Primer set and detection method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression

Abstract

The present invention proposes a primer set and a detection method for rapid detection of industrial pasteurian yeast nitrogen metabolism transport permease gene expression, which belongs to the field of bioengineering and can solve the technical problems of low detection flux, low detection efficiency, long time consumption, etc. in conventional gene quantitative detection methods. The detection method includes extraction of total RNA, preparation of cDNA template by reverse transcription reaction, multiple PCR reaction, capillary electrophoresis, and product fragment analysis, wherein the industrial pasteurian yeast nitrogen metabolism transport permease gene is 30 related genes including Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, etc. The detection method of the present invention has the characteristics of high throughput, convenient operation, high accuracy, etc., and can be applied to the field of industrial pasteurian yeast bioengineering.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a primer set and a detection method for rapidly detecting the expression of industrial Pasteurella nitrogen metabolism transport permease genes. Background Art

[0002] Changes in the composition of wort nutrients, fermentation process parameters, etc. will affect the life metabolism of yeast, leading to changes in yeast fermentation performance and metabolites, thus causing fluctuations in beer flavor. Compared with carbon sources, nitrogen sources are both nutrient components of yeast, affecting fermentation performance indicators such as yeast activity, sugar reduction rate, α-amino nitrogen (Free Amino Nitrogen, FAN) absorption rate, diacetyl and acetaldehyde reduction capacity during brewing, and participate in the formation of flavor substances as a precursor of higher alcohols and esters, affecting the flavor characteristics of the product.

[0003] The composition of wort nitrogen sources is diverse, and the intracellular nitrogen source metabolism is complex and involves multiple regulatory pathways, which has led to its influence on the fermentation performance and flavor metabolism of beer yeast and its regulatory mechanism, which has always been a hot topic and difficulty in the field of beer fermentation. At present, the research on yeast nitrogen metabolism transport and regulatory mechanism is mainly based on the model bacteria Saccharomyces cerevisiae, while there is little research on industrial Pasteurella (S. pastorianus, referred to as Lager yeast).

[0004] For industrial Lager yeast, it has been used for a long time and continuously domesticated in different industrial environments, which has led to different degrees of changes in the genome of the strain, thus deriving the particularity and complexity of the industrial Lager yeast strain. This is also the fundamental reason why different industrial Lager yeast strains show diverse fermentation performance and flavor characteristics. Therefore, the analysis of the genetic information of industrial Lager yeast is the basis for the study of its nitrogen metabolism regulation and the metabolism of key flavor substances.

[0005] However, the fluorescent quantitative PCR method is currently the most commonly used gene quantitative detection method, but it has defects such as low throughput, low detection efficiency, and long time consumption, which makes it impossible to achieve high-throughput rapid detection of related gene expression levels. Therefore, a fast and accurate detection method is needed to simultaneously detect multiple nitrogen metabolism and transport-related genes in Lager yeast. Summary of the invention

[0006] In view of the technical problems of conventional gene quantitative detection methods such as low detection throughput, low detection efficiency and long detection time, the present invention proposes a primer set and a detection method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression with high throughput, convenient operation and high accuracy.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A multiplex PCR reaction primer set for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression, the primer set is directed to industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Di Thirty primers were designed, including p5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, and Mep3-sb, as shown in SEQ ID NO: 1. ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57 and SEQ ID NO.59.

[0009] The present invention also provides a reverse transcription reaction primer set for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression, characterized in that the primer set is directed to industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat 2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb were designed to obtain 30 primers, as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.52, SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58 and SEQ ID NO.60.

[0010] The present invention also provides a method for rapidly detecting the expression of industrial Pasteurella nitrogen metabolism transport permease gene, comprising the following steps:

[0011] Search for nitrogen metabolism-related genes of Saccharomyces cerevisiae in the NCBI database, and locate the homologous gene sequence information in the genome of industrial Saccharomyces cerevisiae T1-1 through BLASTn sequence comparison, namely, the nitrogen metabolism transport permease gene of industrial Saccharomyces cerevisiae;

[0012] According to the homologous gene sequence information, CLUSTAL W comparison is performed on the same gene from -Sc and -Sb to find non-homologous segments for primer design, and a reverse transcription reaction primer set and a multiplex PCR reaction primer set are obtained;

[0013] Total RNA extraction from industrial Pasteurella;

[0014] The extracted total RNA is used as a template and the reverse transcription reaction primer set is used as a primer to perform a reverse transcription reaction to prepare a cDNA template;

[0015] The reverse transcription reaction product is used as a template and the multiplex PCR reaction primer set is used as a primer to perform a multiplex PCR reaction, and the multiplex PCR reaction product is subjected to electrophoresis and fragment analysis.

[0016] In one embodiment, the nitrogen metabolism-related genes include genes involved in amino acid metabolism regulation, NCR pathway in nitrogen source metabolism, SPS sensing system and TOR signal transduction pathway, and permease genes involved in nitrogen source transport.

[0017] In one embodiment, the industrial Pasteurella nitrogen metabolism transport permease genes include 30 genes including Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc and Mep3-sb.

[0018] In one embodiment, industrial Pasteurella β-actin genes ACT1-Sc and ACT1-Sb are also included as internal reference genes. The internal reference gene primers in the reverse transcription reaction to prepare the cDNA template are shown as SEQ ID NO.62 and SEQ ID NO.64; the internal reference gene primers in the multiple PCR reaction are shown as SEQ ID NO.61 and SEQ ID NO.63.

[0019] In one embodiment, in the reaction system of the multiplex PCR reaction, the total reaction system is 20 μL, including 4.0 μL of 25 mM MgCl2, 4.0 μL of 5× PCR buffer, 0.7 μL of DNA polymerase, 2 μL of multiplex PCR reaction primer set, and 9.3 μL of cDNA template;

[0020] The reaction conditions are:

[0021] S1, pre-denaturation at 95°C for 10 minutes;

[0022] S2, denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds;

[0023] S3, extension at 68-70°C for 1 min, 35 cycles;

[0024] S4: Store at 4°C, with S2 and S3 steps totaling 35 cycles.

