62 results about "Biological studies" patented technology

The application discloses a construction method and application of an ovary cell line of Quasipaa spinosa. The method comprises the following steps: selecting metamorphosis period larva ovary tissue, disinfecting and obtaining the ovary tissue, and then using collagenase I digestion solution to digest the ovary tissue at 26 DEG C for 1 hour; after the tissue block adheres to the wall, using a low-osmotic-pressure complete culture medium to carry out primary culture at 25-27 DEG C, wherein the culture medium is 60% diluted DMEM / F12 culture medium containing 10%-15% fetal bovine serum; when the cell confluence degree is higher than 70%, using trypsin-EDTA to digest the cells for 1-2 minutes to subculture, and the subculture culture medium contains 30% cell conditional culture medium; and finally, the cells are cryopreserved through programmed cooling. The ovary epithelial cell line of Quasipaa spinosa is successfully established for the first time, the survival rate of the cells after recovery is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and in-vitro conservation of germplasm resources.

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 a covalent polypeptide inhibitor targeting protein kinase A, a conjugate, a kit, a pharmaceutical composition and application. The general formula of the covalent polypeptide inhibitor is Z1-Seq-Z2; wherein Seq is an amino acid sequence; the C-terminal of Seq comprises a pseudo-substrate recognition motif recognized by protein kinase A, a non-natural amino acid with a covalent reaction group, and can be covalently crosslinked with a Cys200 residue on a catalytic subunit of protein kinase A; Z1 is selected from hydrogen, an acyl group, a fluorescent group, biotin, an isotopic tag or a biological ortho-reaction group; and Z2 is selected from a hydroxyl group, an amino group, a cell-penetrating peptide or a stabilization modification group. The covalent polypeptide inhibitor can strongly and irreversibly inhibit PKA activity, and can be used as an active probe to specifically label active PKA in a complex biological sample, and is used for biological research and drug development.

This invention provides a method for storing mango seeds, belonging to the field of seed preservation technology. The method involves cutting off mango fruits with embryos, along with their pedicels and branches, 77-84 days after fruit set. A mixture of pyraclostrobin, lambda-cyhalothrin, and chlorothalonil is sprayed on the fruit, pedicels, and branches. After drying, styrene-butadiene latex is applied to the pedicels and branches. Once the latex dries, a mixture of guanidine-copper acetate and benzoyl-pyraclostrobin is sprayed on the fruit, pedicels, and branches again. After drying, the mango fruits with petioles and branches are placed in a sealed box and stored at 10-14°C. The mango variety used is the Golden Phoenix mango. This invention increases the storage period of Golden Phoenix mango seeds from 1.5 months to 5 months, which is of great significance for the genomics and cell biology research of Golden Phoenix mangoes.

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.

The application discloses a monoclonal antibody of a rice field eel Adam8a and a preparation method and application thereof, relates to the technical field of biological antibodies, and fills the research blank of the rice field eel Adam8a. The rice field eel Adam8a monoclonal antibody is high in specificity and titer, simple in preparation process, and controllable in cost, can be widely used for recognizing Adam8a homologous proteins in different fish ovary, and is suitable for being prepared into a fish ovary Adam8a protein detection kit, and is compared with the expression of the protein in different fish, so that the rice field eel Adam8a monoclonal antibody becomes a very convenient detection tool in the biological research of fish ovary development.

The invention relates to ribozyme for labeling biotin on target RNA (Ribonucleic Acid) as well as a screening method and application thereof. The RNA labeling technology is one of core tools for modern molecular biology research and clinical diagnosis. A specific marker is covalently linked to a target RNA molecule, so that accurate tracking, efficient detection and functional analysis of RNA can be realized. A biotin-streptavidin system becomes one of the technical routes which are most widely applied due to extremely high affinity and signal amplification capability of the biotin-streptavidin system. At present, common biotin-labeled RNA technical methods mainly comprise a chemical solid-phase synthesis method, an enzymatic method and a chemical labeling method. However, the methods have the limitations of poor site specificity, low efficiency, complex manipulation and the like in the use process. The engineering ribozyme for catalyzing RNA biotinylation is obtained by taking 12: 0 biotin coenzyme A as a biotin donor through an in-vitro screening technology. The ribozyme provided by the invention can efficiently and specifically carry out biotin labeling on target RNA, the reaction process is simple, and the labeling activity is up to 90% or above.

