52 results about "Molecular cloning" patented technology

The invention belongs to the technical field of biology, and particularly discloses a DNA molecular cloning method based on GgeAgo and application of the DNA molecular cloning method. The method comprises the following steps: shearing a vector plasmid by a cleavage system; mixing a vector plasmid cleavage product with an exogenous fragment with a homologous region, assembling by adopting a recombination-based cloning method, and transforming escherichia coli to generate a recombinant plasmid. The cutting system comprises a guide nucleic acid molecule and a programmable endonuclease GgeAgo or / and a mutant thereof, the GgeAgo cuts plasmids under the guide of the guide nucleic acid molecule to linearize the plasmids, a to-be-inserted exogenous fragment with a homologous region is added, and after escherichia coli is transformed, the exogenous fragment is efficiently and accurately inserted into the target plasmids. The molecular cloning method provided by the invention is high in efficiency, short in time and simple and convenient to operate, and mutation caused by PCR is effectively reduced. The molecular cloning method provided by the invention provides a new thought for an Ago-based molecular cloning technology and a DNA synthesis and assembly technology.

The invention discloses an application of ribonuclease SiRe0902 with high efficiency and thermal stability in specific recognition of a GG site. The amino acid sequence of the gene is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1. The ribonuclease SiRe0902 has very efficient and specific RNA (Ribonucleic Acid) hydrolysis activity, and does not have non-specific cutting on DNA (Deoxyribose Nucleic Acid) at all. In addition, the enzyme is derived from thermophilic archaea, is detected to have good thermal stability, and has very good RNA cleavage activity under the conditions of 37 DEG C and 65 DEG C. Through detection, the enzyme can specifically recognize a GG sequence for cutting, and also has high cutting activity for the GG sequence in a secondary structural region. Therefore, the ribonuclease SiRe0902 is novel ribonuclease with thermal stability, has extremely high RNA hydrolytic activity under normal-temperature and high-temperature conditions, and meets the requirements in the fields of clinic, production, molecular cloning and the like.

The invention discloses a SNP molecular marker for identifying resistance of cucumber to bacterial stem rot and its application, belonging to the field of biotechnology-assisted breeding technology. The site of the SNP marker is at the 10,867,465 bp of the physical position of cucumber chromosome 6, the base of the site is A or G, the cucumber material with the base of the site A is a cucumber material resistant to bacterial stem rot, and the cucumber material with the base of the site G is a cucumber material susceptible to bacterial stem rot. The invention not only lays a foundation for the precise positioning and molecular cloning of the cucumber bacterial stem rot resistance gene BSSR, but also provides an efficient way for the use of molecular markers to assist in the breeding of new cucumber varieties resistant to cucumber bacterial stem rot.

The invention discloses a bone targeting forsythiaside A loaded exosome and a preparation method and application thereof, and relates to the technical field of biomedicine.The preparation method comprises the following steps that bone marrow mesenchymal stem cells are separated and cultured, and GLG1 is over-expressed through a molecular cloning technology to obtain GLG1-BMSCs; the exosome GLG1-Exos of the GLG1-BMSCs is collected through an ultracentrifugation method; and loading forsythiaside A into the exosome through an ultrasonic method and an extrusion method to obtain the GLG1-Exos-FTA. The traditional Chinese medicine active ingredient forsythiaside A is loaded, and forsythiaside A is delivered to bone marrow in a targeted manner, so that the effect of preventing and improving diabetic osteoporosis is achieved; due to the targeting effect, the bioavailability of forsythiaside A can be enhanced, and meanwhile, the curative effect of resisting diabetic osteoporosis is improved. Experiments show that the loading rate of forsythiaside A of the GLG1-Exos-FTA reaches 36.9%, the particle size distribution peak value is 122.4 nm, and the GLG1-Exos-FTA has typical exosome characteristics and bone targeting, and can significantly improve the bone microstructure of diabetic osteoporosis mice.