[0025] In one embodiment, the concentration of SEQ ID NO.2 in the reverse transcription reaction primer set is 500nM, the concentration of SEQ ID NO.4 is 500nM, the concentration of SEQ ID NO.6 is 500nM, the concentration of SEQ ID NO.8 is 500nM, the concentration of SEQ ID NO.10 is 15.625nM, the concentration of SEQ ID NO.12 is 125nM, the concentration of SEQ ID NO.14 is 500nM, the concentration of SEQ ID NO.16 is 15.625nM, the concentration of SEQ ID NO.18 is 500nM, the concentration of SEQ ID NO.20 is 500nM, the concentration of SEQ ID NO.22 is 31.25nM, the concentration of SEQ ID NO.24 is 500nM, the concentration of SEQ ID NO.26 is 7.8125nM, the concentration of SEQ ID NO.28 is 500nM, the concentration of SEQ ID NO.30 is 1500nM, and the concentration of SEQ ID NO. The concentration of SEQ ID NO.32 was 62.5 nM, the concentration of SEQ ID NO.34 was 500 nM, the concentration of SEQ ID NO.36 was 250 nM, the concentration of SEQ ID NO.38 was 500 nM, the concentration of SEQ ID NO.40 was 31.25 nM, the concentration of SEQ ID NO.42 was 1500 nM, the concentration of SEQ ID NO.44 was 500 nM, the concentration of SEQ ID NO.46 was 250 nM, the concentration of SEQ ID NO.48 was 1500 nM, the concentration of SEQ ID NO.50 was 500 nM, the concentration of SEQ ID NO.52 was 1500 nM, the concentration of SEQ ID NO.54 was 500 nM, the concentration of SEQ ID NO.56 was 500 nM, the concentration of SEQ ID NO.58 was 500 nM, and the concentration of SEQ ID NO.60 was 500 nM; the internal reference gene primer SEQ ID The concentration of SEQ ID NO.62 was 500 nM, and the concentration of SEQ ID NO.64 was 500 nM.

[0026] In one embodiment, the concentration of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57 and SEQ ID NO.59 in the multiplex PCR reaction primer set are all 200 nM; the concentration of the internal reference gene primers SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.66 and SEQ ID NO.67 in the multiplex PCR reaction primer set are all 200 nM; The concentration of NO.63 used was 200 nM.

[0027] In one embodiment, the product size of the industrial Pasteurella nitrogen metabolism transport permease gene Bap2-sc is 237 bp, the product size of Bap2-sb is 198 bp, the product size of Bap3-sc is 132 bp, the product size of Bap3-sb is 188 bp, the product size of Gnp1-sc is 214 bp, the product size of Gnp1-sb is 184 bp, the product size of Agp1-sc is 130 bp, and the product size of Agp1-sb is 188 bp. The product size of -sb is 170bp, the product size of Tat1-sc is 192bp, the product size of Tat1-sb is 107bp, the product size of Tat2-sc is 138bp, the product size of Tat2-sb is 313bp, the product size of Dip5-sc is 233bp, the product size of Mup1-sc is 266bp, the product size of Mup1-sb is 272bp, and the product size of Hip1-sc is 22 The product size of Hip1-sb is 179bp, the product size of Lyp1-sc is 248bp, the product size of Lyp1-sb is 256bp, the product size of Alp1-sc is 148bp, the product size of Alp1-sb is 390bp, the product size of Gap1-sc is 295bp, the product size of Gap1-sb is 262bp, the product size of Put4-sc is 334bp, and the product size of Mep1-sc is 148bp. The product size of Mep1-sb is 307bp, the product size of Mep2-sc is 284bp, the product size of Mep2-sb is 241bp, the product size of Mep3-sc is 349bp and the product size of Mep3-sb is 226bp; the product size of the industrial Pasteurella β-actin gene ACT1-Sc is 361bp, and the product size of ACT1-Sb is 382bp.

[0028] Compared with the prior art, the advantages and positive effects of the present invention are:

[0029] 1. The method for rapid detection of industrial Pasteurella nitrogen metabolism, transport and permease gene expression proposed in the present invention adopts GeXP multiple gene expression quantitative analysis technology to detect industrial Pasteurella nitrogen metabolism, transport and permease related genes (Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, The method for expressing the above-mentioned genes (Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc and Mep3-sb) can fully analyze the genetic information of industrial Lager yeast, understand the regulation of nitrogen metabolism and the metabolism of key flavor substances, thereby controlling the expression of nitrogen metabolism transport permease genes during fermentation and improving the flavor stability and consistency of beer;

[0030] 2. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression proposed in the present invention has the characteristics of high detection efficiency and high accuracy, and can complete high-throughput detection of 30 related genes within 2 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The expression level of the industrial Pasteurella nitrogen source metabolism permease gene during the fermentation process provided by the embodiment of the present invention;

[0032] Figure 2 The genes whose expression levels are downregulated in the mid-logarithmic period after the addition of the four key amino acids provided in the embodiments of the present invention;

[0033] Figure 3 Genes whose expression levels are downregulated in the mid-log phase after the addition of the key amino acids Glu, Lys and Val provided in the embodiments of the present invention;

[0034] Figure 4 The genes whose expression levels are downregulated in the mid-log phase after the addition of the key amino acids Lys and Val provided in the embodiments of the present invention;

[0035] Figure 5 The gene whose expression level is downregulated in the mid-logarithmic period after the addition of the key amino acid Val provided in the embodiment of the present invention;

[0036] Figure 6 The genes whose expression levels change in the late logarithmic period after the addition of the four key amino acids provided in the embodiments of the present invention;

