330 results about "Transformation efficiency" patented technology

According to the D-psicose 3-epimerase mutant and the application thereof, the D-psicose 3-epimerase mutant is obtained through protein C-terminal sequence modification, software analysis and sequence alignment of different sources of D-psicose 3-epimerase, the half-life period of the mutant enzyme at 60 DEG C, 70 DEG C and 80 DEG C is prolonged, and the activity of the mutant enzyme is improved. The substrate affinity and conversion efficiency of the enzyme to D-fructose as a substrate for synthesizing D-psicose are improved, and the Tm value of the mutant is 73.23 DEG C and is improved by 11.98 DEG C compared with that of a wild type; the half-life period at the temperature of 60 DEG C is 255.35 min and is 17.60 times that of a wild type, the problem that the half-life period of the wild type is too short at the high temperature is solved, the relative action time of the mutant enzyme is long, the yield of the product D-psicose is remarkably increased by applying the genetically engineered bacterium containing the modified enzyme for biotransformation, the product yield is increased by 4.3%, the equilibrium conversion rate reaches 33%, and the yield of the product is greatly increased. And a green, environment-friendly, sanitary and safe biological preparation method is provided for the preparation process of the D-psicose.

The invention discloses a gene for improving wheat immature embryo transformation efficiency and application thereof, and belongs to the technical field of plant genetic engineering. According to the invention, a new wheat regeneration gene TaDJA gene is found, and the TaDJA gene is over-expressed in a wheat variety 'Kefeng No.3' which is extremely difficult to transform, so that the callus induction rate and the transformation efficiency of wheat immature embryos can be improved; the method is beneficial for integrating exogenous nucleic acid molecules into the genome of the wheat variety difficult to transform, and provides technical support for genetic improvement of the wheat variety difficult to transform.

The invention discloses a pichia pastoris engineering bacterium for synthesizing bakuchiol from de novo by converting methanol as well as construction and application of the pichia pastoris engineering bacterium. A heterologous p-coumaric acid synthesis pathway is introduced to neutral sites of different chromosomes of pichia pastoris, a high-yield p-coumaric acid strain is obtained through gene knockout, overexpression or heterologous expression of tyrosine and phenylalanine synthesis pathway key genes of a shikimic acid pathway, a heterologous bakuchiol biosynthesis pathway is introduced on the basis, and a high-yield p-coumaric acid strain is obtained. After overexpression of bakuchiol synthetase, endogenous overexpression or heterologous expression of MVA pathway key genes and exogenous acetyl coenzyme A supply pathway genes, methanol concentration optimization and bacterial strain His4 gene supplementation, the yield of bakuchiol is effectively increased to 91.2 mg / L and is increased by 59.8 times compared with that of an initial bacterial strain, and the yield of bakuchiol in a 15L fermentation tank reaches 692.8 mg / L. The method has the characteristics of high conversion efficiency, low production cost, convenience in preparation, wide industrial application prospect and the like.

The invention relates to the field of plant genetic engineering, and discloses application of a ZmWOX5b gene in improvement of corn genetic transformation efficiency. By separating the ZmWOX5b gene and constructing a recombinant vector, an expression cassette or a recombinant bacterium containing the gene, genetic transformation is carried out on corn inbred lines B73, B104, Zheng 58, FSJ115 and Xiang 249 by utilizing an agrobacterium-mediated method, and a research result shows that the gene can remarkably improve the transformation efficiency. Compared with a traditional method, the method has the advantages that the efficiency of easily-transformed genotypes is effectively improved, meanwhile, the transformation effect of difficultly-transformed maize inbred lines is greatly improved, and the limitation of receptor genotypes in maize genetic transformation is effectively broken through. The achievement obviously widens the application range of corn genetic transformation, and provides a new tool for efficiently developing gene function research and application of high yield, high quality, disease and pest resistance, stress resistance and the like of corn.

