Plant stress resistance-related protein OsIAA18, encoding gene and application thereof

[0049] Example 1 Acquisition of Rice Stress Resistance-Related Protein OsIAA18 and Its Encoding Gene

[0050] 1. Experimental materials

[0051] Refer to Jan et al. (2013) [Asad Jan, Kyonoshin Maruyama, Daisuke Todaka, Satoshi Kidokoro, Mitsuru Abo, Etsuro Yoshimura, Kazuo Shinozaki, Kazuo Nakashima and Kazuko Yamaguchi-Shinozaki. OsTZF1, a CCCH-Tandem Zinc Finger Protein, Confers Delayed Tolayed Sess Rice by Regulating Stress-Related Genes.Plant Physiology, 2013, 161:1202-1216], the plant leaf material of the rice variety 'Huaidao No. 5' was removed, quick-frozen in liquid nitrogen, and stored at -80°C.

[0052] 2. Extraction and purification of total RNA from leaves

[0053] Take about 2.0 g of the leaves of Huaidao No. 5, grind them into powder in liquid nitrogen, put them into a 10 mL centrifuge tube, and use the Applygen Plant RNA Extraction Kit (Applygen Technologies Inc, Beijing) to extract the total RNA of Huaidao No. 5 leaves. Including: Plant RNA Reagent, plant tissue lysis, RNA isolation, removal of plant polysaccharides and polyphenols; ExtractionReagent, organic extraction to remove protein, DNA, polysaccharides and polyphenols; Plant RNA Aid, removal of plant polysaccharides, polyphenols and secondary metabolites. mRNA was purified from total RNA using the QIAGEN Oligotex Mini mRNA Kit (QIAGEN, GmbH, Germany). Finally, take 1 μL and test its integrity by electrophoresis on 1.2% agarose gel, take another 2 μL and dilute it to 500 μL, use a UV spectrophotometer to test its quality (OD260) and purity (OD260/OD280), and extract the total RNA, detected by non-denaturing gel agarose gel electrophoresis, 28S and 18S bands are clear, and the brightness ratio of the two is 1.5-2:1, indicating that the total RNA is not degraded, and the purified mRNA meets the experimental requirements and can be used for rice OsIAA18 protein Cloning of full-length cDNA.

[0054] 3. Full-length cloning of OsIAA18 protein cDNA

[0055] Primers were designed with the cDNA sequence of OsIAA18 on NCBI (National Center for Biotechnology Information) to carry out full-length cloning of OsIAA18 protein cDNA.

[0056] The primer sequences are as follows:

[0057] OsIAA18-GC-F: 5'-ATGGGGGAGGCGTCGG-3'

[0058] OsIAA18-GC-R: 5'-TCAACACTCAGCAGCTGTTCT-3'

[0059] Using Oligo(dT) reverse transcription of the total RNA of Huaidao No. 5 leaves as a template, high-fidelity FastPfu enzyme was used for PCR amplification. The PCR conditions were 95°C for 1min, followed by 95°C for 20s, 53°C for 20s and 72°C for 1min , for 36 cycles, and finally extended at 72 ° C for 5 min. The PCR amplification product was detected by agarose gel electrophoresis, and an amplified fragment with a length of 1362bp was obtained.

[0060] Based on the results of the above steps, the target cDNA sequence was obtained, the nucleotide sequence of which is shown in the sequence SEQ ID NO 1 in the sequence listing. The sequence SEQ ID NO 1 in the sequence listing is composed of 909 bases, from the 4th to the 906th base at the 5' end is its open reading frame, encoding the amino acid residues shown in the sequence SEQ ID NO 2 in the sequence listing sequence of proteins. The sequence SEQ ID NO 2 in the sequence listing consists of 302 amino acid residues. The gene was named OsIAA18, and the protein encoded by it was named OsIAA18.

[0061] Example 2 Construction of OsIAA18 Gene Overexpression Vector

[0062] The DNA fragment identified correctly by sequencing in Example 1 containing the nucleotide shown in SEQ ID NO 1 in the sequence table was double-digested with BamH I and Sac I, and the DNA fragment was reclaimed with 1% agarose gel, passed through T 4 The recovered OsIAA18 gene fragment was connected with the recombinant vector pCBGUS containing double 35S promoters by DNA ligase, and the recombinant plant expression vector pCAMBIA1301-OsIAA18 containing the rice OsIAA18 gene was obtained through enzyme digestion identification and sequence analysis. The expression vector also contains a gusA reporter gene and a kanamycin resistance marker gene with an intron, such as figure 1 shown.

[0063] Example 3 Transformation of Arabidopsis thaliana with OsIAA18 gene

[0064] The plant expression vector pCAMBIA1301-OsIAA18 of the rice OsIAA18 gene constructed in Example 2 was transformed into Arabidopsis thaliana by the method of dipping flowers, and the specific method is as follows:

[0065] 1. Preparation of Agrobacterium

[0066] (1) Transform pCAMBIA1301-OsIAA18 into Agrobacterium tumefaciens LBA4404 strain (BiovectorCo., LTD) by electric shock method to obtain a recombinant Agrobacterium containing pCAMBIA1301-OsIAA18, and spread it on a plate containing kanamycin resistance to select transformants .

[0067] (2) A single Agrobacterium was picked and inoculated in 5 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), and cultured at 28° C. and 250 rpm for 20 h.

[0068] (3) Transfer 1 mL of bacterial liquid into 20-30 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), culture at 28°C, 250 rpm for about 12 hours, and measure OD 600 ≈ 1.5.

[0069] (4) The cells were collected by centrifugation at 8000rpm, 4°C, 10min, resuspended in Agrobacterium transformation permeate (5% sucrose, 0.05% Silwet L-77, the concentration unit is g/100ml) and diluted to OD600≈0.8.

[0070] 2. Transformation of Arabidopsis by dipping flowers

[0071] (1) Immerse the flowering shoots of Arabidopsis thaliana in the above-mentioned infection solution, stir gently for about 10 seconds, and then take them out. After all the transformation is completed, cover the Arabidopsis thaliana with a fresh-keeping bag to maintain a humid environment, place it horizontally, and avoid Culture in light, remove the fresh-keeping bag after 24 hours and culture upright.

[0072] (2) 4 days after the initial transformation, another transformation can be carried out, repeated twice, and transformed three times in total, so that the flower buds at different stages of development on the inflorescence can be transformed and the transformation efficiency can be improved.

[0073] (3) After growing for about two months, collect the seeds and store them in a refrigerator at 4°C until use.

[0074] The Arabidopsis thaliana transformed by the dipping method flowered and set seeds normally after about two months of growth.