31 results about "Complementation" patented technology

The invention provides a spatial omics data completion method and system based on a variational graph auto-encoder, and relates to the technical field of bioinformatics. Extracting partial common genes in the data pair to obtain a feature matrix; based on the spatial position information, obtaining a first sub-adjacency matrix of cells in the spatial transcriptomics sequencing data; respectively obtaining a second sub-adjacency matrix of the cells in the single-cell RNA sequencing data and a third sub-adjacency matrix of the cells in the data pair based on gene expression similarity; combining the first sub-adjacency matrix, the second sub-adjacency matrix and the third sub-adjacency matrix to obtain a final adjacency matrix; and inputting the feature matrix and the final adjacent matrix into a pre-trained variational graph auto-encoder network to complete the deletion gene of the space transcriptomics. According to the method, the position information and gene expression characteristics between idle data cells can be effectively utilized, errors can be reduced, and meanwhile, the similarity between complementation genes and true values can be improved.

The present application relates to the field of gene editing technology, bioengineering and application of microbial fermentation, and discloses a Pichia pastoris high-efficiency gene editing system, a construction method thereof and application in a high-yield cordycepin engineering strain.The construction of the Pichia pastoris CRISPR-Cas9 gene editing system mainly includes the following steps: the histidine HIS4 nutritional deficiency back complementation is used to realize the resistance-free assembly of KhCas9; the sgRNA-tRNA array and the IIS type restriction endonuclease combination are used to simplify the gene editing operation and realize the simultaneous and high-efficiency editing of multiple genes; and the Brex27 domain is fused with KhCas9 to strengthen the homologous recombination efficiency. By using the gene editing system, through the optimization of methanol assimilation, 3'-AMP and adenosine precursor supply, ATP / NADPH energy supply and the like, the construction of a cordycepin engineering strain without resistance gene residues is realized for the first time, the gene editing efficiency of Pichia pastoris is greatly improved, and a powerful tool is provided for the industrialized production of cordycepin.

The present application relates to the technical field of genetic engineering, and specifically discloses a Chlamydomonas reinhardtii SnRK2.7 gene and application thereof in adaptation to osmotic stress and maintenance of cell survival. The present application first discloses SnRK2.7 the key biological function of the gene in maintaining Chlamydomonas cell homeostasis. By knocking out the gene through CRISPR / Cas9 gene editing technology and adopting an expression frame driven by an endogenous promoter for genetic back complementation, it is confirmed that SnRK2.7 the gene positively regulates cell osmotic stress tolerance and maintains cell long-term survival. In addition, the present application first discovers that the SnRK2.7 protein is specifically located in the Chlamydomonas expansion vesicle, which provides direct spatial evidence for the cell function. The above discovery not only expands the understanding of the osmotic stress signal transduction mechanism of Chlamydomonas, but also provides a new gene target and research tool for the improvement of microalgae stress adaptability, and has important basic research value and biological technology application potential.

The invention discloses a rice disease resistance defense regulation gene SRWD2 as well as an encoding protein and application thereof. According to the invention, a rice disease spot-like mutant tb18 is taken as an experimental material, a rice disease resistance defense regulation gene SRWD2 is separated through strategies such as MutMap positioning and transgene complementation, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the sequence of a protein coded by the gene is shown as SEQ ID NO.2. The biological function of the SRWD2 gene is analyzed, a theoretical basis is provided for clarification of a molecular mechanism of programmed cell death and defense reaction of plants, the gene mutation site is introduced into the plants through gene engineering or conventional means, the disease resistance can be remarkably enhanced, and the gene has important application value in the aspect of disease-resistant variety cultivation of the plants.

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to a CCD4 protein mutant G312S and application thereof. Compared with a wild type CCD4 protein, the mutant has the following mutation: p. G312S, and the NCBI (National Center of Biotechnology Information) accession number of the wild type CCD4 protein is AKO22632.1. According to the invention, the substrate beta-carotene is docked to a CCD4 enzyme activity pocket, a saturated mutant library is constructed, screening is carried out through a bacterial color complementation experiment, and after specific site amino acid mutation, degradation of the substrate beta-carotene is accelerated, so that a certain technical foundation is laid for further cultivation of new varieties of tobacco crops.

