Plant expression vector based on Arabidopsis pri-mir828 gene and its construction and application

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: 2016-02-17
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 pri-mir828 gene and its construction and application
  • Plant expression vector based on Arabidopsis pri-mir828 gene and its construction and application
  • Plant expression vector based on Arabidopsis pri-mir828 gene and its construction and application

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 ( Arabidopsisthaliana ) seeds and wild-type tomato ( Solanum lycopersicum L. Ailsa Craig) seeds.

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

[0043] Enzymes and Chemicals: 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, MarkerDL2000 and MarkerDL2000 were purchased from China TakaRa Company; Tans2K TM PlusIIDNAMarker was purchased from Beijing Quanshijin Company; DNA restriction endonuclease Hin dIII, Eco RI, Pac I was purchased from British NEB Company; TRizol was purchased from Am...

Embodiment 2

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

[0057] 1. Transformation of tomato by Agrobacterium-mediated leaf disc method.

[0058] Aseptic seedling cultivation: Soak tomato seeds with 75% ethanol for 5 minutes, rinse with sterile water after pouring off the ethanol; then rinse with saturated Na 3 PO 4 12H 2 Soak in O solution for 20 minutes, rinse with sterile water once; finally soak in 50% NaClO solution for 10 minutes, and rinse with sterile water for 4-6 times. The sterilized seeds were uniformly inoculated in MSR3 medium (4.4 g L -1 MS+30g·L -1Sucrose+1mL·L -1 R3vitamins+10g·L -1 Agar powder, pH 5.9), placed in an incubator at 26°C (16h light) / 18°C (8h dark) for about 8-10d.

[0059] Pre-cultivation: When the aseptic seedling grows until the cotyledon is fully expanded but the true leaf does not grow, cut off the cotyledon and cut off the two tips of the cotyledon, and place it in MS+2-4D liquid cult...

Embodiment 3

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

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

[0070] miR828 The sequences of the three primers are shown in Table 1, namely miR828stem-loopRT primer, miR828 forward primer and miR828 universal reverse primer. cDNA first-strand synthesis by PrimeScript TM 1stStrandcDNASynthesis Kit (TaKaRa, China) method of synt...

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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 in particular 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 known as microRNA, is a type of endogenous single-stranded non-coding small molecule RNA with a length of about 21-24 nucleotides. miRNAs mediate a new mode of gene expression regulation. In plants, they negatively regulate the transcription or translation of target mRNAs mainly through complementary pairing with target mRNAs at the post-transcriptional level. The expression of miRNA is faster than that of protein gene, and it is not affected by the translation process, so the expression regulation efficiency of target gene is higher. As important regulatory molecules, miRNA-mediated gene expression regulation plays an important role in various physiological and bio...

Claims

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Application Information

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Patent Type & Authority Patents(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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