93 results about "Competent cell" patented technology

The invention discloses a plant immunomodulatory gene RDP as well as an expression vector and application thereof. The inventor finds a plant immunomodulatory factor gene RDP from potatoes, after a corresponding sequence is obtained through cloning, agrobacterium competent cells are transferred into nicotiana benthamiana, it is verified that the plant immunomodulatory factor gene RDP can be successfully expressed in other plants, and it is verified that the pathogenic effects of various phytophthora are good. The gene is proved to be capable of effectively improving the disease resistance of the nicotiana benthamiana after being expressed. Through objective evaluation of an agrobacterium tumefaciens-mediated transient expression system, it is found that the plant immunomodulatory factor RDP can induce plants to generate resistance, the gene can be directly introduced into the plants through a transgenic technology due to the powerful function of the gene, disease-resistant varieties are developed and cultivated, and the gene has great significance in prevention and treatment of plant diseases.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No: 1 and is named as L8. The preparation method of the antibacterial peptide L8 comprises the following steps: S1, obtaining an L8 target gene segment; connecting an L8 target gene segment with a pLC vector to obtain a connection product; transforming the connection product into an escherichia coli DH5-alpha competent cell, and screening positive clones to obtain a pLC-L8 recombinant plasmid; s2, taking the pLC-L8 recombinant plasmid as a substrate, and carrying out cell-free protein synthesis reaction to obtain the antibacterial peptide L8, the cell-free protein synthesis system comprises 175 mM of potassium glutamate, 10 mM of ammonium glutamate, 2.7 mM of potassium oxalate, 33 mM of PEP, 38 mM of compound amino acid, 10 mM of magnesium glutamate, 2% (w / v) PEG-8000, 5 nM of pLC-L8 recombinant plasmid, 11.25 mM of lactose, 33.33% (v / v) of crude extract, 100 mg / mL of escherichia coli tRNA and 2.5 nM of T7 RNA polymerase. The antibacterial peptide L8 is obtained by optimizing the antibacterial peptide LRGG, and compared with the LRGG, the antibacterial peptide L8 subjected to cell-free protein synthesis reaction expression has better antibacterial activity and biological safety.

The invention discloses a type 1 goose astrovirus virus-like particle and a preparation method and a pharmaceutical composition thereof, and the preparation method comprises the following steps: cloning a gene segment for coding type 1 goose astrovirus ORF2 protein to a baculovirus transfer vector, and constructing to obtain a recombinant plasmid; transforming the recombinant plasmid into a DH10Bac competent cell to obtain a recombinant baculovirus rod grain; transfecting the recombinant baculovirus stem into an insect baculovirus expression system to obtain a recombinant baculovirus; and inoculating the recombinant baculovirus into an insect baculovirus expression system, culturing, collecting a cell culture, and purifying to obtain the purified goose astrovirus type 1 virus-like particles. The GAstV-1 VLPs and the vaccine thereof provided by the invention not only solve the technical problem of research and development of the GAstV-1 vaccine, but also have the advantages of clear structure, high safety, good immunogenicity and exact protective efficacy, and have remarkable creativity and industrial application value.

The invention discloses a yeast clone with a pAbAi reporter gene as well as a construction method and application thereof, the yeast clone comprises a bait sequence Bit as shown in SEQ ID NO: 1 and an original promoter Pmin as shown in SEQ ID NO: 2, the promoter of the pAbAi reporter gene is formed by the inserted Bit and Pmin, a pAbAi / -CAAT plasmid as a no-load plasmid as shown in SEQ ID NO: 3 is constructed, the bait sequence as shown in SEQ ID NO: 1 is connected, and the pAbAi reporter gene is obtained. BstBI is used for carrying out single enzyme digestion on pAbAi-Bait / CAAT plasmids, Y1Hgold yeast competent cells are converted, SD / -Ura plates are coated, after the Y1Hgold yeast competent cells grow for 3-5 days, monoclonal colonies are selected and subjected to colony PCR detection, it is confirmed that a bait sequence is successfully integrated into the Y1Hgold yeast competent cells, and subculture is carried out, so that the Y1Hgold yeast is obtained.

