553 results about "Fluorescent protein" patented technology

The invention relates to a fluorescent protein selection marker staygold-levEr gene as well as a construction method and application thereof, and belongs to the technical field of biological engineering. The invention provides a fluorescent protein selection marker staygold-levEr gene for screening an L-valine production strain and a construction method of a gene fragment, compared with other similar gene selection markers, the selection marker provided by the invention has the advantages that the fluorescence intensity is positively correlated with the concentration of L-valine, and the screening marker has the advantages that the screening marker can be used for screening the L-valine production strain; according to the screening marker, the screening efficiency of the L-valine high-yield strain can be obviously improved, the screening time and cost can be saved, the screening efficiency can be improved, and the screening marker has a wide application prospect.

The invention provides a fluorescent protein selection marker staygold-ygjHr gene and application thereof, and belongs to the technical field of biological engineering. The preparation method comprises the following steps: replacing codons of L-valine in nucleotide sequences of a fluorescent protein gene Staygold and a gene ygjH by using a rare codon GTC to respectively obtain a fluorescent protein gene Staygoldr after codon replacement and a gene ygjHr after codon replacement, and connecting the two segments of genes after codon replacement by using a flexible protein peptide to obtain the fluorescent protein selection marker Staygold-ygjHr gene. Experiments prove that the gene can obviously improve the screening efficiency of L-valine high-yield strains.

A super-resolution light-sheet fluorescence microscopy method, comprising: using reversibly photoswitchable fluorescent proteins (RSFPs) to process a biological sample (1000) to be tested; using a light sheet as activation light to be incident onto said biological sample (1000), so as to generate an activated portion in the reversibly photoswitchable fluorescent proteins (RSFPs); using modulated striped structured light as excitation light to be incident onto the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs) towards a surface where the activated portion is located, such that the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs) on a plane where the light sheet is incident emits fluorescence, wherein the activation light and the excitation light have different wavelengths; and collecting a fluorescence image with structured light stripe information as a two-dimensional fluorescence image for super-resolution image processing. Further provided is a super-resolution light-sheet fluorescence microscopy system.

The invention provides a fluorescent protein selection marker staygold-ynaMr gene and application thereof, and belongs to the technical field of biological engineering. The method comprises the following steps: replacing codons of L-isoleucine in nucleotide sequences of a fluorescent protein gene staygold and a gene ynaM by using a rare codon ATA to respectively obtain the fluorescent protein gene staygold after codon replacement and the gene ynaMr after codon replacement, and connecting the two segments of genes after codon replacement by using a flexible protein peptide, and the fluorescent protein selection marker staygold-ynaMr gene is obtained. Experiments prove that the gene can obviously improve the screening efficiency of L-isoleucine high-yield strains.

The invention belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

The invention relates to a rice embryo specific expression transfluorescent protein gene and Malt gene editing integrated vector and a preparation method thereof, the vector comprises a gene editing fragment and a fluorescent protein gene fragment, the sequence of the gene editing fragment is shown as SEQ ID NO.1; the sequence of the fluorescent protein gene segment is as shown in SEQ ID NO. 2; when the vector is applied to a molecular detection technology, a rice induction line with a homozygous embryo-specific expression transfluorescent protein gene and a mutant gene can be screened out, and the time for creating the induction line with the embryo-specific expression transfluorescent protein gene is saved, so that the cost is saved.

The invention discloses a watermelon transgenic screening expression cassette and application thereof, and belongs to the technical field of biology. According to the invention, an expression cassette of a TagRFP gene driven by a d35s promoter is constructed on a pCambia1300 vector, and a recombinant vector is obtained. A recombinant vector is introduced into watermelon cells by utilizing an agrobacterium-mediated genetic transformation technology, and a red fluorescent protein TagRFP gene is integrated into a watermelon genome, so that transgenic watermelons can be screened and identified by taking red fluorescence as an identifier in the whole growth period of watermelon genetic transformation. Compared with a traditional screening method, the screening method has the advantages that the screening process is greatly simplified, and the transgenic watermelon and the non-transgenic watermelon are clearly distinguished, so that the genetic transformation efficiency of the watermelon is remarkably improved.

