Peroxidase gene related to synthesis of procyanidine as well as prokaryotic expression vector and detection method of peroxidase gene

Abstract

The invention relates to the technical field of molecular biology, in particular to a peroxidase gene related to synthesis of procyanidine as well as a prokaryotic expression vector and a detection method of the peroxidase gene. The invention relates to plant peroxidase (POD), which is an oxidoreductase widely existing in plant tissues, in particular to plant peroxidase (POD). The strain can catalyze decomposition of hydrogen peroxide, and participates in physiological processes of cell wall modification, plant defense reaction, oxidative stress regulation and the like. Plant peroxidase belongs to a polygene family, and a plurality of genes for coding the peroxidase exist in different varieties of plants. The invention clones and provides a POD gene, a prokaryotic expression vector and an induction and detection method thereof. Whether POD is successfully induced or not is detected through SDS-PAGE, and finally a rapid and effective prokaryotic expression induction method is provided. The method has the advantages that the expression product of the target gene can be quickly and effectively identified, and the research of gene functions and the development of protein products are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly relates to a peroxidase gene related to proanthocyanidin synthesis, a prokaryotic expression vector thereof, and a detection method. Background Art

[0002] Plant peroxidase (POD) has many functions that have been difficult to determine due to its broad substrate specificity, spatio-temporal expression patterns, and responses to environmental stimuli. Under conditions of catalyzing hydrogen peroxide or its derivatives, heme peroxidases can oxidize a variety of organic and inorganic compounds (López-molina et al., 2003). By comparing amino acid sequences, its superfamily is divided into three categories (Guo et al., 2007). Class I is intracellular peroxidase, including cytochrome C peroxidase (CcP), gene-duplicated bacterial catalase, and ascorbate peroxidase (APX). Class II is secreted fungal enzymes such as manganese peroxidase and lignin peroxidase (LiP) (Morgenstern et al., 2008). Finally, Class III is secreted plant peroxidase (Basu et al., 2006; Hiraga et al., 2001).

[0003] Class III PODs are widely involved in a series of physiological activities during the plant life cycle. Many POD subtypes are present in the plant cell wall and play key roles in many important biological processes during the plant life cycle by binding to a variety of substrates (Shigeto and Tsutsumi, 2016). Their physiological roles are related to cell wall metabolism (Francoz et al., 2015; Zhou et al., 2002), wound healing (Passardi et al., 2004a), fruit growth and ripening (Andrews et al., 2000; Aydin and Kadioglu, 2001), and auxin metabolism (Kawano et al., 2001). In addition, Class III PODs respond to biotic and abiotic stresses by regulating the levels of various reactive oxygen species. Therefore, studying the functions of Class III POD genes is of great importance for crop genetic breeding.

[0004] Duan Yuqing et al. (2004) found that lotus pod proanthocyanidins have the functions of scavenging free radicals, resisting lipid peroxidation, regulating blood lipids, protecting the heart, etc. They also have a certain protective effect on the skin, can accelerate the biosynthesis of antioxidant enzymes in the skin and serum and improve the activity of enzymes, improve the body's antioxidant capacity, and have a strong regulatory effect on immune function. It also has a significant inhibitory effect on the biosynthesis of melanin, and has anti-melanoma and anti-radiation effects. In previous studies, plant peroxidases can use catechins and other phenolic substances as substrates to polymerize and form dimers in an alkaline environment (Hosny M, et al, 2002). Peroxidases in strawberries can oxidize catechins into dimeric proanthocyanidins, trimeric proanthocyanidins and oligomeric proanthocyanidins (López-Serrano M, et al, 2002; López-Serrano M, et al, 1997).

[0005] Prokaryotic protein expression can be achieved by gene cloning technology, whereby exogenous target genes are expressed in specific prokaryotes or cells by constructing expression vectors and introducing them into expression strains. Prokaryotic expression systems can be used to mass produce recombinant proteins for the production of plant growth regulators, antimicrobial peptides, insect-resistant proteins, etc., thereby increasing crop yield and quality, reducing pesticide use, and ensuring food safety. At the same time, prokaryotic expression systems can also be used to produce industrial enzymes, etc., with the advantages of high efficiency, low cost, and environmental protection, providing a new approach for industrial production.