The application discloses a construction method and application of engineering escherichia coli for producing humulone, and the pTrcHis2B-IDI plasmid and pACYDuet-1-CCL4-VPS-PT1-linker-PT2 plasmid are transferred into engineering escherichia coli Trc-low competent cells to obtain the engineering escherichia coli for producing humulone. The engineering humulone bacteria are constructed based on the Trc-low competent cell host, the pACYDuet-1-CCL4-VPS-PT1-linker-PT2 is combined with the pTrcHis2B-IDI, the side chain precursor activation function of CCL4 is matched with the IDI / DMAPP donor module, the VPS-mediated skeleton construction and the PT1 / PT2-mediated continuous isoprenylation process are further connected, the humulone target path can be more completely operated in the same host, and therefore the specificity, continuity and directional guiding ability of the product biosynthesis are improved.

The application discloses a bacteriocin synthesis regulation gene plnC overexpression and a construction method and application of a recombinant plant lactis bacillus, and has the characteristics that the nucleotide sequence is shown in SEQ ID No:1 in the sequence table, and the construction method steps are as follows: 1) taking the plant lactis bacillus genomic DNA as a template, designing specific primers and performing PCR amplification; 2) purifying and double enzyme cutting the plnC gene fragment, and connecting the linear carrier pMG36e which is also double enzyme cut to obtain a pMG36e-plnC recombination cloning carrier; (3) through an electric transformation method, the pMG36e-plnC is transferred into plant lactis bacillus B1 competent cells to obtain a plnC overexpression strain B1-plnC, and the advantages are that the synthesis of the plant lactis bacillins is promoted, the plnC gene expression amount of the overexpression strain B1-plnC is increased by more than 4.5 times, and the PlnC protein content is increased by more than 1.5 times.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

The invention is applicable to the technical field of genetic engineering, and provides a vaccine for preventing and treating staphylococcus aureus mastitis and application of the vaccine. A recombinant bacillus subtilis construction method comprises the following steps: taking a DNA sequence as shown in SEQ ID NO.3 as a template, and performing PCR amplification to obtain a PCR product sequence; connecting the PCR product to a pHT43 vector, and converting the pHT43 vector into a competent cell DH5alpha, so as to obtain a recombinant plasmid pHT43-cCsa; the method comprises the following steps: transforming a recombinant plasmid pHT43-cCsa into a competent cell of bacillus subtilis WB800N, taking pHT43-F and pHT43-R as primers, and screening a positive transformant through a PCR (Polymerase Chain Reaction) technology; the recombinant bacillus subtilis for expressing the staphylococcus aureus Csa protein is constructed by utilizing a homologous recombination technology, so that pathological injury caused by staphylococcus aureus infection can be reduced, and staphylococcus aureus mastitis of dairy cattle can be prevented.

The invention discloses a crassostrea hongkongensis NKA-alpha protein polyclonal antibody as well as a preparation method and application thereof. The preparation method comprises the following steps: constructing a recombinant expression vector containing an NKA-alpha target gene segment; transforming the recombinant expression vector into competent cells to construct a recombinant expression strain, inducing the recombinant expression strain to express fusion protein, and collecting and purifying to obtain NKA-alpha recombinant protein; carrying out animal immune treatment by taking the NKA-alpha recombinant protein as an antigen, collecting and separating antiserum, and purifying to obtain the Crassostrea hongkongensis NKA-alpha protein polyclonal antibody. The prepared Crassostrea hongkongensis NKA-alpha polyclonal antibody is high in affinity, can specifically recognize NKA-alpha protein in Crassostrea hongkongensis, and can be widely applied to Crassostrea hongkongensis biological immunodetection.

