong ML, Mederano JF. real-time PCR for mRNA quantification. BioTechniques.2005; 39: 75-85). However, in the case of performing a quantitative fluorescence PCR assay, it is necessary to select an appropriate reference gene for normalizing the expression of a target gene in order to avoid variation in the quantitative results. Reference genes, also known as housekeeping genes, are generally thought to be capable of stable expression at different developmental stages, in different tissue types and under different experimental conditions.

The reference gene can be stably expressed under different test conditions, so that the method plays an important role in ensuring the accuracy of the fluorescent quantitative PCR result. As the commonly used reference genes, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TUB, Ubiquitin (UBQ) and 18S ribosomal RNA (18S) have been reported (balance SA. quantification of mRNA using real-time reverse transcription PCR (RT-PCR): strands and plasmids. J Mol Endocrinol.2002; 29: 23-39). However, many studies have shown that these reference genes have significant differences in expression levels in different Plant materials, different Plant tissues and experimental treatments (Li J, Han J, Hu Y, Yang J.Selection of reference genes for quantitative real-time PCR product expression in tree science 2016; 7: 516; Qi S, Yang L, Wen X, Hong Y, Song X, Zhang M, et al. reference gene selection for RT-PCR analysis of lower degree expression in quantitative expression and expression in molecular and expression in Plant specimen K.2016; sample K7: 287, sample gene expression in molecular and expression in molecular expression, K.K. 1. expression in expression of expression in protein, K.K. 1. expression in expression of expression in genes, qian YQ, Han L, Liu JX, Sun ZY. identification of reliable reference genes in buffer loss for access transport relations. Gene. 2014; 547:55-62). Therefore, before performing gene expression studies, it is important to select and evaluate suitable and reliable reference genes in different experimental conditions. The reports about eggplant reference gene screening at present comprise two most stable reference genes SmEf1 alpha and SmTRX (strong, good plum plant, Rough wave, old day, Jingqingmin, Li Zheng xing, Lideming, Sun Bao, Sun light smell, eggplant qRT-PCR reference gene screening and stability analysis under high temperature stress [ J ] gardening reports, 2017,44(03): 475) 486) expressed in eggplant under high temperature stress; furthermore, the results of Kanakachari et al analyzing the stability of reference genes in eggplant at different fruit development stages showed that geNorm considered SAND and TBP to be the most stable combination of reference genes, while NormFinder considered Expressed to be the best reference gene (Kanakachari M, Solanke AU, Prabhakaran N, Ahmad I, Dhandapani G, Jayabalan N, et al. evaluation of Suitable reference genes for normalization of qPCR gene expression vectors in brinjal; solar gene expression from dual expression levels of applied Biochemistry & Biotechnology.2016; 178: 433-50). To date, no relevant report of the reference gene applicable to different fruit development stages under eggplant low-temperature conditions exists. Therefore, screening and evaluating the reference genes of the eggplant in different fruit development periods under the low-temperature condition is beneficial to improving the accuracy of selection of the eggplant low-temperature resistance genes, and lays a foundation for research of eggplant low-temperature resistance molecular mechanisms.

Disclosure of Invention

The invention aims to solve the technical problem of providing a reference gene with high stability in eggplant gene expression analysis under the conditions of low temperature and/or normal temperature.

In order to solve the technical problems, the invention provides an application of any one of the following M1-M3 as an internal reference gene in eggplant gene expression analysis:

m1, a set of two genes consisting of D1 and D4;

M2、D1；

M3、D4；

M1-M3, wherein D4 is a gene encoding a protein having an amino acid sequence of SEQ ID No. 4; the D1 is a gene encoding a protein of which the amino acid sequence is SEQ ID No. 6.

In the application, the D4 can be a DNA molecule with a coding sequence of SEQ ID No. 3; the D1 can be a DNA molecule with a coding sequence of SEQ ID No. 5.

In the above application, the eggplant gene expression analysis may be an analysis of eggplant gene transcription level.

In order to solve the above technical problems, the present invention provides a reagent for eggplant gene expression analysis.

The reagent for eggplant gene expression analysis provided by the invention is any one of the following E1-E3:

e1, consisting of a substance named W4 for detecting the expression level of D4 as claimed in claim 1 or 2 and a substance named W1 for detecting the expression level of D1 as claimed in claim 1 or 2;

e2, substance named W1 for detecting the expression level of D1 as claimed in claim 1 or 2;

e3, substance named W4 for detecting the expression level of D4 as claimed in claim 1 or 2.

In the above reagent, the W4 may be a primer for PCR amplification of the full length of D4 or a fragment thereof, and the W1 may be a primer for PCR amplification of the full length of D1 or a fragment thereof.

In the above reagent, the PCR may be fluorescence quantitative PCR or ordinary quantitative PCR.

In the reagent, the W4 can be a D4 specific primer pair consisting of D4-F and D4-R, the D4-F is a single-stranded DNA shown in SEQ ID No.9, and the D4-R is a single-stranded DNA shown in SEQ ID No. 10;

the W1 can be a D1 specific primer pair consisting of D1-F and D1-R, the D1-F is a single-stranded DNA shown in SEQ ID No.11, and the D1-R is a single-stranded DNA shown in SEQ ID No. 12.

The kit containing the reagent for eggplant gene expression analysis also belongs to the protection scope of the invention.

The kit for eggplant gene expression analysis can contain other reagents required for PCR (such as fluorescent quantitative PCR) besides the reagents.

The application of the reagent or the kit in eggplant gene expression analysis also belongs to the protection scope of the invention.

As used herein, the eggplant gene expression analysis may be an analysis of gene expression in an eggplant fruit, such as an analysis of gene expression in an eggplant fruit under cold conditions and/or under ambient conditions.

