Plant expression vector containing chloroplastic glutamine synthetase genes, construction and use thereof

A technology of plant expression vectors and chloroplasts, applied in the field of plant expression vectors, can solve the problems of low nitrogen utilization, difficulties in meeting the needs of crop growth and development, environmental hazards, etc., and achieve the effect of enhancing nitrogen utilization

Inactive Publication Date: 2009-04-22
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the nitrogen in the soil is difficult to meet the needs of crop growth and development, so in production, the method of applying nitrogen fertilizer is widely used to meet the needs of crops
However, due to ammonia volatilization, nitrate nitrogen loss, and denitrification, the nitrogen utilization rate of the applied nitrogen fertilizer is very low. The national average nitrogen utilization rate in the current season is less than 30%. Soil and water pollution, causing harm to the environment on which people live
However, because the trait of high nitrogen use efficiency is difficult to evaluate in field conditions, it is difficult to select crop varieties with high nitrogen use efficiency by traditional breeding methods. Ability to become the most natural and environmentally friendly way
At present, there is no plant expression vector containing the chloroplast-type GS2 gene present in the Arabidopsis genome regulated by the tomato 1,5-bisphosphate ribulose carboxylase oxygenase small subunit gene (rbcS) and the 35S double promoter. to report

Method used

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  • Plant expression vector containing chloroplastic glutamine synthetase genes, construction and use thereof
  • Plant expression vector containing chloroplastic glutamine synthetase genes, construction and use thereof
  • Plant expression vector containing chloroplastic glutamine synthetase genes, construction and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: GS2 gene cDNA amplification and TA cloning

[0049] First, search for the full-length gene cDNA sequence of Arabidopsis chloroplast type GS2 from GenBank, and design a pair of primers with the following sequence:

[0050] GS25: 5'-caccgcATGcCTCAGATCTTAGCAGCTTC-3'

[0051] GS23: 5'-gaattcTTAAACATTCAAAGAAAGCTTTT-3'

[0052] Primer GS25 at the 5' end, add the characteristic sequence of caccgc at the end, and change the G after ATG to c to form a SphI restriction site; primer GS23 at the 3' end, add an EcoRI site at the end;

[0053] Use TRIzoL Reagent (Invitrogen) to extract total RNA from Arabidopsis thaliana seedlings, take 0.1g of young leaves of Arabidopsis thaliana, add 1ml of TRIzoL RNA extraction solution, grind in a mortar, let stand at room temperature for 5min, and then transfer to a centrifuge tube , add 0.2ml of chloroform, shake and mix, and centrifuge at 12000rpm / min for 15min at 4°C. Transfer the supernatant, add 0.5ml of isopropanol, mix we...

Embodiment 2

[0055] Example 2: Construction of Gateway entry cloning vector pENTR*-PRbcS-*T-GS2

[0056] The construction strategy of the entry cloning vector pENTR*-PRbcS-*T-GS2 is as follows: Figure 4 As indicated, the purified plasmid vectors pENTR*-PrbcS-*T-GFP and pMD18-GS2 were cut with SphI (Fermentas) and EcoRI (Fermentas), and the cut vectors and inserts were separated by agarose gel electrophoresis. The vector fragment pENTR*-PrbcS-*T (4.0kb) produced after pENTR*-PrbcS-*T-GFP was cleaved and the DNA fragment (1.2kb) of the GS2 gene produced by cleavage of pMD18-GS2 were recovered in the gel, and then The DNA fragments of pENTR*-PrbcS-*T and GS2 genes were ligated with the ligase kit of TaKaRa to generate the entry vector pENTR*-PRbcS-*T-GS2. Conversion of high efficiency (10 8 ) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on the LB plate added with kanamycin (Km, 50μg / ml), cultivate overnight at 37°C, and screen f...

Embodiment 3

[0057] Embodiment 3: Construction of GS2 gene plant expression vector pK2-35S-Prbcs-*T-GS2

[0058] The construction strategy of GS2 gene plant expression vector is as follows: Image 6 As shown, pENTR*-PrbcS-*T-GS2 was subcloned into the plant expression vector pK2GW7 (the destination vector of Gateway, purchased from Belgium VIB / Gent Company) through the LR reaction of Gateway technology. The specific method is: use the plasmid extraction kit to purify Gateway’s target vector pK2GW7, add 150ng each of pENTR*-PrbcS-*T-GS2 and pK2GW7 to the LR reaction system of Gateway, 1μl LR ClonaseII Enzyme Mix (Invitrogen), mix After reacting overnight at 25°C, PrbcS-*T-GS2 was integrated into pK2GW7 by the action of integrase to obtain the plant expression vector plasmid pK2-35S-Prbcs-*T-GS2 of GS2. Conversion of high efficiency (10 8 ) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on an LB plate added with spectinomycin (Spe...

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Abstract

The invention discloses a special plant expression vector pK2-35S-Prbcs-*T-GS2 which contains an arabidopsis chloroplast type glutamine synthetase gene (GS2) and can improve the metabolic ability of plant nitrogen. A RT-PCR method is utilized to clone a GS2 gene from a model plant Arabidopsis, a promoter of a Rubisco small subunit with the light-inducible promoter is used to control the overexpression of the GS2 gene in plant leaves, and the GS2 gene is transformed into wild tobacco through a leaf disk transformation method. The experimental result shows that the GS2 gene can perform normal transcription in transgenic tobacco and the growth condition of a strain with the GS2 gene is better than that of a check plant (un-transformed wild type) under low nitrogen nutrition condition, which indicates that the overexpression of the GS2 gene can improve the efficiency of a GS/GOGAT (glutamine synthetase/glutamate synthetase) way to a great extent, thereby improving the capability of the assimilation of the plant nitrogen. The special vector has the advantages that the special vector can be used for molecular breeding of crops, improves the utilization rate of nitrogen and the tolerance to low nitrogen stress, and obtains higher yield under the condition of small nitrogenous fertilizer application or even no nitrogenous fertilizer application.

Description

technical field [0001] The invention belongs to the field of plant engineering, and in particular relates to a plant expression vector pK2-35S-Prbcs-*T-GS2 of a chloroplast-type glutamine synthetase gene GS2, a construction method thereof and its application in transgenic crops. Background technique [0002] Nitrogen is the mineral nutrient element with the largest demand for plants, and it is one of the nutrient elements necessary for the normal growth and development of plants. At the same time, it is also one of the most restricted nutrient elements for agricultural production and ecosystem growth and development (Jones et al., 2005, Soil Biology and Biochemistry, 37:413–423). In order to obtain as much nitrogen as possible for growth and development, plants have formed several ways to obtain nitrogen: soil nitrogen absorption, atmospheric nitrogen fixation (legumes), dry and wet deposition nitrogen absorption, insect predation, etc. , for general plants (non-legume plan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/52A01H1/00
Inventor 李昆志王艺霖陈丽梅赵艳傅冰潘丽峰
Owner KUNMING UNIV OF SCI & TECH
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