[0037] Figure 7 Genes whose expression levels change in the late logarithmic period after the addition of the key amino acids Pro, Lys, and Val provided in the embodiments of the present invention;

[0038] Figure 8 Genes whose expression levels change in the late logarithmic period after the addition of the key amino acids Glu and Lys provided in the embodiments of the present invention;

[0039] Fig. 9 Genes whose expression levels change in the late logarithmic period after the addition of the key amino acids Pro and Lys provided in the embodiments of the present invention;

[0040] Fig.10 Genes whose expression levels change in the late logarithmic period after the addition of the key amino acids Lys and Val provided in the embodiments of the present invention;

[0041] Fig.11 The gene whose expression level changes in the late logarithmic period after the key amino acid Val is added as provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The embodiment of the present invention provides a multiplex PCR reaction primer set for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression, the primer set is directed to industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-s b, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb were designed to obtain 30 primers, as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57 and SEQ ID NO.59.

[0044] The embodiment of the present invention also provides a reverse transcription reaction primer set for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression, the primer set is directed to industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-s b, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb were designed to obtain 30 primers, as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.52, SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58 and SEQ ID NO.60.

[0045] The embodiment of the present invention further provides a method for rapidly detecting the expression of industrial Pasteurella nitrogen metabolism transport permease gene, comprising the following steps:

[0046] S1. Search for nitrogen metabolism-related genes of Saccharomyces cerevisiae in the NCBI database, and use BLASTn sequence comparison to locate the homologous gene sequence information in the genome of industrial Saccharomyces cerevisiae T1-1, namely, the nitrogen metabolism transport permease gene of industrial Saccharomyces cerevisiae;

[0047] S2. Based on the homologous gene sequence information, CLUSTAL W comparison is performed on the same gene from -Sc and -Sb to find non-homologous segments for primer design, thereby obtaining a reverse transcription reaction primer set and a multiplex PCR reaction primer set;

[0048] The above step S2 is specifically as follows: using the gene information of the sequenced industrial Lager yeast TT-1, CLUSTAL W comparison is performed on the same gene from different sources (-Sc / -Sb), and non-homologous segments are found for primer design respectively, and the non-homologous segments are located at the 3' end of the primers as much as possible. Primer design is performed using Primer Primer 5.0 software. The design principle is: the length of the PCR product is 100 to 400 bp, and the difference between the PCR products is at least 3 bp, T m The value was around 55°C, and the secondary structure and mismatch of the primers were avoided as much as possible. On this basis, BLAST sequence comparison was performed to preliminarily verify the specificity of the primers to ensure that there was no homology between the primers and other sequences in the genome to avoid mismatches. Universal primers were added to the upstream and downstream of the designed primers, and all primers were synthesized by Invitrogen.

[0049] S3, total RNA extraction of industrial Pasteurella;

[0050] S4, using the extracted total RNA as a template and the reverse transcription reaction primer set as a primer to perform a reverse transcription reaction to prepare a cDNA template;

[0051] S5. Using the reverse transcription reaction product as a template and the multiplex PCR reaction primer set as a primer to perform a multiplex PCR reaction, and performing electrophoresis and fragment analysis on the multiplex PCR reaction product.

[0052] In the above embodiment, a method for rapidly detecting the expression of nitrogen metabolism transport permease gene of industrial Pasteurella is provided. Since industrial Lager yeast has been used for a long time and continuously domesticated in different industrial environments, the genome of the strain has changed to varying degrees, thereby deriving the particularity and complexity of the industrial Lager yeast strain, which undoubtedly adds technical difficulty to accurately find its nitrogen metabolism transport permease detection gene. Then the technical difficulty that the present invention needs to overcome first lies in this. The present invention searches for the relevant gene sequence of the model fungus Saccharomyces cerevisiae (S.cerevisiae), adopts homologous gene search, sequence alignment and other methods. The formula finally locked on the nitrogen metabolism transport permease detection gene in industrial Lager yeast, but the results showed that there were a large number of related genes, reaching 30. Therefore, the second technical difficulty that the present invention needs to overcome is to achieve high-throughput detection of up to 30 genes. In order to further improve the detection efficiency and achieve high-throughput detection of 30 related genes, the present invention combines the steps of designing multiple PCR primers, multiple rounds of screening, primer concentration screening, etc., and finally achieves the simultaneous detection and analysis of 30 genes, which lays a research foundation for the research on the nitrogen metabolism regulation mechanism and key flavor substance metabolism of industrial Lager yeast, and can be used to guide actual production.

[0053] In order to more clearly and in detail introduce the method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression provided by the embodiment of the present invention, it will be described in conjunction with specific examples below.

[0054] Example 1

[0055] This embodiment also provides a method for rapidly detecting the expression of industrial Pasteurella nitrogen metabolism transport permease genes, which specifically comprises the following steps:

[0056] (1) The nitrogen metabolism-related genes of Saccharomyces cerevisiae were searched in the NCBI database. By BLASTn sequence comparison, the homologous gene sequence information in the genome of industrial Saccharomyces cerevisiae T1-1 was locked, namely, the nitrogen metabolism transport permease genes of industrial Saccharomyces cerevisiae (Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-s b, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc , Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb);

[0057] (2) Based on the full-length sequences of the industrial Pasteurella nitrogen metabolism transport permease gene and the internal reference genes ACT1-sc and ACT1-sb obtained in step (1), Primer Premier 5 software was used to design specific upstream and downstream primers that meet the characteristics of the GeXP multiplex PCR reaction and can detect the expression of the industrial Pasteurella nitrogen source metabolism permease gene, as well as specific upstream and downstream primers as internal reference genes. The primer sequences are shown in Table 1.

