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

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

Example Embodiment

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

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

[0030] GmMYB29-F: ‘5-CGGGTCTTTGGAAACGAA-3’ (SEQIDNO.3)

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

[0032] Prepare PCR reaction solution (50μl system) according to the following component sequence: 25μl 10×PCRBuffer, 9μlddH 2 O, 10μldNTP, 1.5μl primers (SEQIDNO.3 and SEQIDNO.4), 2μl cDNA and 1μl KODFX enzyme. The reaction was carried out on a BIO-RADPTC-200 PCR machine, and the program was denaturation at 94°C for 2min; then at 98°C for 10sec, 53°C for 30sec, 68°C for 2min, a total of 33 cycles; then extended at 68°C...

Example Embodiment

[0033] Example 2. Expression analysis of GmMYB29 in different tissues of soybean

[0034] Soybean variety Jianli Niu Maohuang under normal field growth conditions, take roots, stems, leaves, flowers at full bloom, take pods and seeds 10 days after flowering (10DAF), take 20DAF, 25DAF, 30DAF, 40DAF, 50DAF and fully mature 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 constitutively expressed in soybean is used as the internal reference gene, and the specific primers and TaqMan-MGB probe sequences are as follows:

[0035] Specific primers and probes for amplification of GmMYB29:

[0036] Upstream primer: 5'-GCCAGGAAGAACTGACAATGAGA-3' (SEQIDNO.5)

[0037] Downstream primer: 5'-TGGAATTGGAATCAGAACGTTTAAT-3' (SEQIDNO.6)

[0038] MGB probe: 5'-AGTCCAAGCCAAGTTCCAAAAGAGCCAC-3' (SEQIDNO.7)

[0039] Specific primers for GmIFS2 amplification:

[0040] Upstr...

Example Embodiment

[0046] Example 3, GmMYB29 regulation of isoflavone synthesis key genes IFS2 and CHS8 promoter transient expression activity analysis

[0047] The expression vector for GmMYB29 transcriptional activity analysis in Arabidopsis protoplasts was modified from pAN580 vector. Double digestion with BamHI and NotI restriction enzymes, GFP fragments were removed, Klenow enzyme was used to blunt the ends, and then T4 ligase was used to ligate to construct an intermediate vector. In the multi-cloning site segment of the intermediate vector, clone and connect the GmMYB29 sequence cloned in Example 1 to form the CaMV35S::GmMYB29 recombinant plasmid. After the sequencing is correct, it will be used as the effector vector Effector.

[0048] Select the two restriction sites of XbaI and XmaI to design primers. Use the pGL3 vector as a template to amplify the complete ORF of LUC by PCR. After digestion, recovery, and T4 ligase ligation, the multiple cloning site segment of the intermediate vector str...

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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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