[0006] In view of the current bottleneck problem of unclear candidate polymerases related to proanthocyanidin polymerization, the present invention aims to use the lotus reference genome, lotus transcriptome and phenotypic data of proanthocyanidin content and polymerization degree differences between different varieties to conduct association analysis on lotus Class III POD genes and to mine important POD genes that may be related to proanthocyanidin synthesis or polymerization. A prokaryotic expression system of the gene is constructed in order to provide a certain scientific basis for the in vitro induction of POD genes and the production of the industrial enzyme. Summary of the invention

[0007] The present invention first provides a POD gene related to proanthocyanidin synthesis, wherein the POD gene is a Peroxidase gene, and the Peroxidase gene is Nn_Peroxidase17 with a sequence as shown in SEQ ID NO.1.

[0008] The present invention also provides the use of the above-mentioned POD gene related to proanthocyanidin synthesis in identifying or assisting in identifying the proanthocyanidin synthesis trait of a species.

[0009] In certain embodiments, the species is Lotus.

[0010] The present invention also provides the use of a substance for detecting the POD gene related to proanthocyanidin synthesis as described above in identifying or assisting in identifying the proanthocyanidin synthesis traits of a species, and the substance is a primer, or a probe, or a gene chip, or a gene detection kit.

[0011] The present invention also provides a prokaryotic expression vector, and the prokaryotic expression vector includes the POD gene related to proanthocyanidin synthesis as described above.

[0012] The present invention also provides a detection method for the Peroxidase gene as described above, and the method includes the step of amplifying the Peroxidase gene.

[0013] In some embodiments, the method further includes the steps of obtaining the phenotypic data of different varieties of lotus flowers and performing an association analysis between the phenotypic data and the candidate differential genes.

[0014] In some embodiments, it specifically includes the following steps: (1) Obtaining differential data by performing transcriptome sequencing on different varieties of lotus flowers; (2) Mining all differential genes of the third class of peroxidases in lotus flowers through GO and KEGG enrichment analysis; (3) Performing an association analysis between the differential genes and the phenotypic data of proanthocyanidin content to obtain the target peroxidase gene; (4) Constructing a prokaryotic vector and inducing the protein for the target peroxidase gene.

[0015] The present invention also provides a kit for identifying or assisting in identifying the proanthocyanidin synthesis traits of a species, and the kit contains a substance for detecting the POD gene related to proanthocyanidin synthesis as described above, and the substance is a primer, or a probe, or a gene chip, or a gene detection kit.

[0016] The present invention finally provides the use of the above kit in any one of the following: (A1) Identifying or assisting in identifying the proanthocyanidin synthesis traits of a species; (A2) Identifying or assisting in identifying proanthocyanidin synthesis germplasm resources; (A3) Breeding for proanthocyanidin synthesis; Optionally, the species is lotus flower.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention provides a method for constructing a prokaryotic expression vector of POD and its induction and detection. It includes inserting the target gene into the prokaryotic expression vector, screening the induction expression conditions by gradient, detecting whether POD is successfully induced by SDS-PAGE, and finally providing a rapid and effective prokaryotic expression induction method. The advantage of the present invention is that it can rapidly and effectively identify the expression product of the target gene, which is helpful for the research of gene function and the development of protein products. Brief Description of the Drawings

[0018] Figure 1 Differentially expressed genes (DEGs) in different parts of two different varieties at the same developmental stage.

[0019] Figure 2 Venn diagram of all DEGs.

[0020] Figure 3 Heat map analysis diagram of 42 POD differential genes.

[0021] Figure 4 Correlation network analysis diagram of 42 POD differential genes and the content of procyanidins in lotus.

[0022] Figure 5 Relative expression level diagram of candidate genes. The relative expression level was quantitatively detected by RT-qPCR.

[0023] Figure 6 Determination diagram of procyanidin content.

[0024] Figure 7 Signal peptide prediction diagram of candidate genes.

[0025] Figure 8 Protein expression result diagram at different induction times.

[0026] Figure 9 Detection diagram of catechin / epicatechin monomer content.

[0027] Figure 10 Detection diagram of gallocatechin / epigallocatechin monomer content.

[0028] Figure 11 Detection diagram of type A procyanidin dimer content.

[0029] Figure 12 Detection diagram of type B procyanidin dimer content.

[0030] Figure 13 Detection diagram of type C procyanidin trimer content.