The invention discloses construction and application of an escherichia coli L-tryptophan biosensor, and belongs to the technical field of bioengineering. The L-tryptophan biosensor provided by the invention comprises a mutated leader peptide fragment tnaC-tnaA, a promoter of the mutated leader peptide fragment tnaC-tnaA, a green fluorescent protein coding gene eGFP and a pTrc99a plasmid skeleton, a recombinant strain containing the L-tryptophan biosensor is cultured, the expression of green fluorescent protein in a culture solution is gradually enhanced along with the increase of the concentration of the L-tryptophan, and the fluorescence value of a unit cell shows better correlation when the concentration of the L-tryptophan is 0-1.5 g / L; therefore, when the biosensor is used for detecting the L-tryptophan in the fermentation liquor, the biosensor has the characteristics of simplicity in operation, sensitive response and high threshold value.

The invention provides a genetically engineered bacterium and a construction method of the genetically engineered bacterium, and the genetically engineered bacterium is characterized in that a green fluorescent protein sfGFP is used for mediating protein glutaminase, PG (protein glutaminase), PGF (protein glutaminase), PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF and PGF; eC 3.5. 1.44) is efficiently secreted and expressed in engineering bacteria, and belongs to the technical field of biological engineering. The method comprises the following steps: an obtained sfGFP gene is derived from an NCBI database (GenBank numbering: CP035486.1), an obtained PG zymogen gene PP (Propeptide-Protein glutaminase) is derived from a Chryseobacterium prion prgA gene (GenBank numbering: AB046594.1) in the NCBI database, a recombinant plasmid pHT01 / sfGFP-PP is introduced into bacillus subtilis WB800N to obtain an engineering bacterium, extracellular secretion expression of PP is realized, the sfGFP-PP is activated by trypsin to obtain PG, and the PG is subjected to enzyme activation to obtain the recombinant bacillus subtilis. The expression quantity and the secretion efficiency of the PG in the bacillus subtilis are greatly improved. The recombinant PG produced by the method can generate 23.5 U / mL enzymatic activity under activation of trypsin, can be used for improving protein functional characteristics, and has relatively high industrial production and application values.

The invention discloses an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter gene is shown as SEQ ID NO.1. The oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, is obtained by cloning in an oil palm genome for the first time, and the promoter gene has high transcriptional activity and can drive downstream fluorescent protein mNeonGreen expression. The candidate oil palm endogenous U6 promoter gene can be provided for subsequently establishing a high-efficiency oil palm gene editing technology system based on a CRISPR / cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) system.

The invention provides a probe for cell senescence analysis and a preparation method and application thereof, and belongs to the technical field of cell detection. The invention discloses a probe for cell aging analysis. The probe comprises fusion protein formed by fibronectin RGD and fluorescent protein, the fibronectin RGD is a polypeptide fragment containing an amino acid sequence as shown in SEQ ID NO: 1. The probe is fixed on a solid-phase carrier and then co-incubated with cells to be detected, the condition of senescent cells can be analyzed and judged by detecting the intensity of a fluorescence signal, the purpose of rapid analysis is achieved, the analyzed cells can be recycled and then continuously cultured, the detection cost is low, and the probe is suitable for large-scale popularization and application in the market.

The invention relates to the field of biological detection, in particular to fusion protein, a fluorescent probe and application thereof. The invention provides a fusion protein. The fusion protein comprises an acid-sensitive polypeptide, a circulating rearranged fluorescent protein and a connecting peptide, the amino acid sequence of the connecting peptide comprises one or more of LE, TR, SAG and GT. The fluorescent probe disclosed by the invention breaks through limitation of a traditional detection method, can realize rapid real-time quantitative glycollic acid detection, has good detection characteristics of high cost performance, simplicity and convenience in operation, rapid reaction, high specificity and the like, and is a user-friendly glycollic acid quantitative detection tool.

The invention belongs to the technical field of gene engineering, and relates to a construction method of a mouse model capable of rapidly degrading MSTN protein, which comprises the following steps: sequentially inoculating a 5'homologous arm, 3xEAAAK, an FKBP1A gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein mCherry sequence and a 3 'homologous arm on a Donor vector to obtain a Donor recombinant vector; designing gRNA based on a third exon of the Mstn gene, mixing the gRNA, Cas9 protein and a Donor recombinant vector, injecting the mixture into a mouse fertilized egg, and performing in-vitro culture and transplantation to obtain an F0-generation mouse; and hybridizing the F0-generation mouse with a wild type, or continuously breeding the filial generation to obtain the knock-in mouse model. According to the invention, a dTAG targeted protein degradation technology is utilized, the obtained homozygous knock-in mouse is subjected to dTAG administration, and rapid and reversible degradation and removal of MSTN protein can be realized.