The invention relates to a method for regulating and controlling a flower type by silencing a peony organ gene based on a transient expression system, which belongs to a peony flower type regulation and control method and comprises the following steps of: designing a specific primer aiming at a target gene PsuAG, obtaining a target fragment through PCR (Polymerase Chain Reaction) amplification, and inserting the target fragment into a transient expression vector to construct a gene silencing fusion expression vector; introducing the gene silencing fusion expression vector into an agrobacterium competent cell, preparing an infection solution to infect a peony plant, and injecting the infection solution into a pedicel by using an injector when a flower bud of the peony plant develops to an initial stage of a large wind chime period; when the flower buds grow to the initial stage of the round peach stage, injecting the infection liquid into pedicels and sepal bases; after each time of injection, weak light culture is carried out under a shading condition. The efficient silencing of the key gene PsuAG for regulating stamen petals is realized, and the double petals rate of the peony flower type is improved. The method does not need to depend on a stable genetic transformation system, operation is directly carried out on floral organs of a complete plant, and the gene function verification period is remarkably shortened.

The present application belongs to the technical field of genetic engineering, and relates to a CrtI enzyme mutant, a recombinant plasmid, a recombinant strain and application thereof. The CrtI enzyme mutant provided by the present application is obtained by random mutation of a wild-type CrtI gene through error-prone PCR, and the amino acid sequence thereof is shown as SEQ ID NO. 1. The obtained mutant has multiple site variations in the amino acid sequence, thereby significantly improving the catalytic efficiency and substrate affinity of the enzyme. The present application further constructs a recombinant expression vector containing the CrtI enzyme mutant gene, and transforms the recombinant expression vector into a red Phaffia yeast competent cell, and obtains a high-yield astaxanthin recombinant strain through resistance screening and pigment phenotype screening. Compared with the wild-type CrtI enzyme, the CrtI enzyme mutant of the present application significantly improves the conversion rate of beta-carotene to astaxanthin in the red Phaffia yeast.

The invention discloses a green fluorescence report system for screening a Listeria monocytogenes quorum sensing inhibitor as well as a construction method and application of the green fluorescence report system. The report system is constructed through the following steps: synthesizing a DNA fragment according to a listeria monocytogenes AGR system pII promoter sequence, connecting the DNA fragment with a green fluorescent protein pEGFP plasmid, amplifying an EGFP-pII target fragment through PCR, and connecting the target fragment to a pKSV7 shuttle vector plasmid through homologous recombination to obtain a pKSV7-EGFP-pII plasmid, and then transferring the gene into a competent cell of listeria monocytogenes ATCC BAA-679, and screening to obtain a report system. The report system disclosed by the invention can be used for intuitively evaluating the expression condition of the quorum sensing system of the listeria monocytogenes and screening out the quorum sensing inhibitor.

This invention discloses an engineered bacterium expressing the sqr gene and its application in aerobic remediation of hexavalent chromium pollution. The construction method of the engineered bacterium includes the following steps: S1. Constructing the pEcgRNA-sqr plasmid; S2. Obtaining donor DNA; S3. Preparing electrocompetent cells; S4. Co-transforming the pEcgRNA-sqr plasmid and donor DNA fragment into the electrocompetent cells, performing resistance screening, and obtaining engineered bacteria integrating the sqr gene. This invention utilizes the constructed engineered bacterium expressing SQR coupled with nano-ferrous sulfide to establish a "sulfur metabolism-FeS regeneration" cycle system, accelerating Cr(VI) reduction. Under aerobic / microaerobic conditions, the removal rate of 50 mg / L Cr(VI) in 1 hour can reach over 95%, while simultaneously alleviating the competitive pressure of intracellular reducing power caused by heterologous expression of the sqr gene, improving remediation efficiency and strain growth stability, and achieving a synergistic effect of biochemistry.

This invention constructs pET-32a-CP1 and pET-32a-CP2 expression vectors based on the CP1 and CP2 protein genes of Giardia duodenalis, transforms them into Rosetta (DE3) competent cells, and induces expression using IPTG. After SDS-PAGE analysis of the recombinant protein expression, Ni-NTA affinity chromatography is used to purify the proteins. Two indirect ELISA methods are then established using these as coating antigens. The indirect ELISA methods based on the recombinant proteins CP1 and CP2 of this invention exhibit good specificity, sensitivity, and repeatability, with concordance rates of 98% and 94% with microscopic detection results, respectively.