The application provides a key enzyme involved in glycosylation modification of triterpene saponins, and an application thereof, and belongs to the technical field of genetic engineering. The glycosyltransferase gene is screened based on the omics research of the base plant of traditional Chinese medicine Duanxueliu, i.e. clinopodium gracile, and a pET28a-MBP-CcGT-g51295 prokaryotic expression vector is constructed to successfully obtain the glycosyltransferase. It is found that the glycosyltransferase can catalyze Saikogenin A and Saikogenin F to generate Prosaikogenin A and Prosaikogenin F respectively by using a glycosyl donor UDP-fucose; can catalyze Saikogenin A to generate clinopodiside I and clinopodiside X by using UDP-glucose; can catalyze Prosaikogenin A to generate Saikosaponin B1, Saikosaponin c1 and Saikosaponin X by using UDP-glucose; and can catalyze Prosaikogenin F to generate Saikosaponin A and Buddlejasaponin IV by using UDP-glucose, which reveals the catalytic function of the glycosyltransferase and provides a gene component for the biological research of triterpene saponins, and solves the problem of biological resources of rare saponins.

The present invention belongs to the technical field of genetic engineering, and specifically relates to a copepod luciferase mutant and the use thereof. The mutant is obtained by means of performing one or more mutations on a wild-type copepod luciferase with the signal peptide removed, and can be expressed in a prokaryotic expression system, resulting in an increased luminescence half-life or a signal rise within a certain time range, and the luminescence pattern of the luciferase is converted to glow-type luminescence or luminescence with a sustained increase in luminescence intensity. Some copepod luciferase mutants exhibit enhanced luminescence intensity. The mutant is widely used in biological research for real-time tracking and continuous monitoring, such as for labeling and tracking activities or processes in living organisms. On the basis of the advantage of increased luminescence half-life of the copepod luciferase mutant, the mutant can be used as and / or in the preparation of a glow-type luciferase. The copepod luciferase mutant is more suitable as a reporter gene for in-vivo detection in scientific research, and is conjugated with avidin, a digoxin antibody, etc., for sequencing, thereby shortening the sequencing time and improving the sequencing quality.

The utility model relates to the technical field of plant living body imaging, and discloses a plant living body imaging and simulation illumination device which comprises a box body, when the device is used, an LED plant growth lamp can be turned on firstly through an arranged environment simulation assembly, and light irradiates a plant through a through hole; light is dispersed around plants through the astigmatic lenses arranged on the through holes, so that more real natural light is simulated, meanwhile, natural light such as sunny days, cloudy days and night with moonlight can be simulated through the colored lenses, and an environment closer to the natural environment is created. According to the present invention, with the arrangement of the CCD camera, the weak fluorescence signal can be captured, the specific gene of the living plant can be subjected to fluorescence labeling, and after the EMS mutagenesis treatment, the fluorescence brightness change can be detected in the imaging device so as to find the enhancer or the suppressor, such that the biological research data can be obtained; and a better use prospect can be brought.

The application discloses a composite amplification detection system for body fluid tracing based on mRNA markers and application thereof, high-specificity mRNA markers can realize accurate tracing of five main body fluid types including peripheral blood, saliva, semen, menstrual blood and vaginal secretion, and can make up for the problem that in the previous body fluid tracing system, less markers lead to false positive or false negative problems caused by cross-reaction, and effectively improve the accuracy of single source and mixed body fluid, and old body fluid identification, and improve the applicability and feasibility of the system in forensic practice and biological research. Based on the fluorescence labeling technology, the application realizes stable, specific and efficient composite amplification reaction of multiple high-specificity mRNA markers at the same time, and the number of composite mRNA markers in the system is relatively large, and the identification efficiency of single source and mixed body fluid, and old body fluid is high.

