10 results about "Hygromycin B" patented technology

The invention discloses an oryzias latipe spermatogonial stem cell strain capable of stably expressing Cas9 protein as well as a construction method and application of the oryzias latipe spermatogonial stem cell strain. The oryzias latipe spermatogonial stem cell strain is designed and constructed based on a Tol2 transposon system, wherein the transposon vector comprises a coding sequence of a Cas9 gene, a hygromycin B resistance gene and left and right arm sequences of a Tol2 transposon; constructing a Tol2 transposase expression vector; co-transfecting the transposon vector and the expression vector to the spermatogonial stem cells SG3 of the oryzias latipe; screening in an ESM4 culture medium containing hygromycin B to obtain a drug-resistant cell population capable of stably expressing Cas9; selecting monoclonal colonies under a microscope, and carrying out continuous subculture to obtain an oryzias latipe spermatogonial stem cell strain which stably expresses Cas9 protein and is named as oryzias latipe gonad cells SG3-Cas9. The method has the advantages of short construction period, high efficiency, good expression stability and the like, and provides a reliable cell platform for fish cell level gene editing research.

The invention provides a PEG / CaCl2 mediated Penicillium citrinum genetic transformation method, and belongs to the technical field of microbial genetic transformation, and the method comprises the following steps: adding a target gene segment into Penicillium citrinum protoplast suspension, and carrying out an ice bath reaction; the target gene segment is obtained by taking pJET-hph-02944 plasmid as a template and carrying out PCR (Polymerase Chain Reaction) amplification by adopting a hygromycin B resistance gene primer pair with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; adding a PTC (Positive Temperature Coefficient) buffer solution into the mixed system, uniformly mixing and standing; continuously adding a TB3 liquid culture medium containing ampicillin, and carrying out recovery culture; uniformly mixing the resuscitated protoplast with a molten TB3 solid culture medium, pouring a plate, and carrying out dark culture for the first time; then covering a TB3 solid culture medium containing hygromycin B and ampicillin, carrying out secondary dark culture to obtain a transformant, and transferring the transformant to a PDA plate containing hygromycin B for screening. According to the method, CaCl2-PEG is adopted for mediated transformation of Penicillium citrinum, the transformation efficiency is higher, and the transformation speed is higher.

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.

The invention discloses an eurytopic chlamydomonas reinhardtii expression vector based on a PSAD promoter and a 3 * Flag tag as well as a construction method and application of the eurytopic chlamydomonas reinhardtii expression vector. The nucleotide sequence of the eurytopic chlamydomonas reinhardtii expression vector is shown as SEQ ID No.1; the vector comprises a PSAD promoter, a target gene insertion site EcoR V, a 3 * Flag tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing a skeleton of a pHyg-PSAD-3Flag vector; constructing a sequence fragment fused with a 3 * Flag tag; and connecting the sequence fragment fused with the 3 * Flag tag with a plasmid skeleton fragment through T4 DNA (deoxyribonucleic acid) enzyme so as to construct the pHyg-PSAD-3Flag vector. According to the invention, the expression plasmid of the chlamydomonas pHyg-PSAD-3Flag can be used for efficiently screening algae strains capable of stably expressing target protein.

The application provides a MET2 gene knockout Saccharomyces cerevisiae strain, a construction method and application, and belongs to the technical field of genetic engineering. The MET2 gene knockout Saccharomyces cerevisiae strain is obtained by replacing the MET2 gene in the Saccharomyces cerevisiae strain BSPC040 with a hygromycin B resistance gene and then introducing the pJFE3-XI plasmid. The MET2 gene knockout Saccharomyces cerevisiae strain can significantly improve the glycol alcohol conversion rate of the Saccharomyces cerevisiae BSPC040 strain carrying a xylose metabolic pathway. In the case of consuming the same amount of xylose, compared with the Saccharomyces cerevisiae strain without the MET2 gene knockout, the ethanol yield of the MET2 gene knockout Saccharomyces cerevisiae strain is increased by 23.1%, which provides a new theoretical basis and gene editing strategy for strain optimization in the industrial production of lignocellulosic ethanol.