[0031] Figure 14 Detection diagram of oligomeric procyanidin content OPC. Detailed implementation manners

[0032] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] SEQ ID No.1: >gene18702

[0034] Example 1 Transcriptome Sequencing of Lotus and Heatmap Analysis of Differentially Expressed Peroxidase Genes RNA sequencing was used to analyze the receptacles and seed coats of two lotus varieties, 'Taikonglian 36' and 'Guoqinghong', at three different developmental stages: immature stage (about 10 days after pollination, the seeds are yellow and the embryos are not plump), near-mature stage (about 30 days after pollination, the seeds are greenish-yellow and the embryos are relatively plump), and mature stage (about 45 days after pollination, the seeds are purple-brown and the embryos are plump). Genes were compared according to the criteria of log2(FC) ≥ 2 and false discovery rate (FDR) < 0.01. There were 2,783 differentially expressed genes (DEGs) (1,587 up-regulated and 1,196 down-regulated) between the receptacles of 'Taikonglian 36' and 'Guoqinghong' at the first stage; there were 1,350 DEGs (807 up-regulated and 543 down-regulated) between the seed coats of 'Taikonglian 36' and 'Guoqinghong'. At the second stage, there were 3,149 DEGs (1,989 up-regulated and 1,160 down-regulated) between the receptacles of 'Taikonglian 36' and 'Guoqinghong'; there were 5,854 DEGs (3,267 up-regulated and 2,587 down-regulated) between the seed coats of 'Taikonglian 36' and 'Guoqinghong'. At the third stage, there were 3,118 DEGs (1,723 up-regulated and 1,395 down-regulated) between the receptacles of 'Taikonglian 36' and 'Guoqinghong'; there were 543 DEGs (286 up-regulated and 257 down-regulated) between the seed coats of 'Taikonglian 36' and 'Guoqinghong'. At the second stage, 'Taikonglian 36' and 'Guoqinghong' had the most DEGs in both receptacles and seed coats. See Figure 1 . The Venn diagram showed that the expression levels of 61 genes changed significantly in different parts at different stages. See Figure 2 . Forty-two POD differentially expressed genes were identified by significant heatmap, and differences were found in different parts and different developmental stages. See Figure 3 .

[0035] Example 2 Nn_Peroxidase17 Gene Expression Analysis and Acquisition of Phenotypic Data We extracted proanthocyanidins from the same samples used for RNA sequencing analysis and performed qualitative and quantitative analysis of proanthocyanidin substances by High-Performance Liquid Chromatography (HPLC), obtaining phenotypic data on the proanthocyanidin content in different parts and at different developmental stages of the two varieties. We performed a correlation network analysis on the 42 differentially expressed POD genes and the phenotypic data of lotus proanthocyanidin content determination. See Figure 4 , and found one Nn_Peroxidase17The gene was used as a candidate gene for expression analysis, see Figure 5 and the determination of proanthocyanidin content, see Figure 6 .

[0036] Example 3 Nn_Peroxidase17 Construction and induction of the prokaryotic vector of the gene Through TargetP2.0 (http: / / www.cbs.dtu.dk / services / TargetP / ) and WoLF PSORT (Horton et al., 2007) for Nn_Peroxidase17 signal peptide prediction, it was found that the gene Nn_Peroxidase17 contains a signal peptide, see Figure 7 . To avoid the formation of inclusion bodies of the expressed protein, we truncated the Nn_Peroxidase17 gene by 24 amino acids. The full-length coding region of the truncated Nn_Peroxidase17 was amplified using a high-fidelity enzyme and primers with homologous recombination arms. After the amplified product was recovered, it was ligated to the digested pET-28a vector by homologous recombination. After the recombinant plasmid was sequenced and detected to be correct, 1 μL was taken and added to 50 μL of Escherichia coli BL21 (DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42 °C for 90 s, incubated on ice for 30 min, added 100 μL of antibiotic-free liquid Luria-Bertani (LB) medium, incubated at 37 °C for 30 min, and then added to 10 mL of LB liquid medium containing kanamycin (Kana), and cultured at 37 °C with shaking at 200 r / min overnight. The cultured bacterial solution was expanded at a ratio of 1:100, and cultured at 37 °C with shaking at 200 rpm until the OD600 value reached 1.0 - 1.2. The bacterial solution was transferred to a shaker at 18 °C with shaking at 200 r / min and continued to be cultured for 1 - 2 hours. After the bacterial solution was cooled, 400 μL of 0.5 M isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and induced overnight at 18 °C for 12 h and induced at 37 °C for 4 h, 8 h, 10 h, and 12 h respectively. When induced at 18 °C for 12 h, the induction effect was not obvious; when induced at 37 °C for 4 h, 8 h, 10 h, and 12 h, obvious target bands appeared starting from 4 h, see Figure 8 , which can significantly save experimental time. Nn_Peroxidase17 The optimal induction conditions for the gene are 0.5 M IPTG and induction at 37 °C for 4 h.

