Process for structuring carrier of expressing seven exogenous geng at same time in chloroplast

A technology of chloroplast expression and chloroplast genome, which is applied in the field of plant genetic engineering and can solve problems such as no discovery

Inactive Publication Date: 2006-06-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of recombinant cry2Aa2 protein obtained by this method has reached 45% / TSP, which paves the way for the simultaneous expression of multiple genes by chloroplast genetic engineering, but there are no reports of documents and patents closely related to the subject of the present invention.

Method used

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  • Process for structuring carrier of expressing seven exogenous geng at same time in chloroplast
  • Process for structuring carrier of expressing seven exogenous geng at same time in chloroplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1——Expression of FaeC protein in transgenic plants

[0060] 1. Solutions and reagents

[0061] (1) Plant tissue protein extraction buffer

[0062] 15mM Na 2 CO 3 , 35mM NaHCO 3 , and finally adjust the pH to 9.6;

[0063] (2) Close the buffer

[0064] 0.05% Tween-20, 0.05% NaN 3 , 1 mM EDTA, 0.25% BSA, 0.17M H 3 BO 4 , 0.12M NaCl, adjust the pH to 8.5 with NaOH;

[0065] (3) NPP buffer

[0066] 0.1M Glycine, 1mM MgCl 2 , 1mM ZnCl 2 , and adjust the pH to 10.4 with NaOH.

[0067] 2. Operation steps

[0068] (1) Take 100 mg of transgenic tobacco leaves and non-transgenic tobacco leaves, add liquid nitrogen to quickly grind them, then add an appropriate amount of protein extraction buffer, place at 4°C for 2 hours, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant, which is tobacco of total soluble protein.

[0069] (2) Determine the protein concentration in the total protein of transgenic tobacco and non-transgenic tobacco according...

Embodiment 2

[0074] Example 2—Expression of FaeD protein in transgenic plants

[0075] 1. Solutions and reagents

[0076] (1) Plant tissue protein extraction buffer

[0077] 15mM Na 2 CO 3 , 35mM NaHCO 3 , and finally adjust the pH to 9.6;

[0078] (2) Close the buffer

[0079] 0.05% Tween-20, 0.05% NaN 3 , 1 mM EDTA, 0.25% BSA, 0.17M H 3 BO 4 , 0.12M NaCl, adjust the pH to 8.5 with NaOH;

[0080] (3) NPP buffer

[0081] 0.1M Glycine, 1mM MgCl 2 , 1mM ZnCl 2 , and adjust the pH to 10.4 with NaOH.

[0082] 2. Operation steps

[0083] (1) Take 100 mg of transgenic tobacco leaves and non-transgenic tobacco leaves, add liquid nitrogen to quickly grind them, then add an appropriate amount of protein extraction buffer, place at 4°C for 2 hours, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant, which is tobacco total soluble protein.

[0084] (2) Determine the protein concentration in the total protein of transgenic tobacco and non-transgenic tobacco according to t...

Embodiment 3

[0089] Example 3 - Expression of FaeE protein in transgenic plants

[0090] 1. Solutions and reagents

[0091] (1) Plant tissue protein extraction buffer

[0092] 15mM Na 2 CO 3 , 35mM NaHCO 3 , and finally adjust the pH to 9.6;

[0093] (2) Close the buffer

[0094] 0.05% Tween-20, 0.05% NaN 3 , 1mM EDTA, 0.25% BSA, 0.17M H 3 BO 4 , 0.12M NaCl, adjust the pH to 8.5 with NaOH;

[0095] (3) NPP buffer

[0096] 0.1M Glycine, 1mM MgCl 2 , 1mM ZnCl 2 , and adjust the pH to 10.4 with NaOH.

[0097] 2. Operation steps

[0098] (1) Take 100 mg of transgenic tobacco leaves and non-transgenic tobacco leaves, add liquid nitrogen to quickly grind them, then add an appropriate amount of protein extraction buffer, place at 4°C for 2 hours, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant, which is tobacco of total soluble protein.

[0099] (2) Determine the protein concentration in the total protein of transgenic tobacco and non-transgenic tobacco according ...

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Abstract

The invention relates to a chloroplast simultaneity expression seven exogenous genes vector forming method. It belongs to genetic engineering technique field. It includes the following steps: forming a chloroplast expression vector containing faeC, faeD, faeE, faeF, faeG, faeH, and faeI seven gene orders which form K88ac flagellin gene cluster; extracting coding K88ac wild bacterium C83907 plasmid; using primer 1 whose nucleotide sequence is same with SEQ ID NO: 1 and primer 2 whose same with SEQ ID NO: 2 to process PCR amplification; nucleotide sequence 3018bp is same with SEQ ID NO.5; using primer 3 whose same with SEQ ID NO:3 and primer 4 whose same with SEQ ID NO:4 to process PCR amplification; the nucleotide sequence is same with SEQ ID NO.6; connecting with pMD18-T vector; transforming to escherichia coli DH5 alpha; and gaining escherichia coli clone pTEI with faeCD sequence. The invention overcomes the key defect of polygene technique of plant genetic improvement, and offers a good method of producing effective antibody plant bacterin for baby pig.

Description

technical field [0001] The invention relates to a method in the technical field of plant genetic engineering, in particular to a vector construction method for simultaneously expressing seven foreign genes in chloroplasts. technical background [0002] The use of genetic engineering to improve agronomic traits and the use of plants as bioreactors to produce certain medicinal proteins and medicinal ingredients have become research hotspots in plant genetic engineering. So far, most of the genetic transformation studies have transferred a single exogenous gene into the recipient plant, but some excellent traits are controlled by multiple genes, and some molecular anabolic pathways are also involved by multiple genes. The conventional multi-gene transformation method is to load a single gene into an expression vector, and then transfer it to a recipient plant for offspring hybridization screening to obtain a multi-gene transformant, or perform transgenic operation on the obtain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/09
Inventor 张大兵申慧峰梁婉琪钱炳俊袁政
Owner SHANGHAI JIAO TONG UNIV
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