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Safflower chalcone isomerase (CHI) gene and application thereof

A technology of chalcone isomerase and gene, which is used in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2014-05-28
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no report about the cloning and expression analysis of chalcone isomerase gene in safflower

Method used

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  • Safflower chalcone isomerase (CHI) gene and application thereof
  • Safflower chalcone isomerase (CHI) gene and application thereof
  • Safflower chalcone isomerase (CHI) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Safflower Petal RNA Extraction

[0018] Sow the seeds of safflower "Tacheng safflower" (purchased in Tacheng, Xinjiang) in a nutrient bowl filled with sand, and cultivate them under appropriate conditions in an intelligent artificial climate chamber to collect safflower petals.

[0019] 1) Wrap the tweezers, mortar, pestle and medicine spoon with tinfoil, put them in a 180°C oven for 4 hours for dry heat sterilization, and set aside;

[0020] 2) Treat 1.5mL centrifuge tubes and pipette tips with 0.1% DEPC water overnight, then autoclave at 120°C for 20 minutes, and place them in an oven at 60°C to dry for later use;

[0021] 3) Take about 100 mg of safflower petals, add liquid nitrogen and quickly grind to fine powder, divide into two 1.5mLEP tubes, add 1mL RNAiso Plus to each, mix well, and let stand at room temperature for 5 minutes;

[0022] 4) Centrifuge at 12000rpm for 5min at 4°C, transfer the supernatant to a new 1.5mL centrifuge tube;

[0023] 5) A...

Embodiment 2

[0031] Example 2 Synthesis of the first strand cDNA

[0032] The RNA stored at -80°C was taken, and the concentration of RNA was detected by nanogrop. The concentration of extracted RNA was all around 1000ng / ul. Reverse transcription of cDNA was carried out according to the operating instructions of the reverse transcription kit. The reverse transcription reaction system is shown in Table 1 and Table 2. The cDNA after reverse transcription was stored in a -20°C refrigerator for later use.

[0033]

[0034] Carry out the reaction on the PCR instrument: 65°C, 5min, and quickly cool it on ice.

[0035]

[0036] Reverse transcription reaction conditions: 42°C for 55 minutes, 70°C for 10 minutes, place on ice to cool, obtain cDNA for subsequent reactions, and freeze at -20°C for future use.

Embodiment 3

[0037] Example 3 Cloning of the Middle Fragment of the CHI Gene

[0038] According to the candidate genes selected from the sequencing results of safflower 454 in our laboratory, specific primers (Table 3) were designed to verify the intermediate fragments. According to the middle fragment of the verified CHI gene, design primers (Table 3) for full-length gene cloning. According to the splicing and alignment of the safflower petal 454 sequencing results, the middle fragment sequence of the CHI gene was obtained, and a 209bp middle fragment was amplified by RT-PCR ( figure 2 A), RT-PCR reaction system is shown in Table 4. After gel recovery, it was connected to the cloning vector pEASY-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and further passed bacterial liquid PCR ( figure 2 B) and Eco R I , Han dIII digestion identification ( figure 2 C), the sequencing result is correct.

[0039]

[0040]

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Abstract

The invention discloses a safflower chalcone isomerase (CHI) gene. The RNA (Ribonucleic Acid) of safflower pedals is reversely transcribed into a cDNA (Complementary Deoxyribonucleic Acid), the cDNA is used as a template to carry out 3' and 5' race cloning, the 3' and 5' end sequences of the safflower CHI gene are respectively obtained, a safflower CHI sequence is obtained through splicing of the 3' and 5' end sequences, the whole gene length is 1161bp, and the length of an opening reading frame is 654bp. The safflower CHI gene is named ct-(i)CHI( / i), and the result of the sequence comparison and analysis of the safflower CHI gene on ncbi (National Center for Biotechnology Information) shows that the homology of the gene with a dutchmanspipe root CHI gene reaches 82%. Experiments show that the synthesis of flavone can be promoted by the over-expression ct-(i)CHI( / i) gene, the flavone content can be lowered after the ct-(i)CHI( / i) gene is inhibited, and the safflower CHI gene has wide prospects in the aspect of cultivating high-content flavonoids transgenic plants.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to safflower chalcone isomerase CHI gene and its application. Background technique [0002] safflower( Carthamus tinctorius L.) is an annual herb with dry tubular flowers belonging to Compositae. Safflower has the functions of invigorating blood circulation and promoting menstrual flow, removing blood stasis and healing wounds, purifying toxins and clearing rashes, etc. Its main active ingredients are safflower yellow and safflower red. Safflower yellow is a mixture of various water-soluble chalcones in safflower. It is not only a valuable food coloring, but also has the functions of promoting blood circulation, dredging collaterals, reducing inflammation and analgesia. Symptoms and other hair surface also play an important role. Chalcone isomerase (Chalcone isomerase, CHI; EC5.5.1.6) is one of the key enzymes in the synthetic pathway of plant flavonoids, which i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/84A01H5/00
Inventor 刘秀明李海燕李校堃杨文婷姚娜
Owner JILIN AGRICULTURAL UNIV
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