38 results about "Transformation efficiency" patented technology

This invention discloses a genetic transformation and gene editing method applicable to multiple sesquiterpene species, belonging to the field of plant genetic transformation and gene editing technology. The method uses the cotyledonary nodes of sterile seedlings of the sesquiterpene genus as explants, and introduces an optimized pScEF1α-Cas9 gene editing transformation vector using Agrobacterium infection. Regenerated plants are obtained through recovery culture, selection culture, and rooting culture. The vector contains four gene expression units: pScEF1α:Cas9, pGmU6:sgRNA, pGmUBI:DsRed2, and pCaMV35S:HygR. In vivo screening is further performed using the DsRed2 fluorescent reporter gene, and the gene-edited plants are identified by molecular detection. This method is applicable to common sesquiterpene, stem-nodled sesquiterpene, and spiny sesquiterpene, and has advantages such as strong species versatility, high transformation efficiency, short cycle, visualized screening, and high positive rate, providing efficient technical support for gene function research and genetic improvement of sesquiterpene species.

The application discloses a method for improving genetic transformation rate of high-altitude meadow grass, comprising the following steps: (1) taking mature seeds of high-altitude meadow grass as explants to induce callus, and obtaining the callus; (2) adding a surfactant Silwet L-77 with a mass fraction of 3‰ of the bacterial liquid to the agrobacterium bacterial liquid carrying a hygromycin resistance gene, and obtaining a culture bacterial liquid; (3) placing the regenerated plant in a screening culture medium containing 40 mg / L of hygromycin resistance for culture, screening the survived plants, and obtaining the genetically transformed plants. The method can improve the transformation efficiency of the high-altitude meadow grass by about 4.54 times through adding the surfactant in the genetic transformation process and cooperating with the screening of the obtained differentiation culture medium.

This invention relates to a genetic transformation method for Ningxia wolfberry using germinating seeds as explants, belonging to the field of plant genetic transformation technology. The invention mainly includes the following steps: (1) After disinfecting Ningxia wolfberry seeds, inoculate them onto a germination medium, and obtain germinating seeds through vernalization and dark culture treatment; (2) Activate and culture Agrobacterium rhizogenes and Agrobacterium tumefaciens strains carrying target gene expression frames to obtain a bacterial solution for infection; (3) Take the germinating seeds obtained in step (1) and mix them with the bacterial solution obtained in step (2) for infection, then transfer the infected germinating seeds to a co-culture medium or substrate for co-culture; (4) Cultivate the material after co-culture in step (3) to induce root growth, and detect the expression level of the target gene or reporter gene on the roots or plants to obtain transgenic positive plants. This invention has the advantages of simple operation, significantly shortened transformation cycle, and high transformation efficiency.

The present application relates to a method for transforming corn, comprising: the primary callus obtained by the method for inducing corn callus, using the method for obtaining corn suspension cell line to obtain corn suspension cell line; agrobacterium infection corn suspension cell group in the corn suspension cell line. The method for transforming corn of the present application first induces the primary callus from corn proembryo, quickly obtains the suspension cell line, shortens the experimental period to 40-50 days, is simple and convenient to operate, has good repeatability, simultaneously makes the corn genetic transformation not affected by the season, meets the needs of annual stable transformation; then uses agrobacterium infection to transform the corn suspension cell line, and regenerates seedlings again; the transformation efficiency can reach about 30%, the transgenic positive rate can reach about 90%, and the single copy rate is about 50%; since the pressure on corn explant (proembryo) supply is significantly reduced, the human and material resources consumption required for culturing the explant is greatly reduced, and large-scale genetic transformation is realized.

