Rice OsPPR035 gene as well as encoded protein and application thereof
A rice and gene technology, applied in the field of genetic engineering, can solve the problems affecting the growth, development and production of rice, no harvest, and reduced rice yield, etc., and achieve the effect of increasing the content of proline in cells and enhancing the ability of the rice.
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Embodiment 1
[0048] Cloning of embodiment 1 rice OsPPR035 gene
[0049] 1. Seedling cultivation
[0050] The rice variety Nipponbare was germinated at 30°C for 48 hours, and then sowed in the greenhouse. When the rice leaves were 3-5 pieces, the DNA or RNA was ready to be extracted.
[0051] 2. RNA isolation:
[0052] RNA extraction: freeze the sample in a mortar with liquid nitrogen and grind it into powder, add 1mL TRNzol-A+ reagent to a 2mL EP tube, oscillate fully, place at room temperature for 5min, then add 0.2mL chloroform, shake vigorously After 15 seconds, place at room temperature for 3 minutes; centrifuge at 12,000 rpm for 10 minutes at 4°C, transfer the supernatant to a new 2 mL EP tube, add an equal volume of isopropanol to precipitate RNA, and add 100 μL RNase-free ddH 2 O dissolved. The quality of total RNA was identified by electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0053] 3. Synthesis of first-strand cDNA by reverse transcription...
Embodiment 2
[0065] Example 2 Protein sequence information and homology analysis of rice OsPPR035
[0066] According to the ORF of the new rice OsPPR035 (gene number: Os01g0651100 / LOC_Os01g46230) of the present invention, the amino acid sequence of rice OsPPR035 is deduced, with a total of 598 amino acids and a molecular weight of 64430 Daltons. See SEQ ID NO: 2 for the detailed sequence. Through the comparison of the BLASTP program on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the OsPPR035 protein has typical PLS, E and DYW domains, and belongs to DYW in the PPR protein family subfamily.
[0067] Through multiple sequence alignment of some PPR-encoded proteins in plants, we found that these proteins all contain a conserved PPR domain ( figure 1 ).
Embodiment 3
[0068] Example 3 Expression analysis of rice gene OsPPR035 under adversity stress
[0069] 1. Adversity stress treatment
[0070] Select plump Nipponbare seeds, wash them with distilled water, sterilize with 3% NaClO for 10 minutes and then clean them, accelerate germination at 30°C, transfer the seeds to the germination box for hydroponic growth after the seeds are white, and apply nutrient solution (standard nutrient solution of the International Rice Institute) after the three-leaf stage . Cultivate in a light culture room at 28°C, 16h / 8h, and carry out stress and hormone treatment at the four-leaf stage, including: normal water control (CK), 20% PEG6000, 40mM hydrogen peroxide (H 2 o 2 ), 5 μM abscisic acid (ABA) and 180 mM salt (NaCl). The control and treatment materials were sampled before stress and 0.5h, 2h, 4h, 8h, 24h after stress. All processing and sampling were performed under continuous light conditions.
[0071] 2. RNA extraction and first-strand cDNA synth...
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