As above, the low temperature condition may be a daily minimum temperature of 7 to 17 ℃. The normal temperature condition can be that the daily minimum temperature is 17-28 ℃.

In the reagent or the kit, each primer pair can be independently packaged, and the molar ratio of the two single-stranded DNAs in each primer pair can be 1: 1.

The invention screens 18 candidate internal reference genes from transcriptome sequencing data (data not disclosed) of eggplant fruits (ovaries) based on FPKM values of gene expression levels, and evaluates the expression stability of the candidate internal reference genes in the eggplant fruit development process under the conditions of low temperature and normal temperature. Three commonly used internal reference screening tools (geonorm, BestKeeper and RefFinder) were selected for correlation analysis and showed that D5(sme2.5_08608.1_ g00002.1) had the best expression stability in biological samples at different fruit development stages under low and normal temperature conditions, followed by D1(sme2.5_01136.1_ g00003.1) and D4(sme2.5_00276.1_ g 00016.1). And comparing and analyzing the screened reference genes D5, D4 and D1 with 3 reference genes (SAND, TBP and Expressed) with the best stability in the eggplant fruit development process and 2 reference genes (SmEf1 alpha and SmTRX) with the best stability under the high-temperature condition, and the result shows that the stability of D1, D4 and D5 is better than that of the five control reference genes (SAND, TBP, Expressed, SmEf1 alpha and SmTRX) in a low-temperature sample, a normal-temperature sample and all samples (Table 5). Therefore, the three reference genes screened by the invention are stable and reliable. The three genes D1, D4 and D5 can be used as reference genes for gene expression analysis in fruits under low temperature and/or normal temperature conditions. The three genes D1, D4 and D5 can be used as reference genes individually, and in order to improve the accuracy of the fluorescence quantitative PCR, the three genes D1, D4 and D5 can be combined to be used as a set of reference genes. The invention not only provides reliable reference genes for the standardized quantification of the gene expression levels of the eggplants in different fruit development periods under the low-temperature condition, but also provides reliable reference genes for the standardized quantification of the gene expression levels of the eggplants in different fruit development periods under the normal-temperature condition. The invention further promotes the research of the low-temperature resistance genes of the eggplant in different fruit development periods.

Drawings

FIG. 1 is the agarose gel electrophoresis pattern of the products of PCR of 18 candidate reference gene primers. Wherein, Lane M is marker 50 bp; lanes D1-D18 show the PCR amplification products corresponding to the candidate reference genes with the gene numbers D1-D18 in Table 1, and 50 bp-200 bp show the partial band size of the marker.

FIG. 2 shows melting curves of 18 candidate reference genes. The abscissa is temperature, the ordinate is fluorescence value, and D1-D18 are 18 candidate reference genes of the gene numbers D1-D18 of Table 1.

FIG. 3 is the mean Cq of the 18 candidate reference genes and five reported reference genes in all biological samples. In the figure, the abscissa is candidate reference genes, D1-D18 are 18 candidate reference genes with the gene numbers D1-D18 of table 1, a1 is SAND, a2 is Expressed, A3 is SmEf1 α, a4 is SmTRX, and a5 is TBP; the ordinate is the cq value.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 establishment of reference Gene in Gene expression analysis of eggplant at different fruit development stages under Cold and Normal temperature conditions

1 test Material

Eggplant parthenocarpic line D-10 and eggplant non-parthenocarpic line 03-2 (Li Yan, Liu Zhong, Chen Yu Hui, Zhang Ying, Zhang Wei, Liang Yong. influence of temperature on endogenous hormone content in the development process of eggplant parthenocarpic ovary (fruit.) Chinese vegetables 2013 (22): 32-38) are lines bred by vegetable and flower institute of Chinese academy of agricultural sciences, and the biological material can be obtained by the public from vegetable and flower institute of Chinese academy of agricultural sciences, is only used for repeating relevant experiments of the invention, and cannot be used for other purposes.

D-10 is a low temperature resistant inbred line of the eggplant, the fruit can normally expand to form a seedless fruit under the low temperature condition (the lowest daily temperature is 7-17 ℃ in the flowering and fruit setting period, the average lowest daily temperature is 12.6 ℃, parthenocarpy is shown, no seed exists in the fruit, the fruit normally expands), and the fruit with the seed is formed under the normal temperature condition. 03-2 is a low temperature intolerant inbred line, which can form stiff fruits or fallen flowers and fallen fruits under the low temperature condition (the lowest temperature is 7-17 ℃, the daily average lowest temperature is 12.6 ℃), and can form normal fruits with seeds under the normal temperature condition. Two parts of materials are planted in the vegetable and flower research institute of Chinese academy of agricultural sciences in Peking test field from 2 months to 6 months.

Sowing two seeds of the material in the greenhouse in the middle ten of 2 months, maintaining the temperature at 18-30 ℃ and the relative humidity at 50%, and growing under the natural illumination condition. In the middle and last ten days of 4 months (the Menga has appeared buds but has not yet bloomed), the method is planted in the experimental open field of Peking at the vegetable and flower research institute of Chinese academy of agricultural sciences. From middle and late ten days of 4 months to early 5 months, the lowest daily temperature is 7-17 ℃, D-10 fruits normally expand to form seedless fruits under the temperature condition (T1 for short), and small stiff fruits or fallen flowers and fallen fruits are formed at 03-2; the lowest temperature is 17-28 ℃ from 6 months to 7 months, and the two material fruits normally develop to form the seed fruits under the temperature condition (T2 for short). Two material samples were taken at two temperatures, T1 and T2, at the day of flowering, 2d, 4d, 10d, 15d and 20d of ovaries or fruits after flowering. Three biological repeated samples are taken at each period, the retrieved samples are wrapped by tinfoil paper and then put into liquid nitrogen for quick freezing, and the samples are stored in a refrigerator at the temperature of minus 80 ℃ for standby. The sample taken under the temperature condition of T1 is referred to as a low-temperature sample, the sample taken under the temperature condition of T2 is referred to as a normal-temperature sample, and the group consisting of the low-temperature sample and the normal-temperature sample is referred to as all samples.

