Plant expression vector constructed by dihydroxy acetone synthetase and dihydroxy acetone kinase gene expression cassette in series as well as construction method and application thereof

A technology of plant expression vector and ribulose diphosphate carboxylase, which is applied in the field of plant genetic engineering, can solve problems such as increased workload, low success rate, and difficulty in realizing two gene transformation operations, so as to achieve enhanced efficiency, enhanced resistance The effect of improving the ability of plants to absorb and tolerate exogenous formaldehyde

Inactive Publication Date: 2010-09-22
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of DAS and DAK to construct the formaldehyde assimilation pathway involves the transformation of two genes. If these two genes are subcloned into two plant expression vectors containing different selection marker genes, when making transgenic plants, it is necessary to obtain a single When transgenic plants with two genes are transformed again, double transgenic plants with two genes can be obtained, or transgenic plants with a single gene can be crossed to obtain double transgenic plants. double the workload
In addition, double transgenic plants can also be obtained by co-transformation of two vectors, but the success rate of this method is very low, and it is more difficult to realize the transformation operation of two genes for plants that are difficult to transform

Method used

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  • Plant expression vector constructed by dihydroxy acetone synthetase and dihydroxy acetone kinase gene expression cassette in series as well as construction method and application thereof
  • Plant expression vector constructed by dihydroxy acetone synthetase and dihydroxy acetone kinase gene expression cassette in series as well as construction method and application thereof
  • Plant expression vector constructed by dihydroxy acetone synthetase and dihydroxy acetone kinase gene expression cassette in series as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of entry vector pEN-L4*-PrbcS-*T-DAK-L3*

[0049] (1) Construction of entry vector pEN-L4*-PrbcS-*T-DAK-L3*:

[0050] Use SphI and XhoI to cut pEN-L4*-PrbcS-*T-GFP-L3*(pEN-L4*-PrbcS-*T-GFP-L3* for the construction method, see another patent application of the applicant, patent The application number is 201010110946.5, and the name of the invention: Pathway cloning entry vector pEN-L4*-PrbcS-*T-GFP-L3* and its construction method and application and pENTR*-PrbcS-*T-DAK(pENTR*-PrbcS-*T -For the construction method of DAK, please refer to another patent application of the applicant, Patent Application No. 200810058341.9), to recover the vector pEN-L4*-PrbcS- generated after pEN-L4*-PrbcS-*T-GFP-L3* is cut *T-L3* fragment (4.3kb) and pENTR*-PrbcS-*T-DAK are cleaved to produce the DNA fragment (1.8kb) of the DAK gene, and then use TaKaRa's ligase kit to combine the vector fragment with The DNA fragments of the DAK gene are ligated to obtain the entry vec...

Embodiment 2

[0051] Example 2: Construction of plant expression vector in tandem with DAK and DAS gene expression cassettes

[0052] Through the LR reaction of Gateway, the PrbcS-*T-DAK fragment in the entry vector pEN-L4*-PrbcS-*T-DAK-L3* and pENTR*-PrbcS-*T-DAS(pENTR*-PrbcS-*T -DAK is constructed in another patent application of the applicant, the PrbcS-*T-DAS fragment in application number 200810058341.9) is simultaneously subcloned into the plant expression vector pK7m34GW2-8m21GW3 (Gateway's destination vector, purchased from VIB / Belgium) Gent company), obtained the plant expression vector pK-35S-PrbcS-*T-DAK-PROLD-PrbcS-*T-DAS( image 3 ). The specific method is: Purify Gateway's target vector pK7m34GW2-8m21GW3 with a plasmid extraction kit, and add pEN-L4*-PrbcS-*T-DAK-L3* and pENTR*-PrbcS-*T to the LR reaction system of Gateway -150ng each of DAS and pK7m34GW2-8m21GW3, 1ul LR Clonase plus Enzyme Mix (Invitrogen), mix and react overnight at 25°C, integrate PrbcS-*T-DAK and PrbcS-*T-D...

Embodiment 3

[0053] Example 3: Transformation of Agrobacterium with a plant expression vector consisting of DAK and DAS gene expression cassettes in series

[0054] Prepare Agrobacterium competent cells, and transfer the constructed plant expression vector pK-35S-PrbcS-*T-DAK-PROLD-PrbcS-*T-DAS into Agrobacterium (C58Cl(pPMP90)) by electric pulse method , Select transformants on the plate with spectinomycin. Take a small amount of plasmid into the Agrobacterium competent cells and mix gently; add the mixture to the pre-cooled electrotransformation cup, tap the cup body gently to make the mixture fall to the bottom of the cup; place the electrotransformation cup in the electrotransformation cup In the chute of the instrument (BIO-RAD), use a 1mm electric shock cup and 200 ohm, 2.5kV / 0.2cm parameters for electric shock. Take out the electric transformation cup immediately after the electric shock, quickly add 0.5ml SOC medium, mix well, and transfer to 1.5 ml centrifuge tube; 28℃, 200rpm shake...

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Abstract

The invention particularly relates to a plant expression vector, namely pK-35S-PrbcS-*T-DAK-PROLD-PrbcS-*T-DAS, for improving the capability of assimilating and formaldehyde absorption of plants, as well as a construction method thereof and application thereof in improving the capability of absorbing and resisting exogenous formaldehyde of plants. The plant expression vector, namely pK-35S-PrbcS-*T-DAK-PROLD-PrbcS-*T-DAS, for improving the capability of assimilating and formaldehyde absorption of plants is formed by an optical inducible promoter (PrbcS) containing a tomato 1,5 ribulose diphosphate carboxylase (Rubisco) 3C small subunit gene and a transfer peptide sequence (*T) thereof, a dihydroxy acetone synthetase (DAS) gene of candida boidinii and a dihydroxy acetone kinase (DAK) gene (two genes) of Pichia pastoris. The conversion operation of both the DAS gene and the DAK gene can be completed by performing a conversion test by using the vector for one time.

Description

Technical field: [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a plant expression vector pK-35S-PrbcS-*T-DAK-PROLD-PrbcS-*T-DAS that improves the ability of plants to assimilate and absorb formaldehyde, and a construction method and improvement Application in the ability of plants to absorb and tolerate exogenous formaldehyde. Background technique: [0002] Formaldehyde is a colorless gas with a strong pungent odor. It is easily soluble in water, alcohol and ether. It is widely used in industrial production and is the most widely used chemical raw material in the adhesive industry. With the development of economy and the improvement of people's living standards, building decoration materials made of various raw materials have entered various indoor public places and homes, making formaldehyde the most recognized chemical substance in indoor air pollution. The allowable value of free formaldehyde concentration in ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/52C12N15/54C12N15/66A01H5/00
Inventor 陈丽梅肖素勤潘正波梁峰李莉孙振宋中邦李昆志
Owner KUNMING UNIV OF SCI & TECH
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