CN109609512B - Application of Phalaenopsis PP2A Gene as Internal Reference Gene - Google Patents

Abstract

The invention belongs to the technical field of Phalaenopsis genetic engineering, in particular to the matter of applying for a patent application of a Phalaenopsis PP2A gene as an internal reference gene. The PP2A gene cDNA sequence is 2207 bp in length, including a complete ORF of 1764 bp in length, encoding phosphoprotease 2A (PP2A). Further research on the expression of the PP2A gene under the conditions of normal growth temperature and low temperature stress of Phalaenopsis by real-time quantitative PCR technology showed that the gene expression was relatively stable, and it had the potential to be used as an internal reference gene. When it was further used as an internal reference gene to analyze the expression of PhNAC1 gene, it showed more accurate analysis results. It can lay a good foundation for analysis and application for the screening of cold tolerance genes of Phalaenopsis and the screening of cold tolerance germplasm resources, so it has good practical value and scientific research application significance.

Description

Application of Phalaenopsis PP2A Gene as Internal Reference Gene

technical field

The invention belongs to the technical field of Phalaenopsis genetic engineering, in particular to the matter of applying for a patent application of a Phalaenopsis PP2A gene as an internal reference gene.

Background technique

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotes, consisting of structural subunit A, catalytic subunit C and regulatory subunit B. Controls signal transduction and cellular metabolism. In animals and yeast, PP2A functions in multiple signaling pathways. However, its role in plant signaling pathways is poorly understood, and it is currently found to be mainly involved in the signal transduction of hormones such as abscisic acid, phytogenin, and ethylene.

Phalaenopsis spp. is a general term for the single-stemmed epiphytic orchid of the Orchidaceae Phalaenopsis genus. Because of its beautiful flower shape, colorful flower color, long flowering period and high ornamental value, it has become one of the most popular Chinese New Year flowers for many years. Changpin. Native to tropical and subtropical regions, it likes warmth and aversion to cold, and the optimum temperature for growth is 18 to 28 °C. In winter, it will stop growing below 10 °C. Therefore, low temperature is an important environmental factor affecting the growth of Phalaenopsis. However, in northern my country, the temperature in winter and spring is relatively low. If phalaenopsis needs to be cultivated, it needs to be cultivated in a modern greenhouse. However, the heating cost is high and the energy consumption is huge, which leads to a sharp rise in production costs and restricts the healthy development of the phalaenopsis industry. . Therefore, cold tolerance resources and gene mining are one of the important goals of Phalaenopsis breeding.

Real-time quantitative PCR is one of the more commonly used techniques in the screening of cold tolerance genes in Phalaenopsis. One of the application foundations of real-time fluorescence quantitative PCR technology is the selection of suitable internal reference genes. In the prior art, many reference genes have been reported for Phalaenopsis. However, it is well known that: in principle, the internal reference gene is a gene that is constantly expressed under certain conditions, so as to detect the expression level of the target gene under specific conditions as a reference; but in actual work, it can be stably expressed under any conditions. Internal reference genes are almost non-existent. Therefore, it is necessary to screen the most suitable reference genes for different situations. As far as the screening of cold tolerance genes in Phalaenopsis is concerned, there is no good report that can be used as an internal reference gene in the screening of cold tolerance genes.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a Phalaenopsis PP2A gene. Studies have shown that this gene can be stably expressed in different Phalaenopsis varieties and at different times under normal growth temperature and low temperature stress conditions, so it has the potential to be used as an internal reference gene. The internal reference gene can lay a certain technical foundation for the research of related functional genes of Phalaenopsis and the screening of low temperature resistant Phalaenopsis resources.

The technical solution adopted in this application is described in detail as follows.

Phalaenopsis PP2A gene, the gene expression is relatively stable and consistent under normal growth temperature and low temperature stress conditions, the PP2A gene, its cDNA sequence length is 2207 bp, including a complete, 1764 bp long ORF, the specific cDNA base The sequence is shown in SEQ ID NO.1.

For this gene, when using PCR to amplify, the primer sequences can be designed as follows:

PP2A-F: 5'-TTTGAGYGATTTGTGAAGGC-3',

PP2A-R: 5'-TGGAAAAAMATAACAGCAGG-3'.