This invention discloses a plasmid for gene overexpression in non-pattern *Bacillus laterosporus*. Its leader region contains a methyltransferase expression cassette derived from the *Bacillus laterosporus* i83 genome, enabling stable existence in specific hosts containing the RM system. Based on this, a PidR2 promoter is added to obtain the pMCPidR2 shuttle plasmid. The PidR2 promoter effectively drives target gene expression, the pWV01 replicon ensures plasmid replication in non-pattern hosts, and the chloramphenicol resistance gene is used for positive colony selection. The plasmid contains a TraJ element, allowing it to be introduced into non-pattern microbial hosts via conjugation transfer. This invention also provides a target gene expression strategy in non-pattern microorganisms, achieving high transformation efficiency and high strain yield through conjugation transfer transformation of donor bacteria, conjugation transfer helper bacteria, and recipient bacteria, resulting in stable expression of the target protein. This invention provides an effective vector for efficient and stable gene overexpression in specific non-pattern microorganisms, helping to solve the problem of difficult genetic manipulation of industrial production strains.

This invention provides a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, which employs the Agrobacterium-mediated approach and is based on the optimal Agrobacterium concentration (OD) for stable transformation of oil palm. 600 The optimal Agrobacterium infection time for zygotic embryos was determined by measuring the concentration of acetylsuccinone and co-culture time. The transient GUS expression staining rate was compared between zygotic embryos of different oil palm varieties and at different growth stages. The method described in this invention optimizes infection conditions based on stable transformation parameters, clarifies the optimal explant materials and treatment process, and effectively improves transient transformation efficiency and experimental repeatability. This study establishes for the first time an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtains zygotic embryos most suitable for transient transformation, achieving efficient transient expression of exogenous genes in oil palm zygotic embryos. This provides a rapid and low-cost technical means for studying oil palm gene function.

This invention discloses a PEG-mediated genetic transformation method for *A. anthracnose* protoplasts. Through the preparation, transformation, and transformation of *A. anthracnose* protoplasts, and the screening of transformants, the hygromycin B resistance gene fragment was successfully transferred into the *A. anthracnose* genome, achieving successful genetic transformation of *A. anthracnose* protoplasts. More importantly, this invention establishes a new enzymatic digestion system and improves the culture mechanism in protoplast preparation. During the culture and screening of transformants, a more suitable resistance concentration is used, balancing the stringency of screening with the survival of transformants. This improves transformation efficiency, shortens the overall transformation process, and establishes a more efficient and convenient genetic transformation system for *A. anthracnose* protoplasts.

This invention provides an in vitro cultured bezoar, its preparation method, and its application, belonging to the field of traditional Chinese medicine preparation technology. The preparation method of this invention involves obtaining a bile-resistant, high-β-glucuronidase-active *Escherichia coli* strain through bile gradient acclimation, which is then combined with *Enterococcus faecalis* to form a functional bacterial community. This community is then cultured in vitro through a three-stage process of proliferation, transformation, and crystallization, followed by gradient calcium ion-induced crystallization and vacuum freeze-drying to obtain in vitro cultured bezoar. This invention aims to solve the problems of low conversion efficiency, uneven crystallization, insufficient content of effective components, and unstable product quality in existing technologies. The obtained in vitro cultured bezoar product has significantly higher bilirubin and conjugated bile acid contents than the standards of the *Chinese Pharmacopoeia*, and the process is stable, controllable, and suitable for industrialization.

This invention discloses an enzyme preparation and a method for preparing squalene, belonging to the field of genetic engineering technology. The enzyme preparation includes inositol-2-dehydrogenase and squalene dehydrogenase. The amino acid sequence of inositol-2-dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of squalene dehydrogenase is shown in SEQ ID NO. 8 or SEQ ID NO. 10. This invention mutates wild-type inositol-2-dehydrogenase and wild-type squalene dehydrogenase to obtain specific amino acid sequence mutants: inositol-2-dehydrogenase mutant D270P, squalene dehydrogenase mutant H89L, and squalene dehydrogenase mutant H89L / A294R. These mutants exhibit higher enzyme activity, effectively improving the yield and conversion efficiency of squalene during preparation.

