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Application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole

A technology of propiconazole and cabbage, which is applied in the field of plant genetic engineering, can solve the problems of pesticide residues, shorten the breeding years, hidden dangers and the like, and achieve the effects of increased sensitivity and great application prospects.

Active Publication Date: 2021-11-02
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

For example, Chinese patent CN103650809A discloses a method for controlling early bolting of summer-planted kale and Chinese cabbage, by spraying a mixture of propiconazole + urea at an appropriate concentration on the leaves, combined with scientific nitrogen fertilizer application and rational irrigation, to regulate the soil The moisture and available nitrogen content of the water and available nitrogen to the appropriate level can achieve the effect of controlling the bolting of kale and Chinese cabbage; however, frequent application of propiconazole is likely to cause pesticide residues and bring potential safety hazards; molecular breeding through genetic engineering can greatly shorten the breeding time. years, improve breeding efficiency and crop yield, and will not bring safety hazards; however, there are few reports on the genes that regulate the absorption and accumulation of propiconazole in Chinese cabbage

Method used

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  • Application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole
  • Application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole
  • Application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Yeast-deficient library screening of propiconazole-related transport genes

[0041] 1. Activation of yeast-deficient library

[0042] Transfer the yeast-deficient library from -80°C ultra-low temperature freezer to 4°C freezer to thaw. Use a 96-pin replicator to transfer the mutant strains from a standard 96-deep well plate to a YPD solid plate for activation, and culture at 30°C for 2 days until a large colony is formed.

[0043] 2. Propiconazole screening of yeast-deficient library

[0044] Use a 96-pin replicator to inoculate the mutant strain on the activated plate in a standard 96 deep-well plate containing 200 μL of liquid YPD, shake it in a shaker at 180 rpm at 30°C for 12 hours, and then transfer 25 μL of the bacterial solution to the 96-well plate containing 1 mL of liquid YPD. Continue to activate for 3h in the deep well plate. Pipette 25 μL of the reactivated bacterial solution into a 96-deep well plate containing 1 mL of sterile water. Pipette ...

Embodiment 2

[0050] Example 2 Introducing BraALA3 and BraENT1 Deficient Yeast Sensitivity Experiment to Propiconazole

[0051] 1. Cloning of Chinese cabbage BraALA3 and BraENT1

[0052] 1) Total RNA extraction and cDNA synthesis

[0053] Oil green 701 Chinese cabbage was sown in a seedling tray, and cultured for 4 weeks at 20°C under the conditions of 12 hours of light and 12 hours of darkness. Kit (OMEGA) was used to extract RNA, and reverse transcription was performed according to PrimeScript Reverse Transcription Kit (TaKaRa Biological Co., Ltd., Dalian, China).

[0054] 2) Construct TA vector

[0055] The extracted cDNA was used as a template, and the upstream and downstream homology arm sequences of the BraALA3 gene were amplified by PCR. The cDNA amplification concentration was 5 ng / μL, and the following reaction mixture was used: 5 μL 5×GC buffer; 2 μL dNTPs; 10 μM each primer 1.25 μL; 0.75 μL DMSO; 0.25 μL pusion DNA polymerase; 1 μL gDNA (5ng); 2 0 to 20 μL.

[0056] Wherein,...

Embodiment 3

[0131] Example 3 The W303 strain yeast introduced with BraALA3 and BraENT1 is sensitive to propiconazole

[0132] 1. Yeast overexpression strain construction

[0133] 1) Restriction digestion of pYES2 vector

[0134] Set up the enzyme digestion reaction system: 3 μL BamHI (Takara); 3 μL EcoRI (Takara); 10 μL 10×Mbuffer (Takara); 3 ug complete pYES2 plasmid; deionized H2O to a final volume of 100 μL. Digested at 37°C for 3 hours, and purified by gel extraction kit (OMEGA).

[0135] 2) Amplify flanking sequences

[0136] Extract the constructed A-Bra-ALA3-01, TA-Bra-ALA3-03, TA-Bra-ALA3-09, and TA-BraENT1 as templates, and perform PCR amplification on the BraALA3 and BraENT1 genes. The concentration of the plasmid amplification is 5 ng / μL, use the following reaction mixture: 5 μL 5×GC buffer; 2 μL dNTPs; 1.25 μL each primer at 10 μM; 0.75 μL DMSO; 0.25 μL pusion DNA polymerase; 1 μL gDNA (5 ng); Wherein the homology arm amplification primer sequences of the three homologous ...

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Abstract

The invention discloses an application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole. Propiconazole-related transporter genes are screened through a yeast defect type library, after gene homology comparison, a Bragg ALA3 gene family and a Bragg ENT1 gene family related to propiconazole transport are preliminarily obtained, and after the Bragg ALA3 gene family and the Bragg ENT1 gene family are transferred to defect type yeast and W303 strain yeast, it is found that the sensitivity to propiconazole is remarkably improved; the BraALA3 and BraENT1 gene families are knocked out to find that the flowering Chinese cabbage of the mutant flowering Chinese cabbage is thickened and the whole plant is shortened; the results show that the BraALA3 and BraENT1 gene families have the potential of cultivating good varieties of flowering cabbage, and have great application prospects.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, more specifically, to the application of BraALA3 and BraENT1 gene families in regulating the absorption and accumulation of propiconazole. Background technique [0002] Brassicaceae (Brassicaceae Burnett) is the largest family of Brassica plants, including about 4000 species of plants in more than 400 genera. At the same time, cruciferous plants are also a class of plants with great scientific research value and production significance, including model plants such as Arabidopsis and rapeseed, and many important cultivated crops such as cabbage, cabbage, radish, etc. The medicinal value and nutritional value of cruciferous plants are very high, and the phenols, organic acids, and minerals produced in the body have antioxidant effects; Isothiocyanates have strong anticancer activity and can effectively prevent and treat cancer. [0003] Cabbage heart (Brassica campestris L.ssp.c...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84C12N15/55A01H5/00A01H6/20
CPCC07K14/415C12N15/8218C12N15/8261C12N15/8205C12N9/22
Inventor 林菲王思威徐汉虹林衿蓓陈焯越张娴瑜
Owner SOUTH CHINA AGRI UNIV
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