Brassica campestris nitrate stress reference gene as well as primer and application thereof

Abstract

The invention discloses a brassica chinensis nitrate stress reference gene and primers and application thereof, and relates to the field of molecular biology, the reference gene is a Bc60S RPL4-1 gene, the nucleotide sequence of the Bc60S RPL4-1 gene is shown as SEQ ID NO: 2, and the nucleotide sequence of the Bc60S RPL4-1 gene is shown as SEQ ID NO: 3. A primer for amplifying the reference gene comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 10 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 11. The invention provides a reference gene Bc60S RPL4-1 gene which is highly stable in expression in brassica chinensis nitrate stress research, and the accuracy and reliability of quantitative analysis of brassica chinensis nitrate stress related gene expression are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, and in particular to a Brassica parachinensis nitrate stress reference gene, primers thereof and application. BACKGROUND

[0002] Brassica parachinensis is an important leafy vegetable in China, with short growth cycle and strong adaptability, and is widely cultivated in South China. However, in modern intensive agricultural production, excessive application of nitrogen fertilizer leads to soil nitrate accumulation, which may cause stress to Brassica parachinensis growth and affect its yield and quality. Nitrate is an important nitrogen source for plants, but high concentration of nitrate can inhibit root development, interfere with photosynthesis, and even induce oxidative stress, leading to leaf yellowing and biomass reduction. In addition, excessive accumulation of nitrate in vegetables can also harm human health through the food chain. Therefore, it is of great significance to study the molecular response mechanism of Brassica parachinensis to nitrate stress for optimizing nitrogen fertilizer management and breeding nitrate-tolerant varieties.

[0003] Real-time fluorescence quantitative PCR (RT-qPCR) is a key technology for studying gene expression in plant stress response, but its accuracy is highly dependent on stable expression of reference genes. Reference genes are used to correct the RNA extraction efficiency, reverse transcription differences and amplification bias between samples to ensure the reliability of target gene expression data. However, the expression stability of traditional reference genes (such as Actin 、 GAPDH 、 18S rRNA ) is easily affected by environmental conditions. Previous studies have shown that nitrate stress may interfere with plant basic metabolism, leading to fluctuations in the expression of some commonly used reference genes. If these genes are directly selected for standardization, it may cause data distortion. However, there is no systematic report on the screening of Brassica parachinensis reference genes under nitrate stress, which limits the accuracy of related molecular mechanism research. Therefore, it is urgent to screen suitable nitrate stress reference genes to lay a foundation for subsequent Brassica parachinensis gene expression research. SUMMARY

[0004] The present application provides a Brassica parachinensis nitrate stress reference gene, primers thereof and application to solve the above problems.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows: Technical scheme one: a Brassica parachinensis nitrate stress reference gene, the reference gene is Bc60S RPL4-1 gene, the nucleotide sequence of the Bc60S RPL4-1 gene is shown as SEQ ID NO: 2.

[0006] Technical solution two: a primer for amplifying the above-mentioned internal reference gene, the primer comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 10 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 11.

[0007] Technical solution three: application of the above-mentioned internal reference gene or primer in screening Brassica parachinensis nitrate-tolerant varieties.

[0008] Technical solution four: application of the above-mentioned internal reference gene or primer in expression analysis of related genes under nitrate stress of Brassica parachinensis.

[0009] Compared with the prior art, the beneficial effects of the present application are: The present application provides an internal reference gene with high expression stability in the study of nitrate stress of Brassica parachinensis Bc60S RPL4-1 , which significantly improves the accuracy and reliability of quantitative analysis of nitrate stress-related gene expression of Brassica parachinensis, is suitable for different nitrate-tolerant varieties of Brassica parachinensis, has universality, has important practical application value and scientific significance for in-depth analysis of the molecular mechanism of nitrate stress response of Brassica parachinensis, breeding of new nitrate-tolerant varieties, and provides an important reference for the study of abiotic stress of Brassica parachinensis and other cruciferous plants. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is the verification of the internal reference gene of Brassica parachinensis under nitrate stress conditions. DETAILED DESCRIPTION

[0011] In order to make the purpose and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0012] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods, etc. already existing in the prior art.