The invention relates to the technical field of nucleic acid detection, and discloses a method and a device for detecting and distinguishing ginseng and American ginseng and terminal equipment, and the method comprises the following steps: enriching specific DNA fragments of ginseng and American ginseng by using a multi-enzyme isothermal amplification technology, and screening high-specificity crRNA by combining with a PAM sequence of Cas protein. During detection, a two-channel detection system is constructed by using crRNA, Cas protein and a test probe; after the crRNA accurately recognizes a target sequence in a to-be-tested sample according to a base complementation principle, the trans-cleavage function of the Cas protein is activated, and the test probe is cleaved to form a visual signal. Through two-channel logic, not only can single species be identified, but also a mixed adulteration sample can be identified, the method has the advantages of high sensitivity, strong specificity, constant temperature in the whole process, no need of a precise variable temperature instrument and the like, the problem of interference of near-source species is effectively solved, and simple, convenient and rapid detection of ginseng and American ginseng is realized.

The invention relates to the technical field of plant breeding, discloses a high-quality and high-yield alfalfa directional breeding method and application thereof, and aims to solve the problem that western alfalfa breeding lacks an integrated molecular marker screening system which is combined with a time sequence stress phenotype and can be simultaneously associated with high-protein quality, drought resistance and saline-alkaline characteristics. The method comprises the following steps: constructing association and verification double groups, and performing stress memory activation, ploidy and genetic purity verification and quality preliminary screening; simulating three-stage time sequence stress from a seedling stage to an early overwintering stage, and measuring four-dimensional phenotypic indexes of yield, quality, stress response and habitat adaptation; gWAS analysis is carried out, a double-character co-association marker is mined, and molecular expression, genetic complementation and multi-environment verification are carried out; finally, new germplasm is created through phenotype primary screening, genotype verification, ecological adaptation three-level screening and field trials, the breeding efficiency is improved, the bred variety can stably adapt to the western composite habitat, the high-yield and high-protein characteristics and the soil improvement capacity are both considered, and the field ineffective workload is greatly reduced.

The invention discloses an efficient and accurate wheat parent matching method and system based on the distance between a core germplasm and a genome, and belongs to the technical field of cross breeding. The matching method comprises the following steps: acquiring whole genome SNP genotype data of a breeding population, constructing a core germplasm of which the sample size is smaller than that of an original population but the original genetic diversity is greater than or equal to 95%, analyzing the population genetic structure of the core germplasm, calculating the genome distance between individuals of the core germplasm, and selecting the whole genome SNP genotype data of the breeding population. And screening Top N combinations with maximum and minimum genome distances by taking parents from different genetic subgroups as a primary condition and taking an interval from the top quartile to the maximum value of the genome distance as a primary screening range, and outputting a recommended hybrid combination list. According to the method, quantitative indexes of the genetic population structure and the genome distance are innovatively coupled through an algorithm model, double screening of subpopulation complementation and distance optimization is achieved, decision logic is more scientific, and prediction accuracy is higher.

The invention provides a system and a method for complement expression after migratory locust gene knockout, and belongs to the technical field of insect gene editing, the system for complement expression after migratory locust gene knockout comprises an overexpression plasmid and a living transfection reagent; the overexpression plasmid carries a GAPDH (glyceraldehyde-phosphate dehydrogenase) promoter and a complement gene; the method comprises the following steps: 1) cloning a back-filling gene to a carrier skeleton carrying a GAPDH promoter; 2) respectively dissolving the overexpression plasmid and the living transfection reagent, and then mixing and incubating to obtain a system for complement expression after migratory locust gene knockout; 3, the system is delivered to a migratory locust living body, by means of the system for complement expression after migratory locust gene knockout, the expression quantity of the gene in a migratory locust gene knockout mutant can be specifically complemented, complement expression of the target gene can be detected at the mRNA and protein level, and an effective means is provided for accurately verifying the function of the knockout gene.