2 test methods and results

2.1 Total RNA extraction and cDNA first Strand Synthesis

Total RNA was extracted from all samples using the Quick RNA Isolation Kit (Huayuyo, Beijing, China), and the detailed extraction method was according to the instructions in the Kit. The integrity of the RNA was checked by electrophoresis on a 1% agarose gel to ensure that the RNA was not degraded. And detecting the concentration of RNA by using a BioSpec-nano ultraviolet-visible micro spectrophotometer, wherein the OD260/OD280 of the RNA sample is between 1.9 and 2.2, the OD260/OD230 is not less than 2.0, and only the RNA sample meeting the conditions can be used for synthesizing cDNA.

The detection result of the extracted RNA sample in 1% agarose gel electrophoresis shows that the sample is two clear bands, the ratio of 28S to 18S is close to 2:1, and the dragging phenomenon does not occur, which indicates that the extracted RNA can be used for qualified quality and can be used for the subsequent synthesis of a first strand of cDNA.

First strand cDNA was synthesized using 5 × All-In-One Master Mix kit (applied biosystems abm, Vancouver, Canada), and the detailed synthesis method was also referred to the instructions attached to the kit. All RNA and cDNA samples were set up in three biological replicates. RNA for reverse transcription is uniformly quantified, and is subjected to reverse transcription to form cDNA, and then cDNA stock solution is diluted by 10 times and is stored in a refrigerator at the temperature of minus 20 ℃.

2.2 sources and primer design of candidate reference genes

The group of subjects to which the present inventors were assigned in 2016 obtained transcriptome sequencing data of 60 eggplant biological samples, and 18 genes having a suitable size of fpkm (amplified number of fragments of gold base of transcription sequence of peptides) and a small coefficient of variation among all biological samples were selected as candidate reference genes from among 8 ten thousand and 8 thousand genes. The CDS sequences of these 18 candidate reference genes were used to design primers for fluorescent quantitative PCR. The primer design adopts a PrimerSelect tool in a DNASTAR software package, and the specific parameters are set as follows: t is_mThe value is larger than 55 ℃, the primer length is 18-25bp, and the product length is 100-200 bp. Details of candidate reference genes and primers are shown in Table 1.

TABLE 1 candidate reference genes and primer information

Note: the gene sequence number is ID in Eggplant Genome DataBase.

2.3 real-time fluorescent quantitative PCR amplification

Primers required for the experiments were synthesized by Beijing Huada Gene Co.

Fluorescent quantitative PCR assay in Roche

480 fluorescent quantitative analyzer, Roche

480SYBR Green I Master kit fluorescent quantitative PCR kit. The specific reaction conditions are as follows:

(1) the total reaction volume was 10. mu.l, including 2. mu.l cDNA template (50 ng/. mu.l), 0.25. mu.l (0.5. mu.M) primers, 5. mu.l SYBR Green I Master Mix and 2.5. mu.l of nucleic acid-free water, respectively;

(2) the reaction procedure is as follows: pre-denaturation at 95 ℃ for 10min, amplification for 40 cycles (95 ℃ for 10s, 56 ℃ for 20s, 72 ℃ for 30s in single capture mode), melting at 95 ℃ for 5s, melting at 65 ℃ for 1min, continuous capture mode at 97 ℃ and cooling at 40 ℃ for 10 s.

All reactions of the samples included three biological replicates and three mechanical replicates. And analyzing the amplification efficiency and specificity of the candidate internal reference primers by using a standard curve and a melting curve.

2.4 drawing of Standard Curve

(1) Diluting cDNA solution obtained by reverse transcription by 10 times;

(2) diluting with the diluent as initial solution by 5-fold or 10-fold gradient dilution method (1/5,1/25,1/125,1/625 or 1/10,1/100,1/1000, 1/10000);

(3) three parallel fluorescent quantitative PCR reactions were performed using the above system and reaction program;

(4) and calculating three parallel Cq mean values, drawing standard curves under different template concentrations, and obtaining a curve equation, amplification efficiency and correlation coefficients.

2.5 reliability analysis of candidate reference Gene primers

The specificity of 18 to the candidate reference gene primers was checked by agarose gel electrophoresis at 3% concentration, and the results showed that the target bands were all single bands and matched the expected product size (FIG. 1). Furthermore, the fluorescent quantitative melting curve analysis also showed that their PCR products were single peaks (FIG. 2), indicating no non-specific primer dimer formation. Taken together, 18 pairs of primer sequences for candidate internal controls were shown to be specific and effective.

The analysis result of the standard curve shows that the amplification efficiency of the primers varies from 81.05% (D3) to 99.90% (D11), and meets the requirement of fluorescent quantitative PCR on the amplification efficiency of the primers. The correlation coefficient of the primer standard curve of 18 genes is different from 0.9607(D2) -0.9998(D6, D13) (Table 2), which shows that the template gradient dilution concentration is in a reasonable range, and the linear correlation of the standard curve is better. These primers are therefore reliable.

TABLE 2 internal reference gene primer parameters for the quantification of low temperature resistance-related gene expression in eggplant

2.6 expression stability analysis of candidate reference Gene

To evaluate the expression stability of 18 candidate internal reference genes in biological samples under different fruit development stages and different temperature conditions, 18 candidate internal reference genes were analyzed and stability ranked using two internal reference gene screening software (geonorm and bestkoeper) and an online comprehensive evaluation tool (RefFinder, http:// 150.216.56.64/reference. type). The principles and emphasis of these tools vary, and thus the expression stability results for the same gene may vary (Yim AK, Wong JW, Ku YS, Qin H, Chan TF, Lam HM using RNA-Seq data to expression reference genes available for gene expression in sobee. PLOS ONE. 2015; 10: e0136343: e 136343). And finally, comprehensively analyzing and judging the optimal reference genes under different conditions.