The invention belongs to the technical field of animal reproductive biology and cell engineering, and particularly relates to a fluorescence labeling expression system for visualizing an oocyte nucleus maturation process and application of the fluorescence labeling expression system. The fluorescence labeling expression system comprises an eukaryotic expression vector pVenus-LMNB1; the eukaryotic expression vector pVenus-LMNB1 comprises a nucleotide sequence for coding the fusion protein Venus-LMNB1. According to the invention, a nuclear fiber layer protein stably expressed on a nuclear membrane is selected as a research object, a eukaryotic expression vector pVenus-LMNB1 is constructed by using a molecular cloning technology, an LMNB1 gene containing a green fluorescent label Venus is overexpressed in a porcine oocyte by using a microinjection technology, fluorescent protein labeling of a structural protein on the nuclear membrane is utilized, and the expression vector pVenus-LMNB1 is constructed. The expression positioning condition of the LMNB1 in the maturation process of the porcine oocytes is observed, and a powerful tool is provided for development of in-vitro culture related researches of the porcine oocytes.

The invention discloses a method for rapidly identifying in-vivo parasitic powdery plant pathogens, and relates to the technical field of molecular identification of plant pathogenic fungi, and the method comprises the following steps: 1, collecting samples such as plant leaves or stems with in-vivo parasitic pathogens such as powdery mildew and rust disease; 2, establishing a PCR amplification system; 3, adding an amplification template (powder pathogen) and carrying out PCR amplification; 4, separating, purifying and sequencing PCR products or sequencing molecular cloning transformants; and 5, carrying out nucleic acid database BLAST search on the sequencing sequence, and rapidly identifying the classification of living parasitic pathogen species causing plant diseases. The method can effectively avoid the technical bottleneck and cost expenditure that living parasitic fungi cannot be subjected to in-vitro culture to obtain pure culture thalli or an expensive trace DNA extraction kit is used for extracting genomes, and has important reference significance and practical value for rapid identification of living parasitic pathogenic bacteria capable of generating powder, such as powdery mildew and rust germs.

Molecular cloning method using Type IIS restriction enzymes The invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising "n" inserts, wherein "n" is an integer of at least 1, and each insert comprises a sequence of interest "S" and, wherein the method comprises contacting in a reaction mixture: (i) a receiving backbone, wherein: (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or (b) each end of the receiving backbone comprises a different palindromic overhang; (ii) the n inserts, wherein each insert comprises two different overhangs, wherein the overhangs comprised in the n inserts are different overhangs, and each one is complementary only to another overhang comprised in another insert or to an overhang of the receiving backbone, and is non-palindromic, except the insert's overhang(s) complementary to the palindromic overhang(s) of the receiving backbone, and (iii) a DNA ligase; wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert). In a particular embodiment, at least one of the n inserts and the receiving backbone are obtained upon digestion of one or more polynucleotides and a receiving vector with Type IIS restriction enzyme(s).

The circular RNA molecular medicine has a treatment effect on pulmonary arterial hypertension mice. The ribonucleotide circular RNA molecule hsacirc0008828 is cloned and purified and then is modified by lipidosome, nasal administration is carried out on mice, the ribonucleotide circular RNA molecule hsacirc0008828 is effectively delivered to lung tissues of the mice, the ribonucleotide circular RNA molecule hsacirc0008828 has a treatment effect on pulmonary arterial hypertension model mice, and a novel molecular drug is provided for clinical treatment of pulmonary arterial hypertension. The circular RNA molecule disclosed by the invention is used for treating pulmonary arterial hypertension mice as a lung targeting drug.

This invention relates to the fields of molecular cloning and protein expression, and particularly to a Pichia pastoris engineered strain expressing full-length human type III collagen and its applications. The engineered Pichia pastoris strain is obtained by cloning the enzymatically digested human type III collagen gene sequence into an expression vector to obtain a recombinant expression vector, which is then transformed into Pichia pastoris. This engineered Pichia pastoris strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 6, 2024, with accession number CGMCC NO.32510. The Pichia pastoris engineered strain obtained by this invention can efficiently produce the desired human type III collagen through fermentation, exhibiting excellent characteristics such as high yield and absence of endotoxins.

The application discloses a method for dynamically regulating the content of smell and taste substances in the secondary metabolism process of streptomyces, and relates to the technical field of molecular biology. The method for dynamically regulating the content of smell and taste substances in the secondary metabolism process of streptomyces disclosed by the application determines a first promoter and an sgRNA sequence capable of targeting a first target gene affecting the synthesis of a target compound according to the types of microorganisms and the target compound; a first recombinant plasmid is constructed by using a molecular cloning method; meanwhile, a second promoter for starting the synthesis of the target compound and a gene sequence capable of encoding a Cas protein losing cutting activity are determined, and the two are connected to obtain a regulation unit; the linearized enzyme cutting product after double enzyme cutting of the first recombinant plasmid is connected with the regulation unit to obtain a time-sequential dynamic inhibition recombinant plasmid, and after the recombinant plasmid is introduced into streptomyces, a streptomyces strain with reduced content of smell and taste (geosmin) substances in the secondary metabolism process is obtained.