The application discloses application of protein FoUPE7 in reducing pathogenicity of banana fusarium wilt. The application constructs a knockout plasmid and a back complementation plasmid of a gene (FoUPE7) coding protein FoUPE7 of banana fusarium wilt, transforms corresponding protoplasts by using the knockout plasmid and the back complementation plasmid respectively, and selects FoUPE7 knockout mutants and back complementation mutants to find and analyze pathogenicity. It is found that knockout of the gene FoUPE7 can significantly reduce the pathogenicity of banana fusarium wilt, and the pathogenicity of banana fusarium wilt is restored and equivalent to that of the wild type after back complementation of the gene, which indicates that the protein FoUPE7 is a pathogenicity-related protein of banana fusarium wilt, regulates the pathogenicity of banana fusarium wilt, and can be used as a target for prevention and treatment of banana fusarium wilt. The application provides a new target for prevention and treatment of banana fusarium wilt, and is beneficial to development of a banana fusarium wilt prevention and treatment preparation.

The application belongs to the field of plant breeding and biotechnology, and particularly relates to how to reduce the gelatinization temperature of rice by gene OsGLIP11 and application of the gene in creating new germplasm of rice with low gelatinization temperature. The application discloses a nucleic acid sequence of a coding region of gene OsGLIP11, a protein sequence coded by the gene, a target site nucleic acid sequence of CRISPR-Cas9 knockout, a nucleic acid sequence of a knockout mutant and a protein sequence of the knockout mutant, as shown in a standard nucleotide or amino acid sequence table of ST.26; after the gene OsGLIP11 is knocked out by using CRISPR-Cas9 technology, it is found that the gelatinization temperature of rice of the transgenic plant with the gene OsGLIP11 knocked out is significantly lower than that of a wild type variety; after the transgenic plant with the gene OsGLIP11 knocked out is subjected to genetic function complementation by using transgenic function complementation technology, the gelatinization temperature of rice of the function complementation transgenic plant has no significant difference with that of the wild type variety. The application can create new germplasm of rice with low gelatinization temperature by using the gene OsGLIP11.

The invention discloses a method for preventing and controlling maize chlorotic mottle virus by using an anti-disease gene ZmCDPK7 and application of the anti-disease gene ZmCDPK7, and belongs to the technical field of plant protection. According to the application, through instantaneous silencing of the ZmCDPK7, an overexpression and knockout line of the ZmCDPK7 is constructed, the resistance reaction of the overexpression and knockout line to maize chlorotic mottle virus infection is analyzed, and it is proved that the ZmCDPK7 positively regulates the immunity of maize to the MCMV. In order to further clarify the molecular mechanism of the ZmCDPK7 for regulating and controlling immunity, interaction between the ZmCDPK7 and a key pathogenic factor P31 of the MCMV is discovered and proved through technologies such as yeast two-hybridization and bimolecular fluorescence complementation. ROS detection proves that the ROS content of the ZmCDPK7 knockout strain is higher than that of a wild type after MCMV infection. In biological and abiotic stress reactions, the resistance of the ZmCDPK7 knockout strain to biological and abiotic stress is proved to be reduced.

The invention discloses a rice gene OsLTD1 and application thereof. The CDS sequence of the rice gene OsLTD1 is as shown in SEQ ID NO.1, and the amino acid sequence of encoding protein of the rice gene OsLTD1 is as shown in SEQ ID NO.3. The invention further discloses a preparation method of the rice gene OsLTD1. Through gene localization and function complementation verification technologies, it is identified that function deletion of the OsLTD1 gene can significantly enhance the resistance of rice to rice blast. The function deletion mutant osltd1 of the gene shows leaf yellowing and lesion-like phenotypes from a seedling stage to a tillering stage, and shows broad-spectrum resistance to a plurality of pyricularia grisea physiological races. According to the invention, the broad-spectrum disease-resistant character and the excellent agronomic character are efficiently combined through a visual leaf color marker associated with gene function deletion, a new high-yield and disease-resistant rice variety is finally cultivated, and a new technical scheme and a new gene resource are provided for solving the problem of rice blast prevention and treatment.