Phospho orchid PP2A gene encodes phosphoprotease 2A (PP2A), the protein includes 587 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

The application of the Phalaenopsis PP2A gene as an internal reference gene is used to analyze and evaluate the expression of related functional genes, for example, for the expression analysis of the functional gene NAC domain protein gene ( PhNAC1 ).

The real-time fluorescence quantitative PCR detection and analysis method using the Phalaenopsis PP2A gene as an internal reference gene, during detection and analysis, for the Phalaenopsis PP2A gene, the primer sequences are designed as follows:

qPP2A-F: 5'-TCTGTTGGCTGTGGAAGGAT-3',

qPP2A-R: 5'-AAATCCGACCTGGTAGTTTCTG-3'.

The real-time fluorescence quantitative PCR detection and analysis method of the described Phalaenopsis PP2A gene as an internal reference gene, when detecting and analyzing the expression of PhNAC1 , for the NAC domain protein gene ( PhNAC1 ), the primer sequences are designed as follows:

qPhNAC1-F: 5'-ATCTGAACAAGTGCGAGCCT-3',

qPhNAC1-R: 5'-ATCCTTACCAGTTGCCTTCC-3'.

This application is the first time to mine the PP2A gene cDNA in the low temperature stress transcriptome database of Phalaenopsis. The analysis of the gene shows that the cDNA sequence contains a complete ORF with a length of 1764 bp, encoding phosphoprotease 2A (PP2A). Further research on the expression of the PP2A gene under the conditions of normal growth temperature and low temperature stress of Phalaenopsis by real-time quantitative PCR technology showed that the gene expression was relatively stable, and it had the potential to be used as an internal reference gene. When it was further used as an internal reference gene to analyze the expression of PhNAC1 gene, it showed more accurate analysis results.

On the whole, the PP2A gene provided by the application is relatively stable in expression under low temperature stress, and by utilizing this characteristic, a good analytical application foundation can be laid for the screening of cold tolerance genes of Phalaenopsis and the selection of cold tolerance Phalaenopsis germplasm resources, Therefore, it has good practical value and scientific research application significance.

Description of drawings

Fig. 1 is the melting curve of real-time fluorescence quantitative PCR of Phalaenopsis PP2A gene;

Fig. 2 is the standard curve of real-time fluorescence quantitative PCR of Phalaenopsis PP2A gene;

Figure 3 shows the fluorescence thresholds of PP2A gene in different varieties of Phalaenopsis under low temperature stress at different times and real-time fluorescence quantitative PCR;

(Note: D: Phalaenopsis variety "Big Pepper"; F: Phalaenopsis variety "Fule Sunset"; CK1: normal growth temperature; CK2: 16℃/11℃ after treatment for 3 days; T1: 11℃/6℃ After treatment for 1 day; T2: After treatment at 11℃/6℃ for 2 days; T3: After treatment at 11℃/6℃ for 3 days; T5: After treatment at 11℃/6℃ for 5 days; T7: After treatment at 11℃/6℃ for 7 days; 0 h : The normal growth temperature of the Phalaenopsis variety "Da Chili"; 1 h, 2 h, 4 h, 12 h, 24 h, 48 h: The Phalaenopsis variety "Da Chili" was treated at 4 ℃ for 1 h, 2 h, 4 h, 12 h , 24 h, 48 h);

Fig. 4 is the expression electropherogram of PP2A in different varieties of Phalaenopsis under low temperature stress at different times;

Figure 5 shows the application of PP2A as an internal reference gene in the quantitative expression study of PhNAC1 gene in Phalaenopsis;

Figure 6 shows the application of Actin as an internal reference gene in the quantitative expression study of PhNAC1 gene in Phalaenopsis.

Detailed ways

The application will be further explained below in conjunction with the examples. Before introducing specific embodiments, a brief introduction and description of some experimental backgrounds in the following embodiments are as follows.

biomaterials:

Phalaenenopsis Big Chili and Phalaenenopsis Fuller's Sunset, both of which are common Phalaenopsis cultivars; the materials used in the following examples are taken from the Orchid Engineering Technology Center of Zhengzhou Normal University Inside a smart solar greenhouse;

Synthesis and sequencing of relevant primer sequences were provided by Shanghai Yingjun Biotechnology Company;

Experimental reagents:

（用于实时荧光定量PCR）等，购自TaKaRA公司；Reverse Transcriptase M-MLV reagent (reverse transcription preparation for PCR), PrimeScriptRT reagent Kit With gDNA Eraser (reverse transcription preparation for real-time PCR), SYBR®Premix Ex TaqTM

(for real-time fluorescent quantitative PCR), etc., purchased from TaKaRA;

RNAprep polysaccharide polyphenol plant total RNA extraction kit, etc., purchased from Tiangen Reagent Company;

laboratory apparatus:

Microspectrophotometer (for RNA concentration determination), Quawell Q5000, USA;

Fluorescence quantitative PCR instrument Mastercycler ep realplex2, a product of Eppendorf, Germany;

Plant artificial climate box, USA PERCIVAL E-41HO2.

