Poplar chloroplast site-specific transformation method and DNA sequence for said method

A technology of DNA sequence and chloroplast, which is applied in the field of DNA sequence to achieve the effect of high expression efficiency, good safety, and reduced chance of variation and deformity

Inactive Publication Date: 2004-06-16
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current poplar genetic engineering research and application are limited to nuclear genome manipulation, and there is no report on the genome sequence of poplar chloroplasts and the genetic transformation of chloroplasts

Method used

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  • Poplar chloroplast site-specific transformation method and DNA sequence for said method
  • Poplar chloroplast site-specific transformation method and DNA sequence for said method
  • Poplar chloroplast site-specific transformation method and DNA sequence for said method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning of Two Poplar Chloroplast DNA Fragments and Construction of Chloroplast Transformation Vector

[0030] 1. Amplification and cloning of two chloroplast DNA fragments of poplar

[0031] Design and synthesize the following two pairs of primers:

[0032] P1: 5'-GCG GGT ACC GAG CAG GCT ACC ATG AG-3’

[0033] KpnI

[0034] P2: 5'-CGC GGG CCC CGA GAG GCT TAG TTG ATC-3’

[0035] ApaI

[0036] P3: 5'-GCG GCG GCC GC C CAT GGA ATA AGG TTT GAT-3’

[0037] NotI

[0038] P4: 5'-CGC CCG CGG AAA GCA ACG ACT GGA GTG-3’

[0039] SacII

[0040] Using poplar chloroplast DNA as a template, amplified by P1 and P2 primers, the PCR reaction was carried out on a Perkin-Elmer PE480 DNA amplification instrument, and the reaction program was: 95°C for 5min (one cycle); 95°C for 1min, 48°C 1min, 72°C 1min30sec (30 cycles); 72°C 10min. The PCR amplified product was about 1.7kb. The PCR product purified by phenol-chlo...

Embodiment 2

[0046] Expression of Green Fluorescent Protein (GFP) Gene in Chloroplasts of Poplar and Establishment of Efficient Chloroplast Transformation System

[0047] 1. Carrier:

[0048] Poplar Chloroplast Site-directed Transformation Expression Vector pPZG Containing GFP Gene

[0049]2. Transformation acceptor material:

[0050] Poplar tomentosa (Populus tomentosa Carr.) sterile vaccine

[0051] 3. Gene transformation:

[0052] The leaves of poplar were transformed by gene gun method. First, a large amount of high-quality plasmid DNA was prepared. The preparation method was improved by our laboratory, so that high-quality DNA could be prepared and diluted to 1 μg / μl for transformation. Then prepare microparticle bullets according to conventional methods, bombard the poplar leaves treated with hyperosmosis for 4 hours with Bio-RadPDS-1000 / He gene gun, then treat with hyperosmosis for 16 hours, then transfer to differentiation medium and cultivate for four days, then transfer Diff...

Embodiment 3

[0064] High-efficiency expression of Bt toxin protein gene in chloroplast and acquisition of transgenic insect-resistant poplar

[0065] 1. Carrier:

[0066] Poplar chloroplast site-directed transformation expression vector pPZB containing the Bt cryIA(a) gene cloned by our laboratory

[0067] 2. Transformation acceptor material:

[0068] Poplar tomentosa (Populus tomentosa Carr.) sterile vaccine

[0069] 3. Obtaining of chloroplast transformants:

[0070] The poplar chloroplast transformation system established in Example 2 was used to introduce the Bt gene into poplar chloroplasts and obtain transgenic plants (T0 generation). PCR detection was carried out on the transgenic plants, and the primers were specific primers for amplifying the Bt insecticidal protein gene, and 20 plants were detected as PCR positive plants. These positive plants were subjected to Southern hybridization analysis, and the hybridization probe was Bt insecticidal protein gene cryIA(a) labeled with ...

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Abstract

The present invention provides a method for site-directed transforming exogeneous gene into poplar chloroplast, and said method includes the folloiwng steps: using DNa recombinant fragment constituted by using sequence indicated by SEQ ID No:1 or its fragment which is not less than 1Kb, sequence indicated by SEQ ID No:2 or its fragment which is not less than 1 kb and an exogenous gene; positioning the exogenous gene between SEQ ID No:1 or its fragment and SEQ ID No:2 or its fragment; inserting the obtained DNA recombinant fragment into a cloning vector to obtain the poplar chloroplast genome site-directed recombinant transforming vector, using obtained vector to transformer poplar cell; and breeding the transformed poplar cell to obtain plant. It also provides the DNA sequence used for said method.

Description

technical field [0001] The present invention relates to a new poplar transgenic method, that is, using two sequences derived from poplar chloroplast DNA as homologous recombination fragments to construct a chloroplast-directed transformation vector, which can insert foreign genes into the poplar chloroplast genome at a fixed point The appropriate position, and make it highly expressed in the poplar chloroplast genome. The invention also provides DNA sequences for use in the methods of the invention. Background technique [0002] In the cells of higher plants, the three organelles of nucleus, chloroplast and mitochondria each contain DNA molecules, forming an independent and interrelated genetic system. Since the birth of genetic engineering technology in the early 1970s, the transformation technology of introducing foreign genes into the nucleus has been widely used in the genetic improvement of important crops and the study of molecular biological mechanisms. For decades,...

Claims

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Application Information

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IPC IPC(8): A01H1/06C12N5/10C12N15/11C12N15/63C12N15/82
Inventor 周奕华张丽华陈正华石东乔侯丙凯胡赞民
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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