[0058] Table 1 Specific upstream and downstream primers for industrial Pasteurella nitrogen source metabolism permease-related genes and internal reference genes

[0059]

[0060]

[0061]

[0062] The results of capillary electrophoresis using a single primer showed that the length of the amplified product was consistent with the expected product length, indicating that the designed primers have strong specificity and are suitable for GeXP detection.

[0063] (3) Take a full tank of yeast cell sample from the fermentation tank and quickly extract RNA. The specific steps are as follows:

[0064] (3-1) Place 750 μL of zirconium oxide beads in a 1.5 mL centrifuge tube (with screw cap), approximately 2.5 cm high;

[0065] (3-2) Add lysis buffer (480 μL Lysis Buffer + 48 μL 10% SDS + 480 μL Phenol: Cloroform: IAA) to the yeast collection tube in order and resuspend. Vortex for 10-15 seconds;

[0066] (3-3) Transfer the mixture into the prepared 750 μL zirconium oxide bead centrifuge tube and cap tightly;

[0067] (3-4) Place the centrifuge tube on a vortex shaker for 10 min (maximum speed) to lyse the cells;

[0068] (3-5) Centrifuge at 16000g for 5 min at room temperature and transfer the upper aqueous phase to a 10 mL centrifuge tube;

[0069] (3-6) Add 1.9 mL Binding Buffer and mix well;

[0070] (3-7) Add 1.25 mL of 100% ethanol and mix well;

[0071] (3-8) Take 700 μL of the mixed solution and transfer it to the Filter Cartridge + Collection Tube. Centrifuge at 12000 g for 1 min and discard the collected solution. Repeat this step until all the mixed solution is transferred.

[0072] (3-9) Add 700 μL Wash Solution 1 to the filter cartridge, centrifuge at 12,000 g for 1 min, and discard the collected solution;

[0073] (3-10) Add 500 μL Wash Solution 2 / 3, centrifuge at 12000 g for 1 min, discard the collected solution, and repeat this step;

[0074] (3-11) Centrifuge at 12000 g for 1 min to completely remove the liquid on the membrane and transfer the filter cartridge to a new collection tube;

[0075] (3-12) Add 25-50 μL of elution solution preheated at 95-100°C to the center of the membrane, centrifuge at 12000 g for 1 min, then add another 25-50 μL of elution solution to the center of the membrane, centrifuge at 12000 g for 1 min to obtain RNA collection solution;

[0076] (3-13) DNase I treatment: 50-100 μL RNA sample + 1 / 10th vol 10× DNase I Buffer + 4 μL DNase I, react at 37°C for 30 min. Add 1 / 10th vol DNase Inactivation Reagent (vortex before use), vortex and mix, let stand at room temperature for 5 min, centrifuge at 12000 g for 2-3 min to precipitate the DNase Inactivation Reagent, transfer the RNA supernatant to a new tube for storage, and the purified total RNA can be obtained.

[0077] (4) Reverse transcription reaction to prepare cDNA template

[0078] The purified yeast cell RNA obtained in step (3) was used as a template, using the GenomeLabTM GeXP priming kit from Beckman Coulter, the downstream primers of the multiple primers in Table 1 as specific primers, and the total RNA of yeast cells as a template to synthesize the first chain of cDNA. The reaction system was 20 μL.

[0079] Among them, the GenomeLabTM GeXP start-up kit (product number: PNA85017) contains reverse transcription reaction buffer, reverse transcriptase, PCR reaction buffer, KANr RNA, DNase / RNase-free water, DNAMarker (DSS-400), mineral oil, GeXP loading buffer; DNA polymerase (product number: PNA85022); separation gel (product number: PNA608010); separation buffer (product number: PNA608012), all purchased from Beckman Coulter, cDNA first-chain synthesis reaction system and reaction procedure are shown in Tables 2-3, where control 1 and control 2 are negative controls.

[0080] Table 2 Reaction system for preparing cDNA template by reverse transcription reaction

[0081]

[0082] Table 3 Reaction procedure for preparing cDNA template by reverse transcription reaction

[0083] step Reaction temperature (℃) Reaction time (min) 1 48 1 2 42 60 3 95 5 4 4 save

[0084] (5) Multiplex PCR reaction

[0085] Using the first-strand cDNA synthesized in step (4) as a template and the upstream primer of the multiple primers in Table 1 as a specific primer, a multiplex PCR amplification reaction was performed. Two parallel tubes were set for each sample. The PCR reaction system and reaction procedure are shown in Table 4-5.

[0086] Table 4 Reaction system of multiplex PCR

[0087] Reaction system Addition amount (μL) <![CDATA[25mM MgCl2]]> 4.0 PCR Buffer 5× 4.0 ThermoStart DNA Polymerase 0.7 Primer (forward singlet 200nM) 2 Template cDNA 9.3

[0088] Table 5 Reaction procedures for multiplex PCR

[0089] step Reaction temperature (℃) Reaction time (min) 1 95℃ 10min 2 94℃ 30s 3 55℃ 30s 4 68-70℃ 1min 5 N / A Repeat steps 2-4 for 34 cycles (35 cycles in total) 6 4℃ save

[0090] (6) GeXP capillary electrophoresis detection

[0091] (6-1) Prepare SLS / DSS mixture: Take DSS 400 (molecular weight internal standard -400), add 80 volumes of SLS (loading buffer, formamide), vortex on a vortex shaker for 30 seconds or mix 10 times with a pipette tip for later use;

[0092] (6-1) Add 40 μL of SLS / DSS mixture to each sample well, then add 0.1 μL of multiplex PCR product to the GeXP sample plate (do not allow bubbles to form), and add a drop of paraffin oil to cover the sample;

[0093] (6-1) Add 2 / 3 volume of separation buffer to the corresponding wells of the buffer plate and sample plate;

[0094] (6-1) Enter the Set Up program, enter the sample name, specify the Frag-3 separation method for each sample, specify the default GeXP analysis method, and start running the sample.