The invention provides a genome editing method for animal bifidobacterium. The genome editing method comprises the following steps: constructing plasmids capable of being expressed in bifidobacterium; carrying out demethylation on the plasmid, and carrying out in-vitro methylation modification; the plasmid subjected to methylation modification is electrically transformed into competent cells of animal bifidobacterium; a gene editing mutant strain is obtained through resistance screening and homologous recombination, a'totipotent 'plasmid which can express Cas9 and sgRNA in bifidobacteria and carries a homologous repair template is constructed, the plasmid is protected by simulating a methylation modification system of bifidobacteria in vitro, then the plasmid is electrically transferred into a thallus, and the homologous repair template is obtained. A gene editing mutant strain is obtained through resistance screening and homologous recombination, gene knockout is achieved in animal bifidobacterium, flow gene editing is achieved, methylation modification is conducted on non-methylation plasmids in vitro, and a mutant strain without a selection marker can be obtained. In addition, the invention also provides a genome editing system, electronic equipment and a computer readable medium.

The invention discloses a urate oxidase gene derived from aspergillus cristatus and application thereof, and the urate oxidase gene is characterized in that a natural urate oxidase gene coding region sequence is obtained from the aspergillus cristatus, and the gene is optimized according to the codon preference of escherichia coli to obtain an optimized sequence. Connecting the optimized gene to a carrier through NdeI and BamHI restriction enzyme cutting sites, and constructing a wild type uox recombinant plasmid; the method comprises the following steps: replacing a sequence of uox of aspergillus cristatus for coding 243-site tyrosine (Y) with a sequence for coding lysine (K) through site-specific mutagenesis, constructing mutant Y243K recombinant plasmids, respectively transforming the recombinant plasmids into competent cells, culturing, and adding IPTG to induce target protein expression. And collecting the induced thalli, carrying out ultrasonication and centrifugation to collect the supernatant so as to obtain a crude enzyme, and purifying the crude enzyme through a nickel affinity column so as to obtain the purified UOX protein. Uric acid is used as a substrate to measure the enzyme activity of the purified UOX, and the result shows that the specific enzyme activity of the mutant Y243K is obviously higher than that of a wild type, and the activity is improved by 2.75 times, so that the mutant Y243K has higher application value.

The invention discloses a group of nocardia seriolae mycolic acid synthase genes and a construction and application method of a deletion strain of the nocardia seriolae mycolic acid synthase genes, and belongs to the technical field of disease prevention and control. Comprising the following steps: strain culture and whole genome extraction, specific primer design and gene fragment cloning, deletion plasmid construction, competent cell preparation, electrotransformation and deletion strain screening, and deletion strain characteristic verification. By analyzing the synergistic function of nocardia seriola seriola mycolic acid synthesis key genes, the core effect of the nocardia seriola seriola mycolic acid synthesis key genes in a synthesis pathway is clear to fill the blank of multi-gene association research, and a core theoretical basis is provided for revealing the pathogenic mechanism of nocardia seriola seriola mycolic acid.

The invention provides a method for prokaryotically expressing cat allergen Fel d 1 recombinant protein, which comprises the following steps of: cloning a cat allergen Fel d 1 gene to multiple cloning sites of an expression vector pGEX-4T-1 to obtain a recombinant expression vector pGEX-4T-1-Fel d 1, transforming the recombinant expression vector into escherichia coli Rosetta (DE3) competent cells to construct recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), and carrying out prokaryotic expression on the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) to obtain the cat allergen Fel d 1 recombinant protein. IPTG (isopropyl-beta-d-thiogalactoside) is used for inducing protein expression, and a GST affinity chromatography medium is used for capturing high-purity soluble GST-Fel d 1 fusion protein. According to the invention, a pGEX-4T-1-GST label system is combined with Escherichia coli Rosetta (DE3), so that the problem of soluble expression of the cat allergen Fel d 1 protein is fundamentally solved.

