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Soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as coding gene and application thereof

A technology of transcription factors and coding genes, applied in the field of genetic engineering

Active Publication Date: 2011-08-24
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In soybean, so far, the role of MYB12 has not been reported

Method used

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  • Soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as coding gene and application thereof
  • Soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as coding gene and application thereof
  • Soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, soybean GmMYB12B2 gene cloning

[0026] Total RNA was extracted from the leaves of soybean variety Jilin 32 with RNAiso Reagent (purchased from TaKaRa), and the integrity of the RNA was detected by 1% agarose electrophoresis. Synthesis of cDNA was carried out according to the instructions of Reverse Transcriptase M-MLV (RNase H-). according to GmMYB12b (AB510904) designed primers, 5'-ATGGAGAGGAGTTTATCAGG and 5'-TCACGACAAGTCATGATCAG. Perform PCR amplification according to the following reaction system and conditions: 25 μl system, containing 2.5 μl of 10×PCR Buffer, 2 μl of 2.5 mM dNTP mix, 1 μl of 10 μM primer 1 and primer 2, 1 μl of cDNA, Taq DNA Polymerase (purchased from TIAN GEN , 2.5u / μl) 0.5μl, deionized water 17μl. Reaction conditions: pre-denaturation at 94°C for 8 min; 30 cycles at 94°C for 30 sec, 56°C for 30 sec, and 72°C for 60 sec; post-extension at 72°C for 8 min. Amplified by PCR GmMYB12B2 Composed of 783 base pairs, the reading f...

Embodiment 2

[0027] Embodiment 2, soybean GmMYB12B2 Gene Expression in Prokaryotes

[0028] At the 5' end and 3' end of the gene, we designed Nd and EcoRI The primers for the restriction sites were used for PCR amplification using the correct plasmid pMD18-T-GmMYB12B2 identified by sequencing as a template, and pMD18-T was purchased from TaKaRa Company. Primers are:

[0029] GmMYB12B2-YH-F: 5'-CGACATATGATGGAGAGGAGTTT-3'

[0030] GmMYB12B2-YH-R: 5'-CAGGAATTCTCACGACAAGTCA-3'

[0031] After the amplified product was purified by an agarose gel DNA purification and recovery kit (purchased from Vertejie Company), it was purified with Nd and EcoRI Double digestion, recovery and purification, and the same Nd and EcoRIThe double-digested pET28a(+) (purchased from Novagen) vector was ligated. The ligation product was transformed into Escherichia coli ( E. coli ) DH5α, after verification, was transformed into the host strain Rosetta (DE3) (purchased from Novagen) for expression. ...

Embodiment 3

[0035] Embodiment 3, soybean GmMYB12B2 Gene expression in yeast

[0036] The construction method of the yeast expression vector is the same as that in Example 2. Will GmMYB12B2 Subcloned in pGBKT7 (purchased from clontech) vector, after the correct identification, it was transformed into yeast AH109 (purchased from clontech) for expression.

[0037] Competent yeast preparation and transformation methods were carried out according to the instructions of Matchmaker? One-Hybrid Library Construction & Screening Kit (clontech).

[0038] After the recombinant plasmid pGBKT7-GmMYB12B2 was transferred into yeast AH109, ​​it could grow on the defective medium SD / -Trp / -His / -Ade containing 10mM 3-AT, indicating that GmMYB12B2 It has transcription activation activity and is a transcription activator.

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Abstract

The invention discloses a soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as a coding gene and an application thereof, belonging to the technical field of the gene engineering. The MYB provided by the invention has the name of GmMYB12B2, and the amino acid residue sequence of the MYB is disclosed by SEQ ID No.2; and the nucleotide sequence of the coding gene of the soybean MYB transcription factor is disclosed by SEQ ID No.1. The gene disclosed by the invention has an important meaning on the flavonoid metabolism of improved plants, and especially has an important meaning on culturing high-isoflavone soybean varieties.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a soybean MYB transcription factor, its encoding gene and its application. Background technique [0002] Soybean can not only provide high-quality and rich protein and oil, but also an important source of isoflavones with various biological activities. Studies have found that soybean isoflavones have very important physiological functions and application value. Soy isoflavones are a class of secondary metabolites, which are synthesized by a branch of the phenylpropanoid metabolic pathway, which is ubiquitous throughout the plant kingdom, and it produces other phenols in addition to isoflavones Compounds, such as: lignin, flavonoids, flavonols, proanthocyanidins and anthocyanins, etc. [0003] The metabolic pathway of soybean isoflavones is catalyzed by a variety of enzymes, and is affected by growth factors such as developmental stages, pests and diseases, and the env...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N5/10A01H4/00A01H5/00
Inventor 王庆钰李晓薇王英李景文张艳翟莹张庆林钱丹丹张海军闫帆
Owner JILIN UNIV
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