The invention discloses a Phi29 DNA polymerase mutant with improved thermal stability, a construction method of the Phi29 DNA polymerase mutant and a genetically engineered bacterium. The mutant is obtained by performing single-point mutation on serine at the 527th site on an amino acid sequence shown as SEQ ID NO.2. The invention further discloses a construction method of the Phi29 DNA polymerase mutant. The mutant is realized through a single-point mutation design, and compared with a wild type Phi29 DNA polymerase, the mutant shows better thermal stability in a temperature interval of 40-70 DEG C. The Phi29 DNA polymerase mutant obtained by means of the construction method provided by the invention has better thermal stability. When being used for DNA amplification operation at a relatively high temperature, the fluorescent probe can present relatively high thermal stability, so that the fluorescent probe has relatively high application value and development potential in multiple fields of molecular biology research, clinical diagnosis and the like.

The application discloses a trienomycin photoaffinity probe, a preparation method and application, and comprises the following steps: taking Trienomycinol as a substrate, carrying out a substitution reaction with an iodine-substituted bisaziridine, then removing propylidene protection to obtain a corresponding photoaffinity negative probe; using Fmoc-D-alanine to selectively esterify a hydroxyl group at position 11 of the negative probe, then removing an Fmoc protection group on nitrogen and carrying out condensation with cyclohexanecarboxylic acid to obtain a corresponding photoaffinity positive probe; trienomycin protein target identification comprises the following steps: (1) enrichment of an intermediate Trienomycinol and synthesis of a trienomycin photoaffinity positive probe; (2) target identification of trienomycin by using the photoaffinity probe and molecular biology research on the target. The separated trienomycin intermediate, the synthesized trienomycin probe derivative and the identified trienomycin protein target have the advantages of high originality and high yield.

The invention belongs to the technical field of agriculture, and provides an artificial feed and a method for testing the influence of the artificial feed on the growth and development of tomato leaf miners, the method comprises three artificial feed formulas, and standardized feed blocks are prepared through moist heat sterilization and low-temperature preservation. The method comprises the following steps: subpackaging feed by adopting finger-shaped tubes, inoculating according to the density of 3 larvae in each tube, feeding at 25 + / -1 DEG C, and measuring indexes such as the duration, the survival rate, the pupation rate and the eclosion rate of the larvae. Results show that the difference between the formula I and natural foodstuff is minimum, the larva survival rate, pupation rate, pupa weight, eclosion rate and hatching rate are not obviously different from those of natural tomato leaf feeding, and although the larva and pupa duration is prolonged and the single female egg laying amount is reduced, the complete life history of tomato leaf miners can still be supported. According to the method, indoor stable test insects with consistent physiological status are realized, and reliable insect source guarantee is provided for biological research and prevention and control technology development of the tomato leaf miner.

The present application relates to the technical field of biological research data analysis, and discloses a kind of computing biology research data analysis display method, including extracting total RNA from biological tissue or cell, RNA reverse transcription is cDNA, constructs library, and utilizes high-throughput sequencing platform to sequence library, to obtain a large number of short sequences.The present application can effectively identify abnormal repeat regions by comparing and threshold judging the number of short sequences mapped to the same genomic coordinate interval, so as to determine whether the gene expression difference result is reliable, when the number of abnormal sequences exceeds the preset repeat sequence threshold, by eliminating the influence of repeat sequence operation, i.e. for the case of high FPKM value, remove the completely same and overlapping short sequences, and re-align the updated results, the expression distortion problem caused by repeat counting can be corrected.

