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MYB type transcription factor GmMYB29 of Glycine max as well as encoding gene and application of transcription factor GmMYB29

A transcription factor, soybean technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of not reaching a significant level of increase, increasing accumulation of isoflavones, and difficult survival of transgenic plants.

Active Publication Date: 2016-06-29
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of the study showed that although overexpression of individual enzyme genes in the isoflavone synthesis pathway could increase the accumulation of isoflavones, the increase did not reach a significant level
Although inhibiting the competition pathway can greatly increase the content of isoflavones, it is difficult for transgenic plants to survive under non-laboratory conditions because important metabolic pathways such as flavonoids, anthocyanins and proanthocyanidins in plants are blocked.
The modification of a single gene in the pathway is difficult to achieve the desired results, so the current research is focused on the identification of transcription factors that regulate isoflavone synthesis, and the use of transcription factors to activate or inhibit different structural genes in the isoflavone synthesis pathway to control the production of isoflavones Yield is the main genetic engineering method to regulate isoflavone synthesis in the future
So far, only two MYB transcription factors that regulate isoflavones have been identified in soybean, which still need further research

Method used

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  • MYB type transcription factor GmMYB29 of Glycine max as well as encoding gene and application of transcription factor GmMYB29
  • MYB type transcription factor GmMYB29 of Glycine max as well as encoding gene and application of transcription factor GmMYB29
  • MYB type transcription factor GmMYB29 of Glycine max as well as encoding gene and application of transcription factor GmMYB29

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1, cDNA cloning and identification of soybean GmMYB29 and its coding gene GmMYB29

[0029] The total RNA of leaves of soybean variety Williams82 was extracted with plant total RNA extraction kit (purchased from Tiangen Company), and the integrity of RNA was detected by 1% agarose gel electrophoresis. Synthesis of cDNA refers to TaKaRaPrimerScript TM The RTreagentkitwithgDNAEraser kit explains the operation. Design primers:

[0030] GmMYB29-F: '5-CGGGTCTTTGGAAACGAA-3' (SEQ ID NO.3)

[0031] GmMYB29-R: '5-CGCATTACTCTGGCTGGT-3' (SEQ ID NO.4);

[0032] Prepare PCR reaction solution (50μl system) according to the following components: 25μl 10×PCRBuffer, 9μlddH 2 O, 10 μl ldNTP, 1.5 μl primers (SEQ ID NO.3 and SEQ ID NO.4), 2 μl cDNA and 1 μl KODFX enzyme. The reaction was carried out on a BIO-RADPTC-200 PCR instrument. The program was denaturation at 94°C for 2 minutes; followed by 33 cycles at 98°C for 10 sec, 53°C for 30 sec, and 68°C for 2 min; then extended ...

Embodiment 2

[0033] Example 2, Expression Analysis of GmMYB29 in Different Soybean Tissues

[0034] Under normal field growth conditions for the soybean variety Jianli Niu Maohuang, the roots, stems, leaves, and flowers were taken at the full flowering stage, the pods and seeds were taken 10 days after flowering (10DAF), and the 20DAF, 25DAF, 30DAF, 40DAF, 50DAF and fully mature soybeans were taken. Seeds, all collected samples were quick-frozen in liquid nitrogen and stored at -80°C. The extraction of total RNA and the inversion of cDNA were the same as in Example 1. The Tubulin gene expressed constitutively in soybean was used as an internal reference gene, and the sequences of specific primers and TaqMan-MGB probes were as follows:

[0035] Specific primers and probes for amplifying GmMYB29:

[0036] Upstream primer: 5'-GCCAGGAAGAACTGACAATGAGA-3' (SEQ ID NO.5)

[0037] Downstream primer: 5'-TGGAATTGGAATCAGAACGTTTAAT-3' (SEQ ID NO.6)

[0038] MGB probe: 5'-AGTCCAAGCCAAGTTCCAAAAAGAGCCAC...

Embodiment 3

[0046] Example 3, GmMYB29 regulates the transient expression activity analysis of key genes IFS2 and CHS8 promoters for isoflavone synthesis

[0047] The expression vector for GmMYB29 transcriptional activity analysis in Arabidopsis protoplasts was transformed from pAN580 vector. Double digestion was performed with BamHI and NotI restriction endonucleases, the GFP fragment was removed, the blunt end was treated with Klenow enzyme, and then T4 ligase was used to construct an intermediate vector. In the multiple cloning site segment of the intermediate vector, the GmMYB29 sequence cloned in Example 1 was cloned and connected to form a CaMV35S::GmMYB29 recombinant plasmid, which was used as an effector vector after correct sequencing.

[0048] Choose XbaI and XmaI two enzyme cutting sites to design primers and use the pGL3 vector as a template to amplify the complete ORF of LUC by PCR. After enzyme digestion, recovery, and T4 ligase ligation, the multiple cloning site segment con...

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Abstract

The invention belongs to the field of plant genetic engineering and discloses an MYB type transcription factor GmMYB29 of Glycine max as well as an encoding gene and an application of the transcription factor GmMYB29. The transcription factor GmMYB29 has an amino acid sequence shown as SEQ ID NO.2. An open reading frame of the gene has a DNA sequence shown as SEQ ID NO.1. The transcription factor GmMYB29 increases the content of isoflavones of the Glycine max by promoting driving expression activity of a key structure gene promoter for an isoflavone biosynthetic pathway. The transcription factor GmMYB29 has significant value in breeding of a new Glycine max variety with high content of isoflavones.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a soybean MYB type transcription factor GmMYB29, its coding gene and its application. Background technique [0002] Isoflavones are secondary metabolites unique to higher plants, which cannot be synthesized in animals. Isoflavones play an important role in animals and plants. In animals, it has various biological functions such as estrogen, anti-cancer, anti-oxidation, anti-hemolysis, treatment of cardiovascular disease, lowering cholesterol, preventing osteoporosis, promoting reproduction, lactation and growth. In plants, it can be used as a protective substance to protect the normal growth of plants and resist the invasion of pathogenic bacteria, and it is also necessary for the symbiosis between rhizobia and plants. Isoflavones are mainly distributed in some species of legumes. Although isoflavones with different structural types have been detected in other higher plan...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 喻德跃褚姗姗王娇程浩朱莹
Owner NANJING AGRICULTURAL UNIVERSITY
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