This invention relates to a rice leaf color regulating gene in the field of rice molecular genetic breeding. OsPRDA1 and its applications, the gene OsPRDA1 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2. This invention clones the target gene from a rice albino lethal mutant using Mutmap+ technology. OsPRDA1 The function of the gene was verified using CRISPR / Cas9 gene editing technology and complementation experiments. OsPRDA1 The protein encoded by this gene is located in chloroplasts and participates in rice chloroplast development by regulating the expression of chloroplast genes. This invention can be applied to molecular genetic breeding of rice leaf color traits and is of great significance for further understanding the mechanism of rice chloroplast development and improving rice yield.

The invention relates to a morchella monospore hybridization breeding method, which belongs to the technical field of strain breeding, and comprises the following steps: (1) selecting morchella, obtaining a tissue isolation strain through a tissue isolation method, and obtaining a monospore isolation strain through a monospore isolation method; (2) respectively carrying out mating type gene detection on genomes of the tissue isolation strain and the single spore isolation strain; (3) in the same species, respectively selecting single spore isolation strains only carrying MAT1-1-1 mating type genes and single spore isolation strains only carrying MAT1-2-1 mating type genes for pairing hybridization; and (4) selecting the successful single spore hybrid combination and the corresponding two parent strains to perform cultivation test and agronomic character evaluation, and selecting a hybrid combination with yield and disease resistance superior to those of a single parent. The invention provides a high-efficiency breeding scheme capable of being popularized, reveals a remarkable effect of a mating type gene hybrid combination on the aspects of increasing the yield of morchella esculenta and enhancing disease resistance, and verifies positive regulation and control of mating type gene complementation on the yield and stress resistance.

The invention discloses a sclerotinia sclerotiorum gene SsCRZ1 and application thereof in regulation and control of infection pad formation and pathopoiesis, and belongs to the technical field of microbial genetic engineering. The DNA sequence of the gene SsCRZ1 is as shown in SEQ ID NO: 1, and the amino acid sequence of the protein coded by the gene SsCRZ1 is as shown in SEQ ID NO: 2. Gene knockout and refilling experiments prove that the infection pad forming ability of sclerotinia sclerotiorum and the pathogenicity of sclerotinia sclerotiorum to host plants can be remarkably weakened by deletion of the SsCRZ1 gene, and the functions can be recovered by refilling the gene. Therefore, the SsCRZ1 gene is a gene necessary for formation and pathopoiesis of a sclerotinia sclerotiorum infection pad. According to the invention, the function of the SsCRZ1 gene in sclerotinia sclerotiorum is defined for the first time, and a theoretical basis is provided for the gene as a new target for prevention and control of sclerotiniose.

The application discloses a single-stranded DNA molecular weight standard based on light-controlled self-assembly and a preparation method thereof, and comprises the following steps: S1, chemically synthesizing a DNA sequence containing a photosensitive bridging group, which can self-assemble to form a hairpin structure, and self-assembly can also occur between the hairpin structures; S2, annealing the DNA sequence, and self-assembly of individual DNA sequences to form hairpin structures; S3, mixing the hairpin structures with a nucleic acid ligase, under ultraviolet light, the photosensitive bridging group is broken, the exposed single-stranded sequence is paired with other hairpin structures through base complementation, and cascade self-assembly is realized through the nucleic acid ligase; S4, heat inactivation treatment; S5, ultraviolet light treatment is performed again, so that all the photosensitive bridging groups are broken; and S6, an electrophoresis loading buffer is added, and low-temperature preservation is carried out. The application adopts a one-pot method to rapidly prepare the DNA molecular weight standard, has the advantages of simple process, low manufacturing cost and strong expandability, and has a good application prospect.