The invention discloses recombinant streptomyces mobaraensis as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, smY2019 is used as an original strain, and a CRISPR / cas9 technology is utilized to construct five chassis cells of a GIs gene cluster; on the basis of smY2019-deltaG5, two kinds of chassis cells in which endogenous CRISPR / Cas system genes are knocked out are constructed. And on the basis of the smY2019-deltaG5-C2, further constructing a chassis cell smY2019-deltaG5-C3 of which a non-homologous end connection (NHEJ) repair pathway gene is knocked out. The TGase secretory enzyme activity of the chassis cell smY2019-deltaG5-C3 is improved by 37.3% compared with that of the original strain smY2019, and meanwhile, the transformation efficiency, the growth speed, the protein synthesis capability and the energy metabolism level of the chassis cell are obviously improved compared with that of the original strain smY2019.

The invention provides an AbAIL5 gene and application of the AbAIL5 gene in improvement of genetic transformation efficiency of amorphophallus bulbifer, and belongs to the technical field of biology. The invention provides an AbAIL5 gene. The nucleotide sequence of the AbAIL5 gene is shown as SEQ ID NO: 1. Genetic transformation is performed on the amorphophallus bulbifer somatic embryo by constructing an overexpression vector of the AbAIL5 gene, so that the genetic transformation efficiency of the amorphophallus bulbifer callus can be improved, and candidate genes are provided for application of a large-scale breeding technology and a genetic transformation technology of the amorphophallus bulbifer callus in good varieties.

The invention belongs to the technical field of plant genetic transformation, and particularly relates to a callus induction culture medium for genetic transformation, a culture medium combination and a genetic transformation method. The culture medium composition comprises a callus induction culture medium, a recovery culture medium, a screening culture medium, a differential culture medium and a rooting culture medium, wherein each 1L of the callus induction culture medium is prepared from the following components: 5-8g of tryptone, 5-8g of yeast, 100-120mg of sodium chloride, 0.8-1g of glutamic acid, 250-300mg of monopotassium phosphate, 100-150mg of magnesium sulfate, 0.8-1mu g of biotin, 10-20g of mannitol and the balance of water. By optimizing the culture medium formula and the transformation condition, the induction efficiency, the regeneration efficiency, the transformation efficiency and the plant regeneration capacity of the wheat are improved, and the method can be widely applied to the technical field of agricultural biology, especially in research, development and production of transgenic crops, and is beneficial to rapidly cultivating new varieties with excellent characters.

The invention discloses a TaIMPA1 gene and application of the TaIMPA1 gene in improvement of plant genetic transformation efficiency. The nucleotide sequence of the gene is shown as SEQ ID No: 1 in a sequence table. According to the invention, the key gene TaIMPA1 is cloned from a plant body to delivery for the first time, and the genetic transformation performance is enhanced by remarkably improving the delivery efficiency of exogenous DNA (Deoxyribose Nucleic Acid). The discovery fills up the research blank of plant endogenous delivery regulatory factors, and an original solution is provided for overcoming the core problem of genotype dependence.

The invention discloses a method for improving utilization of synthesis gas of clostridium. The method comprises the following steps: blocking or down-regulating expression of a transcription factor NrdR; and / or enhancing the expression of a transcription factor DeoR. The method provided by the invention effectively improves the utilization and conversion efficiency of the clostridium aerovorans on the carbon gas such as synthesis gas, and has popularization and application values.

The present application relates to the technical field of elicitor peptide MsPep and its application, and discloses the use of elicitor peptide MsPep in improving the genetic transformation efficiency of alfalfa, wherein the application takes the leaf of alfalfa as an explant, introduces an exogenous gene through Agrobacterium tumefaciens, and adds the elicitor peptide MsPep in each stage of genetic transformation, so as to significantly improve the dedifferentiation rate, the out-growth rate, the differentiation rate, the out-bud rate, the rooting rate and the transformation efficiency, and shorten the transformation period, and the present application has application prospect in improving the genetic transformation of alfalfa and other plants.