[0013] Example 1 1. Test materials and treatment Lianzhou F1 (nitrate-tolerant variety) and Jinqiuhong No. 2 (nitrate-sensitive variety) (purchased from Guangzhou Changhe Seed Co., Ltd.) were used as experimental materials. The nitrate stress tolerance of these two varieties has been proven in previous reports (Zhang Ying, Li Haijun, Gao Fucheng, et al. Screening of salt-tolerant varieties of Chinese cabbage and its salt tolerance mechanism [J]. Journal of North China Agricultural University, 2023, 38(S1):170-179.). Sponge blocks were used for seedling cultivation. When the seedlings reached the three-leaf stage, healthy seedlings with uniform growth were selected and transplanted into hydroponic boxes containing 5L of nutrient solution, with 6 seedlings per box. The macronutrient solution used a 1 / 2 Hoagland formula, and the micronutrient solution used a general formula. After transplanting, seedlings were subjected to 80mmol / L nitrate stress treatment for 3 days, of which NO3- - Provided by Ca(NO3)2·4H2O. Leaves were collected from plants subjected to nitrate stress for 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, with 10 plants per treatment and 3 biological replicates.

[0014] 2. Internal reference gene screening and primer design Because of the lack of genomic data for Chinese cabbage, in Arabidopsis thaliana ( Arabidopsis thaliana ) and cabbage ( Brassiceae The internal reference gene sequences were obtained from the genome as reference sequences. TBLASTn was performed on the *Caulis Caulis Peperomia* transcriptome data to obtain the corresponding homologous sequences, resulting in the seven candidate internal reference genes required for this embodiment: BcActin (SEQ ID NO.1) Bc60S RPL4-1 (SEQ ID NO.2) BcGAPDH (SEQ ID NO.3) BcTUB (SEQ ID NO.4) BcUQCC1 (SEQ ID NO.5) BcSDAF2 (SEQ ID NO.6) BcCYP (SEQ ID NO.7). Primer combinations are shown in Table 1.

[0015] Table 1. Information on candidate internal reference genes and primers

[0016] 3. RNA extraction and cDNA synthesis Total RNA was extracted from Chinese cabbage leaves under each treatment according to the Trizol reagent instructions (Beijing TransGen Biotech Co., Ltd.). Genomic DNA was removed using a cDNA synthesis kit (Beijing TransGen Biotech Co., Ltd.), and cDNA was synthesized by reverse transcription. The cDNA was diluted to 100 ng / μL and used as a template for RT-qPCR experiments.

[0017] 4. qRT-PCR Real-time fluorescence quantitative analysis was performed according to the Premix Ex Taq™ (Perfect Real Time) (TaKaRa, Japan) kit. The reaction system was as follows: 10 μL: 2 x TransStart® Green qPCR Super Mix 5 μL, forward and reverse primers (10 μmol·L -1 ) 0.2 μL each, ddH2O 3.1 μL, cDNA (10 ng·μL -1 ) 1.5 μL. The reaction program was as follows: 94°C, 30 s, [94°C, 20 s; 56°C, 30 s; 72°C, 20 s] x 40 cycles; melting curve program: 65°C heating to 90°C, 5 s. Each reaction was repeated three times.

[0018] 5. Data and result analysis After PCR, the gene amplification was analyzed automatically, and the corresponding domain value cycle number, i.e., Ct value, was exported, as shown in Table 2.

[0019] Table 2 Ct average value of candidate internal reference genes in Brassica parachinensis leaves under nitrate stress at different treatment times

[0020] The Ct value obtained after the qRT-PCR reaction was analyzed for the stability of each candidate internal reference gene using GeNorm, Bestkeeper, Normfinder, and ReFinder software. GeNorm software calculated the expression stability M value of each candidate internal reference gene, and the higher the M value, the poorer the stability. NormFinder software calculated the stability value of each candidate internal reference gene by combining the within-group variance and between-group variance, and the smaller the value, the more stable the internal reference gene. Bestkeeper can directly input the Ct value, and by calculating the standard deviation (SD) and coefficient of variation (CV) value, the stability of each internal reference gene is compared, and the smaller the standard deviation and coefficient of variation, the better the stability.

