13 results about "Quantitative PCR analysis" patented technology

The invention belongs to the technical field of biology, and particularly relates to application of hepcidin expression quantity in larimichthys crocea liver as larimichthys crocea visceral white-spot disease resistance evaluation index and a construction method. The hepcidin expression quantity in the larimichthys crocea liver is applied as the larimichthys crocea visceral white-spot disease resistance evaluation index. The construction method of the resistance evaluation index comprises the following steps: taking healthy small yellow croaker intraperitoneal injection pseudomonas proteinus strain bacterial liquid, and determining a half lethal dose in 96 hours; the method comprises the following steps: injecting a half lethal dose of bacterial liquid of pseudomonas proteinus strains into the abdominal cavity of healthy small yellow croaker for 96 hours, and after infection, taking different tissues to carry out hepcidin gene expression fluorescent quantitative PCR analysis; a regression model of gene expression quantity and bacterium loading quantity is established through fluorescence quantification, and the relationship between hepcidin expression quantity and disease resistance is constructed. Accurate disease-resistant phenotype information is provided for development of breeding of visceral white-spot disease-resistant improved varieties of the small yellow croakers, the accuracy of disease-resistant breeding is promoted, early detection of diseases is realized, and prevention and treatment of the diseases are effectively guided.

This invention discloses a method for delaying leaf senescence in Paspalum notatum under salt stress. The method includes the following steps: plant material culture and salt stress treatment; H2O2 content determination; DAB staining; chlorophyll content and Fv / Fm value determination; lipidomics data determination and analysis; endogenous hormone level analysis; real-time quantitative PCR analysis of gene expression levels; and transcriptomics data analysis. This invention discovers that root-derived H2O2 in Paspalum notatum under salt-driven conditions delays leaf senescence by participating in the regulation of jasmonic acid. The role of root-derived H2O2 was studied using diphenylthioiodine (DPI), a specific inhibitor of NADPH oxidase. Furthermore, the influence of the presence or absence of root-derived H2O2 on the expression of genes related to jasmonic acid synthesis precursors was investigated at the gene expression level. Mass spectrometry-based omics technology and data analysis are the first key technologies for implementing this project. Lipidomics, as one of the most important branches of metabolomics, can perform qualitative and quantitative analysis at the level of individual lipid species.

The present invention proposes a method for evaluating the growth of exogenous microorganisms in soil, comprising the following steps: S1, adding exogenous microorganisms and H2 to dry soil 18 O to carry out soil culture test, collect soil samples and extract soil DNA; S2, H2 16 O and H2 18 O. Preparing a liquid culture medium for culturing exogenous microorganisms, collecting the obtained exogenous microorganisms and extracting DNA; S3. Dissolving the soil DNA obtained in S1 and the bacterial DNA sample in S2 in a cesium chloride solution and performing density gradient centrifugation to obtain the distribution ratios of different DNAs along the density gradient; S4. Determining the average density of the exogenous microbial DNA in the soil based on the obtained distribution ratios of the different DNAs to achieve a quantitative evaluation of the growth degree of the exogenous microorganisms; the present invention performs quantitative PCR analysis on unique gene fragments of exogenous microorganisms, which can eliminate the interference of other microorganisms in the soil and perform highly specific analysis of the exogenous microorganisms.

This invention discloses the artificial synthesis of the disease-resistant gene NLP27 and its application, belonging to the field of plant genetic engineering technology. This invention selects a conserved 27-amino acid fragment from Verticillium dahliae NLP1, modifies its nucleotide sequence, and synthesizes the NLP27 gene, which is then overexpressed in potato plants through genetic transformation. Inoculation analysis shows that transgenic plants with the NLP27 gene exhibit high resistance to potato late blight and scab. Quantitative PCR analysis shows that excessively high NLP27 transcript levels significantly inhibit potato plant growth. After screening for expression levels and disease resistance, three transgenic lines with moderate NLP27 expression levels (such as PT1, PT6, and PT7) were obtained. These lines significantly enhanced resistance to potato late blight and scab without sacrificing yield.