2.6.1 transcriptional expression abundance of candidate genes

In the quantitative fluorescent expression analysis, the level of the transcription expression level of a gene can be reflected by the Cq value of the gene. The larger the Cq value, the lower the expression level. The mean Cq values for all biological samples varied from 14.33-22.77, and D5 and D18 had the highest and lowest expression levels, respectively, in these samples (FIG. 3).

2.6.2GeNorm analysis results

The stability of the candidate reference gene was mainly determined by the stability value (M) when performing the geonorm analysis. Genes with an M value of less than 1.5 are considered to be stably expressed, and the smaller the M value, the higher the stability of gene expression (Vanderompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. genome biol. 2002; 3: RESECH 0034.Pubmed: 12184808). Table 3 shows the average stability values calculated for geNorm in the automatic analysis mode.

As shown in Table 3, the stability averages of the candidate reference genes at low temperature, normal temperature and all sample conditions were 0.39-1.10,0.41-0.96 and 0.46-1.04, respectively, and the stability averages were less than 1.5. Among all samples and the low temperature samples, D1 and D2 are the most stable two reference genes, and the corresponding M values are 0.46 and 0.39, respectively. D4 and D5 are the most stable reference genes under the condition of normal temperature, and the corresponding M is 0.41. In addition, D18 was the most unstable reference gene in all sample data sets, with corresponding M values of 1.10, 0.96 and 1.04, respectively. According to the analysis result of geonorm, the three sample cases D5, D4, D1 and D2 were considered in combination as the most stable-performing reference genes.

TABLE 3 mean values M of stability of candidate reference genes and stability ranking

GeNorm can be based on the paired variance (V) of the normalization factor of the candidate gene_n/V_n+1) To determine the number of the most suitable reference genes required. Default value is Vn/n +1 ═ 0.15. If Vn/n +1<0.15 indicates that the combination of n genes as reference genes is stable enough and the n +1 th gene is not required to be introduced. As shown in Table 4, the pair-wise variation values V of the low-temperature samples and all the samples_2/3Both are less than 0.15, indicating that only two reference genes are required in the two sample sets; and the pair-wise variation value V of the normal temperature sample_2/3Greater than 0.15 and V_3/4Less than 0.15, which indicates that three reference genes in the sample set can be accurately used for gene expression research.

TABLE 4 pairwise variation values of candidate reference genes

2.6.3BestKeeper analysis results

The BestKeeper can be used for analyzing the expression stability of the candidate reference genes, the software evaluates the expression stability of the candidate reference genes in a specific biological sample by calculating a Standard Deviation (SD) and a Coefficient of Variation (CV), the smaller the Standard Deviation (SD) and the Coefficient of Variation (CV), the more stable the gene expression is, the SD value of the candidate genes is more than 1, and the candidate genes are filtered, in the test, the average Cq value of 18 candidate reference genes in all biological samples is from 14.33 to 22.77, all the Cq values are in the same order of magnitude, so the stability of the candidate reference genes is judged according to the SD value, and the smaller the Standard Deviation (SD), the more stable the gene expression is. The SD calculated by BestKeeper is shown in table 5. The first three are D5, D1 and D4 in low temperature condition according to the order of SD value from small to large; d10, D5 and D8 are arranged at the first three positions under the normal temperature condition; the first three of all samples were D5, D1 and D10. It can be seen that D5 is the most stable reference gene in all samples under low temperature conditions, D10 is the most stable reference gene under normal temperature conditions, and D5 is also very stable, and ranks second. BestKeeper analysis showed that D5 was the most stable gene when the three samples were combined.

2.6.4RefFinder analysis results

The results of stability ranking of these 18 candidate reference genes by geonorm and bestkoeper are different (table 3, table 5). For comprehensive evaluation of the stability of these genes, M was calculated by RefFinder₂These genes were ranked. The results show that D5 is most stably expressed in three sets of low temperature, normal temperature and all samples, D4 and D1 are reference genes ranked second or third, 3 genes of D5, D1 and D4 are stably positioned at the first 3 positions in the stable ranking of each sample set, and are 3 genes which are most stably expressed in all samples at low temperature and normal temperature in 18 candidate reference genes. And the M value is less than 1.5 according to the geNorm result, and the SD is less than 1 according to the BestKeeper result, and the 3 genes are better reference genes.