Provided herein are methods of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors that include detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof, and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set. Related methods, compositions, kits, systems, and computer readable media are also provided.

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

The invention discloses a method for high-throughput screening of key genes in a 1, 5-pentamethylene diamine biosynthetic pathway of escherichia coli, and the high-throughput screening is carried out by using a 1, 5-pentamethylene diamine biosensor. The 1, 5-pentamethylene diamine biosensor is a recombinant plasmid obtained by integrating a lysine decarboxylase coding gene cadA and a promoter trc thereof, and a green fluorescent protein (GFP) coding gene and a promoter pyrF thereof into a plasmid skeleton. A fluorescent quantitative flow cytometer is used for screening single-cell thalli with high yield of 1, 5-pentanediamine according to the fluorescence intensity of GFP, key genes can be rapidly screened out from a large number of genes to be screened in a high-throughput mode, and the screening workload and time are greatly saved. According to the application of the key gene in the biosynthetic pathway of the escherichia coli 1, 5-pentanediamine screened by the method disclosed by the invention, the yield of the 1, 5-pentanediamine produced by fermenting the escherichia coli KA30 can be greatly improved, the substrate conversion rate in the fermentation process is improved, the production cost of the 1, 5-pentanediamine is reduced, and the economic benefit is improved.

The invention relates to a biosensor capable of efficiently responding to vanillin, and belongs to the technical field of bioengineering. An aldehyde response transcription factor YqhC from Escherichia coli and a promoter element PyqhD regulated by the aldehyde response transcription factor YqhC are used for establishing a vanillin response RFP report system, a yqhC gene is subjected to semi-rational design, a mutation library is constructed through saturated mutation, vanillin is used as a substrate, and a non-background fluorescence response, a non-background fluorescence response function and a non-background fluorescence response function are obtained by screening response concentration and response threshold values. The YqhC mutant has a wider substrate detection range (0-10 mM) and higher response strength (167 times), and is named as YqhC-V18. The biosensor containing a vanillin response element (YqhC-V18) and a fluorescent protein report element (RFP) is further obtained, and can be applied to detection of a sample containing vanillin, or screening of a vanillin production strain, or regulation and control of the production process of vanillin.

The present invention relates to a recombinant human elastin protein, and its preparation method and application. The recombinant human elastin protein comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1, as well as one or more of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. The amino acid sequence of the recombinant human elastin protein is shown in SEQ ID NO.2. The nucleotide sequence is a recombinant plasmid, a recombinant vector, or a recombinant bacterium. The preparation method comprises: synthesizing the nucleotide sequence as shown in SEQ ID NO.1, constructing a recombinant protein, inducing expression, centrifuging, purifying, and drying to obtain the recombinant human elastin protein as shown in SEQ ID NO.2. The application is to detect and evaluate the anti-aging, anti-wrinkle, and firming effects of the recombinant human elastin protein, as well as in cosmetics. The preparation method of the present invention is simple, the protein expression level is high, and the cosmetic product has anti-aging, anti-wrinkle, and firming effects.

The invention belongs to the field of biotechnology and medicine, and particularly relates to mRNA (messenger Ribonucleic Acid) for coding red fluorescent protein and application thereof. The mRNA for coding the red fluorescent protein comprises an ORF sequence of the red fluorescent protein, and the nucleotide sequences of the red fluorescent protein are shown as SEQ ID NO: 5-13. According to the mRNA for coding the red fluorescent protein provided by the invention, the mRNA for coding the red fluorescent protein has relatively high translation efficiency by reasonably optimizing the nucleic acid sequence of the functional region, so that the output of the red fluorescent protein is increased, and the mRNA for coding the red fluorescent protein has a wide application prospect in the field of gene drug development.

