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Polymerase chain reaction (PCR) primer for cloning Actin gene of phalaris arundinacea linn and method

A technology of thorn grass and gene, which is applied in the field of molecular biology to achieve the effects of improving efficiency, shortening breeding years, and speeding up the breeding process

Inactive Publication Date: 2012-07-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the study of Actin gene

Method used

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  • Polymerase chain reaction (PCR) primer for cloning Actin gene of phalaris arundinacea linn and method
  • Polymerase chain reaction (PCR) primer for cloning Actin gene of phalaris arundinacea linn and method
  • Polymerase chain reaction (PCR) primer for cloning Actin gene of phalaris arundinacea linn and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Preparation of test materials

[0037] Pre-cooling and sterilizing the seeds of the weed grass, and planting them in plastic flowerpots after germination. The seedlings were grown for 30 days and used to extract RNA.

[0038] 2. Extraction of RNA

[0039] (1) Weigh about 0.2g of Lemongrass leaves, grind them into fine powder with liquid nitrogen, and divide them into two 1.5mL EP tubes (as fast as possible to avoid RNA degradation);

[0040] (2) Add 1mL TRIzol (Total RNA Extraction Reagent) to each tube, shake vigorously to make it evenly mixed, and leave it at room temperature for 5min (note that the amount of sample should not exceed 10% of TRIzol);

[0041] (3) When the content of protein, fat and polysaccharide in the sample is high, centrifuge at 12000rpm for 10min at 4°C, and suck the supernatant into a clean 1.5mL EP tube;

[0042] (4) Add 0.2mL chloroform to each tube, vibrate vigorously for 15 seconds, place at room temperature for 2-3min, and centrifuge at...

Embodiment 2

[0070] According to the cDNA sequences of the Actin genes of plants closely related to S. chinensis that have been registered on GenBank, they mainly include the sequences of rice, maize, stargrass, ryegrass and Leymus chinensis. The conserved regions of these cDNA sequences were analyzed with DNAMAN software, the specificity and other indicators of the primers were checked with Premier Primer 5.0, primers were designed, and primers were synthesized by Shanghai Sangong. Screen the primer pairs with good specificity among the designed primers and continue to do recovery conversion and linking experiments. The specific primers screened by the present invention are as follows:

[0071] Forward primer: 5′GACCGAGCGTGGTTACTC 3′

[0072] Reverse primer: 5′GCACCTCCAATCCAGACA 3′

[0073] The cDNA obtained by reverse transcription in Example 1 was used as a template, and the above-mentioned primer sequence was used as a PCR reaction primer to carry out PCR amplification. The amplifica...

Embodiment 3

[0078] 1. The PCR gel electrophoresis band in Example 2 is subjected to gel cutting to recover the target fragment.

[0079] (1) After the DNA fragments to be recovered are separated by electrophoresis, cut out the required DNA fragments from the agarose gel, put them in a 1.5mL centrifuge tube, and centrifuge at a low speed.

[0080] (2) Add 3 times the volume of sol solution, 50°C metal bath (heating block) sol, and gently flip the centrifuge tube until the agarose gel is completely dissolved.

[0081] (3) After the gel solution is cooled to room temperature, add it to an adsorption column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0082] (4) Add 700 μl of rinse solution to the adsorption column, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0083] (5) Add 500 ...

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Abstract

The invention provides a pair of polymerase chain reaction (PCR) primers for cloning an Actin gene of phalaris arundinacea linn and a method, which belong to the field of molecular biology. Nucleotide sequences of the pair of PRC primers for cloning the Actin gene of the phalaris arundinacea linn are indicated as SEQ ID NO.1 and SEQ ID NO.2. The invention further provides the method for cloning the Actin gene of the phalaris arundinacea linn. The method includes extracting total ribonucleic acid (RNA) in a glass blade tissue of the phalaris arundinacea linn, and the RNA is inverse-transcripted into complementary deoxyribose nucleic acid (cDNA). The special primers are used for conducting a PCR to obtain a partial segment of the Actin gene with sequence length as 453bp. The gene segment can be used as an internal reference gene and provide convenience for research of other genes. The method has the advantages of being high in flexibility, strong in specificity, simple and fast and having good application value in the aspect of molecular technology breeding of the phalaris arundinacea linn.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a PCR primer used for cloning the Heliotrope Actin gene and a method for cloning the Heliotrope Actin gene. Background technique [0002] Phalaris arundinacea, also known as Phalaris arundinacea, is a plant of the Gramineae genus Phalaris. Because of its strong stress resistance, high yield and nutritional value, it is widely used in forage grass, artificial wetland plants, papermaking raw materials and biomass energy. Grass is a perennial large erect C 3 Plants with strong root system and rhizomes, the root system depth reaches more than 3m. Compared with other gramineous forage grasses, Limegrass has the characteristics of high biological yield, low maintenance cost, strong stress resistance, high wood fiber content, easy management and harvesting, etc. It is a renewable energy source, and can be cultivated continuously in northern climate conditions. It takes 10-12 years to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12N15/29C12Q1/68
Inventor 张蕴薇丛丽丽杨富裕张新全于晓丹刘斯佳李洪超
Owner CHINA AGRI UNIV
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