The present application relates to the field of biotechnology, and particularly relates to a method for evaluating DNA methylation transformation efficiency. The present application can simply, quickly, directly and comprehensively evaluate DNA methylation transformation efficiency of the sample to be tested by detecting the methylation and non-methylation ΔCt values of four genes GNAS, GPR1, PAX6 and Actin respectively through conventional fluorescent PCR primer design, and calculating (2 ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1). The method provided by the present application can be used for evaluation and quality control before carrying out methylation experiment or constructing methylation sequencing library, and the sample with low transformation efficiency can be detected in time to avoid carrying out subsequent experiment and causing resource waste such as experimental cost.

The invention discloses a method for increasing protein content and amino acid content of phaeodactylum tricornutum and application. According to the method, a DPH gene in wild type phaeodactylum tricornutum is knocked out through a gene knockout technology, so that the total protein content and the amino acid content of the phaeodactylum tricornutum are increased, a method for knocking out the DPH gene in the wild type phaeodactylum tricornutum comprises the steps that the DPH gene is knocked out in a targeted mode through a CRISPR / Cas9 editing carrier, the nucleotide sequence of gRNA of the DPH gene is knocked out in a targeted mode through the CRISPR / Cas9 editing carrier is shown as SEQ ID No.3, and the content of the total protein in the wild type phaeodactylum tricornutum is increased. The total protein content and the essential amino acid content of the DPH gene knockout phaeodactylum tricornutum strain are remarkably improved compared with those of a wild type strain. The transformed algal strain can be used as a high-quality protein source to be applied to the field of aquaculture, the nutritional value and the transformation efficiency of feed are effectively improved, and the growth performance and the health condition of bred animals are improved. Wide market prospects and important economic values are realized.

The application relates to the field of plant genetic engineering, and specifically discloses a high-efficiency gene transformation system based on the stem tip meristem of a yellowing seedling of Zhongmian 113. The core components of the system include L1 medium suitable for Zhongmian 113, Agrobacterium GV3101 carrying a target gene, a special resuspension solution containing acetosyringone and Silwet-L77, and special primers for overexpression and gene editing; and the preparation method is as follows: seed disinfection and germination are performed to obtain a yellowing seedling, the stem tip is cut in an X shape to expose the meristem, Agrobacterium infection is performed, and then dark culture and light culture are performed, and after transplanting and cultivation to the true leaf stage, positive plants are screened and identified through PCR; the system can be used for gene transformation of Zhongmian 113 and improvement of high yield, high quality, disease resistance and the like, and has the advantages of strong adaptability, high transformation efficiency and short cycle; in addition, the preparation method of the application does not need callus regeneration, is simple and convenient to operate, has a standardized process, is good in repeatability and stability, and can quickly realize transgenic seed acquisition.

The present disclosure relates to a high throughput, scalable system for electroporation of biological cells. The two part system comprises an electroporation reaction array that interfaces with a control unit, providing electrical pulse, temperature control, and mixing of the sample contents. The control unit automates the entire process; electroporation, cell recovery and outgrowth are performed in a electroporation array assembly. The bottom of each reaction well of the electroporation array assembly contains a pair of coplanar electrodes. The bottom surface containing the electrodes is treated in a way to render it hydrophilic. This results in increased wetting of the electrodes thereby increasing transformation efficiency. The electrode configuration allows for processing of smaller sample volumes, reducing the consumption of expensive biological reagents by several orders of magnitude compared to conventional cuvette-based electroporation devices.

The application discloses ZmHSCF1 protein and application of an encoding gene thereof in regulation of callus proliferation and plant regeneration. ZmHSCF1 It is found through agrobacterium infection of maize young embryos by using a vector containing an expressible ZmHSCF1 The overexpression of the gene can promote callus formation and proliferation, and can significantly improve the callus formation rate, the proportion of resistant callus, the regeneration efficiency and the transformation efficiency, and especially, the obtaining of transgenic positive genetic materials of a maize breeding parent, a difficult-to-transform inbred line, is realized. The application has important significance for improving the transformation efficiency and the number of high-quality transformants of plants, especially important maize breeding parents and other stubborn maize inbred lines, and for realizing more accurate, rapid and low-cost creation of breeding new materials by using agrobacterium-mediated transgenic or gene editing dominant molecular design breeding.