The invention belongs to the technical field of bioengineering and analytical chemistry, and provides a probe eZinCh-2 modified plasmid based on FRET (Fluorescence Resonance Energy Transfer) and application of the probe eZinCh-2 modified plasmid in zinc ion detection. According to the invention, the plasmid is constructed through molecular cloning and homologous recombination technologies, and is microinjected into a zebrafish embryo, so that a transgenic strain capable of stably inheriting and expressing the probe protein is successfully obtained; as a living body biological tool, the zebrafish of the strain is combined with a confocal microscope spectrum FRET technology, and real-time and quantitative monitoring and visual imaging of dynamic changes of zinc ions at living body, cell and tissue levels are achieved. The method solves the problem that real-time dynamic visual monitoring of the zinc ions cannot be realized in a living body model in the prior art, and is suitable for real-time microscopic quantitative analysis of the content of the zinc ions in model organisms such as zebra fish and the like.

The invention provides an UTR element Hh and a construction method and application thereof, and relates to the technical field of mRNA. Ribosome load prediction and secondary structure optimization are respectively carried out on 5 'UTR of a gene of which the NCBI number is AF339412.1 and 5' UTR of a gene of which the NCBI number is MK475748.1 through a PaddleHelix platform, and an inhibitory hairpin structure is avoided by the obtained optimized sequences, so that the luciferase expression quantity is obviously improved compared with that of natural UTR. On the basis of sequence optimization, HBB and hbb are directionally assembled through a molecular cloning technology, an HBB-hbb chimera (Hh) is constructed, the DNA sequence of the Hh is as shown in SEQ NO.1, the RNA sequence of the Hh is as shown in SEQ NO.2, and the protein expression quantity is further improved.

The present invention discloses a glycosyltransferase and its application in the production of glycosides. The present invention clones a glycosyltransferase from a low-temperature resistant Bacillus subtilis using molecular cloning technology, constructs an expression vector with the cloned glycosyltransferase molecule and successfully expresses it in vitro. The enzyme activity test shows that the glycosyltransferase expressed in vitro can catalyze rebaudioside A to rebaudioside D, and the glycosyltransferase has the characteristic of low-temperature resistance, can still catalyze the reaction under the condition of 25°C, has a wide pH value reaction range, and can achieve the catalytic reaction within the range of pH6-8. The new glycosyltransferase disclosed in the present invention has the advantages of low-temperature resistance and wide pH value reaction, and is suitable for the production of various glycoside glycosylation products.

The invention discloses an antibiotic-free screening gene expression system, plasmid, engineering bacteria and application thereof, and relates to the technical field of bioengineering. The expression system provided by the invention comprises a first strain, a second strain, a third strain and plasmids of antibiotic-free screening genes, the first strain, the second strain and the third strain are escherichia coli strains with a thyA gene knocked out, the first strain is used for molecular cloning, the second strain is used for expression of target protein, and the third strain is used for biosynthesis of natural products such as polyketone and non-cellulose peptide; a thyA complementary gene expression cassette is inserted into the plasmid of the antibiotic-free screening gene, and a resistance gene is knocked out; the thyA complementary gene expression box contains a thyA gene.

The invention belongs to the field of molecular biology, and particularly provides a PCR (Polymerase Chain Reaction) amplification method capable of directly generating sticky end products in a PCR process and application. The novel double-stranded sticky end primer is matched with the high-fidelity polymerase without 5 '-3' end excision enzyme activity, and a DNA product with a sticky end structure can be generated in one step in the PCR process, so that the efficiency of connecting DNA in molecular cloning or sequencing library building is remarkably improved.

The present invention relates to the field of protein labeling or tracing, and particularly to a composition and method for labeling or tracing a target protein. The method first analyzes the structure of the target protein and identifies a loop sequence on the protein surface that faces outward. Then, through molecular cloning methods, a small tag (such as an HA tag) with flexible amino acid linker peptides at both ends is inserted into the middle of the loop sequence. Finally, a nanobody fused to the small tag and fused to a fluorescent protein is expressed. This method utilizes the nanobody's ability to specifically recognize the target protein surface tag to achieve intracellular tracing of the target protein.