The application discloses a method for constructing a directional SNP mutant in Neurospora crassa, and the method comprises the following steps: designing and synthesizing a forward primer containing a target SNP and a reverse primer located downstream of a 3'-UTR of a gene, and amplifying a gene segment A containing a mutation site from genomic DNA as a template; respectively amplifying homologous arm segments B and C located upstream and downstream of the segment A, wherein a reverse primer of the segment B comprises a reverse complementary sequence of a forward primer of the segment A; amplifying a hygromycin B resistance gene from a plasmid pCSN44 as a template hph , wherein primers at two ends of the hygromycin B resistance gene are respectively reverse complementary to the reverse primer of the segment A and the forward primer of the segment C; fusing the segments B, A, hph and C into a fusion segment B-A-hph-C through fusion PCR; transforming the fusion segment into the Neurospora crassa through electroporation, and obtaining an SNP site-directed mutant strain through hygromycin resistance screening and first-generation sequencing verification. The application has the advantages of simple operation, high mutation efficiency and accurate site, and provides a reliable technical means for gene function research of the Neurospora crassa.

The invention relates to the technical field of agricultural biology, and provides an efficient and stable canna edulis germplasm resource in-vitro preservation method which comprises the following steps: step 1, selecting healthy and pollution-free sterile canna edulis test-tube plantlets; 2, NAA, BA, kanamycin, hygromycin B, sucrose and agar are added to a B5 culture medium formula, and a culture medium is obtained; 3, the test-tube plantlets are inoculated into a culture medium, the culture medium is placed in an artificial climate box to be cultured, and subculture is conducted once every 6-8 months (the generations are controlled within 6 generations); 4, in the culture process, the growth condition of the test-tube plantlet is recorded regularly, and meanwhile, a conventional culture medium without a growth inhibitor is set as a control group. By optimizing a culture medium formula and controlling culture conditions, excessive growth of test-tube plantlets is effectively inhibited, formation of short and strong seedlings is promoted, and functions of root systems and leaves are enhanced, so that long-term, stable and efficient germplasm resource preservation is realized, the transplanting survival rate is remarkably increased, and the method has important application value.

The application discloses a method for establishing a genetic transformation system of Ophiocordyceps xuefengensis, and belongs to the technical field of microbial genetic transformation. The application provides a method for preparing Ophiocordyceps xuefengensis protoplasts and constructing a PEG-CaCl2 mediated genetic transformation system, and a screening system with hygromycin B as a resistance marker. The hph gene of hygromycin B is successfully transformed into the Ophiocordyceps xuefengensis bacteria by PEG mediation, and the target gene can still be detected after the transformants are transferred for 4 generations, which indicates that the genetic transformation system is successfully constructed. Through genetic transformation, the specific functions and mechanisms of specific genes in the development process of Ophiocordyceps xuefengensis fruiting bodies can be explored through gene knockout or overexpression experiments.

The invention discloses a hansenula polymorpha mediated RNA (Ribonucleic Acid) interference system as well as a construction method and application thereof. The RNA interference system comprises an shRNA (short hairpin ribonucleic acid) interference plasmid pGAPZ DUOX-shRNA (short hairpin ribonucleic acid) and a dsRNA (double-stranded ribonucleic acid) interference plasmid pGAPZ Tubulin alpha-1-dsRNA (double-stranded ribonucleic acid); in the shRNA interference plasmid and the dsRNA interference plasmid, a hygromycin B resistance marker and a CEN / ARS-AmpR sequence are inserted into a pGAPZ A vector; the shRNA interference plasmid further comprises a DUOX-shRNA expression element, and the DUOX-shRNA expression element comprises a shRNA expression element The dsRNA interference plasmid is characterized in that the dsRNA interference plasmid further comprises a Tubulin alpha <-1 > dsRNA expression element. The RNA interference system disclosed by the invention can stably express exogenous double-stranded RNA, and yeast modified based on the RNA interference system can effectively deliver the double-stranded RNA into pest bodies.

The invention relates to a high constitutive activity-based PSAD strong promoter driven exogenous gene expression vector system and a construction method and application thereof. The nucleotide sequence of the system is shown as SEQ ID No.1; the main sequence of the vector system is sequentially composed of the following components: a PSAD promoter, an exogenous gene insertion site EcoR V, a 3 * HA tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B (Hyg +) screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing an outer edge skeleton sequence fragment of the vector system; synthesizing a DNA fragment of the PSAD promoter; and carrying out in-vitro homologous recombination splicing on the skeleton sequence fragment and a DNA fragment of a PSAD promoter, so as to finally obtain the pHyg-PSAD-3HA expression vector. The high-constitutive strong promoter PSAD is added, so that transcription and expression of an exogenous gene can be efficiently driven, and the gene expression is quicker and more stable.