[0037] Example 4 Nn_Peroxidase17 Functional verification of the gene The positive bacterial solution with correct sequence sequencing was amplified and cultured, and then plasmid DNA was extracted. The Agrobacterium tumefaciens GV3101 was transformed with the positive plasmid. For the acquisition of transgenic tobacco, the recombinant plasmid was transferred into the receptor tobacco plants by the Agrobacterium-mediated leaf disc transformation method. After co-cultivation, differentiation culture, strong shoot culture and rooting culture to obtain the plants, the DNA of transgenic tobacco leaves was extracted for the identification of positive seedlings. The leaves of positive seedlings were ground into powder in liquid nitrogen, and the total proanthocyanidins were ultrasonically extracted with analytical pure methanol, and then the crude extract was extracted with ethyl acetate, and the oligomeric proanthocyanidins powder was obtained by rotary evaporation. The oligomeric proanthocyanidins powder was dissolved in chromatographically pure methanol and fixed volume to a 25 mL volumetric flask. 1 mL of the fixed volume solution was taken and repeated 3 times in the liquid phase bottle for ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) detection. Nn_Peroxidase17 In the overexpression lines of the gene, the content of proanthocyanidins A changed, which was 1.36 times that of wild-type tobacco, indicating that Nn_Peroxidase17 the gene may be related to the oxidative polymerization of proanthocyanidin A dimers, see Figures 9 - 14 .

[0038] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A peroxidase gene related to proanthocyanidin synthesis, characterized in that, The peroxidase gene is Nn_Peroxidase17 with a sequence as shown in SEQ ID NO.

1.

2. Use of the peroxidase gene related to proanthocyanidin synthesis according to claim 1 in identifying or assisting in identifying the proanthocyanidin synthesis traits of a species.

3. The application according to claim 2, wherein: The species is lotus.

4. Use of a substance for detecting the peroxidase gene related to proanthocyanidin synthesis according to claim 1 in identifying or assisting in identifying the proanthocyanidin synthesis traits of a species; the substance is a primer, or a probe, or a gene chip, or a gene detection kit.

5. A prokaryotic expression vector, characterized in that, The prokaryotic expression vector includes the peroxidase gene related to proanthocyanidin synthesis according to claim 1.

6. A detection method for the peroxidase gene as described in claim 1, characterized in that, The method includes the step of amplifying the peroxidase gene.

7. The detection method according to claim 6, wherein: The method further includes the steps of obtaining the phenotypic data of different varieties of lotus and performing correlation analysis between the phenotypic data and candidate differential genes.

8. The detection method according to claim 7, wherein Specifically, it includes the following steps: (1) Obtaining differential data by performing transcriptome sequencing on different varieties of lotus; (2) Mining all differential genes of the third class of peroxidases in lotus through GO and KEGG enrichment analysis; (3) Performing correlation analysis between the differential genes and the phenotypic data of proanthocyanidin content to obtain the target peroxidase gene; (4) Constructing a prokaryotic vector and inducing protein for the target peroxidase gene.

9. A kit for identifying or assisting in the identification of the proanthocyanidin synthesis trait of a species, characterized in that: The kit contains a substance for detecting the peroxidase gene related to proanthocyanidin synthesis according to claim 1, and the substance is a primer, or a probe, or a gene chip, or a gene detection kit.

10. Use of the kit according to claim 9 in any one of the following: (A1) Identifying or assisting in identifying the proanthocyanidin synthesis traits of a species; (A2) Identifying or assisting in identifying proanthocyanidin synthesis germplasm resources; (A3) Breeding for proanthocyanidin synthesis.

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