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Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene

A plant expression vector, glutamine technology, applied in the field of plant genetic engineering

Inactive Publication Date: 2009-04-15
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report on the plant expression vector containing the cytoplasmic GS1 gene present in the Arabidopsis genome

Method used

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  • Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene
  • Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene
  • Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Extraction and detection of Arabidopsis total RNA

[0052] In the present invention, Arabidopsis thaliana (Arabidopsis thaliana, ecotype, Columbia) total RNA was prepared using the TRNzol Reagent (purchased from invitrogen company) method. Take 0.1g of fresh leaf tissue, quickly and fully grind it into powder in liquid nitrogen, add 1ml of TRIzoL RNA extraction solution, mix well, let stand at room temperature for 5min, add 0.2ml of chloroform, shake vigorously and mix well, centrifuge at 12000rpm at 4°C for 15min , transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol, mix well and place at room temperature for 10 min. Centrifuge at 12000rpm for 10min at 4°C, discard the supernatant, wash the pellet once with 1ml of 75% ethanol, dry the pellet in vacuum, add 20μl of diethylpyrocarbonate (DEPC) treated water to fully dissolve the RNA, take 2μl of total RNA and wash with 1.2% agar Glucose gel electrophoresis detection, the results...

Embodiment 2

[0053] Example 2: Amplification and TA cloning of GS1 gene

[0054] GS1 gene amplification and TA cloning strategies such as figure 2 As shown, first search the full-length cDNA sequence of Arabidopsis thaliana cytoplasmic GS1 coding region from GenBank, and design a pair of primers with the following sequence:

[0055] GS 15: 5'-CA CCATGG CTTCACTTGCAGATTTAATC-3'

[0056] GS 13: 5'- GAATTC TCATGGTTTCCAAAGGATTGTGG-3'

[0057] Primer GS15 at the 5' end adds the CACC characteristic sequence to the 5' end, and thus generates an NcoI restriction site; primer GS13 at the 3' end adds an EcoRI site restriction end.

[0058] Using Arabidopsis total RNA as a template, using RevertAid TM - MuLV Reverse Transcriptase Kit (Fermentas) for cDNA synthesis. Take 0.1-0.5μg of total plant RNA, 50ng of oligo(dT), 1μl of 10mM dNTP mix, make up to 10μl with DEPC-treated water, mix well, then centrifuge briefly to collect it at the bottom of the tube, and place it in a constant temperature d...

Embodiment 3

[0060] Example 3: Construction of Gateway entry cloning vector pENTR*-PrbcS-GS1

[0061] The construction strategy of pENTR*-PrbcS-GS1 is as follows Figure 4 As shown, the purified plasmid vectors pENTR*-PrbcS-*T-GFP (patent application number is 200710066422.9) and pMD18-GS1 were cut with NcoI (Fermentas) and EcoRI (Fermentas), and separated by agarose gel electrophoresis. The vector and insert fragments, the vector fragment pENTR*-PrbcS (3.8kb) produced after pENTR*-PrbcS-*T-GFP was cleaved and the GS1 gene produced by cleavage of pMD18-GS1 were recovered from the gel The DNA fragment (1.0kb), and then use the ligase kit of TaKaRa to connect the DNA fragment of pENTR*-PrbcS and GS1 gene to generate the entry vector pENTR*-PrbcS-GS1. Use the ligation reaction mixture to transform high-efficiency (108) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), and spread the transformed Escherichia coli on an LB plate added with kanamycin (Km, 50 μg / ml), Cultivate o...

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Abstract

The invention relates to a special plant expression vector pH2-35S-PrbcS-GS1 which comprises an arabidopsis thaliana cytoplasm glutamine synthetase gene GS1 and can improve the utilization rate of a plant nitrogen element. A method of RT-PCR is used for cloning the GS1 gene from the arabidopsis thaliana of a model plant, a photoinduction type promotor (the promotor of a a small subunit Rubisco) is used for controlling the excessive expression of the GS1 gene in a plant leaf and a leaf disc conversion method is used for transferring the GS1 gene into a pPZP221-PrbcS-Dof1 type transgene tobacco. An experiment result shows that the GS1 gene can be normally transferred in the transgene tobacco; under the nutrition condition of low nitrogen and the growing conditions of indoor irradiation for 24 hours of 2000LUX and 25 DEG C, the growing situation of the plant transferred with the single gene of Dof1 is (the expression of the gene is controlled by the photoinduction type promotor Prbcs) is a little better than that of a contrast tobacco (a wild type without transgene); after being transferred under the natural growing condition of a green house, the growing situation of the tobacco which is simultaneously transferred with the GS1 gene and the Dof1 gene shows remarkable growing advantages than that of the contrast plant; and therefore, simultaneously and excessively expressing the GS1 gene and the Dof1 gene, can improve the efficiency of the GS / GOGAT (glutamine synthetase / glutamic acid synthetase) approaches in the leaf more extensively, thereby improving the utilization rate of the plant nitrogen element. The vector can be broadly applied to the molecule breeding of crops, improving the utilization rate of the plant nitrogen element thereof and the durability to the nutrition condition of low nitrogen and being capable of obtaining a higher yield under the conditions of applying less fertilizers and even not applying the fertilizers.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a plant expression vector pH2-35S-PrbcS-GS1 of Arabidopsis cytoplasmic glutamine synthetase gene GS1, its construction method and application. Background technique [0002] Nitrogen is the main limiting nutrient element for plant growth. Since the nitrogen in cultivated soil is usually difficult to meet the nitrogen demand of crops, people often meet the needs of crop growth by applying a large amount of nitrogen fertilizer to the soil. Nitrogen fertilizers are usually produced under high temperature and high pressure conditions, which consume a lot of energy. However, only about 30% of the nitrogen used in chemical fertilizers is absorbed and utilized by crops, and most of the rest enters the groundwater, causing nitrogen loss and causing water and environmental pollution. , Long-term application of chemical fertilizers also causes soil compaction, which seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/52A01H1/00A01H4/00
Inventor 李昆志王莎莎陈丽梅赵艳傅冰王艺霖潘丽峰
Owner KUNMING UNIV OF SCI & TECH
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