The invention provides an anti-mycoplasma hyopneumoniae nano antibody and application thereof, and belongs to the technical field of biology. The amino acid sequence of the nano antibody is as shown in SEQ ID NO: 2. The invention also provides a coding gene of the nano antibody, a recombinant vector containing the coding gene, a recombinant strain and a recombinant cell. The preparation method of the nano-antibody comprises the following steps: cloning a coding gene of the nano-antibody into a vector, and then converting into a competent cell to obtain a recombinant strain for expressing the nano-antibody; after IPTG induced expression is adopted, the nano antibody is obtained. According to the present invention, the mycoplasma hyopneumoniae-oriented nano-antibody with the metabolic inhibition effect is firstly found, the metabolic inhibition valence is 105, the mycoplasma hyopneumoniae-oriented nano-antibody is used for preventing and / or treating mycoplasma hyopneumoniae infection, the effect is significant, and the use of antibiotics is reduced. The preparation method of the anti-mycoplasma hyopneumoniae nano antibody is simple, the cost is low, the yield reaches 70 mg / L, and the anti-mycoplasma hyopneumoniae nano antibody is suitable for large-scale production.

This invention relates to the field of recombinant protein technology, specifically to an α-hemolysin recombinant protein and its prokaryotic expression method. A method for preparing a high-purity, tag-free α-hemolysin monomer is as follows: an expression vector encoding the fusion protein of SEQ ID NO.7 is constructed and transformed into competent cells to obtain engineered bacteria; after culturing and inducing expression, the bacterial cells are lysed and centrifuged to obtain the supernatant; the fusion protein eluate is obtained by a first affinity chromatography on a nickel column, dialyzed, and digested with HisSUMO protease; a second nickel column chromatography is performed, and the flow-through is collected to obtain a high-purity, tag-free α-hemolysin monomer. This technical solution can solve the technical problems of existing prokaryotic expression of α-hemolysin recombinant proteins, such as severe inclusion body formation, complex purification processes, insufficient product activity, and large batch-to-batch variability, thereby obtaining a high-purity, highly active, and structurally intact α-hemolysin monomer, which has ideal application value.

The application belongs to the technical field of biotechnology, and particularly relates to a Clostridium perfringens bacteriophage lytic enzyme produced by Bacillus subtilis, a preparation method and application thereof, an optimized Clostridium perfringens bacteriophage lytic enzyme coding nucleic acid sequence according to the codon bias of Bacillus subtilis, and an optimized Clostridium perfringens bacteriophage lytic enzyme coding nucleic acid sequence as shown in SEQ ID NO. 1. The preparation method of the Clostridium perfringens bacteriophage lytic enzyme is as follows: firstly, a recombinant expression vector containing a Clostridium perfringens bacteriophage lytic enzyme gene is constructed, and then the vector is transferred into a Bacillus subtilis competent cell to obtain a Bacillus subtilis strain recombinant engineering bacterium. After the recombinant strain is induced to express, a fermentation liquor with Clostridium perfringens bacteriophage lytic enzyme activity can be obtained. The fermentation liquor can specifically inhibit the growth of Clostridium perfringens, and provides a safe, efficient and orally-taken preparation for treating animal diseases caused by Clostridium perfringens.

A bacteriophage lytic enzyme and its application. The name of the bacteriophage lytic enzyme is PlyNJ3; the gene fragment encoding the lytic enzyme PlyNJ3 is amplified from streptococcus suis by PCR, and a prokaryotic expression vector plasmid pET32a-PlyNJ3 containing the PlyNJ3 gene is constructed, the recombinant plasmid is obtained by transforming DH5 alpha competent cells, and the protein is massively expressed in an escherichia coli expression system. Then, the lytic enzyme PlyNJ3 is purified by affinity purification medium, the high-efficiency expression of the lytic enzyme PlyNJ3 in vitro is realized, and the lytic effect of the lytic enzyme PlyNJ3 on different serotypes of streptococcus suis, streptococcus agalactiae and other common streptococci is verified in vitro. The bacteriophage lytic enzyme PlyNJ3 prepared by the application can selectively lyse target pathogenic bacteria, and has a good application prospect in the development of new bacteriophage drugs.

