CN102965383A - Method for cloning and identifying wheat glutamine synthetase gene - Google Patents

Abstract

The invention discloses a method for cloning and identifying wheat glutamine synthetase gene. The method comprises the following steps: extracting total RNA (ribonucleic acid) of wheat tissues by utilizing a Trizol kit, carrying out DNaseI processing, obtaining constant volume with DEPC treating water, and detecting the completeness of RNA by utilizing 1% agarose gel electrophoresis; conducting cDNA synthesis by adopting a 20mumL reaction system, and carrying out warm bath for 1h at 42 DEG C; cloning the total-length cDNA of GS1; cloning total-length cDNA of GS2; by utilizing a method for identifying the gene expression of the wheat GS1 and GS2 through semi-quantitative PCR, carrying out PCR amplification by utilizing a primer in a table 1 through the obtained cDNA, and identifying the product by using agarose gel electrophoresis. According to the method, the total-length cDNA of the GS1 and GS2 are cloned from leaves of common wheat cultivated variety for the first time, the transcription and expression characteristics of the GS1 and GS2 in different tissues of wheat can be explored, thereby not only establishing a basis for further research on the regulation mode of wheat in growth process and action in nitrogen utilization, but also providing materials for breeding high-efficiency nitrogen variety through a transgenosis method.

Description

The cloning and identification method of one grow wheat glutamine synthetase gene

Technical field

The invention belongs to gene clone and authenticate technology field, relate in particular to the cloning and identification method of a grow wheat glutamine synthetase gene.This gene involved in plant nitrogen asimilation, transfer and utilization are for providing material by the efficient kind of transgenic method seed selection nitrogen.

Background technology

Nitrogen is the principal element that affects growth and development of plants, no matter is directed to the ammonia that discharges in nitrate, ammonium ion, microorganism fixed nitrogen or the vegetable metabolic process, all must just can assimilate into amino acid through glutamine synthetase (GS) catalysis.The GS gene of many plants such as barley, corn, paddy rice, Arabidopis thaliana has been cloned, and the GS functional study has become one of study hotspot that improves nitrogen utilization efficiency.Two class GS are arranged in the higher plant, and a class is positioned in the cytosol, is called cytosolic GS, i.e. GS1, transfer and the recycling of nitrogenous source when storing the transhipment of nitrogenous source and leaf senile when mainly participating in seed germination; Another kind of being positioned in the plastid is called plastid type GS, i.e. GS2 mainly participates in the assimilation process of the ammonia (elementary nitrogen) that photorespiration, nitrate reductase produce ^[Forefathers studies show that the cigarette seedling of pea GS1 conversion can improve the photosynthetic rate of blade when nitrogen is coerced, and increase the biological yield of plant, promote transfer and the recycling (Fuentes, 2001) of nitrogen.The grain number per spike and the thousand seed weight that turn the GS1 gene corn increase, and nitrogen utilization efficiency improves (Martin, 20006).Efficiently express GS1 and GS2 gene and can improve the patience that paddy rice lacks soil nitrogen, promote absorption and the recycling (Sun Hui etc., 2005) of nitrogen.

Summary of the invention

On the previous research work basis, we design full-length cDNA primer and the corresponding PCR circulation of clone wheat GS1 and GS2 gene, have obtained the full-length cDNA of GS1 and GS2 gene.And the design Auele Specific Primer, utilize sxemiquantitative PCR to identify GS1 and the expression status of GS2 in the wheat different tissues.

The embodiment of the invention is achieved in that the cloning and identification method of a grow wheat glutamine synthetase gene, and the method may further comprise the steps:

Utilize the Trizol test kit to extract the total RNA of Wheat Tissue, DEPC processed the water constant volume after DNaseI processed, and utilized 1% agarose gel electrophoresis to detect the integrity of RNA;

The synthetic 20 μ L reaction systems that adopt of cDNA, 42 ℃ of temperature are bathed 1h;

The GS1 full length cDNA clone;

The GS2 full length cDNA clone;

Utilize sxemiquantitative PCR to identify the method for wheat GS1 and GS2 genetic expression, by obtaining cDNA, utilize the primer in the table 1 to carry out pcr amplification, product is identified in agarose gel electrophoresis.

Further, the synthetic 20 μ L reaction systems that adopt of cDNA are: RNA, 2 μ g; M-MLV, 200U; DNTP, 125pmol; OligodT, 100pmol; RNasin, 25U.