[0095] (7) Multiple primer optimization

[0096] Optimization of reverse primer concentration: For genes with too strong signals, reduce the reverse primer concentration in the reverse transcription reaction to adjust the signal intensity.

[0097] a. According to the signal intensity, the genes were divided into two groups, one for the non-adjustment group and the other for the adjustment group, and the reverse primer working solutions were prepared for each group;

[0098] b. Prepare 8 gradients of diluted primer working solutions by diluting the adjusted primers in a 2-fold mode, and determine the dilution multiple for each primer;

[0099] c. Combine the non-adjusted group and the adjusted group primers with determined primer dilution concentration to form a reverse primer set and establish a reverse transcription reaction;

[0100] d. Use the reverse transcription product and forward primer set to establish a multiplex PCR reaction;

[0101] e. Measure the appropriate concentration of each primer in the GeXP system;

[0102] f. Use the adjusted reverse primer concentration to perform reverse transcription PCR reaction, repeatedly verify the optimization results, and adjust the reverse primer concentration according to the reverse transcription PCR results and experimental needs;

[0103] g. Optimized multiplex reactions can use standard GeXP protocols.

[0104] (8) Gene expression analysis

[0105] The optimized multiple primers can be used to detect the gene expression level of the sample. The expression level calculation formula is as follows:

[0106] Relative gene transcription amount = target gene expression / (reference gene * KAN r expression level).

[0107] The final optimized concentrations of downstream primers are shown in the following table.

[0108] Table 6 Downstream (reverse) primer concentrations of each gene

[0109] Gene name Reverse primer concentration (nm) Lyp1-sc 250 Gap1-sb 250 Gnp1-sb 125 Hip1-sc 62.5 Tat2-sc 31.25 Alp1-sc 31.25 Agp1-sb 15.625 Gnp1-sc 15.625 Dip5-sc 7.8125 Agp1-sc 500 Gap1-sc 500 Hip1-sb 500 Lyp1-sb 500 Mep1-sc 500 Mep2-sb 500 Mep2-sc 500 Mep3-sb 500 Mep3-sc 500 Mup1-sc 500 Tat1-sb 500 Tat1-sc 500 Act1-sb 500 Act1-sc 500 Bap2-sb 500 Bap2-sc 500 Bap3-sb 500 Bap3-sc 500 Mup1-sb 1500 Tat2-sb 1500 Put4-sc 1500 Mep1-sb 1500 Alp1-sb 1500

[0110] Figure 1 The expression levels of industrial Pasteurella nitrogen source metabolism permease genes during fermentation. Figure 1 It can be seen that this method can detect the expression levels of 30 genes simultaneously.

[0111] Example 2

[0112] This example uses the multiple expression analysis method of nitrogen metabolism genes as described in Example 1 to study the effect of key amino acid addition on the expression of nitrogen metabolism genes in Lager yeast, specifically:

[0113] During the fermentation process of different amino acid addition experiments, the transcription analysis of 30 nitrogen source transport permease genes was performed on yeast at different growth stages (mid-logarithmic growth: fermentation 48h; late-logarithmic growth: fermentation 72h). The specific analysis results are shown in Figure 2-Figure 5 .

[0114] Depend on Figure 2It can be seen that in the middle of logarithmic growth, the expression of ALP1-Sb gene was inhibited in both the experimental and control. The addition of key amino acids caused the expression of nitrogen source transport permease genes to change. After adding four key amino acids, the expression of 10 genes decreased significantly compared with the control (AGP1-Sc, ALP1-Sc, HIP1-Sb, GNP1-Sc / Sb, MEP2-Sc / -Sb, MEP3-Sb, DIP5-Sc, MUP1-Sc); Figure 3 The analysis showed that the expression levels of three genes (HIP1-Sc, MEP3-Sc, TAT2-Sc) decreased significantly after the addition of Glu, Lys and Val (Glu: p<0.05; Lys: p<0.05; Val: p<0.01), and there was no significant difference between the control and the addition of Pro. Figure 4 Analysis showed that the expression levels of four genes (BAP2-Sb, PUT4-Sc, TAT2-Sb, MEP1-Sb) decreased after the addition of Lys and Val (p<0.05); Figure 5 Analysis showed that the expression levels of AGP1-Sb and GAP1-Sc genes were significantly reduced when Val was added (p<0.01).

[0115] In addition, this example also analyzes the changes in the expression of related genes in the late logarithmic growth period. The specific results are as follows: Figure 6-Figure 11 Specifically, in the late logarithmic growth period, the transcription of nitrogen source transport permease genes was found to be significantly different from the control after adding key amino acids. Figure 6-Figure 7 The results showed that the four key amino acids still significantly inhibited the expression of most nitrogen source transport and permease genes (TAT1-Sb, AGP1-Sc, ALP1-Sc, HIP1-Sc, GNP1-Sc / Sb, MEP3-Sb, DIP5-Sc) in the late logarithmic growth period, but Glu significantly increased the expression of TAT2-Sb, PUT4-Sc, MEP3-Sc, BAP2-Sc, MUP1-Sb, GAP1-Sc and LYP1-Sc genes, and Pro significantly increased the expression of MEP3-Sc and AGP1-Sb genes, thereby weakening the inhibitory effect on nitrogen metabolism in yeast.