The invention relates to the field of microbial genetic transformation, and discloses a preparation method of Ilyonectria robus fungus protoplast and a construction method of a genetic transformation system.The preparation method comprises the following steps that S11, I.robus hyphae are prepared, after I.robus is cultured on a PDA solid culture medium for 14 days, the I.robus hyphae are inoculated to a CMC liquid culture medium to be subjected to shake-flask culture, and spores are filtered and collected; diluting the spore concentration, inoculating into a YEPD liquid culture medium, carrying out shake-flask culture, filtering and collecting hyphae; and S12, preparing enzymatic hydrolysate, namely adding the hyphae prepared in the step S11 into the enzymatic hydrolysate prepared in the step S12, carrying out shake flask enzymolysis, filtering, centrifuging filtrate, discarding supernate, adding a 0.7 M NaCl solution, centrifuging, and discarding the supernate, so as to obtain the I.robus fungus protoplast. The protoplasts with proper quantity and good quality can be efficiently obtained, and the transformation efficiency of a subsequent genetic transformation system is improved; and the constructed genetic transformation system can be used for construction of a mutant library of the I.robusta strain, interaction of a host plant and the I.robusta strain and research on the pathogenesis of the I.robusta strain.

The present disclosure generally relates to an evolved cytidine deaminase derived from a cytidine deaminase, and a method of editing DNA using the same. In some aspects, the present disclosure describes directed evolution of a TadA-derived adenosine deaminase (TadA-CD) for cytidine deamination. In some embodiments, the TadA-CD comprises a plurality of mutations as compared to the parent TadA variant. In some embodiments, the TadA-CD is fused to a programmable DNA binding protein. Other aspects of the present disclosure generally relate to a cytosine base editor (CBE) comprising a programmable DNA binding protein and a TadA-CD. In some embodiments, the disclosed cytosine base editor has an improved conversion efficiency and a reduced off-target editing frequency compared to a naturally occurring CBE. Polynucleotides, vectors, and kits for generating and delivering CBE are also provided. Also provided are cells containing such vectors and CBE. In addition, methods of treatment comprising administration of CBE are also provided.

The invention belongs to the technical field of plant biology, and discloses a wild soybean genetic transformation method which comprises the following steps: S1, disinfecting wild soybean seeds; s2, the backs of the disinfected wild soybean seeds are cut once, the cut wild soybean seeds are placed on a culture medium, and imbibition seeds are obtained; s3, seed coats of the wild soybean seeds are removed, growing points of embryo tips are exposed through dissection, and explants are obtained; s4, putting the explant into an agrobacterium infection solution containing target plasmids, and carrying out vacuum infiltration in a vacuum environment; and S5, after releasing vacuum, standing and infecting in an agrobacterium infection solution for 3-16 hours. The glycine soja genetic transformation method can effectively improve the transformation efficiency of glycine soja.

The invention discloses an agrobacterium tumefaciens-mediated phalaenopsis genetic transformation method, a complete plant is obtained through protocorm-like body induction, protocorm-like body proliferation, genetic transformation and protocorm-like body differentiation seedling, the method has the characteristics of simple and feasible process, short cultivation period, high transformation efficiency and the like, the obtained protocorm-like body can be stored for a long time, and the method is suitable for large-scale popularization and application. And the material is a good acceptor material, and can meet the requirements of genetic transformation research and annual production of seedlings of phalaenopsis amabilis.

The invention discloses an induction method for obtaining a transgenic persea americana composite plant without tissue culture, and belongs to the technical field of persea americana biology. The induction method provided by the invention comprises the following steps: germinating avocado seeds by using quartz sand to obtain avocado seedlings, infecting rootless seedlings from the avocado seedlings by adopting a vacuum infiltration method, and inserting the rootless seedlings subjected to dip dyeing into a sterilized coco coir matrix for culturing to obtain the avocado composite plant. According to the method, an efficient avocado genetic transformation system mediated by agrobacterium rhizogenes is established, tissue culture is not needed, rootless seedlings are adopted as materials, the transformation efficiency is improved, meanwhile, consumption of experimental equipment, consumables and manpower is reduced, the problem that the avocado genetic transformation system is not established yet is solved, and the method is suitable for popularization and application. The technical effect of quickly, efficiently, simply and easily obtaining the transgenic material with low consumption is achieved, and a more convenient and efficient way is provided for genetic transformation of avocados.