Further, GS1 full length cDNA clone method is:

GS1 total length forward primer GS1QF:5 '-CTGCAGAAAGCACCACCCCCACC-3 ' and reverse primer GS1QR:5 '-CAGCTGGTGTGGTACCACGGCAGC-3 '.PCR circulation: 94 ℃ of denaturations, 2min; 94 ℃ of sex change, 45s; Anneal 61 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min.The PCR product is identified in 1% agarose gel electrophoresis.Utilize dna gel to reclaim test kit and reclaim purpose band (Figure 1A), connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

Further, GS2 full length cDNA clone method is:

GS2 total length forward primer GS2QF:5 '-CTTCCCTCCCTCCTCTCCTC-3 ' and reverse primer GS2QR:5 '-TATCCTTTTAATAACGTAGTTC TCCG-3 ';

PCR circulation: 94 ℃ of denaturations, 2min; 94 ℃ of sex change, 45s; Anneal 56 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min;

The PCR product is identified in 1% agarose gel electrophoresis;

Utilize dna gel to reclaim test kit and reclaim purpose band (Figure 1B), connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

Further, utilize sxemiquantitative PCR to identify that the method for wheat GS1 and GS2 genetic expression is:

By obtaining cDNA, utilize primer to carry out pcr amplification: 94 ℃ of denaturations, 2min, 94 ℃ of sex change, 30s; Anneal 54 ℃ 30s; Extend 72 ℃, 30s; 28 circulations of increasing are extended 72 ℃, 5min, and product is identified in 1% agarose gel electrophoresis.

Further, described design of primers is:

(1) design of primers of GS1 full-length cDNA; Forward primer GS1QF:5 '-CTGCAGAAAGCACCACCCCCACC-3 ' and reverse primer GS1QR:5 '-CAGCTGGTGTGGTACCACGGCAGC-3 ',

(2) design of primers of GS2 full-length cDNA; Forward primer GS2QF:5 '-CTTCCCTCCCTCCTCTCCTC-3 ' and reverse primer GS2QR:5 '-TATCCTTTTAATAACGTAGTTCTCCG-3 '

(3) GS1 sxemiquantitative PCR Auele Specific Primer.Forward primer 5 '-TCCTGTGGAAGCCCTGAAGC-3 ' and reverse primer 5 '-CGACGATGATGCGACCTACCTAA-3 ';

(4) GS2 sxemiquantitative PCR Auele Specific Primer.Forward primer 5 '-GAACGGAGGCTGACAGGGCTAC-3 ' and reverse primer 5 '-ACAAGAATGGACGACGGACGAAC-3 '.

The present invention has cloned GS1 and GS2 first from common wheat Cultivar blade full-length cDNA, GS1 and GS2 have been explored at wheat different tissues transcription and expression characteristic, not only for further its control methods in the Growth of Wheat process of research and the effect in the nitrogen utilization are laid a good foundation.More provide material by the efficient kind of transgenic method seed selection nitrogen.

Description of drawings

Fig. 1 is the pcr amplification figure of the wheat glutamine synthetase full-length cDNA that provides of the embodiment of the invention.A, GS1 gene amplification; B, GS2 gene amplification.

Fig. 2 is that the sxemiquantitative PCR that utilizes that the embodiment of the invention provides identifies the expression characteristic of wheat glutamine synthetase gene in the Wheat Seedling blade.A, GS1 gene; B, GS2 gene.1-6 represent respectively wheat emerge after 2,4,8,20,28 and 30 days.

Embodiment

In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.

On the previous research work basis, we design full-length cDNA primer and the corresponding PCR circulation of clone wheat GS1 and GS2 gene, have obtained the full-length cDNA of GS1 and GS2 gene.And the design Auele Specific Primer, utilize sxemiquantitative PCR to identify GS1 and the expression status of GS2 in the wheat different tissues.

The cloning and identification of wheat glutamine synthetase gene of the present invention is achieved through the following technical solutions:

1, utilize the Trizol test kit to extract the total RNA of Wheat Tissue, DEPC processed the water constant volume after DNaseI processed, and utilized 1% agarose gel electrophoresis to detect the integrity of RNA.

2, synthetic 20 μ L reaction systems (RNA, the 2 μ g of adopting of cDNA; M-MLV, 200U; DNTP, 125pmol; OligodT, 100pmol; RNasin, 25U), 42 ℃ of temperature are bathed 1h.

3, GS1 full length cDNA clone.GS1 total length forward primer GS1QF:5 '-CTGCAGAAAGCACCACCCCCACC-3 ' and reverse primer GS1QR:5 '-CAGCTGGTGTGGTACCACGGCAGC-3 '.PCR circulation: 94 ℃ of denaturations, 2mi n; 94 ℃ of sex change, 45s; Anneal 61 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min.The PCR product is identified in 1% agarose gel electrophoresis.Utilize dna gel to reclaim test kit and reclaim the PCR product, connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

4, GS2 full length cDNA clone.GS2 total length forward primer GS2QF:5 '-CTTCCCTCCCTCCTCTCCTC-3 ' and reverse primer GS2QR:5 '-TATCCTTTTAATAACGTAGTTCTCCG-3 '.PCR circulation: 94 ℃ of denaturations, 2min; 94 ℃ of sex change, 45s; Anneal 56 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min.The PCR product is identified in 1% agarose gel electrophoresis.Utilize dna gel to reclaim test kit and reclaim the PCR product, connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

5, utilize sxemiquantitative PCR to identify the method for wheat GS1 and GS2 genetic expression.Obtain cDNA by step 1 and 2, utilize the primer in the table 1 to carry out pcr amplification: 94 ℃ of denaturations, 2min, 94 ℃ of sex change, 30s; Anneal 54 ℃ 30s; Extend 72 ℃, 30s; 28 circulations of increasing are extended 72 ℃, 5min, and product is identified in 1% agarose gel electrophoresis.