The present invention discloses the application of the ERS1 gene in regulating the thermosensitive sterility of rice, including obtaining a sterile mutant by gene editing the wild type of medium indica 3037 and naming it ers1-2 (glutamyl-tRNA synthetase), and further verifying the genetic relationship between the ERS1 gene and the sterile phenotype through complementation experiments. The mutant exhibits a typical thermosensitive sterile phenotype, with a fruit set rate of only 20% at high temperature and a fruit set rate of 70% at low temperature. Further cell biological analysis of the mutant found that the pollen development and meiotic process of the mutant were severely delayed at high temperature, leading to male sterility, and these defects can be restored at low temperature, thereby giving the mutant the characteristics of thermosensitive sterility. The mutation of the ERS1 gene has created a new thermosensitive sterile line in rice, which has great potential in rice hybrid breeding.

The invention discloses a rice disease resistance defense regulation gene ECDR1 as well as an encoding protein and application thereof. According to the invention, a rice disease spot-like mutant ecdr1 is taken as an experimental material, a rice disease resistance defense regulation gene ECDR1 is separated through strategies such as map-based cloning and transgene complementation, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the sequence of a protein coded by the gene is as shown in SEQ ID NO.2. According to the invention, the biological function of the ECDR1 gene is preliminarily analyzed, and a theoretical basis is provided for clarification of a molecular mechanism of plant programmed cell death and defensive reaction; particularly, the gene mutation site is introduced into a plant through gene engineering or a conventional means, the disease resistance can be remarkably enhanced, and the gene mutation site has important application value in the aspect of cultivation of disease-resistant varieties of the plant.

The invention relates to the technical field of recommendation methods, in particular to a multi-target recommendation method based on auxiliary evolution, which comprises the following steps of: firstly, calculating a comprehensive score of each individual, sorting the individuals, and storing the individuals in a candidate set; selecting individuals from the candidate set to form a main parent population P and auxiliary parent populations P'and P '', and performing crossover operation and mutation operation on the parent populations P, P 'and P' 'to form populations P2, P' 2 and P '' 2; elite strategy is carried out on the populations P2, P '2 and P' '2, and redundant populations are eliminated; according to a sorting result and a calculation result, selecting a corresponding number of individuals and storing the individuals in the parent populations P3, P '3 and P' '3; using a parent population P3; the individuals in the parent populations P, P 'and P' 'replace the individuals in the parent populations P, P 'and P' ', when the three parent populations evolve a certain algebra T1, knowledge migration is carried out among the three parent populations to form advantage complementation, when the three parent populations evolve a certain algebra T2, migration learning is carried out among the users to reduce the situation of falling into local optimum, loop iteration processing is carried out according to the content, and the probability of falling into local optimum is reduced. And after an iteration stopping condition is met, stopping iteration, and outputting a recommendation result. By screening the data, the data processing precision can be effectively improved, and recommendation of two targets of accuracy and diversity is realized.

The present application belongs to the field of plant molecular biology and genetic engineering technology, and particularly relates to a lily anthocyanin synthesis regulation gene LvWRKY75 and application thereof, and the present application has obtained an overexpression vector and a silencing expression vector of the gene. The overexpression LvWRKY75 gene is transformed into wild-type Arabidopsis, and after obtaining the third generation of transgenic plants, the expression amount of the LvWRKY75 gene and downstream target genes in the transgenic lines is detected, and it is proved that the LvWRKY75 gene has the effect of promoting anthocyanin synthesis. The yeast one-hybrid technology, gel retardation experiment and double-molecule fluorescence complementation experiment prove that the LvWRKY75 gene plays a positive regulation role in the regulation of Westin red lily color, and these findings provide important experimental and theoretical basis for further understanding the molecular mechanism and genetic regulation network of plant flower color formation.

