Application of ossec3b gene in controlling drought resistance in rice
A technology of drought resistance and genetics, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of alleviating food shortage
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Embodiment 1
[0025] Example 1: Amplification of OsSEC3B gene and construction of overexpression vector
[0026] The gene OsSEC3B (gene accession number LOC_Os11g17600) required by the present invention was amplified by RT-PCR to obtain the full-length sequence of the OsSEC3B gene. The specific operation is as follows:
[0027] 1) Extracting RNA from the seedling roots of the japonica rice variety Zhonghua 11 (ZH11);
[0028] 2) Synthesize cDNA by reverse transcription and amplify the OsSEC3B gene:
[0029] The system used in PCR is 20 μl, and the specific recipe is: 1 μl of cDNA first-strand template, 2 μl of 10xPCR buffer, 1.6 μl of 10mM dNTP, 2.5mM Mg2+1.5μl, 0.4μl of forward primer and reverse primer, 0.2 μl of LATaq enzyme μl, add water to 20 μl (PCR buffer, dNTP, Mg2+, LATaq enzyme, etc. used were all purchased from Treasure Bioengineering Dalian Co., Ltd.).
[0030] Forward primer: OsSEC3B-OEF: 5'-TACGAACGATAGCCGGTACCATGGCGCGGTCGAGCGCGGA-3';
[0031] Reverse primer: OsSEC3B-OER: ...
Embodiment 2
[0036] Example 2: Construction of OsSEC3B CRISPR vector
[0037] The applicant constructed the OsSEC3B gene CRISPR knockout vector, and studied the function of the gene from the phenotype of the transgenic plant:
[0038] The CRISPR vector construction method is derived from the article Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA processing system, as follows. The sequences of the primers in the following steps are the same as those in the published article unless otherwise noted:
[0039] (1) Primer design: first enter the OsSEC3B gene LOC number: LOC_Os11g17600 on the rice gene database website (http: / / www.ricedata.cn / gene / ), and download the amino acid sequence of the gene; https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) input amino acid sequence, predicted to contain SEC3 superfamily domain, the position of the domain is 56-149 and 226-858 amino acids, Use this as a reference to design primers; select species on the CRISPR-P website (h...
Embodiment 3
[0049] Example 3: Transformation of plasmid vectors and positive detection of transgenic plants
[0050] The above-mentioned CRISPR vectors pRGEB32-sec3b and pU1301-SEC3B were transformed into the rice variety "Zhonghua 11" (a publicly used one provided by the China Rice Research Institute) through the Agrobacterium-mediated rice genetic transformation method (the specific steps are described below). rice varieties), through pre-cultivation, infection, co-cultivation, selection of hygromycin-resistant calli, differentiation, rooting, training of seedlings, testing, and transplanting to obtain transgenic plants. The rice (Zhonghua 11) genetic transformation method (system) mediated by the above-mentioned Agrobacterium was reported by Hiei et al. (Hiei et al., Efficient transformation of rice, Oryza sativaL., mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA, Plant J, 6:271-282, 1994).
[0051] The specific genetic transformation steps of this embo...
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