[0116] Based on the results of the transcription levels of yeast nitrogen source transport permease at different growth stages, the four key amino acids inhibited the absorption of nitrogen sources by Lager yeast by inhibiting the expression of nitrogen source transport permease genes, among which Lys and Val had the strongest inhibitory effects and Pro had the weakest inhibitory effect; in the late logarithmic growth stage of yeast, the inhibitory effects of Glu and Pro weakened. SEQUENCE LISTING <110> Tsingtao Brewery Co., Ltd. <120> Primer set and detection method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression <130> 20220519 <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> DNA <213> Bap2-sc <400> 1 aggtgacact atagaatacc tgctgcgcta gtaatt 36 <210> 2 <211> 39 <212> DNA <213> Bap2-Sc <400> 2 gtacgactca ctatagggat ctaaaggcaa aaccgttag 39 <210> 3 <211> 37 <212> DNA <213> Bap2-Sb <400> 3 aggtgacact atagaatacg gtgagacaga gtttatt 37 <210> 4 <211> 36 <212> DNA <213> Bap2-Sb <400> 4 gtacgactca ctatagggaa agccgaagag tcacca 36 <210> 5 <211> 35 <212> DNA <213> Bap3-Sc <400> 5 aggtgacact atagaatatc tcggtgcggc ataaa 35 <210> 6 <211> 37 <212> DNA <213> Bap3-Sc <400> 6 gtacgactca ctatagggat tcacaaggtt agggtca 37 <210> 7 <211> 36 <212> DNA <213> Bap3-Sb <400> 7 aggtgacact atagaatacg ggtttatcat cctatc 36 <210> 8 <211> 37 <212> DNA <213> Bap3-Sb <400> 8 gtacgactca ctatagggac gcagtcacaa gaataaa 37 <210> 9 <211> 36 <212> DNA <213> Gnp1-Sc <400> 9 aggtgacact atagaataat ggtaacgacc ttgacg 36 <210> 10 <211> 38 <212> DNA <213> Gnp1-Sc <400> 10 gtacgactca ctatagggaa cctccgtaag taagatgc 38 <210> 11 <211> 38 <212> DNA <213> Gnp1-Sb <400> 11 aggtgacact atagaatagg tttcaacact tacccttc 38 <210> 12 <211> 36 <212> DNA <213> Gnp1-Sb <400> 12 gtacgactca ctatagggaa tcgactttca cggtcc 36 <210> 13 <211> 39 <212> DNA <213> Agp1-Sc <400> 13 aggtgacact atagaataga tcgacctaga ttctcatag 39 <210> 14 <211> 36 <212> DNA <213> Agp1-Sc <400> 14 gtacgactca ctatagggat aaggtccgtt cctcaa 36 <210> 15 <211> 35 <212> DNA <213> Agp1-Sb <400> 15 aggtgacact atagaatact gccgaacagt ccaac 35 <210> 16 <211> 38 <212> DNA <213> Agp1-Sb <400> 16 gtacgactca ctatagggaa atagttggtc ggagttgt 38 <210> 17 <211> 36 <212> DNA <213> Tat1-Sc <400> 17 aggtgacact atagaataat gctgcattca gctacg 36 <210> 18 <211> 37 <212> DNA <213> Tat1-Sc <400> 18 gtacgactca ctatagggaa acaagaaagc aaaccaa 37 <210> 19 <211> 36 <212> DNA <213> Tat1-Sb <400> 19 aggtgacact atagaatagg acgaggtggg atacag 36 <210> 20 <211> 39 <212> DNA <213> Tat1-Sb <400> 20 gtacgactca ctatagggaa aataacgcaa tataactg 39 <210> 21 <211> 36 <212> DNA <213> Tat2-Sc <400> 21 aggtgacact atagaatagg gcgttctatc tgttct 36 <210> 22 <211> 37 <212> DNA <213> Tat2-Sc <400> 22 gtacgactca ctatagggac acttggtaag cctttgg 37 <210> 23 <211> 36 <212> DNA <213> Tat2-Sb <400> 23 aggtgacact atagaataag gaacggaaat ccacat 36 <210> 24 <211> 37 <212> DNA <213> Tat2-Sb <400> 24 gtacgactca ctatagggac gatggcttta ccactac 37 <210> 25 <211> 36 <212> DNA <213> Dip5-Sc <400> 25 aggtgacact atagaatatt acctcccaac caactt 36 <210> 26 <211> 39 <212> DNA <213> Dip5-Sc <400> 26 gtacgactca ctatagggag gattagaccc aacattacc 39 <210> 27 <211> 35 <212> DNA <213> Mup1-Sc <400> 27 aggtgacact atagaatagc tccttacgtt ccacc 35 <210> 28 <211> 38 <212> DNA <213> Mup1-Sc <400> 28 gtacgactca ctatagggac gccaatagtg tcatcata 38 <210> 29 <211> 36 <212> DNA <213> Mup1-Sb <400> 29 aggtgacact atagaatatt tccctcttct tccaca 36 <210> 30 <211> 37 <212> DNA <213> Mup1-Sb <400> 30 gtacgactca ctatagggat caaagcaagt cccacag 37 <210> 31 <211> 37 <212> DNA <213> Hip1-Sc <400> 31 aggtgacact atagaatata ttgggcagat gtagttg 37 <210> 32 <211> 37 <212> DNA <213> Hip1-Sc <400> 32 gtacgactca ctatagggag gaagcgatct gaagagg 37 <210> 33 <211> 36 <212> DNA <213> Hip1-Sb <400> 33 aggtgacact atagaatagg ctggcgtaaa tcaaac 36 <210> 34 <211> 36 <212> DNA <213> Hip1-Sb <400> 34 gtacgactca ctatagggaa ccgacggcta aggtca 36 <210> 35 <211> 36 <212> DNA <213> Lyp1-Sc <400> 35 aggtgacact atagaataag aagcccacta tgagga 36 <210> 36 <211> 37 <212> DNA <213> Lyp1-Sc <400> 36 gtacgactca ctatagggac gtagccatct caccaag 37 <210> 37 <211> 35 <212> DNA <213> Lyp1-Sb <400> 37 aggtgacact atagaatata tgccgtggaa gctca 35 <210> 38 <211> 37 <212> DNA <213> Lyp1-Sb <400> 38 gtacgactca ctatagggag gacccaccac aaacaat 37 <210> 39 <211> 36 <212> DNA <213> Alp1-Sc <400> 39 aggtgacact atagaatata agtgccgctt actctg 36 <210> 40 <211> 37 <212> DNA <213> Alp1-Sc <400> 40 gtacgactca ctatagggaa ctcgttgcca ccttgtt 37 <210> 41 <211> 37 <212> DNA <213> Alp1-Sb <400> 41 aggtgacact atagaatata ttcatgggaa ccgtcat 37 <210> 42 <211> 37 <212> DNA <213> Alp1-Sb <400> 42 gtacgactca ctatagggag attatgaacc cgagcag 37 <210> 43 <211> 36 <212> DNA <213> Gap1-Sc <400> 43 aggtgacact atagaatact gccagtgaat ccgtag 36 <210> 44 <211> 36 <212> DNA <213> Gap1-Sc <400> 44 gtacgactca ctatagggac gacagataac acggca 36 <210> 45 <211> 36 <212> DNA <213> Gap1-Sb <400> 45 aggtgacact atagaatatt gttacttccg ccttct 36 <210> 46 <211> 36 <212> DNA <213> Gap1-Sb <400> 46 gtacgactca ctatagggat gacaaaggga gaagca 36 <210> 47 <211> 35 <212> DNA <213> Put4-Sc <400> 47 aggtgacact atagaatacg ccaatgcgtt tatgt 35 <210> 48 <211> 37 <212> DNA <213> Put4-Sc <400> 48 gtacgactca ctatagggag gcaatctgtc gtagagg 37 <210> 49 <211> 36 <212> DNA <213> Mep1-Sc <400> 49 aggtgacact atagaataga agatgcctac cctgag 36 <210> 50 <211> 37 <212> DNA <213> Mep1-Sc <400> 50 gtacgactca ctataggac caagagtaaa caaaccc 37 <210> 51 <211> 36 <212> DNA <213> Mep1-Sb <400> 51 aggtgacact atagaataac tcaggactgg cgagaa 36 <210> 52 <211> 38 <212> DNA <213> Mep1-Sb <400> 52 38 <210> 53 <211> 37 <212> DNA <213> Mep2-Sc <400> 53 aggtgacact atagaatagc aggctcttac attagtc 37 <210> 54 <211> 37 <212> DNA <213> Mep2-Sc <400> 54 gtacgactca ctatagggag ggatgtaagc agtggat 37 <210> 55 <211> 37 <212> DNA <213> Mep2-Sb <400> 55 aggtgacact atagaatagt tgtaatctcg ctgttga 37 <210> 56 <211> 39 <212> DNA <213> Mep2-Sb <400> 56 gtacgactca ctatagggag ataaccgact tgtttgtag 39 <210> 57 <211> 35 <212> DNA <213> Mep3-Sc <400> 57 aggtgacact atagaatagg caacttggat tcatt 35 <210> 58 <211> 36 <212> DNA <213> Mep3-Sc <400> 58 gtacgactca ctatagggaa accagccaca gcactt 36 <210> 59 <211> 37 <212> DNA <213> Mep3-Sb <400> 59 aggtgacact atagaatata catccaaatc gcataca 37 <210> 60 <211> 40 <212> DNA <213> Mep3-Sb <400> 60 gtacgactca ctatagggag taatcccttc taacttccac 40 <210> 61 <211> 35 <212> DNA <213> Act1-Sca <400> 61 aggtgacact atagaataac gctcctcgtg ctgtc 35 <210> 62 <211> 37 <212> DNA <213> Act1-Sca <400> 62 gtacgactca ctatagggaa gaaggctgga acgttaa 37 <210> 63 <211> 36 <212> DNA <213> Act1-Sba <400> 63 aggtgacact atagaataga ttctggtgac ggtgtt 36 <210> 64 <211> 36 <212> DNA <213> Act1-Sba <400> 64 gtacgactca ctatagggaa cccaagacgg aaggat 36 <210> 65 <211> 48 <212> DNA <213> CAnr b <400> 65 aggtgacact atagaataat catcagcatt gcattcgatt cctgtttg 48 <210> 66 <211> 39 <212> DNA <213> KANr b <400> 66 gtacgactca ctatagggaa ttccgactcg tccaacatc 39