The invention discloses a listeria monocytogenes multi-epitope fusion antigen, a listeria monocytogenes multi-epitope fusion antigen-based nano-particle and application of the listeria monocytogenes multi-epitope fusion antigen-based nano-particle. Amino acid sequences of important antigen protein gene encoding proteins of LM antigens are analyzed, dominant antigen epitopes are screened out and connected in series according to a certain permutation and combination, a multi-epitope fusion antigen gene LM-MeAg is constructed and synthesized, and the listeria monocytogenes multi-epitope fusion antigen-based nano-particle is obtained. And further expressing the LM multi-epitope fusion antigen LM-MeAg gene in escherichia coli, and constructing a pT-LM-MeAg5 recombinant plasmid through a molecular cloning technology. According to the invention, dominant antigen epitopes of important antigens LLO, ActA, InlB and MPL of LM are assembled together in series according to a certain permutation and combination through nucleotides for coding flexible peptide amino acids, an LM multi-epitope fusion antigen gene is constructed, and an LM-MeAg5-PLGA nano-particle molecule with strong immunogenicity of the LM multi-epitope fusion antigen protein is further prepared. The method can be used for researching, developing and preparing efficient and safe anti-LM vaccines or drugs.

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying high / low tartronic acid content of cucumber fruits and application of the SNP molecular marker, and belongs to the technical field of biotechnology-assisted breeding, the site of the SNP marker is the 4th, 352nd and 029bp of the physical position of a cucumber chromosome 4, the basic group of the site is C or T, and the basic group of the site is C or T; the cucumber material with the basic group at the site T is a cucumber material with high content of tartronic acid in the fruit, and the cucumber material with the basic group at the site C is a cucumber material with low content of tartronic acid in the fruit. The invention not only lays a foundation for fine positioning and molecular cloning of the tartronic acid content character of the cucumber fruit, but also provides an efficient way for the molecular marker-assisted breeding of a new cucumber variety with high tartronic acid content.

The present application relates to the recombinant microorganism for producing 3-amino propanol by fermentation method and its construction method and application. Specifically, the present application provides a construction method of the recombinant microorganism for producing 3-amino propanol, which is introducing the coding gene of omega-transaminase into the microorganism producing 3-hydroxy propionaldehyde, wherein the omega-transaminase can catalyze the conversion of 3-hydroxy propionaldehyde into 3-amino propanol in the presence of amino donor. The present application introduces the omega-transaminase capable of catalyzing the synthesis of 3-amino propanol with 3-hydroxy propionaldehyde as the substrate into the microorganism producing 3-hydroxy propionaldehyde based on gene editing, molecular cloning and other technologies, which first creates a brand new full-path biosynthesis process route of 3-amino propanol.

The invention belongs to the field of gene engineering, and relates to a preparation method and application of novel halophilic archaea chitinase. The method comprises the following steps: firstly, performing database comparison based on a genome of halophilic archaea HN-29 to obtain a chitinase gene chiDHap; amplifying by utilizing a PCR (Polymerase Chain Reaction) technology to obtain a target gene segment; then, a target gene segment is connected with a pTA04 vector through a molecular cloning technology, and a recombinant plasmid pTA04-chiDHap is obtained; the method comprises the following steps of: preparing a Hv-YPC liquid culture medium, converting the Hv-YPC liquid culture medium into a halophilic archaea expression host, inoculating the Hv-YPC liquid culture medium with the halophilic archaea expression host, culturing, centrifuging, collecting thalli, re-suspending by using a cell lysis solution, ultrasonically crushing, centrifuging, collecting supernate, and finally purifying to obtain chitinase. The obtained chitinase has good enzymatic characteristics and stability, and can tolerate a variety of metal ions, organic solvents and surfactants; the method can be applied to degradation of chitin, and degradation products can be applied to preparation of functional foods.

The application discloses application of insect adult cytochrome b5 in pest control and belongs to the field of plant protection. Through a molecular cloning technique, the application successfully obtains full-length sequences of two Cyt b5 genes Cyt b5v1 and Cyt b5v2, and confirms that the two genes are in a high expression state in a whitefly resistant population. Double-stranded RNAs, namely dsCyt b5v1 and dsCyt b5v2, are synthesized by using an RNA interference (RNAi) technique, and after feeding treatment, the expression of target genes is significantly inhibited, and the sensitivity of the whitefly to different neonicotinoid insecticides is effectively improved. The application has the core advantages of low toxicity, high efficiency and high safety while realizing high insecticidal activity, and provides important technical support for a green pest control system in a field.