Example 1

In this example, the acquisition process and sequence structure characteristics of the Phalaenopsis internal reference gene PP2A are briefly described as follows.

(1) Low temperature stress treatment and cDNA template preparation

In the previous experimental design, in order to obtain genes whose expression is relatively stable under low temperature stress conditions and under normal growth, the inventors carried out low temperature stress treatment on the related Phalaenopsis materials. The specific treatment methods are as follows:

In the greenhouse, two biennial Phalaenopsis varieties of the same growth period, "Big Pepper" (represented by D in the following) and "Fu Le Sunset" (represented by F in the following), were selected in the same growth period, and the normal temperature in the plant artificial climate box was 26 ℃/22℃ pre-cultured for 15 d (culture conditions: light 60 μmol·m ^-2 ·s ^-1 , light-dark ratio 12 h/12 h, relative humidity 70-90%), and then treated with low temperature stress for different time, each treatment 3 strains with three biological replicates;

Low temperature stress treatment method: The gradual cooling method was adopted to simulate the natural state, that is, the first day and night temperature was 20°C/16°C for 3 days, then 16°C/11°C for 3 days, and finally 11°C/6°C for 7 days; The changes of the temperature are all increased or decreased by 1°C per hour.

The leaves at normal growth temperature were sampled as control 1 (CK1), and the samples were taken as control 2 (CK2) after treatment at 16°C/11°C for 3 days, and samples were taken at 11°C/6°C for 1 day (T1) and 2 days (T2). , 3 d (T3), 5 d (T5) and 7 d (T7) sampling.

Another group of Phalaenopsis cultivar "Da Chili" was taken and treated at 4°C for different times: 1 h, 2 h, 4 h, 12 h, 24 h, and 48 h, and the leaves at normal growth temperature were used as the control (0 h). sampling.

For the samples, 21 total RNA (DCK1, DCK2, DT1, DT2, DT3, DT5, DT7, and FCK1, FCK2, FT1, FT2, FT3, FT5, FT7, 0 h, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h), and the integrity of the extracted total RNA was identified by agarose gel electrophoresis At the same time, the A260/A280 value and A260/A230 value of the total RNA were determined, and the concentration of the extracted total RNA was determined.

The results of electrophoresis showed that the 28S, 18S and 5S bands were clear, and the brightness of the 28S band was about twice that of the 18S. The assay results showed that A260/A280 were between 1.8 and 2.1, and the total RNA concentration was between 200 and 350 ng/µL. These results indicated that the extracted total RNA was of good quality and could be used for subsequent reverse transcription cDNA preparation.

Further, referring to the instructions, the first strand of cDNA reverse transcribed using PrimeScript RT reagent Kit with gDNA Eraser kit is used as a template for real-time fluorescence quantitative PCR reaction, which can be used directly, or the prepared cDNA can be stored at -20°C for later use.

(2) Transcriptome sequencing and acquisition of PP2A gene

RNA-seq sequencing was performed on leaves of two Phalaenopsis phalaenopsis cultivated under normal growth temperature and low temperature treatment conditions with different treatment time, and the cDNA sequence of PP2A gene was obtained from the transcriptome result database. The sequencing and analysis results showed that: PP2A gene cDNA was complete It is 2207 bp long and contains a complete ORF, which is 1764 bp long and encodes protein phosphatase 2A (PP2A).

It needs to be explained that, according to the results of the previous physiological experiments, the inventors determined that the Phalaenopsis "Big Pepper" is a cold-resistant variety, while "Fule Sunset" is a non-cold-resistant variety. Transcriptomes of different treatments under stress were sequenced. In the process of sequencing and comparison, it was found that the PP2A gene was not affected by the variety and treatment time, that is, the expression levels of the two varieties were basically the same at different times, so it was speculated that this gene could be used as an internal reference gene. In order to further confirm this experimental speculation, the inventors conducted a subsequent real-time quantitative PCR experiment (see Example 2), which further proved the presumed result.