The invention relates to the technical field of insect molecular biology, and particularly provides a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) reference gene of small blue-and-white chamaecyparis and application thereof. The method comprises the following steps: selecting seven candidate reference genes for fluorescent quantitative PCR (Polymerase Chain Reaction) analysis by taking different tissues of the small blue-and-white turtle as materials, performing gene stability evaluation on the Ct value of each candidate gene by adopting a delta Ct method, geNorm, NormFinder, BestKeeper and RefFinder, and screening to obtain the fluorescent quantitative PCR reference gene ribosomal protein S3 (RPS3) and ribosomal protein L31 (RPL31) which are stably expressed in different tissues of the small blue-and-white turtle. The reference gene is suitable for fluorescent quantitative PCR of different tissues of small blue-and-white chamaecyparis, and provides a basic support for subsequent gene function research.

The invention relates to the technical field of plantago asiatic L. gene engineering, and particularly provides a plantago asiatic L. real-time fluorescent quantitative PCR (Polymerase Chain Reaction) reference gene and application thereof. According to the method, tender leaves, old leaves, stems, flowers and roots of plantago asiatica are selected as experimental materials, candidate reference genes are screened through a plantago asiatica transcriptome, and then the gene expression stability is analyzed through real-time fluorescent quantitative PCR analysis, dissolution curve analysis, amplification efficiency analysis and various software (geNorm, NormFinder, BestKeeper, delta Ct and RefFinder). Reference genes, namely eIF and Cycl genes, suitable for gene quantification of different tissue parts of plantago asiatica are screened out, and the sequences of the eIF and Cycl genes are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively. The reference gene with stable expression is obtained, is suitable for analyzing the expression quantity of the gene at different parts of the plantago asiatica, makes up for the current situation that the plantago asiatica lacks the reference gene, and provides a basis for the gene expression quantity and function analysis in the plantago asiatica.

The invention provides a reference gene screening method for fluorogenic quantitative PCR (Polymerase Chain Reaction) analysis of peach-necked longhorn beetles and application of the reference gene screening method. The specific scheme is as follows: RPL13 and RPL10 are selected as reference gene combinations in different development stages, and alpha-tubulin and SOD are selected as reference gene combinations in different tissues. The nucleotide sequences are respectively ID 1, ID 2, ID 3 and ID 4. In addition, the invention also provides different primer sequences used when the stably expressed reference gene is used for qRT-PCR (quantitative reverse transcription-polymerase chain reaction) analysis. The method comprises the following steps: selecting 10 candidate genes, applying a qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology, combining GeNorm, BestKeeper, a delta Ct method, NormFinder and RefFinder software for analysis, and screening out a reference gene combination which is stably expressed under different experimental conditions. The screening system provides important technical support for the gene function research of the peach-necked longhorn beetles, and provides a scientific basis for the research of the peach-necked longhorn beetles by utilizing molecular biology.

The present invention discloses a kind of reference gene and construction method of real-time fluorescence quantitative PCR for analyzing gene expression under heat stress of indica rice, and belongs to the field of plant genetic engineering technology. The nucleotide sequences of 3 reference genes of the present invention are shown in sequence table SEQ ID NO.1-10;The flag leaf of II excellent 838 and its parent after high temperature stress is subjected to transcriptome high-throughput sequencing;Select 5 candidate reference genes from the sequencing results, carry out fluorescence quantitative PCR amplification on rice cDNA template, according to the expression difference of candidate genes in different rice samples, screen to the most suitable reference gene;Use the 3 reference genes screened together to calibrate and standardize the expression of target gene, can more accurately detect the expression change of indica rice gene, ensure the sensitivity and accuracy of fluorescence quantitative PCR;Use heat stress response gene to carry out method verification, find that the relative quantitative expression value of each gene in indica rice sample is consistent with the RPKM value and TPM value of sequencing result very well.