Claims

1. A multiplex PCR primer set for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression, characterized in that: A reaction primer set comprising 30 primers designed for industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb, respectively as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23 ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57 and SEQ ID NO.59; and A reverse transcription reaction primer set comprising 30 primers designed for industrial Pasteurella nitrogen metabolism transport permease genes Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc, Mep3-sb, as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.52, SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58 and SEQ ID NO.

60.

2. A method for rapidly detecting the expression of industrial Pasteurella nitrogen metabolism transport permease genes, characterized in that: The following steps are involved: Search for nitrogen metabolism-related genes of Saccharomyces cerevisiae in the NCBI database, and locate the homologous gene sequence information in the genome of industrial Saccharomyces cerevisiae T1-1 through BLASTn sequence comparison, namely, the nitrogen metabolism transport permease gene of industrial Saccharomyces cerevisiae; According to the homologous gene sequence information, CLUSTAL W comparison is performed on the same gene from -Sc and -Sb to find non-homologous segments for primer design, thereby obtaining the multiplex PCR primer set as claimed in claim 1; Total RNA extraction from industrial Pasteurella; The extracted total RNA is used as a template and the reverse transcription reaction primer set is used as a primer to perform a reverse transcription reaction to prepare a cDNA template; The reverse transcription reaction product is used as a template and the multiplex PCR reaction primer set is used as a primer to perform a multiplex PCR reaction, and the multiplex PCR reaction product is subjected to electrophoresis and fragment analysis.

3. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 2, characterized in that: The nitrogen metabolism-related genes include genes involved in amino acid metabolism regulation, NCR pathway in nitrogen source metabolism, SPS sensing system and TOR signal transduction pathway, and permease genes involved in nitrogen source transport.

4. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 2, characterized in that: The industrial Pasteurella nitrogen metabolism, transport and permease genes include 30 genes including Bap2-sc, Bap2-sb, Bap3-sc, Bap3-sb, Gnp1-sc, Gnp1-sb, Agp1-sc, Agp1-sb, Tat1-sc, Tat1-sb, Tat2-sc, Tat2-sb, Dip5-sc, Mup1-sc, Mup1-sb, Hip1-sc, Hip1-sb, Lyp1-sc, Lyp1-sb, Alp1-sc, Alp1-sb, Gap1-sc, Gap1-sb, Put4-sc, Mep1-sc, Mep1-sb, Mep2-sc, Mep2-sb, Mep3-sc and Mep3-sb.

5. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 2, characterized in that: It also includes industrial Pasteurella β-actin genes ACT1-Sc and ACT1-Sb as internal reference genes. The reverse primers of ACT1-Sc and ACT1-Sb are shown in SEQ ID NO.62 and SEQ ID NO.64 respectively; the forward primers of ACT1-Sc and ACT1-Sb are shown in SEQ ID NO.61 and SEQ ID NO.63 respectively.

6. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 2, characterized in that: In the reaction system of the multiplex PCR reaction, the total reaction system is 20 μL, including 4.0 μL of 25 mM MgCl2, 4.0 μL of 5×PCR buffer, 0.7 μL of DNA polymerase, 2 μL of multiplex PCR reaction primer set, and 9.3 μL of cDNA template; The reaction conditions are: S1, pre-denaturation at 95°C for 10 minutes; S2, denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds; S3, extension at 68-70°C for 1 min, 35 cycles; S4: Store at 4°C, wherein steps S2 and S3 are repeated for 35 cycles in total.

7. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 5, characterized in that: In the reverse transcription reaction primer set, the concentration of SEQ ID NO.2 is 500nM, the concentration of SEQ ID NO.4 is 500nM, the concentration of SEQ ID NO.6 is 500nM, the concentration of SEQ ID NO.8 is 500nM, the concentration of SEQ ID NO.10 is 15.625nM, the concentration of SEQ ID NO.12 is 125nM, the concentration of SEQ ID NO.14 is 500nM, the concentration of SEQ ID NO.16 is 15.625nM, the concentration of SEQ ID NO.18 is 500nM, the concentration of SEQ ID NO.20 is 500nM, the concentration of SEQ ID NO.22 is 31.25nM, the concentration of SEQ ID NO.24 is 500nM, the concentration of SEQ ID NO.26 is 7.8125nM, the concentration of SEQ ID NO.28 is 500nM, the concentration of SEQ ID NO.30 is 1500nM, and the concentration of SEQ ID NO. The concentration of SEQ ID NO.32 is 62.5 nM, the concentration of SEQ ID NO.34 is 500 nM, the concentration of SEQ ID NO.36 is 250 nM, the concentration of SEQ ID NO.38 is 500 nM, the concentration of SEQ ID NO.40 is 31.25 nM, the concentration of SEQ ID NO.42 is 1500 nM, the concentration of SEQ ID NO.44 is 500 nM, the concentration of SEQ ID NO.46 is 250 nM, the concentration of SEQ ID NO.48 is 1500 nM, the concentration of SEQ ID NO.50 is 500 nM, the concentration of SEQ ID NO.52 is 1500 nM, the concentration of SEQ ID NO.54 is 500 nM, the concentration of SEQ ID NO.56 is 500 nM, the concentration of SEQ ID NO.58 is 500 nM, and the concentration of SEQ ID NO.60 is 500 nM; the reverse primers of ACT1-Sc and ACT1-Sb are SEQ ID The concentrations used for both SEQ ID NO.62 and SEQ ID NO.64 were 500 nM.

8. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to claim 5, characterized in that: In the multiplex PCR reaction primer set, the usage concentration of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57 and SEQ ID NO.59 are all 200 nM; the forward primers SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64 of ACT1-Sc and ACT1-Sb are all 200 nM; The concentration of NO.63 used was 200 nM.

9. The method for rapid detection of industrial Pasteurella nitrogen metabolism transport permease gene expression according to any one of claims 2 to 8, characterized in that: The product size of the industrial Pasteurella nitrogen metabolism transport permease gene Bap2-sc is 237 bp, the product size of Bap2-sb is 198 bp, the product size of Bap3-sc is 132 bp, the product size of Bap3-sb is 188 bp, the product size of Gnp1-sc is 214 bp, the product size of Gnp1-sb is 184 bp, the product size of Agp1-sc is 130 bp, the product size of Agp1-sb is The product size of Tat1-sc is 192bp, the product size of Tat1-sb is 107bp, the product size of Tat2-sc is 138bp, the product size of Tat2-sb is 313bp, the product size of Dip5-sc is 233bp, the product size of Mup1-sc is 266bp, the product size of Mup1-sb is 272bp, the product size of Hip1-sc is 221bp, The product size of Hip1-sb is 179bp, the product size of Lyp1-sc is 248bp, the product size of Lyp1-sb is 256bp, the product size of Alp1-sc is 148bp, the product size of Alp1-sb is 390bp, the product size of Gap1-sc is 295bp, the product size of Gap1-sb is 262bp, the product size of Put4-sc is 334bp, the product size of Mep1-sc is 307bp, the product size of Mep1-sb is 366bp, the product size of Mep2-sc is 284bp, the product size of Mep2-sb is 241bp, the product size of Mep3-sc is 349bp and the product size of Mep3-sb is 226bp; the product size of the industrial Pasteurella β-actin gene ACT1-Sc is 361bp, and the product size of ACT1-Sb is 382bp.

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