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Rape respiration metabolism-related gene BnAOX1 and application thereof

A gene sequence and amino acid technology, applied in the field of BnAOX1, an enzyme gene related to the respiratory metabolism of rapeseed, can solve the problems of unclear signal regulation mechanism and physiological significance, superficial research, etc.

Active Publication Date: 2012-08-01
武汉中油种业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various environmental stresses can change the operation of the plant cyanide resistance pathway, but these studies are still very superficial, and the signal regulation mechanism and physiological significance of its operation are still unclear

Method used

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  • Rape respiration metabolism-related gene BnAOX1 and application thereof
  • Rape respiration metabolism-related gene BnAOX1 and application thereof
  • Rape respiration metabolism-related gene BnAOX1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: BnAOX1, AtAOX1 cDNA coding region sequence

[0033] A method for preparing rapeseed respiratory metabolism-related gene BnAOX1, the steps of which are:

[0034] Search the published Arabidopsis gene sequence in the NCBI database, BLAST the rapeseed EST sequence on the NCBI website, and design primers on both sides of the gene coding sequence based on the Arabidopsis sequence (BnAOX1F: forward primer: [5'-atgatgatgagtcgctacgg-3 '], reverse primer: [5'-tcaatgatccaattggag-3'] is used to amplify the corresponding sequence from the silique cDNA of rapeseed 15 days. The amplification primers in Arabidopsis are: AtAOX1 (arabidopsis website) forward primer : [5'-atgatgatgagtcgtcgcta-3'], reverse primer: [5'-tcaatgatatccaatgggagc-3'], amplified directly in Arabidopsis 10-day silique cDNA.

[0035] 1. Extract rapeseed and Arabidopsis mRNA.

[0036] For RNA extraction (TRIZOL™ Kit for RNA extraction), 100 mg of material was ground with liquid nitrogen.

[0037] A....

Embodiment 2

[0045] Embodiment 2: Construction of transgene expression vector and transformation of Arabidopsis:

[0046] After the gene sequence amplified by PCR was connected to the TOPO entry vector (invitrogen company), it was transformed into competent cell DH5α (invitrogen company), and spectinase screening was carried out. For primers, refer to Example 1 for the sequence of primers. 1) Amplify and identify positively inserted clones. The plasmid is recombined with Pearleygate 100 (Invitrogen) after a small amount of preparation, and transformed into competent cells DH5α, screened with kanamycin, and the inserted fragment Through PCR identification of carrier primers (35S promoter sequence primers, CACGTCTTCAAAGCAAGTGGA) and gene primers (gene downstream primers, see Example 1 for the primer sequence), see the schematic diagram figure 1 .

[0047] Arabidopsis transformation process:

[0048] Reagent preparation:

[0049] Infiltration medium (1L): 1 / 2xMurashige-Skoog; 5% (mass rati...

Embodiment 3

[0056] Example 3: Screening and verification of transgenic Arabidopsis:

[0057] Screening of transformants:

[0058] The vernalized Arabidopsis seeds were planted in the artificial soil watered with the supersaturated Huabao No. 2 (commercially purchased) nutrient solution, and covered with plastic wrap. After two days, the light was exposed, and the film was removed after three days.

[0059] Conditions of artificial culture room: relative humidity 80%, constant temperature 20-24°C, light intensity 80-200umol / M2 / S, light cycle 8h dark, 16h light culture. About a week, spray herbicide (glufosinate) to screen positive plants.

[0060] PCR identification:

[0061] (1) Extraction of transformed plant total DNA for PCR:

[0062] A. 70% (volume ratio) ethanol scrub blade, weigh about 100mg

[0063] B. Add 600ul of extraction buffer (0.2M Tris-Cl, 0.25NaCl, 25mM EDTA, 0.5% (mass ratio) SDS, pH 7.5), and grind rapidly at room temperature.

[0064] C. Vortex in a 1.5ml Ependorf...

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Abstract

The invention discloses rape respiration metabolism-related gene BnAOX1 and an application thereof. Gene expression difference screening experiments show that the expression level of the BnAOX1 gene in rape high-oil parents and high-oil F2 mixed populations is higher than that of low-oil parents and low-oil F2 mixed populations. A sequence of a rape genome homologous with the BnAOX1 gene is searched from a sequencing database according to an Arabidopis thaliana AOX1 gene sequence on an arabidopsis website and is spliced, and primers at two sides of the gene coding region sequence are designed. A parent zy036cDNA first strand is used as a template to carry out RT-PCR amplification, and the resulting amplified fragment is sequenced to obtain the BnAOX1 gene coding region sequence and mutantalleles or derivatives which are generated through adding, substituting, inserting or deleting one or more nucleotides. Transgene results with Arabidopis thaliana as an acceptor confirm that the overexpression of such gene allows the oil content of Arabidopis thaliana grains to be improved and the grain weight of the grains to be increased. So, such gene has a very good application prospect in oil production breeding of rape and other oil crops.

Description

technical field [0001] The present invention relates to the field of plant genetic engineering, more specifically relates to a rapeseed respiratory metabolism-related enzyme gene BnAOX1, and also relates to the application of a rapeseed respiratory metabolism-related enzyme gene BnAOX1 in improving the yield and oil content of oil crops. Background technique [0002] Rapeseed is one of the most important sources of edible vegetable oil and feed protein in the world, and is also the most important raw material for biodiesel in Europe. Rapeseed is the oil crop with the largest planting area in my country, and it is also the fifth largest crop in my country after rice, wheat, corn and soybean. Rapeseed oil is the main edible oil for Chinese residents, accounting for more than 40% of the total domestic vegetable oil consumption. As the world's largest vegetable oil consumption country, my country's self-sufficiency rate of vegetable oil is less than 40%. In order to solve thes...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02A01H5/00
CPCC12N9/0006C12N15/8247C12Y101/00C12N15/8261Y02A40/146
Inventor 王汉中华玮刘静刘贵华王新发胡志勇詹高淼
Owner 武汉中油种业科技有限公司
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