The invention discloses a soybean genetic transformation method for improving positive transformation rate, which comprises the following steps of: infecting a soybean explant with a recombinant expression vector containing a target gene and a marker gene through agrobacterium liquid; an ALS inhibitor is added into a bud induction culture medium and a bud elongation culture medium to improve screening culture of the positive conversion rate of the explants, and a transformant is obtained. According to the soybean genetic transformation method which takes the gene cloned from the cynodon dactylon as the marker gene and the ALS inhibitor as the screening agent, the positive transformation rate is increased to 6% or above, the maximum is 12.05%, and the soybean transformation efficiency is remarkably improved.

The invention discloses a method for establishing a pea genetic transformation system. The method comprises the following steps: step 1, transforming agrobacterium competent cells by using a plant expression vector; step 2, preparing and storing agrobacterium liquid; step 3, preparation of aseptic pea seedlings; step 4, infection and co-culture of the explants; step 5, restoring culture; step 6, bud induction culture; step 7, carrying out bud elongation culture; and step 8, rooting culture. According to the method, the pea variety Zhongwan No.6 which is easily subjected to a genetic transformation test is screened, the test verifies that the optimal explant is the cotyledonary node, and the bud induction rate of the cotyledonary node is 80% or above. According to the method, the problem of rooting difficulty is solved, a complete regenerated plant is obtained, the pea plant with whitening phenotype is successfully obtained by transforming the CRISPR / Cas9 gene editing carrier of the targeted pea PDS gene, the genetic transformation efficiency is improved to 5% or above, and the genetic transformation efficiency of peas is greatly improved.

The invention discloses a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using an improved leaf disc conversion method, and belongs to the technical field of biology. The invention provides a culture medium composition according to the requirement of woody plant tissue culture nutrients, the requirement of apple leaf regeneration can be met, calluses and regenerated buds with high differentiation capacity are induced, and the problem of low genetic transformation efficiency of apples is solved. An improved leaf disc conversion method based on the culture medium package can significantly improve the editing efficiency of an apple CRISPR / Cas9 system, can efficiently deliver a CRISPR / Cas9 knockout carrier, obtains homozygote and heterozygote plants of different mutation types including deletion, replacement, insertion and the like, and has the editing efficiency as high as 92.3%. The method provided by the invention provides a new thought for analyzing genetic mechanisms of apples, improving agronomic characters and performing germplasm innovation by utilizing a gene editing technology.

The invention discloses a high-throughput genetic transformation method of agrobacterium tumefaciens-mediated maize immature embryos and application of the high-throughput genetic transformation method. According to the method, uniconazole is added into a differentiation / regeneration subculture medium, an optimization system suitable for corn immature embryo genetic transformation is established, and efficient and stable genetic transformation of corn can be effectively achieved in an agrobacterium tumefaciens-mediated mode. Analysis on transformation data of transformation vectors in a year before and after optimization shows that the regeneration plant obtaining proportion is obviously improved to 20.04% after optimization from 4.5% before optimization; the annual average conversion efficiency is obviously improved from 2.19% before optimization to 7.48% after optimization; and the average conversion period is shortened from 112 days before optimization to 93 days after optimization.