The present application relates to the field of biotechnology, and particularly relates to a SNP molecular marker related to feed conversion efficiency of Ningxiang pigs and application thereof. The molecular marker comprises SNP1 and SNP2. The SNP1 molecular marker corresponds to the 97685244th site from the 5' end of chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or G. The SNP2 molecular marker corresponds to the 97782417th site from the 5' end of chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or C. The present application obtains a molecular marker significantly related to the feed conversion efficiency of Ningxiang pigs. The present application can provide guidance for the breeding of Ningxiang pigs by using the molecular marker, and can accurately and efficiently predict the feed conversion efficiency and typing, identify and screen pigs with high feed conversion efficiency and excellent meat quality, and improve the breeding efficiency.

The application provides a method for efficiently and stably transforming algae strains of Porphyra yezoensis by Agrobacterium mediation, and belongs to the technical field of plant transgenesis. The application uses a mixed suspension of leaf thallus fragments, single spores and leaf thallus seedlings of Porphyra yezoensis as a transformation receptor, and establishes an efficient and stable genetic transformation technology through steps such as optimization of an infection solution and gradient screening culture. The application effectively solves the technical problems of low genetic transformation efficiency, poor repeatability and difficulty in screening of transformants of the existing Porphyra yezoensis, and the transformation efficiency can reach 30% to 40%, and the genetic stability of positive plants is close to 100%, thereby providing a reliable technical platform for gene function verification, trait improvement and molecular breeding of Porphyra yezoensis, and having important scientific research value and application prospect.

This invention relates to a novel engineered restriction-modification system for salt-tolerant Jeju bacteria to improve the efficiency of exogenous DNA transformation, belonging to the fields of bioengineering, metabolic engineering, and synthetic biology. This invention involves in-depth analysis of the whole-genome methylation map of Jeju bacteria to identify its unique methylation motif SAGCTS, and further identifies the corresponding restriction endonuclease Sen. Based on this, the restriction-modification system of Jeju bacteria is edited and modified, thereby improving the efficiency of exogenous DNA transformation.

The application belongs to the technical field of enzyme engineering, and relates to a glycoside hydrolase mutant and application thereof. The application successfully obtains a high-temperature stable glycoside hydrolase mutant Bgl8-4X (M332S / H177A / N367P / L254K) through a semi-rational modification strategy, and significantly improves the conversion efficiency of CK. In a high-temperature condition of 70 DEG C, the CK yield of Bgl8-4X reaches 1311.02% of that of the wild-type Bgl8 when a crude ginsenoside extract is used as a substrate, and the application provides a potential candidate enzyme for solving the poor thermal stability difference in multi-enzyme combination and the industrial high-temperature environment adaptation problem.

This application provides a genetically engineered bacterium, its preparation method, and its application. The preparation method of the genetically engineered bacterium includes: knocking out any one or more of the following genes from Bacillus licheniformis 2709: Dmm1, Dmm2, Dcm1, Dcm2, or ngoF, to obtain the aforementioned genetically engineered bacterium. The genetically engineered bacterium prepared using the above method exhibits high transformation efficiency for exogenous recombinant plasmids (e.g., pjoe8999, PMA5, or phy300), effectively overcoming the limitations of Bacillus licheniformis as a genetic tool due to transformation difficulties.

This invention discloses a high-yield Pichia pastoris engineered strain of 11α-hydroxycanrenone and its applications, belonging to the fields of genetic engineering and biocatalysis. Using Pichia pastoris GS115 as the host, and based on the heterologous expression of steroidal 11α-hydroxylase CYP68J5, superoxide dismutase (SOD) and / or catalase (Cat) were overexpressed to construct recombinant engineered strains GS115-CYP68J5-SOD, GS115-CYP68J5-Cat, and GS115-CYP68J5-SOD-Cat. By reducing intracellular peroxidation levels through antioxidant enzymes, the excessive oxidative degradation of the product 11α-hydroxycanrenone was inhibited. The engineered strain co-expressing SOD and Cat achieved a product yield of 82%, which was 5.1 times higher than the control group. The engineered strain constructed in this invention exhibits high transformation efficiency and excellent product yield. The process is simple and easily industrialized, and can be widely applied to the efficient biosynthesis of 11α-hydroxycanrenone.