The present invention provides a method for screening cleavage sites and its application. The method comprises the steps of: S1, database establishment: using computer language to write a program, predicting that after the intein is embedded in each of two adjacent amino acid residues in the amino acid sequence, a splicing reaction occurs to cut itself off, and the amino acid sequence formed after the adjacent peptide segments are connected constitutes a protein database; S2, experiment: inserting the intein sequence fragment into the gene fragment, molecular cloning is performed, and the peptide segment is obtained after translation, and the peptide segment is detected by mass spectrometry to see whether it contains a labeled amino acid sequence, and compared with the protein database to realize the confirmation of the cleavage site. The method of the present invention first constructs a protein database by computer programming, and then conducts experiments and realizes the detection and confirmation of the cleavage site by mass spectrometry; the final detection is realized by mass spectrometry, replacing high-throughput screening, and it is expanded to be able to search for cleavage sites for any active protein.

The invention discloses a serine protease inhibitor SPINK8 as well as a preparation method and application thereof in acute lung injury caused by sepsis, and belongs to the fields of molecular biology, functional protein technology and biological medicine. The amino acid sequence of the SPINK8 protein is as shown in SEQ ID No. 1. According to the invention, the SPINK8 recombinant vector is constructed through molecular cloning for the first time, and the reSPINK8 recombinant protein is obtained through prokaryotic expression and purification. When the acute lung injury caused by sepsis occurs, the reSPINK8 protein can remarkably inhibit lung inflammatory response and improve the pathological state of the lung, and has immunoregulation and protection effects on the acute lung injury caused by sepsis. In view of the advantages that SPINK8 is small in molecular weight, easy to express and purify and easy to prepare medicines, the SPINK8 has important application value in prevention and treatment of sepsis acute lung injury and targeted medicine screening.

This invention belongs to the field of genetic engineering and relates to a method for preparing amylase derived from halophilic archaea and its application; based on halophilic archaea... Haloarcula sp. ZJK10-12、 Haladaptatus The genes for α-amylase, maltose amylase, and glucoamylase were obtained from the whole genome sequence of sp. ZJK29-3. Recombinant plasmids were constructed using primer design and molecular cloning, and transformed into prokaryotic host bacteria to obtain the corresponding recombinant strains. The bacteria were enriched and cultured in Hv-YPC liquid medium, and the cells were collected and lysed by sonication. α-amylase, maltose amylase, and glucoamylase were obtained using nickel column affinity chromatography. The amylases exhibit excellent enzymatic properties and outstanding salt tolerance. Their application in the fermentation of high-salt, dilute soy sauce can improve raw material utilization and product quality, demonstrating good industrial applicability and promotional value.

The application discloses a sequencing data calibration system and method based on microbial amplicon sequencing, and belongs to the field of microbial detection and bioinformatics analysis. The application takes ddPCR technology as the core, first screens common human intestinal microorganisms, designs and obtains 14 reconstructed sequences containing bacterial 16S and fungal 18S / ITS, prepares high-purity DNA standard substances through chemical synthesis and molecular cloning, completes standardization and uniformity verification, then designs high-specificity primers and probes, optimizes ddPCR reaction conditions, establishes a calibration method, completes uncertainty evaluation through joint calibration of 9 laboratories, and prepares multi-target amplicon abundance standard substances. The application realizes the transformation of amplicon sequencing data from relative quantification to absolute quantification, corrects the system error of the whole sequencing process, improves the comparability and reliability of cross-platform and cross-batch data, and provides technical support for the standardization of microbiome research.

The application discloses a chimeric antigen receptor co-stimulatory signal library carrier and a method for enrichment screening of a proliferation phenotype thereof, and belongs to the technical fields of chimeric antigen receptor library carrier preparation and enrichment screening. The application provides a chimeric antigen receptor co-stimulatory signal library fragment, which comprises, from upstream to downstream, a nucleic acid sequence of an upstream library amplification primer region, a first type IIS restriction enzyme recognition region, a co-stimulatory signal library region, a second type IIS restriction enzyme recognition region, a negative screening marker element region, a second type IIS restriction enzyme recognition region, a barcode region, a first type IIS restriction enzyme recognition region, and a downstream library amplification primer region. A skeleton carrier for constructing the chimeric antigen receptor co-stimulatory signal library fragment and a CAR-T cell library enrichment screening method are also provided. The recombination design strategy of the library is more diverse and flexible, the molecular cloning process is more simple and efficient, and the enrichment screening process is more rapid and suitable for important indicators influencing the clinical curative effect of CAR-T.

