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Plant expression vector based on Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof

A technology of pot2-at-pri-mir828 and plant expression vector, which is applied in the field of bioengineering and can solve problems such as poor results

Inactive Publication Date: 2014-03-05
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In tomato plants, overexpression of a single heterologous enzyme gene and regulatory gene involved in the flavonoid biosynthetic pathway to increase fruit anthocyanin content is less effective
However, there is no report on the application of miRNA regulation strategy to improve the synthesis and accumulation of anthocyanins in common tomato.

Method used

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  • Plant expression vector based on Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof
  • Plant expression vector based on Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof
  • Plant expression vector based on Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of plant expression vector pC2300-pOT2-At-pri-miR828.

[0041] Plant material: Columbia ecotype (Columbia-0) wild type Arabidopsis ( Arabidopsis thaliana ) Seed and wild type tomato ( Solanum lycopersicum L. Ailsa Craig) seeds.

[0042] Strains and plasmids: Escherichia coli ( E.coli ) Strain DH5α, Agrobacterium strain GV3101, modified cloning vector pOT2 with 35s promoter, and modified with Pac I single restriction site expression vector pC2300.

[0043] Enzymes and chemical reagents: LA Taq DNA polymerase, common Taq DNA polymerase, T 4 DNA ligase, purification and recovery kit, plasmid extraction kit, reverse transcription kit, fluorescence quantification kit, Marker DL2000 and Marker DL2000 were all purchased from Takara, China; Tans2K TM Plus II DNA Marker was purchased from Beijing Quanshijin Company; DNA restriction endonuclease Hin d III, Eco R I, Pac I was purchased from NEB, UK; TRizol was purchased from Invitrogen, USA. The PC...

Embodiment 2

[0056] Example 2: Genetic transformation of tomato by plant expression vector pC2300-pOT2-At-pri-miR828.

[0057] 1. Agrobacterium-mediated transformation of tomato with leaf disc method.

[0058] Sterile seedling culture: first soak tomato seeds with 75% ethanol for 5 minutes, pour out the ethanol and rinse with sterile water; then use saturated Na 3 PO 4 ·12H 2 Soak in O solution for 20 minutes, and rinse once with sterile water; finally, soak in 50% NaClO solution for 10 minutes, and rinse with sterile water 4-6 times. Evenly inoculate the sterilized seeds in MSR3 medium (4.4g·L -1 MS+30g·L -1 Sucrose+1mL·L -1 R3 vitamins+10g·L -1 Agar powder, pH 5.9), placed in an incubator at 26°C (16h light) / 18°C (8h dark) for about 8-10 days.

[0059] Pre-culture: When the aseptic seedling grows until the cotyledons are fully expanded but the true leaves do not grow out, cut the cotyledons and cut off the two tips of the cotyledons, and place them in MS+2-4D liquid culture medium (4.4g·L -1 ...

Embodiment 3

[0068] Example 3: Real-time fluorescent quantitative PCR analysis of transgenic tomato plants.

[0069] The tender leaves of one transgenic plant were selected from the two miR828 overexpression transgenic tomato lines that were positive by PCR, and the total RNA was extracted with TRizol and treated with DNaseⅠ. RNA concentration and quality were identified with a nucleic acid protein detector (Nanodrop ND-1000, thermo) and electrophoresis. Take tomato Sly - U6 As an internal reference gene, the expression of miR828 gene was detected by real-time fluorescence quantitative method; Sly-CAC It is an internal reference gene to detect miR828 target genes Sly - myb-like1 (SGN320618) Expression level.

[0070] miR828 The sequences of the three primers are shown in Table 1, namely miR828 stem-loop RT primer, miR828 forward primer and miR828 universal reverse primer. The synthesis of the first strand of cDNA is based on PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa, China) m...

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Abstract

The invention discloses a plant expression vector pC2300-pOT2-At-pri-miR828 based on an Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof. The plant expression vector pC2300-pOT2-At-pri-miR828 contains the At-pri-miR828 gene and a pOT2 cloning vector CaMV35s promoter, and has a nucleotide sequence shown in SEQ ID NO. 5. After plants are subjected to transfection by the plant expression vector disclosed by the invention, plants with high expression of the At-pri-miR828 gene can be obtained, and the biosynthesis of anthocyanin in the plants is regulated and controlled.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a plant recombinant expression vector of Arabidopsis pri-miR828 gene, and the construction and application of the recombinant expression vector. Background technique [0002] MicroRNA (miRNA), also called microRNA, is a type of endogenous single-stranded non-coding small RNA with a length of approximately 21-24 nucleotides. miRNA mediates a new mode of gene expression regulation. In plants, it negatively regulates the transcription or translation of target mRNA through complementary pairing with target mRNA at the post-transcriptional level. The expression of miRNA is faster than that of protein genes and is not affected by the translation process, so the expression and regulation of target genes are more efficient. As an important regulatory molecule, miRNA-mediated gene expression regulation plays an important role in a variety of physiological and biochemical pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 贾小云贺立恒丁娜刘慧沈洁金雷皓李芳唐贵良
Owner SHANXI AGRI UNIV
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