Although studies have also reported the transcriptional expression levels of genes analyzed using an internal reference gene as a reference (Deng X, Zhou S, Hu W, Feng J, Zhang F, Chen L, Huang C, Luo Q, He Y, Yang G, et al (2013); Ectopic expression of what TaCIPK14, encoding a calceirin B-like protein-interacting protein enzyme kinase, contrast saline and color expression in. thiobacillus Plant,149(3): 367) 377; YaT, Peng H, Whitaker BD, Juric WM (2013), Difference expression of calcium/calcium-regulated SRs protein to stress biological and stress expression (1322, 2016) (500. thiobacillus strain) and cucumber strain, 18; Huang K, Skyo, Sk, K, but more Accurate and reliable results using multiple internal reference genes (Vanderomere J, De Press K, Pattyyn F, Poppe B, Van Roy N, De Paepe A, Speleman F.2002; Accurate knowledge of real-time quantification RT-PCR data by geographic knowledge of multiple internal control genes. genome Biology, 3(7): research0034.0031-research 0034.0011; Artico S, Nardeli, BrilhandO, gross-De-Sa, Alves-ferreria M (2010), Identification and evaluation of new reference genes in genetic engineering for use in clinical diagnosis of real-time outcome of real-time RT-quantification RTBMC Plant Biol,10: 49; wangying, Chenyajuan, Dingliumna, Weijianhua, Wangzhongzhi 2016. screening of genes for stable expression of different tissues and organs of Populus mauritiana plant physiology report (08): 1312) 1320. The assay was based on paired variance values (V) of candidate genes calculated by the geNorm software_n/V_n+1) (Table 3) it can be seen that (V) is present at low temperature and under all sample conditions₂/V₃< 0.15) thus the standard quantification requirements are met by using 2 reference genes, so that 2 genes arbitrarily selected from D5, D4 and D1 can be used as reference genes for expression quantification by combining, and the combinable genes comprise D5+ D4, D5+ D1 and D4+ D1. Under the condition of normal temperature (V)₃/V₄< 0.15), therefore, 3 reference genes can meet the requirements of standard quantification, and D5+ D4+ D1 is the best reference gene combination for expression analysis under the condition of normal temperature.

TABLE 5 stability ranking of 18 candidate reference genes by BestKeeper and RefFinder

Note: the genes with high to low stability are ranked outside the brackets, and the values inside the brackets are the SD value (BestKeeper) and the composite ranking value (RefFinder) corresponding to the genes outside the brackets, respectively.

2.7 comparison of stability of reference Gene

To further confirm the stability of the selected reference genes D5, D1 and D4 in this experiment, five reported reference genes-SAND, TBP, Expressed, SmEf1 α and SmTRX-and reference genes D5, D1 and D4 of the present invention were selected and tested according to the methods of steps 2.1, 2.3-2.6. Wherein, the primers of D5, D1 and D4 are shown in Table 7, the primer of D5 consists of D5-F and D5-R, D5-F is the single-stranded DNA shown in SEQ ID No.7, and D5-R is the single-stranded DNA shown in SEQ ID No. 8; the primer of D4 consists of D4-F and D4-R, D4-F is the single-stranded DNA shown in SEQ ID No.9, D4-R is the single-stranded DNA shown in SEQ ID No. 10; the primer of D1 consists of D1-F and D1-R, D1-F is the single-stranded DNA shown in SEQ ID No.11, and D1-R is the single-stranded DNA shown in SEQ ID No. 12.

According to previous reports, SAND, TBP and Expressed are most stably Expressed During the development of different fruits of eggplant (Kanakachari M, Solanke AU, Prahakaran N, Ahmad I, Dhandapani G, Jayabalan N, et al. evaluation of competent reference genes for normalization of qPCR gene expression students in brinjal (Solanum Melongana L.)) Dual fruit reduction stage. The primer sequences for these three genes are provided directly herein.

SmEf1 alpha and SmTRX are two most stable internal reference genes expressed in eggplants under high temperature stress (huge strength, plum planting well, Rootuobo, old and far-aged people, Jingqingmin, Li Zhengxing, Lideming, Sun Baojuan, Sun Leo, Sunwu, eggplant qRT-PCR internal reference gene screening and stability analysis under high temperature stress [ J ] horticultural science report, 2017,44(03): 475) 486); the primer sequences for these two genes are provided directly in the article.

The expression of the five control reference genes of SAND, TBP, Expressed, SmEf1 α and SmTRX in this experiment in all biological samples at step 2.6.1 is shown in FIG. 3, with an average cq value of 16.78-22.58, which is significantly higher than the average cq values of the three genes D1, D4 and D5, indicating that the expression abundance of the five control reference genes of SAND, TBP, Expressed, SmEf1 α and SmTRX is lower than that of the three reference genes D1, D4 and D5.

The five control reference genes SAND, TBP, Expressed, SmEf1 alpha and SmTRX were also compared for stability with the three reference genes D5, D1 and D4 screened in accordance with the present invention using three assays, getnorm, Bestkeeper and Refinder. The results showed that the stability of D1, D4, and D5 was superior to that of five control reference genes of SAND, TBP, Expressed, SmEf1 α, and SmTRX in all samples, whether at low temperature (table 6). Therefore, the three reference genes screened by the invention are stable and reliable. The three genes D1, D4 and D5 can be used as reference genes for gene expression analysis in fruits under low temperature and/or normal temperature conditions.

The three genes D1, D4 and D5 can be used as reference genes individually, and in order to improve the accuracy of the fluorescence quantitative PCR, the three genes D1, D4 and D5 can be combined to be used as a set of reference genes.

TABLE 6 stability comparison of the 8 selected reference genes

TABLE 7 primer sequences of reference genes of the present invention

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> vegetable and flower institute of Chinese academy of agricultural sciences

<120> reagent for eggplant Gene expression analysis and reference Gene used therefor

<130> GNCFH182202

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 1170

<212> DNA

<213> eggplant (Solanum Melongena L.)