The invention discloses an AlGaN / GaN HEMT biosensor for CTC detection and a preparation method of the AlGaN / GaN HEMT biosensor. The biosensor comprises an AlGaN / GaN HEMT device and a TDN-polypeptide probe attached to the surface of the device. The TDN-polypeptide probe is formed by bonding TDN with a polypeptide probe through a base pair; the method comprises the following steps: self-assembling four single-stranded DNA sequences by adopting a DNA origami technology to form TDN, connecting the top end of the TDN with a polypeptide probe through base pair complementation, and fixing the tail end of the TDN on the surface of a chemically modified AlGaN / GaN HEMT device through sulfydryl to realize interface construction of the AlGaN / GaN HEMT device and the TDN-polypeptide probe so as to prepare the AlGaN / GaN HEMT biosensor for CTC detection. The biosensor has the characteristics of rapidness, no mark, high sensitivity, high specificity, simplicity and convenience in operation and low cost, and can be specifically combined with EGFR on the surface of a CTC cell membrane to realize high-specificity CTC detection.

The invention relates to a rice thermo-sensitive dual-purpose genic male sterile line fertility transformation starting point temperature regulation gene OsRqc1 containing a tms5 gene and application of the gene. Polynucleotide forming the fertility transformation starting point temperature gene OsRqc1 of the thermo-sensitive dual-purpose genic male sterile line has a base sequence as shown in SEQ ID No: 1 in a sequence table, the gene encodes OsRqc1 protein, and the protein has an amino acid sequence as shown in SEQ ID No: 2 in the sequence table. The inventor performs gene knockout on a strain 1S through a Crispr-cas9 technology to obtain a mutant osrqc1 of which the fertility transformation starting point temperature is increased, and the fertility transformation starting point temperature of the mutant is accurately identified by utilizing an artificial climate box. In addition, a genetic complementation experiment proves that OsRqc1 participates in regulation and control of the fertility transformation starting point temperature.

The invention discloses a rice disease resistance defense regulation gene OsPP2C41 as well as an encoding protein and application thereof. According to the invention, a rice disease spot-like mutant ospp2c411 is taken as an experimental material, a rice disease resistance defense regulation gene OsPP2C41 is separated through strategies such as MutMap positioning and transgene complementation, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the sequence of a protein coded by the gene is shown as SEQ ID NO.2. The invention further discloses a preparation method of the rice disease resistance defense regulation gene OsPP2C41. The biological function of the OsPP2C41 gene is preliminarily analyzed, and a theoretical basis is provided for clarification of a molecular mechanism of plant programmed cell death and defensive reaction; particularly, the gene mutation site is introduced into a plant through gene engineering or a conventional means, the disease resistance can be remarkably enhanced, and the gene mutation site has important application value in the aspect of cultivation of disease-resistant varieties of the plant.

A method of expressing an agronomically or non-agronomically beneficial trait in a plant plastid comprising expressing an exogenous nucleic acid in the plant to produce non-photosynthetic mutant plants, and using callus grown from the mutant plants as recipients for introduction of a construct having a functional copy of the mutated gene and a gene conferring an agronomically or non-agronomically beneficial trait. Embodiments provide for mutations in chloroplast-encoded genes, as well as mutations in nuclear-encoded genes targeted to the chloroplast that are required for photosynthesis, plants and plant parts produced from such methods, as well as kits for performing the methods as described.

The invention relates to a multi-gene pyramiding breeding method suitable for animal populations, the method adopts a minimum rotation maximum complementation (mRMC) gene pyramiding method, compared with the prior art, all individuals in the population can stably carry n target genes (ideal populations) at the same time by needing fewer generations, the number of generations is reduced, the number of generations is reduced, the number of generations is reduced, and the number of generations is reduced. And it is ensured that the group still contains n families. According to the method, the gene polymerization efficiency is effectively improved, and the method shows superiority in the aspects of controlling the population inbreeding level, maintaining the population genetic diversity, maintaining the background character genetic progress and the like. The method. The method can be combined with breeding technologies such as genome selection, marker-assisted selection, comprehensive selection indexes and the like, provides an efficient, controllable and programmable general technical framework for polygene pyramiding breeding of livestock and poultry such as pigs, cattle, sheep, chickens and the like, and has important application value for accelerating the molecular design breeding process and breeding new varieties with high resistance and high yield.

