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General expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification

A technology for expression vectors, mitochondria, applied in the direction of using vectors to introduce foreign genetic material, microorganisms, microorganism-based methods, etc.

Inactive Publication Date: 2011-02-16
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of molecular biology operation in the genetic transformation of plant mitochondria, and to provide a self-constructed plant mitochondria universal expression vector pSK-E-P / T and its source of key elements and promoter activity detection

Method used

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  • General expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification
  • General expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification
  • General expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Discovery of cucumber mitochondrial plasmid pC3 and cloning of its sequence

[0024] The inventor discovered for the first time in the world that there is a 550bp circular DNA plasmid in the mitochondrial genome of some cucumber varieties such as "Jinyan No. And named pC3. The mitochondrial genomic DNA of "Jinyan No. 4" cucumber was extracted, and the plasmid pC3 was isolated and purified, then digested with S1 nuclease, smoothed at the end and treated with adenine, and then connected to T-Vector. The long pC3 was sequenced, and the length was 537bp. Sequence analysis found that it contained unique common restriction endonuclease SmaI and XbaI sites. The purified plasmid pC3 was digested with SmaI and cloned into the vector pUC18 to construct the pUC-pC3 / SmaI recombinant plasmid. The full-length pC3 cloned by sequencing was 550bp (see Sequence Listing Sequence 1 for details). pC3 is currently the smallest found in the world Mitochondrial circular DNA plasmi...

Embodiment 2

[0025] Example 2: Acquisition and cloning of the upstream promoter sequence of cucumber mitochondrial gene cob

[0026] According to the cucumber mitochondrial cytochrome B gene (GenBank accession numberAF288044) published by GenBank, using http / / www.fruitfly.org eukaryotic promoter prediction and PlantCARE software, the transcriptional promoter range upstream of the gene was analyzed within 1006bp (AF288044 :2041-3047), and design upstream primer pCMP1 and downstream primer pCMP2 in this interval (see primer sequence 2 for details), and use the extracted total mitochondrial DNA of cucumber as a template for PCR amplification. The PCR amplified product was cloned into the recombinant plasmid pUC-pC3 / smaI with cucumber mitochondrial plasmid pC3 through BamH I and Pst I, and the new recombinant was named pUC-pC3-CMP (see the appendix for details). figure 1) . The cloned 995bp length sequence was compared with GenBank, which proved to be the expected promoter sequence of cucumb...

Embodiment 3

[0027] Example 3: Acquisition and Cloning of the Downstream Transcription Terminator Sequence of Cucumber Mitochondrial Gene ATP9

[0028] According to the cucumber mitochondrial atp9 gene (GenBank accession number AF288043) published by GenBank, using www.softberry.com terminator analysis software, the transcription terminator was determined to be the downstream 896bp sequence of the gene (AF288043:9530-10426), and primers pCMT1 and pCMT2 (see primer sequence 3 for details), PCR amplification was carried out with the extracted cucumber mitochondrial total DNA as a template, and the PCR amplification product was cloned into the pSK vector after being digested with EcoR I and Kpn I, and the formed recombinant was named pSK- CMT (see the attached manual for details figure 2 ), the cloned 896bp length sequence was compared with GenBank, which proved to be the expected terminator sequence of cucumber mitochondrial atp9 gene.

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Abstract

The invention relates to general expression vector pSK-E-P / T construction for plant mitochondria and promoter activity identification. Cucumber mitochondria plasmids pC3 (sequence 1) are discovered for the first time, a gene cob upstream promoter sequence (sequence 2) and a gene atp9 downstream terminator sequence (sequence 3) of the cucumber mitochondria obtained through PCR (polymerase chain reaction) are constructed into a general expression vector pSK-E-P / T (sequence 4) of the plant mitochondria, and the vector also carries replicons copied in escherichia coli and Ampr resistance genes so as to facilitate cloning of exogenous genes and recon screening. Measurement of enzyme activity of promoter -1acZ report gene fusion plasmids in the escherichia coli and fluorescence microscope observation of psk-E-GFP for transforming the green fluorescence of the escherichia coli prove that the promoter on the expression vector has transcriptional activity. The invention provides a novel molecular biology technology tool for researching important life science hotspot problems such as cell apoptosis and the like related with the mitochondria by a mitochondria transformation path.

Description

【Technical field】: [0001] The invention belongs to the technical field of plant genetic engineering, and mainly relates to the construction strategy and implementation of the universal expression vector pSK-E-P / T for plant mitochondria, including the pC3 sequence of the mitochondrial plasmid pC3 from "Jinyan No. 4" cucumber variety, one of the key elements in vector construction. , gene cob promoter sequence and gene atp9 terminator sequence, as well as promoter activity identification and related methods. 【Background technique】: [0002] Mitochondria are important organelles of plants, which are related to important life phenomena such as plant energy metabolism, photorespiration, male sterility and cell apoptosis. Male sterility (CMS) exists widely in flowering plants, and the cultivation of CMS strains is a key link in traditional cross-breeding of higher plants and plays a pivotal role in agricultural production. Recent studies have shown that CMS is related to mtDNA an...

Claims

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Application Information

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IPC IPC(8): C12R1/19C12N1/21C12N15/113C12N15/82
Inventor 白艳玲周腊梅董荣徐海津张秀明乔明强
Owner NANKAI UNIV
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