Soybean gene GmTCM1 and obtaining method and application thereof
A gene and soybean technology, applied in the field of soybean gene GmTCM1 and its acquisition, can solve the problems of small variation range, long cycle, difficulty in obtaining new soybean varieties with high isoflavones, etc.
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Embodiment 1
[0046] Embodiment 1, the acquisition of GmTCM1 gene and the construction of plant expression vector
[0047] Step 1: Total RNA was extracted from soybean leaves of the variety Zhongdou 27, and the first strand of cDNA was synthesized by reverse transcription.
[0048]The reverse transcription steps are as follows: After mixing 2 μL RNA, 1 μL oligo(dT) (50 μM) and 1 μL Super Pure dNTP (10 mM), add DPEC water to 10 μL, incubate the tube at 65 ° C for 5 min, and then cool on ice; Mix 4 μL 5xPrimeScript II buffer, 0.5 μL RNase-Free DNase and 1 μL PrimeScript II RTase, add DPEC water to 20 μL, incubate the mixture at 42 °C for 1 h, then incubate at 95 °C for 5 min, then cool on ice until use.
[0049] Step 2: Search the Phytozome v11.0 database for the full-length sequence of the gene whose accession number is Glyma.20G114200 (GmTCM1), as shown in SEQ ID No.4. According to the sequence of the Glyma.20G114200 (GmTCM1) gene, a pair of cloning primers GmTCM1-S / A was designed, and th...
Embodiment 2
[0070] Embodiment 2, the application of GmTCM1 in anti-biotic stress
[0071] 1. Agrobacterium rhizogenes-mediated transformation of hairy roots
[0072] The plasmid of the expression vector pCAMBIA3301-GmTCM1 was transferred into the competent cells of Agrobacterium rhizogenes, and the susceptible soybean variety Dongnong 50 was used as the recipient for transformation. The specific method is as follows:
[0073] 1) Recipient soybean culture
[0074] Mix the soil and vermiculite in a ratio of 1:1 into a small pot, and water thoroughly before planting. The seeds of soybean variety Dongnong 50, which has a smooth appearance and no insects biting, were evenly planted in small pots, and cultured in dark for 4 days in a cultivation room with suitable temperature conditions.
[0075] 2) Preparation of Agrobacterium rhizogenes infection solution
[0076] The day before the cultivation of Agrobacterium rhizogenes, the transformed bacteria carrying the pCAMBIA3301-GmTCM1 plasmid we...
Embodiment 4
[0092] Example 4, the application of GmTCM1 in resisting abiotic stress
[0093] 1) Transformation of Arabidopsis thaliana with GmTCM1 gene and screening after transformation
[0094] Transform the GmTCM1 gene into wild-type Arabidopsis thaliana, harvest the seeds, sow the transgenic Arabidopsis seeds on MS medium containing 5 mg / L glufosinate-ammonium for screening, and obtain PPT-resistant Arabidopsis with normal growth Five mustard plants were transplanted into nutrient soil and placed in a constant temperature incubator at 22°C for cultivation.
[0095] 2) PCR detection of transgenic Arabidopsis
[0096] The Arabidopsis thaliana rosette leaves of the overexpressed plants transfected with the GmTCM1 gene were selected to extract DNA, and primers 3 and 4 were used for PCR identification, among which the target fragments with a size of 695bp and 400bp were detected, and were identified in 5 PPT positive plants. Two positive Arabidopsis plants were harvested, and each genera...
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