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Tobacco chloroplast genome recombination hotspot short-sequence NtcpRH23

A genome and tobacco leaf technology, applied in the field of plant genetic engineering, can solve the problems of increasing the difficulty of molecular operations and the cumbersome construction process of chloroplast transformation vectors

Inactive Publication Date: 2011-10-19
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This needs to be done on the basis of the sequencing of the plant chloroplast genome, and the construction process of the chloroplast transformation vector is cumbersome, and the longer homologous fragments increase the difficulty of molecular operations

Method used

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  • Tobacco chloroplast genome recombination hotspot short-sequence NtcpRH23
  • Tobacco chloroplast genome recombination hotspot short-sequence NtcpRH23
  • Tobacco chloroplast genome recombination hotspot short-sequence NtcpRH23

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Southern hybridization proves that recombination events occur in the genomic DNA of transgenic tobacco chloroplasts

[0012] In the study of tobacco chloroplast transgenesis, we found that the introduction of exogenous DNA led to the recombination of tobacco chloroplast genome, resulting in recombinant DNA. use Bam The chloroplast genomes of wild-type tobacco and transgenic tobacco were digested with H I and hybridized with exogenous genes as probes (see figure 1 ), multiple hybridization bands of the same size appeared in the chloroplast genome DNA of four independently obtained transgenic tobacco materials T3, T8, I1 and I10, in contrast, there was only one distinct hybridization band in the chloroplast genome of wild-type TW, This indicated that site-specific recombination events occurred in the transgenic tobacco chloroplast genome, and presumed that the recombination occurred at a relatively fixed position.

Embodiment 2

[0013] Example 2: Cloning and Sequence Analysis of Transgenic Tobacco Chloroplast Recombinant Fragments

[0014] In order to further determine and analyze the chloroplast genomic DNA recombination site and formation mechanism of recombined transgenic tobacco, the chloroplast DNA of transgenic tobacco was extracted and used Bam HI digestion, followed by agarose gel electrophoresis, the electrophoresis band of the recombinant DNA fragment was recovered, and then ligated with the plasmid vector. Nucleotide sequence analysis and alignment of 3.2kb chloroplast recombined DNA fragments (sequence 1 in the sequence listing) revealed that the recombination was triggered by a short sequence of 23bp, named NtcpRH23. NtcpRH23 is a short recombination hotspot sequence that has not been reported in the world so far. The significance and value of the discovery of the recombination function of this sequence is that it can be used to construct a new chloroplast expression vector, breaking thr...

Embodiment 3

[0015] Embodiment 3: PCR verification triggers transgenic tobacco chloroplast genomic DNA recombination by NtcpRH23

[0016] In order to further prove the function of the recombination hotspot short sequence NtcpRH23 analyzed by the hybridization and sequencing results, the schematic diagram of DNA recombination in the transgenic tobacco chloroplast genome is triggered by the NtcpRH23 sequence. Downstream design of a pair of amplification primers, if transgenic tobacco does occur figure 2 Indicating chloroplast genomic DNA recombination, transgenic tobacco T3 and T8 can amplify the expected product of 1.2kb length, and transgenic tobacco I1 and I10 can amplify the expected product of 1.3kb length; if the chloroplast genome of transgenic tobacco does not recombine, the amplified Amplified products were not available beyond 50kb. image 3 The experimental results are based on figure 1 and figure 2 The analysis conclusion obtained is completely consistent with the expected l...

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Abstract

The invention relates to a tobacco chloroplast genome recombination hotspot short-sequence NtcpRH23. According to the invention, the obtaining of chloroplast-converted tobacco material is employed as a basis, and a discovery is that: a segment of 23bp short-sequence NtcpRH23 on the constructed carrier can be subject to high-frequency recombinations with upstream direct repeats. According to PCR verifications, recombination events occur in four obtained conversion materials, and tobacco chloroplast genome recombinants are produced. The discovery of the segment of sequence assists in providing a novel molecular biological tool for the construction of chloroplast conversion novel carrier, and for the researches of relevant biological hot problems of chloroplast genome copying, chloroplast genome recombination, and chloroplast genome restoring mechanism.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and mainly relates to the 23bp tobacco chloroplast recombination hotspot short sequence NtcpRH23, and methods for obtaining and verifying the same. Background technique [0002] The chloroplast of higher plants is an important organelle and an important place for photosynthesis in plants. Chloroplasts are genetically semi-autonomous, have their own genomic DNA, and can replicate themselves. Chloroplast DNA has a set of interdependent and influencing DNA replication, recombination and repair pathways. It has been reported that homologous recombination, an important part of chloroplast genome recombination, is mediated by RecA protein, and its research in prokaryotes has shown that the substrate sequence recognized by RecA-mediated homologous recombination is larger than 50bp. However, some related studies have shown that the 16bp direct repeat sequence on the chloroplast genome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82
Inventor 王勇王丹白艳玲朱晔荣任聪戎清清魏利静
Owner NANKAI UNIV
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