The invention relates to a horizontal gel electrophoretic separation-online imaging device. The device comprises a device body, and a bottom plate assembly, a horizontal electrophoresis constant temperature module and a photographing module are arranged in the device body. The horizontal electrophoresis constant-temperature module comprises a tray assembly, a horizontal electrophoresis constant-temperature assembly, an electrode assembly and a driving assembly. The driving assembly is used for driving the tray assembly to enter and exit from the device body, the horizontal electrophoresis constant-temperature assembly is used for loading horizontal electrophoresis gel, and the electrode assembly is rotationally connected with the horizontal electrophoresis constant-temperature assembly. The photographing module comprises a frame assembly, a camera assembly and a light source assembly. The camera assembly is used for collecting a horizontal electrophoresis gel image, and the light source assembly is arranged on a light path between the camera assembly and the horizontal electrophoresis constant temperature module and is used for providing an excitation light source for horizontal electrophoresis gel. Compared with the prior art, the method is suitable for scenes of clinical diagnosis, molecular biology research and the like, and has the advantages of low cost, simplicity and convenience in operation, high detection quality and high stability.

The invention discloses a method for constructing a genetic transformation system of cymbidium kanran, which belongs to the technical field of plant genetic engineering, and comprises the following steps of: carrying out monoclonal propagation on rhizomes after germination of cymbidium kanran seeds to obtain vegetative propagules with stable characters, taking the vegetative propagules as transformation receptors, taking GFP (Green Fluorescent Protein) as marker genes, and constructing the genetic transformation system of cymbidium kanran. The method comprises the following steps: adding agrobacterium EHA105 into a YEB liquid culture medium containing 50 mg / L of kanamycin and 25 mg / L of rifampicin as a dip dyeing solution; placing the injured transformation receptor in dip dyeing for transformation, and then carrying out regeneration culture. The invention relates to a method for carrying out genetic transformation on cymbidium kanran by utilizing the innovative receptor and the screening culture medium, and the method can realize efficient and stable genetic transformation without genotype dependence and can solve the problem that the genetic transformation of cymbidium kanran varieties is difficult to carry out. According to the invention, an efficient genetic transformation and regeneration system of cymbidium kanran is established for the first time, and a foundation is laid for verification of related functional genes of cymbidium kanran and further development of biological research of cymbidium transgenic breeding and the like.

The invention discloses a blocking agent for eliminating environmental DNA (Deoxyribose Nucleic Acid) of human pollution and a detection method of the environmental DNA, and the blocking agent plays a role in inhibiting amplification of a human source sequence by specifically hybridizing with a human source template and blocking primer annealing or DNA polymerase extension. The invention further provides application of the CRISPR-Cas12a technology to in-vitro removal of human-derived DNA pollution, the method can reduce the sequencing cost and the time cost of bioinformatics processing without affecting detection of target biological groups, and the method is mainly used for treating human pollution in marine environment DNA. And the method can also be used for treating human-derived DNA pollution in similar molecular biology research and application.

The invention discloses a biological sample whole RNA normal-temperature long-term preservative as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preservative comprises the following components: a tissue fixing agent, a tissue penetrating agent, a reactive biological sample encapsulating agent, a silicification blocking agent, a pH regulator, an ion concentration regulator and water. The preservative can be applied to bacteria, such as escherichia coli and staphylococcus aureus; a cell, such as an HEK293 cell, an ID8 cell; tissues such as mouse liver tissues and human tumor tissues; organs such as kidneys and hearts of small animals; the whole RNA of a complete organism, such as earthworms, fruit flies and other biological samples with different sizes can be stored at normal temperature for a long time, so that the RNA extraction efficiency and quality can be ensured, the method is particularly suitable for normal-temperature transfer and long-term storage of field collected samples and clinical submitted samples, and the RNA extraction efficiency and quality in subsequent molecular experiments can be ensured; the method is of great significance to molecular biology research and clinical diagnosis.