<400> 1

atgggtaaag agaagactca catcagcatt gtggtcattg gccatgtcga ctctggaaag 60

tcgactacta ctggtcactt gatctataag cttggtggta ttgacaagcg tgttattgag 120

aggttcgaga aggaagctgc tgagatgaac aagaggtcat tcaagtatgc ctgggtgctt 180

gacaagctta aggctgaacg tgagcgtggt atcaccattg atattgcttt gtggaagttt 240

gagaccacta agtactactg cactgtgatt gatgcccccg gacacaggga ctttatcaag 300

aacatgatca ctggtacctc ccaggctgac tgtgctgtcc tgattattga ctccaccact 360

ggtggtttcg aagctggtat ctctaaagat ggatatgtgt catatgctaa gtcttgtctg 420

tgtggttttg agaggtctac taaccttgac tggtacaagg gaccaaccct ccttgaggct 480

cttgaccaga ttaatgagcc caagaggcca tcagacaagc ccctccgtct tccacttcag 540

gatgtttaca agattggtgg tattggaact gtccctgttg gtcgtgtaga gactggtatt 600

atcaagcctg gtatggttgt gacctttggc ccttctggtt tgacaactga agtcaagtct 660

gtagagatgc accacgaagc tctccaggag gcactccctg gtgacaatgt tgggttcaat 720

gttaagaatg ttgctgttaa ggatcttaag cgtggttatg ttgcatcaaa ctccaaggat 780

gacccagcca aggaagcggc cagcttcacc gcccaggtca tcatcatgaa ccatccaggc 840

cagattggaa acggatatgc cccagtgctg gattgccaca cctcccacat tgctgtcaag 900

tttgctgaga tcttgaccaa gattgacagg cgttcaggta aggaactcga gaaggagcct 960

aagttcttga agaatggtga tgctggtatg gttaagatga ttcccaccaa gcccatggtt 1020

gtcgagacct ttgctgagta cccaccattg ggtcgttttg ctgtgagaga catgaggcaa 1080

acagttgctg ttggtgttgt caagaacgtt gaaaagaagg acccaactgg tgccaaggtg 1140

accaaggctg cccagaagaa gggaaagtga 1170

<210> 2

<211> 389

<212> PRT

<213> eggplant (Solanum Melongena L.)

<400> 2

Met Gly Lys Glu Lys Thr His Ile Ser Ile Val Val Ile Gly His Val

1 5 10 15

Asp Ser Gly Lys Ser Thr Thr Thr Gly His Leu Ile Tyr Lys Leu Gly

20 25 30

Gly Ile Asp Lys Arg Val Ile Glu Arg Phe Glu Lys Glu Ala Ala Glu

35 40 45

Met Asn Lys Arg Ser Phe Lys Tyr Ala Trp Val Leu Asp Lys Leu Lys

50 55 60

Ala Glu Arg Glu Arg Gly Ile Thr Ile Asp Ile Ala Leu Trp Lys Phe

65 70 75 80

Glu Thr Thr Lys Tyr Tyr Cys Thr Val Ile Asp Ala Pro Gly His Arg

85 90 95

Asp Phe Ile Lys Asn Met Ile Thr Gly Thr Ser Gln Ala Asp Cys Ala

100 105 110

Val Leu Ile Ile Asp Ser Thr Thr Gly Gly Phe Glu Ala Gly Ile Ser

115 120 125

Lys Asp Gly Tyr Val Ser Tyr Ala Lys Ser Cys Leu Cys Gly Phe Glu

130 135 140

Arg Ser Thr Asn Leu Asp Trp Tyr Lys Gly Pro Thr Leu Leu Glu Ala

145 150 155 160

Leu Asp Gln Ile Asn Glu Pro Lys Arg Pro Ser Asp Lys Pro Leu Arg

165 170 175

Leu Pro Leu Gln Asp Val Tyr Lys Ile Gly Gly Ile Gly Thr Val Pro

180 185 190

Val Gly Arg Val Glu Thr Gly Ile Ile Lys Pro Gly Met Val Val Thr

195 200 205

Phe Gly Pro Ser Gly Leu Thr Thr Glu Val Lys Ser Val Glu Met His

210 215 220

His Glu Ala Leu Gln Glu Ala Leu Pro Gly Asp Asn Val Gly Phe Asn

225 230 235 240

Val Lys Asn Val Ala Val Lys Asp Leu Lys Arg Gly Tyr Val Ala Ser

245 250 255

Asn Ser Lys Asp Asp Pro Ala Lys Glu Ala Ala Ser Phe Thr Ala Gln

260 265 270

Val Ile Ile Met Asn His Pro Gly Gln Ile Gly Asn Gly Tyr Ala Pro

275 280 285

Val Leu Asp Cys His Thr Ser His Ile Ala Val Lys Phe Ala Glu Ile

290 295 300

Leu Thr Lys Ile Asp Arg Arg Ser Gly Lys Glu Leu Glu Lys Glu Pro

305 310 315 320

Lys Phe Leu Lys Asn Gly Asp Ala Gly Met Val Lys Met Ile Pro Thr

325 330 335

Lys Pro Met Val Val Glu Thr Phe Ala Glu Tyr Pro Pro Leu Gly Arg

340 345 350

Phe Ala Val Arg Asp Met Arg Gln Thr Val Ala Val Gly Val Val Lys

355 360 365

Asn Val Glu Lys Lys Asp Pro Thr Gly Ala Lys Val Thr Lys Ala Ala

370 375 380

Gln Lys Lys Gly Lys

385

<210> 3

<211> 639

<212> DNA

<213> eggplant (Solanum Melongena L.)

<400> 3

atggacgcag gtgtagtagc cgccccccct gctgccaatg caaaggaaga gaacaaggtt 60

cacaccgatg tcatgctttt caatcgctgg agctatgatg atgttcagat caatgacatg 120

tctgttgagg attacatcac agcaactcct aacaagcatc cagtttacat gccacacaca 180

gctggtagat accaggccaa gcgtttcagg aaggcccagt gcccaattgt tgagaggctc 240

accaattctc tcatgatgca cggaaggaac aatggaaaga agctaatggc tgttcgcatt 300

ataaagcatg caatggagat cattcatttg ttgacagacc aaaacccaat tcaagtcatt 360

gttgatgctg ttatcaacag tgggccaagg gaagatgcaa cacgtattgg ttcagctggt 420

gttgtgagac gtcaagccgt tgatatttct ccacttcgtc gtgttaacca ggcaatttat 480

ttgctgacaa ctggtgcacg tgagagtgct ttcaggaaca tcaagaccat agctgaatgc 540

cttgcagatg aactcatcaa tgctgccaag ggttcttcaa atagctatgc cattaagaag 600

aaggatgaga ttgaaagggt tgccaaggcc aatcgttaa 639

<210> 4

<211> 212

<212> PRT

<213> eggplant (Solanum Melongena L.)