The application belongs to the technical field of biotechnology, and particularly discloses a DNA molecular cloning method based on GgeAgo and application thereof. The method comprises the following steps: a vector plasmid is cut by a cutting system; the cutting product of the vector plasmid is mixed with an exogenous fragment with a homologous region, and then a recombination-based cloning method is used for assembly, followed by transformation of Escherichia coli to generate a recombination plasmid. The cutting system comprises a guide nucleic acid molecule and a programmable endonuclease GgeAgo or / and a mutant thereof. The GgeAgo cuts the plasmid under the guidance of the guide nucleic acid molecule to linearize the plasmid, an exogenous fragment with a homologous region is added, and then Escherichia coli is transformed to realize efficient and accurate insertion of the exogenous fragment into the target plasmid. The molecular cloning method provided by the application has high efficiency, short time and simple operation, and effectively reduces the mutation caused by PCR. The molecular cloning method provided by the application provides a new idea for the Ago-based molecular cloning technology and the synthesis and assembly technology of DNA.

The present invention discloses a preparation method and application of a Aeromonas veronii mutant strain, belonging to the technical field of microbiology. The preparation method comprises the following steps: S1, extracting the whole genome of Aeromonas veronii; S2, constructing primers used for knocking out the cheZ / mshK / aerA genes; S3, using the extracted whole genome of Aeromonas veronii as a template, and using the primers to amplify the upstream and downstream homologous arms of the target gene and perform overlap extension to obtain the deleted cheZ / mshK / aerA gene fragment; S4, introducing the deleted cheZ / mshK / aerA gene fragment into the suicide plasmid pRE112 to obtain the recombinant plasmid pRE112-ΔCMA; S5, transferring the recombinant plasmid pRE112-ΔCMA into WM3064 competent cells, constructing a homologous recombinant mutant strain through conjugation transfer, and then screening by sucrose pressure to obtain the Aeromonas veronii mutant strain ΔCMA. The present invention uses molecular cloning technology, Overlap PCR technology and homologous recombination technology to perform in-frame deletion mutation on the cheZ / mshK / aerA genes of Aeromonas veronii by using a suicide vector, and obtains a stable hereditary Aeromonas veronii mutant strain ΔCM with three-gene deletion.

The application discloses a beta-1, 6-glycosidic bond hydrolytic enzyme and application thereof in traditional Chinese medicine resources. The method screens a key gene sequence with a function of hydrolyzing beta-1, 6-glycosidic bond from a Candida parapsilosis L7 genome, and names the key gene sequence as CoExg1. A gene engineering bacterium capable of efficiently heterologously expressing CoExg1 is constructed by using a molecular cloning technology. After fermentation and enzyme production, an enzyme catalytic reaction system is established, and a traditional Chinese medicine active molecule in mogroside V is directionally converted, beta-1, 6-glycosidic bonds in different regions of a substrate structure are selectively hydrolyzed, and therefore, efficient and phased synthesis of different products such as sianoside I and mogroside IIIE is realized. In addition, CoExg1 can also hydrolyze beta-glycosidic bonds in the structures of icariin and lupeol, and directionally synthesize icariin II and lupeol, thereby providing important technical support for high-value utilization of traditional Chinese medicine resources and quality improvement and efficiency enhancement of traditional Chinese medicine glycosides.

The application discloses application of a high-efficiency and heat-stable ribozyme SiRe_0902 in specific recognition of a GG site. The amino acid sequence of the ribozyme SiRe_0902 is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 1. The ribozyme SiRe_0902 has very high and specific hydrolysis activity on RNA, and has no non-specific cutting on DNA. In addition, the enzyme is derived from a thermophilic archaea, and is detected to have good heat stability and very good RNA cutting activity under conditions of 37 DEG C and 65 DEG C. The enzyme is detected to be capable of specifically recognizing and cutting a GG sequence, and also has high cutting activity on a GG sequence in a secondary structure region. Therefore, the ribozyme SiRe_0902 is a new heat-stable ribozyme, and has very high RNA hydrolysis activity under normal temperature and high temperature conditions, and meets the needs of clinical, production and molecular cloning fields.