Provided is a kit for rapid detection of avian leukosis virus subgroup J (ALV-J) based on a CRISPR / Cas13a system. The method is based on the combination of the CRISPR / Cas13a system and recombinase aided amplification (RAA) for ALV-J detection. An oligonucleotide probe is designed as a substrate for CRISPR / Cas13a trans-cleavage and produces a detectable signal. The method can substantially improve detection sensitivity by amplifying a detection signal twice by RAA and T7 transcription. The detection method further exhibits excellent specificity, allowing for clear differentiate from other avian viruses. It does not require expensive experimental equipment and special laboratory environment, and it is rapid and efficient. The method is of great significance for biological research and on-site detection of ALV-J.

Methods, articles, and fluidic systems for sorting and growing organoids are generally provided. Organoids may be useful for various applications, such as certain types of biological studies, or for testing the biocompatibility and performance of drugs. However, conventional methods often struggle to identify, isolate, and use organoids. In some embodiments, using the droplet-based methods and associated articles and fluidic systems provided herein, organoids may be selected, isolated, and studied.

The invention discloses a method for high-throughput extraction of DNA from cotton, and belongs to the technical field of plant biology. The invention provides a lysis buffer solution for high-flux extraction of DNA (deoxyribonucleic acid) from cotton, which comprises the following components: 50-80 g / L of NaOH, 2-4 g / L of NaCl, 0.1 g / L of SDS (sodium dodecyl sulfate), 0.8-1.5 g / L of EDTA-2Na. 2H2O, 10-30 g / L of PVP (polyvinyl pyrrolidone), 2 g / L of gelatin and 8-12 g / L of ascorbic acid, and all the components are dissolved in deionized water. According to the high-throughput DNA extraction method for the cotton tissues, efficient DNA extraction of the cotton tissues can be realized, the extraction steps are greatly simplified, high-throughput extraction can be carried out by a single person in a laboratory, DNA for PCR amplification is efficiently obtained, reliable sample support is provided for subsequent molecular biology research, and a convenient tool is provided for researchers.

The invention discloses a tissue culture method of floating moss, which comprises the following steps: disinfecting thalli of the floating moss as explants by 3-7% NaClO for 4-6 minutes to obtain sterile materials of the floating moss, and comparing the growth speeds and growth conditions of the floating moss on four different culture mediums. The invention discloses an optimal disinfection method and an optimal growth and rapid propagation culture medium of floating moss, establishes a tissue culture and rapid propagation system of floating moss, and lays a foundation for subsequent establishment of a genetic transformation system of floating moss and cell biology research.

The invention relates to a double-color fluorescence-based gel electrophoresis detection method, device, medium and kit, and the method comprises the following steps: marking a standard sample with a first fluorescent marker through covalent modification, carrying out mixed pre-dyeing on a test sample and a non-specifically bound second fluorescent dye, and loading the standard sample and the test sample to the same gel lane for synchronous electrophoresis; after electrophoresis, a dual-channel fluorescence imaging system is used for synchronously acquiring exclusive images corresponding to the two fluorescent substances; based on the migration position of the standard sample, calculating a fluorescence signal intensity proportion to eliminate crosstalk, establishing a fragment length and migration position fitting model, and finally accurately calculating the length of a target nucleic acid fragment in the test sample; according to the method, errors between lanes are eliminated through synchronous detection of the same lane, the operation process is simplified, the detection accuracy and efficiency are remarkably improved, and the method is suitable for separation and analysis of biological macromolecules such as DNA and RNA and can be widely applied to the fields of molecular biology research, clinical diagnosis and the like.

The application belongs to the technical field of biomedical research and medicine, and particularly relates to a non-natural amino acid based on trifluoromethyl photodefluorination-acyl fluoride exchange cross-linking and application thereof. The non-natural amino acid structure of the application is shown as formula I. The non-natural amino acid has a new molecular structure and function, and can realize multiple purposes in biological research and biomedical applications. The non-natural amino acid of the application can be applied to many fields, such as medical detection, medical diagnosis, biological macromolecule therapeutic drugs, biological mechanism research, chemical biology research, environmental detection, etc., and has very good practical value and actual significance.