<400> 4

Met Asp Ala Gly Val Val Ala Ala Pro Pro Ala Ala Asn Ala Lys Glu

1 5 10 15

Glu Asn Lys Val His Thr Asp Val Met Leu Phe Asn Arg Trp Ser Tyr

20 25 30

Asp Asp Val Gln Ile Asn Asp Met Ser Val Glu Asp Tyr Ile Thr Ala

35 40 45

Thr Pro Asn Lys His Pro Val Tyr Met Pro His Thr Ala Gly Arg Tyr

50 55 60

Gln Ala Lys Arg Phe Arg Lys Ala Gln Cys Pro Ile Val Glu Arg Leu

65 70 75 80

Thr Asn Ser Leu Met Met His Gly Arg Asn Asn Gly Lys Lys Leu Met

85 90 95

Ala Val Arg Ile Ile Lys His Ala Met Glu Ile Ile His Leu Leu Thr

100 105 110

Asp Gln Asn Pro Ile Gln Val Ile Val Asp Ala Val Ile Asn Ser Gly

115 120 125

Pro Arg Glu Asp Ala Thr Arg Ile Gly Ser Ala Gly Val Val Arg Arg

130 135 140

Gln Ala Val Asp Ile Ser Pro Leu Arg Arg Val Asn Gln Ala Ile Tyr

145 150 155 160

Leu Leu Thr Thr Gly Ala Arg Glu Ser Ala Phe Arg Asn Ile Lys Thr

165 170 175

Ile Ala Glu Cys Leu Ala Asp Glu Leu Ile Asn Ala Ala Lys Gly Ser

180 185 190

Ser Asn Ser Tyr Ala Ile Lys Lys Lys Asp Glu Ile Glu Arg Val Ala

195 200 205

Lys Ala Asn Arg

210

<210> 5

<211> 1383

<212> DNA

<213> eggplant (Solanum Melongena L.)

<400> 5

atgcggaggt taaatcgact cagcctttgc aaatatgaca cagtccccac taagggtggc 60

aacgagatca gaaccaaaac tggtatcacc ggatcggact ttaccgaaac cggaccgata 120

ccggtaataa aaggacaaaa tttctctaga accgtaccga tattggcagt accgggaaat 180

tttaacatta ccttactggt ccggactttg tttaacgttg caatggcaga tatgaaccac 240

cacccaactg ttttccagaa ggcttctaac cagctgcatt tgagctcaag tctatcccaa 300

gatgtccatg ggcgctatgg aggtgttcag cctgctcttt accagaggca ttttgcttat 360

ggcaactact ccaatgcagg actgctgaga ggccaagcca ctcaggatct atcattgatc 420

acctcaaatg cctcacctgt gtttgtgcag gctcctcaag agaaaggatt tgcagctttt 480

gctactgact ttctcatggg tggagtttct gctgctgtat caaagactgc tgctgcccct 540

attgagcgtg tgaaactttt gattcaaaat caagatgaga tgctcaaggc tggtaggctc 600

tctgaaccat acaagggaat tggcgattgt ttcagcagaa caattaagga tgaaggtgtc 660

atgtctttat ggaggggaaa cactgccaat gttatccgtt atttccccac tcaggccctg 720

aactttgcat tcaaggacta tttcaagaga ctcttcaact tcaagaagga ccgtgatggt 780

tactggaagt ggtttgccgg caaccttgca tcaggtggtg ctgctggtgc ttcctctttg 840

ttctttgtct actccttgga ctatgctcgt acccgtctcg ctaatgacgc caaagcttca 900

aagaagggag gtgagaggca gttcaatggt ttggttgatg tctacaggaa gacactcaaa 960

tctgatggaa ttgctggtct ataccgtgga ttcaacattt catgtgttgg tatcattgtt 1020

taccgtggtt tgtactttgg aatgtatgac tcgttgaagc ctgtcctcct gactggaagc 1080

ctgcaggata gtttctttgc tagctttgga cttggttggc tcatcaccaa tggtgctggt 1140

cttgcttctt acccaatcga tacagtaaga agaagaatga tgatgacatc tggtgaggca 1200

gttaagtaca agagctcgct cgatgcattc agccagattg ttaagaatga gggagccaaa 1260

tctctgttca agggtgccgg tgctaacatc ctccgagctg ttgctggtgc tggtgtgttg 1320

gctggatatg acaagcttca ggttcttgtt ttgggaaaga aatatggatc cggtggtgcc 1380

taa 1383

<210> 6

<211> 460

<212> PRT

<213> eggplant (Solanum Melongena L.)