It should also be noted that further BLAST analysis showed that the PP2A gene sequence of the Phalaenopsis phalaenopsis and the registered Xiaolanyu Phalaenopsis PP2A (GenBank accession number: XM_020743358) gene sequence are 99% identical, and the ORF region has 15 bases. Different bases, the encoded amino acid sequence is 99% identical to Xiaolanyu Phalaenopsis PP2A, and there is 1 amino acid difference, but the expression characteristics and specific uses of Xiaolanyu Phalaenopsis PP2A in the prior art are lack of further research and use. discussed.

Example 2

On the basis of Example 1, this example mainly studies and analyzes the stability of transcription and expression of Phalaenopsis PP2A gene under normal growth temperature and low temperature stress conditions. The specific experimental situation is briefly described as follows.

(1) Real-time fluorescent quantitative PCR primer design

Based on the sequencing results of Example 1, the PP2A gene real-time fluorescent quantitative PCR primer sequences were designed as follows:

qPP2A-F: 5'-TCTGTTGGCTGTGGAAGGAT-3',

qPP2A-R: 5'-AAATCCGACCTGGTAGTTTCTG-3';

It should be noted that the length of the amplified product based on the primer sequence is 186 bp.

(2) Real-time fluorescence quantitative PCR reaction

Take equal amounts of 21 cDNA samples of the leaves of different varieties of Phalaenopsis prepared in Example 1, different low temperature treatment conditions and times, mix them evenly as a standard, and dilute 10 times, 100 times, 1000 times and 10000 times respectively. The undiluted samples were used as 5 gradients, and the cDNAs of the 5 gradients were used as templates, and the primers designed in step (1) were used for real-time fluorescent quantitative PCR reaction (3 repetitions);

In the fluorescence quantitative PCR reaction, the 20.0 μL reaction system is designed as follows:

2×SYBR Premix Ex TaqⅡ, 10.0 μL;

Primers F and R (both 10 mmol/L), 0.8 μL each;

cDNA template, 2.0 μL;

ddH ₂ O, 6.4 μL;

The reaction conditions were: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s, for 40 cycles.

The melting curve and standard curve are automatically generated by the real-time fluorescence quantitative PCR instrument. The melting curve is shown in Figure 1. The analysis shows that the melting curves of the real-time fluorescent quantitative PCR of the PP2A gene are all single peaks, indicating that the designed primers are specific and the products are single. The standard curve is shown in Figure 2. The analysis shows that the standard curve of PP2A gene real-time fluorescence quantitative PCR is y=-3.299x+20.17, the amplification efficiency is 101%, and the correlation coefficient is 0.999.

Using the sample cDNAs of 21 different varieties of Phalaenopsis in Example 1 with different low temperature treatments as templates, a real-time quantitative PCR reaction was carried out. 23.42, indicating that the results of real-time quantitative PCR with PP2A primers are reliable, and the PP2A gene can be used for real-time quantitative PCR research under normal growth temperature and low temperature treatment conditions of Phalaenopsis.

The reaction product was further detected by 1.5% agarose gel electrophoresis. The results are shown in Figure 4. It can be seen that the brightness of the PP2A gene in the 21 samples is basically the same, indicating that the expression in these 21 samples is relatively stable.

Example 3

Based on the results of Example 2, it can be judged that the Phalaenopsis PP2A gene has the potential to be used as an internal reference gene. Therefore, the inventors used the Phalaenopsis PP2A gene as an internal reference gene, and took the NAC domain protein gene PhNAC1 as an example to study and analyze the expression characteristics of this gene in leaves of two different varieties of Phalaenopsis at different treatment times under low temperature stress conditions. The experimental procedure is briefly described as follows.

(1) Primer design

When real-time fluorescent quantitative PCR detects the NAC domain protein gene ( PhNAC1 ), the primers are designed as follows:

qPhNAC1-F: 5'-ATCTGAACAAGTGCGAGCCT-3',

qPhNAC1-R: 5'-ATCCTTACCAGTTGCCTTCC-3';

It should be noted that when the primer sequence is used for amplification, the length of the amplified product is 155 bp.