The present application belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene in papaya fluorescent quantitative PCR analysis and a screening method and application thereof. The present application relates to 18S rRNA, Actin, TUB, GAPDH and EF-1 alpha 5 internal reference genes, and the internal reference genes can be applied to the research on gene expression in different varieties, different periods and different tissues of papaya, and provide more accurate gene expression quantification standards for subsequent experimental research on papaya.

The invention discloses an analysis device based on gene detection and a detection method, and relates to the field of biological detection. The invention relates to a fluorescent quantitative PCR analysis device, and aims to solve the problems that the existing fluorescent quantitative PCR device is low in heating efficiency, a reaction system is difficult to quickly reach a target temperature, the detection period is prolonged, the consistency of PCR reaction is influenced and the like, the analysis device comprises a reaction chamber module, a reaction chamber rotary table module, a single chip microcomputer module and the like, and regular room-temperature air is conveyed through a top cover fan; a heating wire in a heating middle cylinder is heated into hot air, the hot air is uniformly dispersed by a bottom fan, all reaction holes are uniformly heated, the hot air passes through the reaction holes and is discharged out of a reaction chamber from a hollow position of an elastic gland, a top cover is connected with the bottom of a machine body through a hydraulic rod, and fluorescence data of all the reaction holes are collected through a fluorescence detection module; the temperature of the reaction chamber is controlled through the single-chip microcomputer module, the rotating speed and the position of the servo motor are adjusted, the time sequence of a fluorescence collection optical system is matched, and the device is suitable for the fields of material protection under high-temperature or variable-temperature working conditions.

The invention belongs to the technical field of plant gene engineering, and particularly relates to a reference gene in papaya fluorescent quantitative PCR (Polymerase Chain Reaction) analysis as well as a screening method and application thereof. The invention relates to five reference genes including 18S rRNA, Actin, TUB, GAPDH and EF-1 alpha, and the reference genes can be applied to research on gene expression of papaya in different varieties, different periods and different tissue ranges, so that a more accurate gene expression quantitative standard is provided for subsequent papaya experimental research.

This invention discloses a barley stripe disease resistance gene. Rdg2b This invention relates to the field of bioengineering technology and its applications. It describes the cloning of [specific species / organisms] from Ganbei No. 2 barley. Rdg2b The complete CDS segment of the gene was extracted, and an overexpression vector was constructed. Agrobacterium-mediated transformation was then performed to obtain overexpression-positive lines. Infection experiments with *Strombus striatum* showed that, compared to the wild type, [the expression was positive]. Rdg2b Overexpression lines showed significantly enhanced disease resistance. Real-time quantitative PCR analysis revealed that overexpression lines exhibited... Rdg2b Gene expression was significantly upregulated, confirming Rdg2b This invention positively regulates barley disease resistance. It provides important genetic resources for molecular breeding of barley for disease resistance.

The invention discloses reference genes for real-time fluorescent quantitative PCR detection of different tissues of rhizoma bletillae in different growth periods, and belongs to the technical field of plant molecular biology, the reference genes are BsGAPDH and BsEIF, the nucleotide sequence of the BsGAPDH is as shown in SEQ ID NO.1, and the nucleotide sequence of the BsEIF is as shown in SEQ ID NO.2. Three different tissues of rhizoma bletillae in seven growth periods are used as experimental materials, and the reference genes are used for real-time fluorescent quantitative PCR detection of different tissues of rhizoma bletillae in seven growth periods. The reference genes BsGAPDH and BsEIF with stable expression are obtained through fluorescent quantitative PCR (polymerase chain reaction) analysis and gene expression stability analysis by various technologies, and the two genes are stably expressed in different tissues in different growth periods of rhizoma bletillae and can be used as reference genes for researching gene expression in different tissues in different growth periods of rhizoma bletillae. The method can lay a foundation for functional research of genes related to accumulation of traditional Chinese medicinal components of rhizoma bletillae, and has relatively good practical value and scientific research application value.