The invention discloses a single-stranded DNA molecular weight standard based on light-operated self-assembly and a preparation method thereof.The preparation method comprises the following steps that S1, a DNA sequence containing a photosensitive bridging group is chemically synthesized, the DNA sequence can be self-assembled to form hairpin structures, and the hairpin structures can also be self-assembled; s2, carrying out annealing treatment on the DNA sequence, and carrying out self-assembly on a single DNA sequence to form a hairpin structure; s3, mixing the hairpin structure with nucleic acid ligase, breaking the photosensitive bridging group under the irradiation of ultraviolet light, complementarily pairing the exposed single-chain sequence with other hairpin structures through bases, and realizing cascade self-assembly through the nucleic acid ligase; s4, carrying out thermal inactivation treatment; s5, carrying out ultraviolet irradiation treatment again to break all the photosensitive bridging groups; and S6, adding an electrophoresis loading buffer solution, and storing at low temperature. The DNA molecular weight standard is rapidly prepared through the one-pot method, and the method has the advantages of being simple in process, low in manufacturing cost, high in expansibility and the like and has good application prospects.

The invention belongs to the technical field of cell replication inhibition, and particularly relates to a method for inhibiting replication of human parvovirus B19 in cells. According to the method, the aim of inhibiting the replication of the human parvovirus B19 in the cells can be remarkably achieved by inhibiting CRM1 or mutating any one of the 255th to 263th amino acids and / or the 285th to 297th amino acids in an NS1 protein amino acid sequence. For example, by taking an NS1 defective genome M20-NS1 as a core and combining with pHelper through a functional complementation experiment, it is found that wild type NS1 (3 * FlagB19-NS1) can restore virus DNA replication, RNA transcription and generation of filial generation virus particles; and the mutant blocks the CRM1-mediated nuclear output pathway, so that the virus genome replication level is reduced by 30%-95%, the RNA transcription is reduced by 20%-80%, and the generation efficiency of filial generation virus particles is reduced by 86%.

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to a CCD4 protein mutant P301L and application thereof. Compared with a wild type CCD4 protein, the mutant has the following mutation: p. P301L, and the NCBI (National Center of Biotechnology Information) accession number of the wild type CCD4 protein is AKO22632.1. According to the invention, the substrate beta-carotene is docked to a CCD4 enzyme activity pocket, a saturated mutant library is constructed, screening is carried out through a bacterial color complementation experiment, and after specific site amino acid mutation, degradation of the substrate beta-carotene is accelerated, so that a certain technical foundation is laid for further cultivation of new varieties of tobacco crops.

The application relates to a mutant enzyme of 3-phosphoglyceroyltransferase, a coding gene and application thereof, and belongs to the fields of biochemistry, molecular biology and metabolism. 58 GPAT9 mutant genes are constructed by using a site-directed mutagenesis technique, and the influence of single and multiple amino acid site changes on GPAT9 enzyme activity is analyzed by combining a GPAT-specific yeast genetic complementation method. It is found that the changes of six amino acid residues (85, 114, 119, 230, 237 and 322) located outside the acyltransferase conserved domain in AtGPAT9 can significantly affect the enzyme activity. There is interaction among the amino acids, for example, the simultaneous mutation of Y85W / N119H / S237N of the three sites can greatly increase the activity of AtGPAT9, accelerate the growth of yeast and promote the synthesis of triacylglycerol, and the triacylglycerol content in the yeast cells expressing the mutant enzyme is increased by 45.7% compared with that of the yeast cells expressing the wild type BnGPAT9.