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Potato pin II gene promoter and application thereof

A promoter and potato technology, applied in the fields of genetic engineering and molecular biology, can solve problems affecting the promotion of transgenic crops, etc., and achieve the effect of strong promoter activity

Inactive Publication Date: 2015-02-11
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the CaMV35S promoter is derived from a virus, it affects the promotion of transgenic crops to a certain extent from the perspective of biosafety

Method used

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  • Potato pin II gene promoter and application thereof
  • Potato pin II gene promoter and application thereof
  • Potato pin II gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning of Potato pinⅡ Gene Promoter

[0022] According to the accession number X04118, query the nucleic acid sequence of the potato protease inhibitor Ⅱ gene in GenBank. According to the nucleic acid sequence, primers are designed to amplify the potato pinⅡ promoter fragment. The designed primers are: forward primer 5'-AACTGCAGCCCAATTCAAAGAACTTG-3' (SEQ ID No.2), a PstI restriction site was introduced at the 5' end, and reverse primer 5'-CGCGGATCCTTAATTAGTACTGCCTCT-3' (SEQ ID No.3) , the 5' end introduces a BamHI restriction site. Using potato genomic DNA as a template, the pinⅡ promoter sequence was amplified by PCR to obtain a 928bp PCR product ( figure 1 ).

[0023] PCR reaction system:

[0024]

[0025] PCR reaction program:

[0026]

[0027] The amplified PCR product was connected to the pGEM-T easy vector carrier (Promega Company), and the amplified fragment was sequenced using the T7 and SP6 primers on the carrier. The sequencing results sh...

Embodiment 2

[0028] Example 2 Construction of pCAMBIA1300-pinⅡ-GUS expression vector

[0029] Plasmid pBI221 (Shanghai Beinuo Biotechnology Co., Ltd.) was digested with HindIII and EcoRI, the 35S-GUS-NOS fragment was recovered, and connected to pCAMBIA1300 (Shanghai Beinuo Biotechnology Co., Ltd.) digested with HindIII and EcoRI to construct Plasmid pCAMBIA1300-221. The T vector containing the pinII promoter fragment was double-digested with PstI and BamHI, and the digested product was run on agarose gel electrophoresis to recover a 928bp fragment. At the same time, the pCAMBIA1300-221 vector was digested with Pst1 and BamH1, and the digested product was run on agarose gel electrophoresis to recover a 11kb fragment. The recovered promoter fragment and vector fragment were ligated with T4 DNA ligase (Promega Company), so that the potato pinⅡ promoter replaced the CaMV35S promoter driving the expression of GUS gene on the pCAMBIA1300-221 vector, and the vector was named pCAMBIA1300-pinⅡ-GUS...

Embodiment 3

[0030] Embodiment 3 pCAMBIA1300-pinⅡ-GUS expression vector transforms tobacco and Arabidopsis

[0031] 1.1 Transformation of tobacco with pCAMBIA1300-pinⅡ-GUS expression vector

[0032] Take 200 μL of Agrobacterium LBA4404 competent cells, add 1 μg of the plasmid DNA constructed in Example 2, and use pCAMBIA1300-221 as a positive control. Quickly freeze in liquid nitrogen for 1 minute, 37°C water bath for 5 minutes, ice bath for 2 minutes, then add 1 ml YEB medium, shake at 120 rpm at 28°C for 4 hours; centrifuge at 4000 rpm for 2 minutes, discard the supernatant, add 0.1 ml YEB culture Cells were resuspended in the base, spread on YEB plates containing 100 μg / mL kanamycin, 125 μg / mL streptomycin and 50 μg / mL rifampicin, and cultured at 28°C for about 48 hours. Pick a single colony grown on the plate, inoculate it in YEB liquid medium containing 100 μg / mL kanamycin, 125 μg / mL streptomycin and 50 μg / mL rifampicin, and culture overnight at 28°C with shaking. PCR amplification ...

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Abstract

The invention relates to a potato pin II gene promoter and application thereof. A nucleotide sequence of the promoter is the nucleotide sequence shown in SEQ ID No.1, or the nucleotide sequence derived from the nucleotide sequence shown in SEQ ID No.1 by replacing, deleting and / or adding one or several nucleotides and having the same function as the nucleotide sequence shown in SEQ ID No.1. The function of the potato pin II gene promoter is verified through analysis on the structure and sequence of the potato pin II gene promoter starting from cloning the potato pin II gene promoter. The promoter is constructed into a GUS (glucuronidase) fusion expression vector for converting tobaccos and arabidopsis thaliana. GUS histochemical stain and GUS enzyme assay results show that the promoter provided by the invention can start the expression of GUS genes and has strong promoter activity, thus a new promoter element is provided for research of plant stress-tolerance gene engineering, and the foundation is laid for genetic improvement of plants and industrialization study.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a potato pinII gene promoter and its application. Background technique [0002] With the development of life sciences, genomics, bioinformatics and other disciplines, the research on transgenic technology is changing with each passing day, and the planting area of ​​transgenic crops is increasing year by year. The expression strength of the target gene in transgenic crops largely affects the application of transgenic crops. There are many factors that affect gene expression, mainly in the following five aspects: nucleic acid structure, transcription strength, post-transcriptional modification, translation efficiency and post-translational modification, among which transcription strength plays an important role. It is the promoter that regulates the strength of gene transcription, so the development of strong promoters is of great si...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/84C12N5/10A01H5/00
Inventor 王国英张生学刘允军
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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