The invention relates to the field of transformation methods and application thereof, in particular to an agrobacterium tumefaciens-mediated pea leaf injection transient transformation method and application thereof, which comprises the following steps: introducing an exogenous gene into pea leaf cells through an injection method by using an agrobacterium tumefaciens-mediated gene transformation system in pea leaves, and realizing transient expression of the exogenous gene; according to the pea leaf injection transient transformation system based on agrobacterium tumefaciens mediation provided by the invention, through an optimized technical scheme, the problems of low transformation efficiency, unstable exogenous gene expression, high operation complexity and the like in the prior art are solved, and remarkable beneficial effects are brought to plant gene function research and commercial application; through an efficient, stable, simple and convenient pea leaf instantaneous transformation technology, many problems in the prior art are solved, the efficiency of gene function research and commercial application is improved, and a powerful technical support is provided for further development of plant molecular biology and genetic engineering technologies.

The invention discloses a bergamot alkene synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and bioengineering. According to the method, bergamotene synthase SanSyn which is obtained from plant clausena lansium and can catalyze FPP to generate alpha-trans-bergamotene is taken as an object, semi-rational design and site-specific mutagenesis are carried out on the bergamotene synthase SanSyn, a series of obtained SanSyn mutants are remarkably improved in the aspect of FPP substrate conversion efficiency, and the optimal bergamotene synthase mutant SanSynI273C-L514I has the advantages that the optimal bergamotene synthase mutant SanSynI273C-L514I can be used for catalyzing the FPP to generate alpha-trans-bergamotene; the unit cell productivity of the alpha-trans-bergamotene generated by catalyzing FPP is 6.9 times of that of a wild type, and the yield is 6.8 times of that of the wild type and reaches 43.58 mg / L. According to the bergamot alkene synthase mutant, the performance of alpha-trans-bergamot alkene synthesized by microorganisms is enhanced, and an effective technical route is provided for green production of industrial high-purity bergamot alkene.

The invention discloses a rubus cochinchinensis efficient genetic transformation method based on RUBY reporter gene and agrobacterium rhizogenes mediation, which realizes that the transformation period is shortened to be within 15 days and the transformation efficiency is improved to be more than 60% through direct explant treatment under a non-tissue culture condition. The method comprises the following specific steps: 1) preparing an explant material; (2) constructing an RUBY carrier and transforming agrobacterium rhizogenes; (3) infecting the explant by utilizing agrobacterium rhizogenes carrying a target gene; (4) co-culturing after infection and carrying out RUBY visual screening; 5) positive root molecular biology verification; (6) inducing positive roots into buds; according to the method, induction of the positive callus, the positive root and the positive bud of the rubus coreanus stem segment under the non-tissue culture condition is successfully achieved, a stable and efficient genetic transformation system is constructed, the method has the remarkable advantages of being easy and convenient to operate, low in cost, wide in applicability, high in reproducibility and the like, and a key technical support is provided for molecular breeding of the rubus coreanus.

The invention relates to an osmanthus protoplast preparation method and an instantaneous conversion method. The preparation method of the sweet-scented osmanthus protoplast comprises the following steps: collecting different tissues of fresh sweet-scented osmanthus, performing surface disinfection and sterilization, cutting into thin strips or slices, adding enzymatic hydrolysate for enzymolysis, filtering, centrifuging, washing with a W5 solution, and re-suspending. The instantaneous transformation method of the osmanthus protoplast comprises the following steps: adjusting the concentration of the osmanthus protoplast by using an MMG solution, adding plasmids for mixing, adding a PEG solution for transformation incubation, adding a W5 solution for washing and centrifuging, and detecting the instantaneous transformation efficiency of the osmanthus protoplast. According to the method, a high-quality protoplast separation system of the leaves, the stems and the petals of the sweet-scented osmanthus is successfully established, efficient conversion of mesophyll and petal protoplast is realized for the first time, and a reliable technical platform is provided for molecular biology research of the sweet-scented osmanthus.

