Plant low nitrogen stress resistant related protein GmDUF-CBS and encoding gene and application thereof
A nitrogen stress, gene technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve the problem of low nitrogen utilization rate
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Embodiment 1
[0038] Embodiment 1, acquisition of soybean protein GmDUF-CBS and its coding gene and recombinant expression vector
[0039] The soybean variety "Pohuang" (Glycine max(L.)Merr.) was cultured under normal conditions until the first three-primary compound leaf grew out, and the total RNA was extracted and reverse-transcribed to obtain cDNA, which was used as As a template, under the guidance of primers PF and PR, amplify with the conventional PCR method. After the reaction, the PCR amplification product is subjected to 1% agarose gel electrophoresis detection, and a DNA fragment of about 1.5kb is recovered and purified; The vector pCXSN (contains the lethal gene ccdB on the T-DNA fragment of the vector pCXSN, with XcmI restriction recognition sequences on both sides. The vector was purchased from The Arabidopsis Biological Resource Center (ABRC; website: http: / / abrc.osu.edu / ), the product catalog number is Vector: 5019471951) was digested with XcmⅠ, and the vector skeleton fra...
Embodiment 2
[0043] Embodiment 2, the acquisition of recombinant Agrobacterium tumefaciens
[0044] The recombinant vector pCXSN-GmDUF-CBS obtained in Example 1 was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method (document: Amanda M Davis, Anthony Hall, Andrew J Millar, Chiarina Darrah and Seth J Davis, Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana, 2009, 5:310.1186 / 1746-4811-5-3; the public can obtain from the Institute of Oil Crops, Chinese Academy of Agricultural Sciences), and obtain the root containing the recombinant vector pCXSN-GmDUF-CBS Agrobacterium carcinoma GV3101, the recombinant Agrobacterium was named GV3101 / pCXSN-GmDUF-CBS;
[0045] The empty vector pCXSN was transformed into Agrobacterium tumefaciens GV3101 by freeze-thawing method to obtain Agrobacterium tumefaciens GV3101 containing the empty vector pCXSN, and the recombinant Agrobacterium was named GV3101 / pCXSN.
Embodiment 3
[0046] Embodiment 3, the acquisition and identification of transgenic Arabidopsis
[0047] 1. Obtaining transgenic Arabidopsis
[0048] Using the two recombinant Agrobacteria obtained in Example 2, the flower bud soaking method was used to transform the Columbia ecotype Arabidopsis Columbia-0 (hereinafter referred to as wild-type Arabidopsis WT) (documents: Xia T, Xiao D, Liu D, Chai W, Gong Q, Wang NN. Heterologous expression of ATG8c from soybean confers tolerance to nitrogen deficiency and increases yieldin Arabidopsis. PLoS One. 2012;7(5):e37217; publicly available from Institute of Oil Crops, Chinese Academy of Agricultural Sciences), obtained T 3 Generation of 8 Arabidopsis lines homozygous transgenic GmDUF-CBS, T 3 Generation of 5 Arabidopsis lines that were homozygously transformed into empty vectors, the specific method is as follows:
[0049] 1. Take the recombinant Agrobacterium GV3101 / pCXSN-GmDUF-CBS or GV3101 / pCXSN and culture it with YEP medium containing rifa...
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