The invention discloses a recombinant Pasteurella multocida-sourced uridine diphosphate glucuronide transferase (PmHS2) mutant and a preparation method thereof, and relates to the technical field of biology, the mutant is obtained by cloning a coding gene to a pET26b (+) vector, transforming BL21 (DE3) Escherichia coli competent cells, and then transforming the recombinant Pasteurella multocida-sourced uridine diphosphate glucuronide transferase (PmHS2) to a pET26b (+) vector. And carrying out IPTG (isopropyl-beta-d-thiogalactoside) induced culture, ultrasonication and nickel affinity chromatography purification to obtain high-purity protein. Compared with wild type recombinant PmHS2, the activity of N-acetyl glucosamino transferase of the mutant is improved by 70%, the residual activity is 67.1% after the mutant is stored at room temperature for 5 days, the soluble expression quantity is improved by 6 times, and the mutant and the preparation method have the advantages of high catalytic efficiency, high stability and high solubility, and the mutant and the preparation method are mature in process, controllable in cost and suitable for industrial production. The method can be efficiently used in the fields of heparin and derivative synthesis, traditional Chinese medicine active ingredient glycosylation and the like, is suitable for large-scale production and has a wide application prospect.

This invention discloses the Clostridium difficile phage recombinant lyase Lys51, its preparation method, and its application, relating to the field of genetic engineering technology. The invention involves introducing the recombinant plasmid pET-28a-SUMO-Lys51 into Escherichia coli BL21(DE3) competent cells via heat shock transformation; screening to obtain the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51; constructing the recombinant plasmid pET-28a-SUMO-Lys51 and the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51, further inducing the expression of Clostridium difficile phage recombinant lyase Lys51 in the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51, and then purifying it to achieve the complete process for preparing Clostridium difficile phage recombinant lyase Lys51.

The invention belongs to the technical field of molecular biology, and discloses a saline-alkaline resistant gene MdLAC12 for enhancing apple and application thereof, an extraction method of the saline-alkaline resistant gene MdLAC12 for enhancing apple comprises the following steps: step 1, extracting total RNA (Ribonucleic Acid) from an apple rootstock M9-T337 root system, and taking cDNA (Complementary Deoxyribonucleic Acid) obtained by reverse transcription as a cloning template; 2, designing an MdLAC12 primer according to an apple genome database GDDH13, and carrying out a conventional polymerase chain reaction (PCR); and step 3, carrying out homologous recombination on the obtained PCR product of the MdLAC12 and pMD18-T, transforming a connection product into an escherichia coli DH5 alpha competent cell, and selecting resistant bacterial plaque for sequencing analysis to obtain a CDs sequence of the MdLAC12. The method is helpful for breeding excellent saline-alkaline tolerant apple rootstocks, and can also improve utilization of saline-alkaline land in a prebiotic area. The method has the advantages of simplicity in operation, high saline-alkaline tolerance and low production cost, and can be widely applied to the saline-alkaline tolerance germplasm resource breeding process.