[0021] 6. Stability analysis result The expression stability M value of each candidate gene was calculated by GeNorm software, and the smaller the M value, the higher the stability; on the contrary, the larger the M value, the less stable. The stability analysis by GeNorm software from strong to weak is as follows: Bc60S RPL4-1 = BcSDAF2 > BcGAPDH > BcActin > BcCYP > BcTUB > BcUQCC1 .

[0022] NormFinder software combined with the variance within the group and the variance between groups to calculate the stability of each candidate reference gene M, M value is smaller, the more stable reference gene; otherwise M value is higher stability is worse. NormFinder software stability analysis from strong to weak order: Bc60S RPL4-1 BcSDAF2 BcActin BcGAPDH BcTUB BcCYP BcUQCC1

[0023] Bestkeeper software can directly input Ct value, by calculating the standard deviation and coefficient of variation value size comparison of each reference gene stability, the smaller the standard deviation and coefficient of variation, the better stability. Bestkeeper software stability analysis from strong to weak order: Bc60S RPL4-1 BcSDAF2 BcActin BcGAPDH BcTUB BcCYP BcUQCC1

[0024] Using ReFinder software to GeNorm, NormFinder, Bestkeeper method analysis of stability ranking of geometric mean, get the comprehensive index ranking, the smaller the index, the more stable expression of reference genes. The results are shown in Table 3, the stability of the gene under high temperature stress: Bc60S RPL4-1 BcSDAF2 BcActin BcGAPDH BcTUB BcCYP BcUQCC1

[0025] Table 3 expression stability of candidate reference genes in Brassica parachinensis leaves under nitrate stress at different times

[0026] 7, reference gene fluorescence quantitative PCR verification experiment In order to further verify the stability of the selected candidate reference genes, the first stability ranking Bc60S RPL4-1 and the second stability ranking BcSDAF2 as a reference gene, while the worst stability BcCYP and BcUQCC1 as a control, analysis of Brassica parachinensis nitrate transporter gene BcNRT1;1 gene expression pattern, the existing research has proved BcNRT1;1 ​​​​​​​​​​​​​​​​​​​​​In the cabbage heart under nitrate stress induced significant up-regulation (Zhang Y P. Cloning and expression characteristics of cabbage nitrate transporter gene NRTs[D]. South China Agricultural University, 2016.), the primer sequence of the gene is: F:5'-CCAACGACCTCGTCTCAT-3'(SEQ ID NO:22) R:5'-CCTTCACTCCTCCAGTTCCTT-3'(SEQ ID NO:23).

[0027] The results are shown in Figure 1 and Bc60S RPL4-1 and BcSDAF2 As the internal reference gene, the expression of the target gene BcNRT1;1 In Lianzhou F1 and Jinqiuhong No. 2 varieties showed a rising trend first and then decreased; but BcCYP and BcUQCC1 When the internal reference gene, the expression mode is different from Bc60S RPL4-1 and BcSDAF2 As a reference, and the results of previous studies are also different.

[0028] Comprehensive results above, the present experiment verified Bc60S RPL4-1 In cabbage nitrate stress related gene expression analysis as a reference gene stability and reliability.

[0029] The above only for the preferred embodiments of the present application, and not to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application, should be included in the scope of protection of the present application.

Claims

1. A Brassica napus nitrate stress reference gene, characterized in that: The internal reference gene is Bc60S RPL4-1 The nucleotide sequence of the gene is shown as SEQ ID NO:

2. Bc60S RPL4-1 The nucleotide sequence of the gene is shown as SEQ ID NO:

2.

2. Primer for amplifying the internal control gene according to claim 1, characterized in that: The primers comprise a forward primer with a nucleotide sequence as shown in SEQ ID NO. 10 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

11.

3. The use of the reference gene of claim 1 or the primers of claim 2 in screening nitrate-tolerant varieties of Brassica parachinensis.

4. The use of the reference gene of claim 1 or the primers of claim 2 in analyzing the expression of related genes under nitrate stress of Brassica parachinensis.

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