The invention discloses a recombinant rabies virus genome plasmid for high-sensitivity expression of red fluorescent protein, a recombinant rabies virus as well as a preparation method and application of the recombinant rabies virus. The preparation method specifically comprises the following steps: (1) constructing the recombinant rabies virus genome plasmid for high-sensitivity expression of red fluorescent protein as shown in SEQ ID NO.2; (2) preparing a recombinant rabies virus for expressing the red fluorescent protein with high sensitivity; and (3) application of the recombinant rabies virus for high-sensitivity expression of the red fluorescent protein in neural circuit tracing. The recombinant rabies virus capable of highly sensitively expressing the red fluorescent protein can efficiently mark an upstream neural network in a retrograde manner, so that the problem of low efficiency of expressing red fluorescence in the existing method is solved; the method has wide application value and market prospect in the aspects of neural circuit structure and function analysis, neuron sparse marking, vaccine and drug research and development, disease model establishment, neural network loss and reconstruction detection, virus replication or pathogenic mechanism research and the like.

The invention discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for targeted knockout of Pten and Trp53 genes, and the recombinant plasmid comprises an EPI vector system. The recombinant plasmid takes an ori element as a replication start site, and sequentially comprises an sgRNA sequence of a targeted Trp53 gene and Pten controlled by double U6 promoters, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, an orip element and an EBNA1 protein expression element. The invention also discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and the established ovarian cancer cell model. By adopting the method to construct the ovarian cancer cell model, the period from cell editing to animal tumor formation is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.

The invention discloses a fluorescence sensor protein design method, a product thereof and a detection method, and belongs to the technical field of biology. The design method of the fluorescent sensor protein is characterized in that the distance between amino acids at two ends of an allosteric domain of the protein is less than 10, and the distance change is more than 2. Based on the design method, the invention also provides a fluorescent sensor protein, a gene thereof, a recombinant expression vector, a transformant, a fluorescent sensor and a method for high-throughput real-time detection of intracellular metabolites, and the fluorescent sensor protein comprises allosteric sterol O-acyltransferase and circulating fluorescent protein cpEGFP; the allosteric sterol O-acyltransferase has an amino acid sequence as shown in SEQ ID NO. 1; the circulating fluorescent protein cpEGFP has an amino acid sequence as shown in SEQ ID NO. 2. The fluorescence sensor protein is applied to concentration detection of intracellular sterol substances, detection is more real-time, and a more accurate tool is provided for analysis of related metabolic flow.

The invention discloses application of phenazine-1-carboxylic acid or medicinal salt thereof in preparation of drugs for inhibiting African swine fever virus. According to the invention, a double-report virus rASFV-Glucc / EGFP for co-expressing green fluorescent protein and Gaussian luciferase is used, and screening from 246 natural small molecule compounds finds that phenazine-1-carboxylic acid has a remarkable inhibition effect on the replication of ASFV; the inhibition effect of the PCA is further evaluated, and it is found that the half maximum cytotoxicity concentration of the PCA to primary porcine alveolar macrophages of target cells is 470.5 mu M, and the half maximum inhibition concentration of the PCA to ASFV in PAMs is 1.59 mu M. According to a dose-dependent inhibition experiment result, when the PCA concentration is 25 [mu] M, the ASFV inhibition effect can reach 100 times or more. The invention has application prospects in preparation of anti-ASFV drugs or preparations and the like.

The invention discloses a construction method and application of a probiotic engineering bacterium EcNpAm targeting animal intestinal tracts and improving inflammation. According to the present invention, the heterologous expression of the immunomodulatory protein Amuc1100 is successfully achieved through the gene engineering technology by using the Escherichia coli Niss1917 (EcN) as the host and using the anaerobic inducible plasmid pnirBMisL as the expression vector, and the novel engineering bacterium EcNpAm suitable for the animal intestinal anaerobic environment is constructed; furthermore, the expression efficiency of the target protein is represented by fusion expression of green fluorescent protein (GFP). The engineering bacterium is applied to prevention and treatment of animal intestinal inflammation, and successfully realizes targeted delivery of therapeutic protein to intestinal tracts of mice. Experiments show that EcNpAm can effectively relieve typical symptoms such as weight loss, colon shortening, pathological injury, colitis and the like of mice induced by dextran sodium sulfate (DSS) and protect integrity and functions of an intestinal barrier structure.