This invention provides a method and system for the transformation of scientific research results based on a large-scale research model of birth defects in children. It relates to the field of computer application technology within bioinformatics. The method involves performing correlation mapping on input research data on birth defects, generating a cross-data source data correlation network using the large-scale research model, and performing research discovery mining processing accordingly. This generates a package of research discovery mining results, which are then screened for transformation potential. A transformation potential screening report is generated by combining relevant biological knowledge bases and clinical translation rules. Based on the transformation goals that meet the screening criteria, a scientific research result transformation plan is generated. Finally, a scientific research result transformation implementation link is established based on the transformation plan, generating a transformation implementation plan and matching transformation tasks with clinical research institutions and scientific validation platforms. This ensures the smooth implementation of scientific research result transformation and improves transformation efficiency and success rate.

This invention discloses a high-efficiency instantaneous microRNAs This invention relates to the field of plant gene transformation technology, specifically silencing methods and their applications. It provides a prokaryotic expression vector that significantly simplifies the recombinant vector construction process and offers a pair of all... microRNA Applicable primer pairs for constructing target-specific primers. microRNA Recombinant vectors; provided microRNAs Transient silencing methods can be used in plants such as melons where genetic transformation systems are immature, have unstable effects, long experimental cycles, and low efficiency. microRNAs The silent method overcomes existing technological bottlenecks and has the advantages of not damaging plant tissues, simple operation, high conversion efficiency, rapid onset of action, good conversion effect, and low conversion cost; it can also control the conversion duration by controlling the number of sprays; it provides an efficient and flexible technical solution for gene function analysis of melons and other crops.

Disclosed is a gene for boosting genetic transformation efficiency of wheat and use thereof, which belongs to the field of plant genetic engineering technology. The use of a TaHRF1 gene and an encoded protein thereof can promote integration of an exogenous nucleic acid molecule into a genome of a plant of interest, and boost efficiency of the integration of the nucleic acid molecule, and the transformed plant grows and develops normally. The plant of interest includes, but is not limited to, monocotyledonous plants such as wheat. The present disclosure provides a new and important genetic resource for overcoming the genotype dependence in genetic transformation of wheat.

The present application relates to the field of biotechnology and molecular diagnosis, and particularly relates to a methylation detection pretreatment kit and method for protecting DNA integrity, which can solve the problem that DNA degradation is serious in traditional bisulfite conversion technology, and conversion efficiency and DNA integrity are difficult to be obtained simultaneously, the kit comprises a conversion solution, a protection solution, a combination solution, a desulfonation solution, a washing solution and an elution solution, the conversion solution contains 2.5-4 mol / L bisulfite and linear polyethylene polyamine, the protection solution contains an organic denaturant and a polyphenol antioxidant, the DNA melting temperature is reduced by the organic denaturant, the linear polyethylene polyamine accelerates the conversion reaction, and the desulfonation system with low water activity is combined to complete the bisulfite conversion under mild conditions of 50-70 DEG C; the present application can realize the conversion efficiency of greater than 99.5% of unmethylated cytosine, greatly reduce DNA degradation, significantly improve the long fragment DNA recovery rate and the detection sensitivity of trace samples, and is suitable for the methylation detection of three generations of long read sequencing and clinical trace samples.

This invention discloses a highly efficient method for transient silencing of plant functional genes and its applications, belonging to the field of plant genetic transformation technology. The transient silencing method for plant functional genes provided by this invention can be used to study functional genes in plants such as melons where genetic transformation systems are immature, have unstable effects, long experimental cycles, and low efficiency. It overcomes technical bottlenecks and has advantages such as not damaging plant tissues, simple operation, high transformation efficiency, rapid onset of action, good transformation effect, and low transformation cost. Furthermore, the duration of transformation can be controlled by adjusting the number of applications (applying or spraying). This provides an efficient and flexible technical solution for gene function analysis in melons and other crops.

