Real-time quantitative PCR reference gene of tilia miqueliana under different tissues and stress treatment conditions as well as screening method and application of real-time quantitative PCR reference gene

A treatment condition, real-time quantitative technology, applied in the field of plant genetic engineering

Pending Publication Date: 2022-03-18
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Real-time quantitative PCR reference gene of tilia miqueliana under different tissues and stress treatment conditions as well as screening method and application of real-time quantitative PCR reference gene
  • Real-time quantitative PCR reference gene of tilia miqueliana under different tissues and stress treatment conditions as well as screening method and application of real-time quantitative PCR reference gene
  • Real-time quantitative PCR reference gene of tilia miqueliana under different tissues and stress treatment conditions as well as screening method and application of real-time quantitative PCR reference gene

Examples

Experimental program
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Effect test

Embodiment 1

[0031] This example is the extraction and verification of 15 Nanjing Tilia candidate internal reference gene sequences described in the present invention.

[0032] RNA from 6 tissues of Tilia Nanjing, stems, leaves, bracts, flowers, and fruits were extracted respectively, and mixed in equal amounts to construct a full-length cDNA library. The full-length transcriptome sequencing data of Tilia Nanjing were obtained through transcriptome sequencing and data analysis. According to the 18srRNA, ACT1, AP47, AQP, EF1α, GAPDH, HIS, PP2α, RPS13, SAMDC, SKIP, TUA, TUB, UBC and UBQ10 gene sequences in the TAIR Arabidopsis database, the sequence numbers are shown in Table 1 and obtained by local Blast Homologous sequences in the full-length transcriptome sequence of Tilia Nanjing. The CDS regions of the above 15 homologous sequences of Tilia Nanjing were analyzed using bioXM software, and primers were designed at both ends of the CDS regions using oligo7 software. PCR reaction was used ...

Embodiment 2

[0036] This embodiment provides fluorescent quantitative PCR primers for amplifying the 15 candidate internal reference genes in the embodiment.

[0037] According to the 15 candidate internal reference gene sequences in Example 1, primers were designed using oligo 7 software. The design principles follow the following principles: (1) The length of the amplification product is 100bp-250bp; (2) The length of the primer is between 15-22bp; (3) The melting temperature (Tm) is between 57-60°C; (3) GC The content is between 40-60%; (4) No hairpin structure, primer-dimer.

[0038] After the primer design was completed, the primers were preliminarily screened by PCR amplification. The PCR reaction system was 10 μl in total, including 1 μl of cDNA template and 0.5 mM of upstream and downstream primers. The PCR reaction program is: (1) pre-denaturation: 94°C, 5min; (2) denaturation: 94°C-10s, (3) annealing: 56°C-10s, (4) extension: 72°C-10s, a total of 35 cycles ; (5) Final extension...

Embodiment 3

[0042] This example provides materials and processing methods for screening candidate internal reference genes of Tilia Nanjing.

[0043] Different tissue samples: 9 tissues collected from 15-year-old mature plants of Tilia Nanjing, including young roots, young stems, adult leaves, bracts, flower buds, flowers (in full bloom), fruits, immature seeds and mature seeds, 3 for each tissue Biological repeat. Select Nanjing Tilia tissue culture seedlings with consistent growth, and carry out different stress treatments: (1) high temperature stress plants are placed in a light incubator, the photoperiod is set to 16h light / 8h dark, and the temperature is set to 42°C; (2) low temperature stress plants Placed in a light incubator, the photoperiod is set to 16h light / 8h dark, and the temperature is set to 4°C; (3) Water stress potted plants are flooded, and the water surface is about 2cm higher than the surface of the substrate; (4) Drought stress Nanjing Tilia The tissue-cultured seed...

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Abstract

The invention discloses a real-time quantitative PCR (Polymerase Chain Reaction) internal reference group gene of tilia miqueliana under different tissues and stress treatment conditions as well as a screening method and application thereof, and belongs to the technical field of plant genetic engineering. The 15 reference genes disclosed by the invention are derived from full-length transcriptome sequences of different tissues of tilia miqueliana, 15 pairs of specific real-time quantitative PCR (Polymerase Chain Reaction) primers are designed according to sequence information, and real-time quantitative PCR amplification is respectively carried out on different tissues of tilia miqueliana and plant materials subjected to stress treatment to obtain Ct values of the reference genes; and sorting the stability of the candidate reference genes from strong to weak, finally comprehensively evaluating the sorting result, and screening to obtain the most stable reference genes and combinations under different tissue and stress treatment conditions. The reference gene obtained through screening and the primer thereof have the advantages of being high in specificity and wide in applicability, and the blank of tilia miqueliana reference gene research is filled.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a real-time quantitative PCR internal reference gene and its screening method and application under different tissues of Nanjing Tilia and stress treatment conditions. Background technique [0002] Nanjing linden is an important native tree species in my country, and it is also an excellent garden ornamental tree species and urban street tree. At the same time, Nanjing Tilia is a multi-purpose economic tree species that gathers nectar, fragrant flowers, wood, fiber and medicine. With the gradual development of cultivation and promotion work, the cultivation of Tilia in Nanjing has begun to take shape nationwide. However, the research field of genetic breeding and functional genes of this genus is still blank. In Nanjing Tilia, only the key enzyme HMGR coding gene in the terpenoid synthesis pathway of medicinal ingredients was isolated and identified, and the act...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11C12Q1/6806G16B25/20G16B30/00
CPCC12Q1/6895C12Q1/6851C12Q1/6806G16B25/20G16B30/00C12Q2600/166C12Q2600/158C12Q2600/13C12Q2531/113C12Q2561/113C12Q2535/122C12Q2563/107
Inventor 王欢利汤诗杰黄犀严灵君王仲伟罗会婷
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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