The invention discloses a construction method of genetically engineered bacteria X33-H-CZS-4 for expressing fusion peptide H-CZS-4, which comprises the following steps: step 1, preparing a fragment with an HSAD2-D3 tag, step 2, preparing a fragment containing a complete CZS-4 coding sequence, assembling the fragment containing the complete CZS-4 coding sequence and the fragment in the step 1 in escherichia coli Top10 competent cells to construct an expression plasmid pPICZalphaA-HSAD2-D3-CZS-4, and step 3, constructing the expression plasmid pPICZalphaA-HSAD2-D3-CZS-4 by using the expression plasmid pPICZalphaA-HSAD2-D3-CZS-4. The method comprises the following steps: 1, carrying out enzyme digestion on the expression plasmid pPICZalphaA-HSAD2-D3-CZS-4, 2, carrying out amplification culture on the expression plasmid pPICZalphaA-HSAD2-D3-CZS-4 to prepare pichia pastoris competent cells, 3, carrying out amplification culture on pichia pastoris X33 to prepare pichia pastoris competent cells, and 4, carrying out enzyme digestion linearization on the obtained expression plasmid pPICZalphaA-HSAD2-D3-CZS-4, and carrying out transformation on the expression plasmid pPICZalphaA-HSAD2-D3-CZS-4 and the pichia pastoris competent cells to obtain the According to the present invention, the peptide H-CZS-4 with the HSAD2-D3 tag is integrated into the pichia pastoris X33, such that the genetically engineered bacterium X33-H-CZS-4 expressing the fusion peptide H-CZS-4 is successfully constructed;

The invention discloses a tea tree cold-resistant gene editing and molecular marker-assisted breeding method, and relates to the field of genetic engineering breeding and molecular breeding, and the method comprises the following steps: designing a CAPS molecular marker specific primer according to an SNP site Chr7: 221281848, carrying out PCR amplification on DNA of a tea tree, carrying out enzyme digestion on a PCR amplification product by using TaqI to detect the genotype, and selecting a CC genotype tea tree to induce callus; the method comprises the following steps: inserting a CsWRKY19 gene into a pBI121 vector to obtain a CsWRKY19 gene overexpression vector; transferring the overexpression vector into an agrobacterium competent cell, and infecting the callus; screening, proliferating and differentiating the infected calluses, carrying out PCR (Polymerase Chain Reaction) screening to obtain positive seedlings, and culturing to obtain cold-resistant tea tree plants; according to the breeding method, the technical bottlenecks of long period, low efficiency and poor accuracy in cold-resistant breeding of the tea trees are broken through through a double-track collaborative breeding strategy of gene editing enhancement and molecular marker screening.

The invention discloses a multi-copy expression construction method of an insecticidal fusion peptide U10-MYRTX-Mri1a-GNA with bacteriostatic activity in pichia pastoris, which comprises the following steps: 1, carrying out reverse PCR (Polymerase Chain Reaction) on a plasmid pGAPZalpha A, introducing a Hind III restriction enzyme cutting site to obtain a plasmid pGAPZalpha A-Hind III, 2, constructing a fragment containing a U10-MYRTX-Mri1a gene, and 3, carrying out multi-copy expression on the fragment containing the U10-MYRTX-Mri1a gene to obtain the insecticidal fusion peptide U10-MYRTX-Mri1a gene. The method comprises the following steps: 1, constructing a U10-MYRTX-Mri1a gene fragment, 2, constructing a U10-MYRTX-Mri1a gene fragment, 3, introducing a GGGSAAA linker and a snowflake agglutinin (GNA)-6 * His label to the C end of the U10-MYRTX-Mri1a gene fragment, 4, constructing a multi-copy expression vector pGAPZalpha A-HindIII-Mira1-GNA * 3, 5, preparing a pichia pastoris competent cell X33, and 6, carrying out electrochemical conversion to obtain a genetically engineered bacterium X33-pGAPZalpha A-HindIII-Mira1-GNA * 3. According to the invention, a linearized plasmid is transferred to pichia pastoris X33, so that a genetically engineered bacterium for expressing the fusion peptide U10-MYRTX-Mri1a-GNA is obtained.

The application belongs to the technical field of biology, and discloses a construction method and application of a subtilin expression strain, wherein the recombinant strain is obtained by transfecting a plasmid carrying a spaBTCSIFEGRK whole gene sequence of bacillus subtilis ATCC6633 and containing or not containing an original promoter into BS168 competent cells, so as to obtain a recombinant strain with high expression of subtilin, wherein the recombinant strain has very high safety, a clear genetic background, is easy to be high-density fermented, and has strong protein secretion capacity compared with the original strain.