The application provides a method for inducing hairy roots of Erythrina variegata by Agrobacterium rhizogenes, and belongs to the technical field of genetic engineering. By optimizing seed germination methods, Agrobacterium rhizogenes infection methods, Agrobacterium rhizogenes types, injection amounts of bacterial liquid, concentrations of bacterial liquid, and bacteriostatic concentrations, a method for inducing hairy roots of Erythrina variegata is developed, which has high seed germination efficiency, high transformation efficiency, short cycle, convenient screening, and low cost. The hairy roots of Erythrina variegata induced by the method grow rapidly and are genetically stable, and can be used as ideal materials for gene function verification of Erythrina variegata; meanwhile, the hairy roots of Erythrina variegata can provide a root-derived cell factory for industrialized production of active ingredients such as erythrin and polysaccharide.

The present application belongs to the field of biotechnology, and provides a method for inducing transgenic hairy roots of Toona sinensis. The present application successfully uses Agrobacterium rhizogenes transformation for the first time to induce transgenic hairy roots of Toona sinensis, directly observes the efficiency of the transgenic hairy roots through the red RUBY gene and the GUS reporter gene carried by the vector, and calculates the transformation efficiency to be more than 90% according to the strains. The uvGFP gene with a strong green fluorescent signal is successfully transformed, and can be used for the verification and screening of the transgenic hairy roots. The transgenic Toona sinensis tissue is obtained under non-tissue culture conditions, the operation process is simple, the technology is simple, the cost is low, the efficiency is high, the complete transgenic plant can be further generated through the transgenic hairy roots, and the difficulty in the verification of the gene function of Toona sinensis is overcome.

The application belongs to the technical field of genetic engineering, and particularly relates to a transport gene NtABCG6 gene and application thereof in promoting genetic transformation efficiency of multiple species. The nucleotide sequence of the NtABCG6 gene is shown as SEQ ID No. 1. By expressing the NtABCG6 gene, the transition of callus to bud differentiation can be significantly accelerated, and the period from agrobacterium infection to obtaining a rooting positive plant is shortened. Stable regeneration promotion effects are exhibited in Nicotiana tabacum, tomato and potato, and a new strategy is provided for constructing a high-efficiency, stable and low-intervention plant genetic transformation system.

The invention belongs to the technical field of biology, and provides a method for identifying a ribosome DNA autonomous replication sequence and application. A ribosome DNA sequence of a target bacterium is obtained by adopting a three-section amplification theory, free plasmids are constructed, the target bacterium is transformed, and the ribosome DNA autonomous replication sequence of the target bacterium is screened by determining the transformation efficiency and evaluating the existence form and loss rate of the plasmids. According to the invention, the DNA autonomous replication sequence is identified from ribosome DNA, which is easier than that from genomes, especially for non-model organisms. The technology belongs to an enabling technology, and solves the problems of high complexity and low success rate of the prior art. In addition, a three-section amplification strategy is adopted to identify the independent replication sequence of the ribosome DNA, so that the method is simpler, more convenient and quicker than the existing method, the identification of the independent replication sequence of multiple groups of species is facilitated, genetic tools are perfected, and the technical development and application development of microorganisms are assisted.

This invention discloses the application of TaWIP2 protein and its encoding gene in improving the genetic transformation efficiency and yield of wheat, belonging to the field of plant genetic engineering technology. The amino acid sequence of the TaWIP2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the TaWIP2 protein encoding gene is shown in SEQ ID NO.1. This invention discovers a novel TaWIP2 protein and its encoding gene involved in regulating wheat regeneration in the wheat genome, and overexpression of this protein... TaWIP2 While improving the efficiency of wheat genetic transformation, genes can also increase wheat yield, providing new genetic resources for breaking through the bottleneck of wheat genetic transformation, accelerating research on wheat gene function, and improving crop agronomic traits.