<400> 6

Met Arg Arg Leu Asn Arg Leu Ser Leu Cys Lys Tyr Asp Thr Val Pro

1 5 10 15

Thr Lys Gly Gly Asn Glu Ile Arg Thr Lys Thr Gly Ile Thr Gly Ser

20 25 30

Asp Phe Thr Glu Thr Gly Pro Ile Pro Val Ile Lys Gly Gln Asn Phe

35 40 45

Ser Arg Thr Val Pro Ile Leu Ala Val Pro Gly Asn Phe Asn Ile Thr

50 55 60

Leu Leu Val Arg Thr Leu Phe Asn Val Ala Met Ala Asp Met Asn His

65 70 75 80

His Pro Thr Val Phe Gln Lys Ala Ser Asn Gln Leu His Leu Ser Ser

85 90 95

Ser Leu Ser Gln Asp Val His Gly Arg Tyr Gly Gly Val Gln Pro Ala

100 105 110

Leu Tyr Gln Arg His Phe Ala Tyr Gly Asn Tyr Ser Asn Ala Gly Leu

115 120 125

Leu Arg Gly Gln Ala Thr Gln Asp Leu Ser Leu Ile Thr Ser Asn Ala

130 135 140

Ser Pro Val Phe Val Gln Ala Pro Gln Glu Lys Gly Phe Ala Ala Phe

145 150 155 160

Ala Thr Asp Phe Leu Met Gly Gly Val Ser Ala Ala Val Ser Lys Thr

165 170 175

Ala Ala Ala Pro Ile Glu Arg Val Lys Leu Leu Ile Gln Asn Gln Asp

180 185 190

Glu Met Leu Lys Ala Gly Arg Leu Ser Glu Pro Tyr Lys Gly Ile Gly

195 200 205

Asp Cys Phe Ser Arg Thr Ile Lys Asp Glu Gly Val Met Ser Leu Trp

210 215 220

Arg Gly Asn Thr Ala Asn Val Ile Arg Tyr Phe Pro Thr Gln Ala Leu

225 230 235 240

Asn Phe Ala Phe Lys Asp Tyr Phe Lys Arg Leu Phe Asn Phe Lys Lys

245 250 255

Asp Arg Asp Gly Tyr Trp Lys Trp Phe Ala Gly Asn Leu Ala Ser Gly

260 265 270

Gly Ala Ala Gly Ala Ser Ser Leu Phe Phe Val Tyr Ser Leu Asp Tyr

275 280 285

Ala Arg Thr Arg Leu Ala Asn Asp Ala Lys Ala Ser Lys Lys Gly Gly

290 295 300

Glu Arg Gln Phe Asn Gly Leu Val Asp Val Tyr Arg Lys Thr Leu Lys

305 310 315 320

Ser Asp Gly Ile Ala Gly Leu Tyr Arg Gly Phe Asn Ile Ser Cys Val

325 330 335

Gly Ile Ile Val Tyr Arg Gly Leu Tyr Phe Gly Met Tyr Asp Ser Leu

340 345 350

Lys Pro Val Leu Leu Thr Gly Ser Leu Gln Asp Ser Phe Phe Ala Ser

355 360 365

Phe Gly Leu Gly Trp Leu Ile Thr Asn Gly Ala Gly Leu Ala Ser Tyr

370 375 380

Pro Ile Asp Thr Val Arg Arg Arg Met Met Met Thr Ser Gly Glu Ala

385 390 395 400

Val Lys Tyr Lys Ser Ser Leu Asp Ala Phe Ser Gln Ile Val Lys Asn

405 410 415

Glu Gly Ala Lys Ser Leu Phe Lys Gly Ala Gly Ala Asn Ile Leu Arg

420 425 430

Ala Val Ala Gly Ala Gly Val Leu Ala Gly Tyr Asp Lys Leu Gln Val

435 440 445

Leu Val Leu Gly Lys Lys Tyr Gly Ser Gly Gly Ala

450 455 460

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gggtaaagag aagactcaca tcag 24

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cacccaggca tacttgaatg ac 22

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ccaggccaag cgtttcagga 20

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

cccttggccc actgttgata ac 22

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ggtgctgctg gtgcttcctc ttt 23

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

aactgcctct cacctccctt cttt 24

Claims (6)

1. The application of any one of the following M1-M3 as an internal reference gene in eggplant gene expression analysis:

m1, a set of two genes consisting of D1 and D4;

M2、D1；

M3、D4；

M1-M3, wherein D4 is a gene encoding a protein having an amino acid sequence of SEQ ID No. 4; d1 is a gene encoding a protein of which the amino acid sequence is SEQ ID No. 6;

the eggplant gene expression analysis is gene expression analysis in eggplant fruits under a low-temperature condition and/or a normal-temperature condition.

2. Use according to claim 1, characterized in that: the D4 is a DNA molecule with a coding sequence of SEQ ID No. 3; the D1 is a DNA molecule with a coding sequence of SEQ ID No. 5.

3. An agent for eggplant gene expression analysis, which is any one of the following E1-E3:

e1, consisting of a primer set named W4 for detecting the expression level of D4 as described in claim 1 or 2 and a primer set named W1 for detecting the expression level of D1 as described in claim 1 or 2;

e2, primer pair for detecting the expression level of D1 described in claim 1 or 2 under the name of W1;

e3, primer pair for detecting the expression level of D4 described in claim 1 or 2 under the name of W4;

the eggplant gene expression analysis is gene expression analysis in eggplant fruits under a low-temperature condition and/or a normal-temperature condition;

the W4 is a D4 specific primer pair consisting of D4-F and D4-R, the D4-F is a single-stranded DNA shown in SEQ ID No.9, and the D4-R is a single-stranded DNA shown in SEQ ID No. 10;

the W1 is a D1 specific primer pair consisting of D1-F and D1-R, the D1-F is a single-stranded DNA shown in SEQ ID No.11, and the D1-R is a single-stranded DNA shown in SEQ ID No. 12.

4. A kit for eggplant gene expression analysis containing the reagent of claim 3;

the eggplant gene expression analysis is gene expression analysis in eggplant fruits under a low-temperature condition and/or a normal-temperature condition.

5. Use of the agent of claim 3 in eggplant gene expression analysis;

the eggplant gene expression analysis is gene expression analysis in eggplant fruits under a low-temperature condition and/or a normal-temperature condition.

6. Use of the kit of claim 4 for the analysis of gene expression in eggplant;

the eggplant gene expression analysis is gene expression analysis in eggplant fruits under a low-temperature condition and/or a normal-temperature condition.

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