This invention discloses an inoculum and a method for the instantaneous transformation of camphor trees, relating to the field of instantaneous transformation technology of camphor trees. The method for the instantaneous transformation of camphor trees is characterized by: camphor tree cultivation, taking camphor leaves; the camphor leaves being washed and then inoculated with the aforementioned inoculum. The viability of Agrobacterium varies significantly at different growth stages. The amplified bacterial solutions at different growth stages are centrifuged, and the concentration of the inoculum is adjusted using a lysate. After inoculating the camphor leaves, they are placed in an incubator for co-cultivation, and the Ca2+ level is measured. 2+ The fluorescence intensity analysis clarified key technologies such as suitable infection methods, Agrobacterium state, infection solution concentration, and co-culture time for camphor trees, which is of great value for promoting the smooth development of molecular biology research and molecular breeding of camphor trees.

The invention belongs to the technical field of fluorescent probes, and discloses a thiocarbonate-based hydrogen polysulfide fluorescent probe, a preparation method and application, the fluorescent probe can respond to H2Sn with high selectivity and high sensitivity, can resist thiol interference, has obviously increased fluorescence intensity in the presence of H2Sn, can be used for qualitative and quantitative detection of H2Sn, and can be applied to detection of H2Sn. The method is successfully applied to living cell and type 2 diabetes mouse imaging, a feasible means is provided for explaining H2Sn participating in physiological and pathological effects in vivo, and a valuable tool is provided for promoting H2Sn biological research and guiding future probe development.

A device, system, and method are provided for accessing and sampling target tissues in body cavities. In some embodiments, the device includes a sheath with a sampling lumen for needle passage, a distal flange for anchoring, and optional features like balloons, cuffs, and suction catheters. The system may incorporate a sampling needle, possibly with an SPME coating. Insertion methods use guidewire and dilator techniques for minimally invasive placement. This enables repeated, low-risk sampling for diagnostic and research applications.

The invention discloses a construction method and application of an ovarian cell line of hapalogenys nitens. The method comprises the following steps: selecting a metamorphosis stage larva ovarian tissue, disinfecting and obtaining, and digesting for 1 hour at 26 DEG C by using collagenase I digestive juice; after the tissue block adheres to the wall, a low-osmotic-pressure complete medium is used for primary culture at the temperature of 25-27 DEG C, and the medium is a 60% diluted DMEM / F12 medium containing 10%-15% of fetal calf serum; after the cell confluence degree exceeds 70%, trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) is used for digesting for 1-2 minutes for passage, and a passage culture medium contains 30% of a cell condition culture medium; and finally performing programmed cooling and cryopreservation. According to the method, the ovarian epithelial cell line of the hapalogenys nitens is successfully established for the first time, the cell recovery survival rate is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and germplasm resource in-vitro preservation.

The application discloses a kind of Vibration Mountain beard toad hind leg / toe tissue cells and its quick acquisition method and application.The method comprises: the body surface disinfection of Vibration Mountain beard individual, cut and take hind leg or toe tissue, and put back after wound disinfection;After tissue is soaked in 75% alcohol and washed with PBS containing penicillin, streptomycin and amphotericin B, it is temporarily stored in 4 ℃ temporary storage solution;After tissue is cut, it is sequentially digested with trypsin and type I collagenase;In the optimized low permeability medium, primary culture is carried out at 25-27 ℃;When cell confluence reaches 60%-70%, it is passaged using trypsin-EDTA digestion;Freezing and recovery.The application realizes the non-lethal cell of Vibration Mountain beard for the first time Quick acquisition, cell first time migration time is only 3 days, grow into monolayer only 12-17 days, pollution rate is reduced to 5.6%, recovery survival rate reaches 76.19%, and provides efficient and reliable technical support for endangered amphibian germplasm resource preservation and biological research.