(2) Real-time fluorescence quantitative PCR detection

The 21 phalaenopsis cDNA samples obtained in Example 1 were used as templates, and qPhNAC1-F and qPhNAC1-R of the PhNAC1 gene were used as primers for real-time quantitative PCR detection (3 repetitions); the 20.0 μL reaction system was designed as follows:

2×SYBR Premix Ex TaqⅡ, 10.0 μL;

Primers F and R (both 10 mmol/L), 0.8 μL each;

cDNA template, 2.0 μL;

ddH ₂ O, 6.4 μL;

The reaction conditions were: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s, for 40 cycles.

The relative expression of PhNAC1 gene was calculated by 2- ^ΔΔCt method.

The experimental results are shown in Figure 5. It can be seen from the analysis that the relative expression of PhNAC1 gene increased significantly in CK2 under the low temperature treatment conditions of 11℃/6℃ for the two Phalaenopsis cultivars, “Da Chili” and “Fule Xiyang”; when the gradual cooling process ended, 11 After 1 d of treatment at ℃/6℃ (T1), the expression levels decreased, and then began to increase gradually, reaching the highest on the 5th day (T5), and then decreased again on the 7th day (T7) and "rich". Happy Sunset" continues to rise. Under the low temperature treatment condition of 4 ℃, the expression level of "Big Pepper" cultivar decreased within 4 h, and then increased gradually at 12 h, 24 h and 48 h.

Example 4

In order to further investigate the accuracy of the results when PP2A is used as the internal reference gene of Phalaenopsis, with reference to the operation of Example 3, this application uses the commonly used Actin as the internal reference gene, and the expression of the NAC domain protein gene ( PhNAC1 ) of Phalaenopsis is carried out. The analysis and related processes are briefly described as follows.

The Actin primers used in the real-time fluorescent quantitative PCR reaction were designed as follows:

qActin-F: 5'-GTTCTTTCCCTATATGCTAGTGGC-3',

qActin-R: 5'-GAAGGATGGCATGAGGAAGTG-3'.

The PCR reaction system, reaction conditions and the calculation method of the relative gene expression are the same as those in Example 3.

The experimental results are shown in Figure 6. It can be seen from the analysis that when Actin was used as the internal reference gene, the relative expression levels of PhNAC1 gene under different low-temperature treatment times of the two Phalaenopsis cultivars were basically consistent with the overall trend of the change in PhNAC1 gene expression when PP2A was used as the internal reference gene (but the individual growth rate was different). There are also subtle differences in stages). In general, the results of related functional gene analysis using PP2A as an internal reference gene are more reliable.

SEQUENCE LISTING

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Claims (5)

1. Butterfly orchidPP2AThe application of the gene as an internal reference gene is characterized in that the gene has stable and consistent expression under normal growth temperature and low temperature stress conditions, and can be used for analyzing and evaluating the expression condition of related functional genes under normal growth or low temperature stress conditions; the above-mentionedPP2AThe cDNA sequence of the gene is 2207 bp in length, and comprises a complete ORF 1764 bp in length, and the specific cDNA base sequence is shown as SEQ ID NO. 1.

2. The moth orchid of claim 1PP2AUse of a gene as an internal reference gene, wherein the functional gene is a NAC domain protein genePhNAC1。

3. Utilize moth orchidPP2AThe real-time fluorescent quantitative PCR detection and analysis method of the gene as the reference gene is characterized in that the detection and analysis aim at the phalaenopsis PP2AThe gene and primer sequence are designed as follows:

qPP2A-F：5'-TCTGTTGGCTGTGGAAGGAT-3'，

qPP2A-R：5'-AAATCCGACCTGGTAGTTTCTG-3'。

4. use of moth orchid as claimed in claim 3PP2AA real-time fluorescent quantitative PCR detection and analysis method using a gene as an internal reference gene, characterized in that the detection and analysisPhNAC1When expressed, against NAC domain protein genePhNAC1The primer sequence is designed as follows:

qPhNAC1-F：5'-ATCTGAACAAGTGCGAGCCT-3'，

qPhNAC1-R：5'-ATCCTTACCAGTTGCCTTCC-3'。

5. preparation of phalaenopsisPP2AThe PCR amplification method of the gene is characterized in that during PCR amplification, the primer sequence is designed as follows:

PP2A-F：5’-TTTGAGYGATTTGTGAAGGC-3’，

PP2A-R：5’-TGGAAAAAMATAACAGCAGG-3’。

Images