Table 1 sxemiquantitative PCR primer

The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.

Claims (6)

1. the cloning and identification method of a grow wheat glutamine synthetase gene is characterized in that the method may further comprise the steps:

Utilize the Trizol test kit to extract the total RNA of Wheat Tissue, DEPC processed the water constant volume after DNaseI processed, and utilized 1% agarose gel electrophoresis to detect the integrity of RNA;

The synthetic 20 μ L reaction systems that adopt of cDNA, 42 ℃ of temperature are bathed 1h;

The GS1 full length cDNA clone;

The GS2 full length cDNA clone;

Utilize sxemiquantitative PCR to identify the method for wheat GS1 and GS2 genetic expression, by obtaining cDNA, utilize the primer in the table 1 to carry out pcr amplification, product is identified in agarose gel electrophoresis.

2. the cloning and identification method of wheat glutamine synthetase gene as claimed in claim 1 is characterized in that, the synthetic 20 μ L reaction systems that adopt of cDNA are: RNA, 2 μ g; M-MLV, 200U; DNTP, 125pmol; OligodT, 100pmol; RNasin, 25U.

3. the cloning and identification method of wheat glutamine synthetase gene as claimed in claim 1 is characterized in that, GS1 full length cDNA clone method is:

GS1 total length forward primer GS1QF:5 '-CTGCAGAAAGCACCACCCCCACC-3 ' and reverse primer GS1QR:5 '-CAGCTGGTGTGGTACCACGGCAGC-3 '.PCR circulation: 94 ℃ of denaturations, 2min; 94 ℃ of sex change, 45s; Anneal 61 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min.The PCR product is identified in 1% agarose gel electrophoresis; Utilize dna gel to reclaim test kit and reclaim the PCR product, connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

4. the cloning and identification method of wheat glutamine synthetase gene as claimed in claim 1 is characterized in that, GS2 full length cDNA clone method is:

GS2 total length forward primer GS2QF:5 ' CTTCCCTCCCTCCTCTCCTC-3 ' and reverse primer GS2QR:5 '-TATCCTTTTAATAACGTAGTTC TCCG-3 ';

PCR circulation: 94 ℃ of denaturations, 2min; 94 ℃ of sex change, 45s; Anneal 56 ℃ 45s; Extend 72 ℃, 10min; 30 circulations of increasing, 4 ℃, 5min;

The PCR product is identified in 1% agarose gel electrophoresis;

Utilize dna gel to reclaim test kit and reclaim the PCR product, connect the PMD-19 carrier, transform intestinal bacteria TOP10, select positive bacterium colony order-checking and identify.

5. the cloning and identification method of wheat glutamine synthetase gene as claimed in claim 1 is characterized in that, utilizes sxemiquantitative PCR to identify that the method for wheat GS1 and GS2 genetic expression is:

By obtaining cDNA, utilize primer to carry out pcr amplification: 94 ℃ of denaturations, 2min, 94 ℃ of sex change, 30s; Anneal 54 ℃ 30s; Extend 72 ℃, 30s; 28 circulations of increasing are extended 72 ℃, 5min, and product is identified in 1% agarose gel electrophoresis.

6. the cloning and identification method of wheat glutamine synthetase gene as claimed in claim 5 is characterized in that, described design of primers is:

(1) design of primers of GS1 full-length cDNA; Forward primer GS1QF:5 '-CTGCAGAAAGCACCACCCCCACC-3 ' and reverse primer GS1QR:5 '-CAGCTGGTGTGGTACCACGGCAGC-3 ',

(2) design of primers of GS2 full-length cDNA; Forward primer GS2QF:5 '-CTTCCCTCCCTCCTCTCCTC-3 ' and reverse primer GS2QR:5 '-TATCCTTTTAATAACGTAGTTCTCCG-3 '

(3) GS1 sxemiquantitative PCR Auele Specific Primer; Forward primer 5 '-TCCTGTGGAAGCCCTGAAGC-3 ' and reverse primer 5 '-CGACGATGATGCGACCTACCTAA-3 ';

(4) GS2 sxemiquantitative PCR Auele Specific Primer; Forward primer 5 '-GAACGGAGGCTGACAGGGCTAC-3 ' and reverse primer 5 '-ACAAGAATGGACGACGGACGAAC-3 '.

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