The invention discloses an Nfkb1 gene overexpression cell line and a construction method thereof, and relates to the field of gene engineering. The constructed recombinant lentiviral expression vector comprises a first LTR, a CMV promoter, an Nfkb1 gene, a Flag polypeptide tag, an EF-1a promoter, a green fluorescent protein gene, a shearing element, a screening gene and a nucleic acid fragment of a second LTR which are sequentially arranged from the 5'end to the 3 'end; co-transfecting a host cell by using the recombinant lentivirus expression vector and lentivirus packaging helper plasmid, and carrying out virus packaging to obtain an Nfkb1-overexpressed recombinant lentivirus; the HT22 cell line overexpressing the Nfkb1 is obtained by infecting the HT22 cell with the recombinant lentivirus overexpressing the Nfkb1, and screening the HT22 cell line overexpressing the Nfkb1 by using a resistance gene. Recombinant lentivirus is used as a vector, HT22 cells are infected with the recombinant lentivirus vector, a cell line capable of stably overexpressing the Nfkb1 gene is obtained through screening, and the effect of the Nfkb1 in neuroinflammation is conveniently researched.

The present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

The invention discloses a rapid degreasing method for a biological tissue sample, which comprises the following steps: sequentially carrying out fixing, tissue dehydration, azeotropic degreasing, tissue rehydration and other treatments on the sample, and the degreasing solution is composed of alcohols and hydrocarbons according to a mass ratio of 1: 1-1: 2. According to the rapid degreasing method provided by the invention, the lipid in the tissue is efficiently extracted through mixed azeotropy of the alcohol and hydrocarbon organic reagents, so that the tissue is transparent and transparent; the problem of poor degreasing in tissues is solved by utilizing the high permeability characteristic of the micromolecular organic reagent; the dissolving capacity to phospholipid and cholesterol is improved by mixing organic reagents; through azeotropy, the degreasing efficiency is improved, the degreasing time is shortened, the internal permeability effect of the tissue is improved, and internal and external uniform permeability of the tissue is realized; by adjusting the components of the organic reagent, boiling rapid degreasing at low temperature is realized, and the retention of a fluorescence signal is improved; the tissue treated by the method is transparent and small in deformation, a fluorescent protein signal can be maintained, and the immunofluorescence staining effect can be improved.

The invention provides a recombinant lentivirus capable of indicating bovine spermatogonial stem cell differentiation, a spermatogonial stem cell line and application, and belongs to the technical field of cell biology. The invention provides a method for constructing a spermatogonial stem cell line for indicating bovine sperm differentiation on the basis of a lentivirus transduction technology, and a visual detection system for differentiating spermatogonial stem cells into spermatoblasts is constructed by regulating expression of mCherry protein by utilizing a PRM1 promoter. According to the method, the expression of PRM1 is indicated by fluorescent protein mCherry, so that the differentiation process of the bovine spermatogonial stem cells is indicated, and a good tool is provided for the induction of differentiation of the bovine spermatogonial stem cells to sperms, the research of a development mechanism and the optimization of a differentiation system of the bovine spermatogonial stem cells. The method is a new way for solving the neck clamping problem that good-variety bovine sperms are difficult to obtain in scientific research and production, can also be used for optimization of a spermatogonial stem cell differentiation system, sorting and purification of sperms, even downstream research of drug screening and the like, and has a good application prospect.

The invention discloses a double-report plasmid system and a construction method and application thereof. The double-report plasmid system comprises a translation report plasmid and an error folding response plasmid; the translation reporter plasmid comprises a target protein gene sequence, an mRNA secondary structure sequence and a luciferase gene sequence; the misfolding response plasmid comprises a heat shock pressure promoter and a fluorescent protein gene. The double-report plasmid system can quantitatively and visually detect translation characteristics and wrong folding conditions of the recombinant protein, the whole operation process is carried out based on a 96-well plate, the double-report plasmid system has the characteristics of low cost, high timeliness, high flux and the like, and the recombinant protein can be rapidly and efficiently soluble (positive: complete soluble; negative: completely insoluble; and in an intermediate state, the solubility is relatively poor), so that the efficiency of recombinant protein expression research is greatly improved.

The invention relates to a transgenic fluorescent protein gene specifically expressed by a rice embryo, a vector and a construction method and application of the transgenic fluorescent protein gene. The gene has a fragment of a nucleotide sequence as shown in SEQ ID NO.1; according to the invention, the fluorescent protein gene is combined with the promoter for embryo specific expression, so that the fluorescent protein gene is expressed in the seed embryo, and the diploid seed can be identified in the early stage of the seed through a fluorescent protein observation system, so that the purposes of early screening and removing the diploid seed are achieved.