The application discloses application of SbWOX gene and SbBBM gene in corn breeding and a corn genetic transformation method, and relates to a bioengineering technology.The application provides a nucleic acid molecule with co-expression of SbWOX protein gene and SbBBM protein gene, and corresponding gene co-expression vectors and microbial transformants.The application also provides application of the co-expression of the SbWOX gene and the SbBBM gene in the corn genetic transformation method.The application obtains corresponding genes and mutant proteins based on the SbWOX gene and the SbBBM gene of sorghum, the two genes are co-expressed in the corn genetic transformation process, even without adding plant growth hormones in the antibiotic screening medium in the tissue culture process, the plant genetic transformation process can be normally completed, and the transformation efficiency and the regeneration efficiency of the corn can be remarkably improved, the transformation rate reaches 10.42%, and the differentiation rate reaches 32.89%.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method of perennial elymus nutans. According to the genetic transformation method of elymus nutans, mature embryos serve as explants for inducing callus formation, the method has the advantages that material obtaining is convenient, operation is simple and easy, the supply problem of explant material obtaining is solved, and meanwhile the physiological state of the mature embryos is consistent, so that the repeatability of experiments is good; in addition, the infection liquid contains the acetosyringone, and the acetosyringone (AS, Acetosyringone) can promote the growth of agrobacterium tumefaciens and enhance the activity of the agrobacterium tumefaciens, so that the conversion efficiency is improved; the browning phenomenon of the calluses is effectively inhibited through sterilization treatment, the normal growth of the calluses can be recovered, the transgenic elymus nutans plants are obtained through the technical scheme, and the research of researchers on the gene functions of the elymus nutans can be greatly promoted.

This invention discloses the application of the GhATG18a gene in accelerating callus induction in cotton. The invention constructs a pCAMBIA2300-GhATG18a overexpression vector from the full-length CDS fragment of this gene, then introduces it into Agrobacterium, and transforms cotton using Agrobacterium-mediated transformation to obtain transgenic lines. qRT-PCR results show that, compared with the CCRI24 control material, the expression level of the GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 is significantly upregulated. Phenotypic observation and histological sections both show that the OE-GhATG18a line overexpressing this gene accelerates the process of somatic cell dedifferentiation and callus induction in cotton, thereby improving the genetic transformation efficiency of cotton. Therefore, overexpressing this gene and applying it to commercial cotton varieties that are difficult to genetically transform can help accelerate the callus induction process, thereby improving the genetic transformation efficiency and providing technical support for breeding high-yield and stress-resistant high-quality cotton germplasm.

The invention belongs to the technical field of microbial genetic transformation, and particularly relates to a method for high-yield production of pleurotus ostreatus transformants, a genetic transformation system and application of the genetic transformation system. Through optimization of protoplast preparation conditions, the number of the obtained protoplast is 4.95 * 10 < 8 > CFU.mL <-1 >, and the regeneration rate of the protoplast in an OM-YEPS regeneration culture medium is 12.7%. A method for producing high-yield pleurotus ostreatus transformants is researched by using a PEG-mediated protoplast transformation method, a set of efficient and stable genetic transformation and screening method is established, about 200-300 positive transformants with stably expressed exogenous genes are obtained, and the transformation efficiency is 65.38%. The genetic transformation system of the high-yield Pleurotus ostreatus transformant, constructed by the invention, lays a solid technical foundation for subsequent gene function verification, metabolic engineering transformation and molecular breeding with excellent characters, and further accelerates the biological research and industrial application process of Pleurotus ostreatus and even other macro fungi.

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 present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

The invention belongs to the technical field of plant cytology, and particularly relates to a method for improving the transformation efficiency of plant protoplast, which comprises the following steps: preparing enzymatic hydrolysate: each liter of enzymatic hydrolysate contains 20 mmol of KCl, 5-20 g of cellulase, 2-6 g of macerozyme, 10 mmol of CaCl2, 1 g of BSA and the balance of water; performing leaf enzymolysis; obtaining a protoplast; preparing a competent protoplast; resuspending the MMG solution; and transforming and incubating the protoplast resuspension by using the target plasmid. According to the method, the high-quality protoplast can be obtained, and when the protoplast is applied to downstream experiments, the conversion efficiency and stability of the experiments can be remarkably improved.