A wheat hydrolyzed hydroxycinnamoyl-CoA ester protein tamag1166 and its coding gene and application

A technology of cinnamoyl coenzyme and tamag1166, which is applied in the field of genetic engineering to achieve the effect of improving resistance to scab

Active Publication Date: 2017-10-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TaMAG1166 protein of the present invention is a hydrolyzed hydroxycinnamoyl-CoA ester protein derived from wheat. Before the present invention was proposed, the application of the hydrolyzed hydroxycinnamoyl-CoA ester-related protein and its coding gene in wheat scab resistance Not yet reported

Method used

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  • A wheat hydrolyzed hydroxycinnamoyl-CoA ester protein tamag1166 and its coding gene and application
  • A wheat hydrolyzed hydroxycinnamoyl-CoA ester protein tamag1166 and its coding gene and application
  • A wheat hydrolyzed hydroxycinnamoyl-CoA ester protein tamag1166 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Obtaining TaMAG1166 protein.

[0025] During the flowering stage of the scab-resistant material Wangshuibai, the gibberella spore liquid was sprayed to the ears with a sprayer, and the spore liquid used was the conidia of strong pathogenic gibberella strains F4, F15, F17 and F34 in Jiangsu Province After inoculation, put the ears in plastic bags immediately to keep the humidity to ensure the onset of Gibberella. Wheat ears were taken 6 hours, 12 hours and 24 hours after inoculation, and inoculated with water as a control. Immediately after taking the materials, they were frozen with liquid nitrogen. Take 700mg of frozen ears of wheat, add 70mg of PVP, grind into powder in liquid nitrogen, add 5mL of 10% trichloroacetic acid / acetone, shake and mix, and precipitate at -20°C for 1 hour; centrifuge at 15,000g at 4°C for 15min, and resuspend the precipitate Place in 5 mL of cold acetone at -20°C for 2 hours; centrifuge at 15,000 g at 4°C for 15 minutes, suspend t...

Embodiment 2

[0026] Example 2: Obtaining the cDNA sequence encoding TaMAG1166 protein.

[0027] The total RNA from the panicle of the disease-resistant wheat germplasm Wangshuibai was extracted with the Trizol kit from Yingjun Company, reverse-transcribed with the reverse transcription kit from Promega Company, and single-stranded cDNA was synthesized. Using cDNA as a template, P1-F: 5'-GAGGAACTATGCTTCAACCCGC-3' (SEQ ID NO.3) and P1-R: 5'-TTCGCCCATCCAAAGTCTGG-3' (SEQ ID NO.4) as primers, PCR amplification, amplification system 25μL, including 5ng template, 5pmol each of P1 primer, 5nmol each of dATP, dTTP, dCTP and dGTP, 37.3nmol MgCl 2 , 0.5 units of DNA polymerase, 1x PCR buffer. The procedure of amplification is: denaturation at 94°C for 3 minutes; 30 cycles, denaturation at 94°C for 20 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute; finally extension at 72°C for 5 minutes. Through PCR amplification, a 1341bp nucleotide sequence containing an open reading fr...

Embodiment 3

[0028] Embodiment 3: the scab resistance of transgenic TaMAG1166 wheat is improved

[0029]Using the panicle cDNA of Wangshuibai at the flowering stage as a template, primer P2 was used for PCR amplification. The pair of P2 primers is as follows: P2-F: 5'-AGTCTCTAGATTATTTGCCGACATGTCCTTGG-3' (SEQ ID NO.5); P2-R: 5 '-ATATTTCTAGACCCAAACATCAATCTCCTTCTCG-3' (SEQ ID NO. 6). After the amplified PCR product was digested with the restriction endonuclease XbaI, it was ligated with the PBI121 expression vector that had been digested with the same restriction endonuclease to obtain the overexpression plasmid of TaMAG1166 promoted by the CaMV35S promoter carrier. The constructed vector was transformed into DH5α Escherichia coli strain by heat shock method, and positive clones were obtained by screening with LB medium containing 50 μg / mL kanamycin. Referring to the materials and methods in the article published by Weeks in Plant Physiol No. 102, page 1077-1084 in 1993, the callus produced...

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PUM

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Abstract

The invention discloses a wheat hydrolysis hydroxy cinnamoyl coenzyme A ester protein TaMAG1166 as well as a coding gene and application thereof. The amino acid sequence of the wheat hydrolysis hydroxy cinnamoyl coenzyme A ester protein TaMAG1166 is as shown in SEQ ID NO:2; and the gene nucleotide sequence of coding the wheat hydrolysis hydroxy cinnamoyl coenzyme A ester protein TaMAG1166 is as shown in SEQ ID NO:1. The gibberellic disease resistance of wheat can be enhanced by transferring the coding gene of the wheat hydrolysis hydroxy cinnamoyl coenzyme A ester protein TaMAG1166 provided by the invention into the wheat through a gene engineering method. Because the coding gene is a self endogenous gene of the food crop wheat, the excessive expression of the coding gene can not influence the food safety of a plant, and the coding gene can be applied to crop breeding.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a wheat hydrolyzed hydroxycinnamoyl-CoA ester protein TaMAG1166, its coding gene and application. Background technique [0002] Wheat scab is a worldwide disease caused by Fusarium graminearum Schwabe. The disease leads to a substantial reduction in wheat yield and seriously affects the quality of wheat. Although the disease can be controlled by biological control and chemical control, the most economical and effective method is to use biotechnology to breed scab-resistant varieties. [0003] Hydrolyzing hydroxycinnamoyl CoA quinatetransferase (HQT) can control the biosynthesis and transfer of major phenolic compounds such as lignin and chlorogenic acid (Hoffmann et al. (2003). Chlorogenic acid is In the process of plant phenylpropanoid metabolism, the phenolic compounds produced by the esterification of caffeic acid and quinic acid can increase the level of phenolic compounds ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N1/21A01H5/00
CPCC07K14/415C12N15/8282
Inventor 马正强贾海燕刘世蓉徐文绮
Owner NANJING AGRICULTURAL UNIVERSITY
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