osqr1 protein and its coding gene and application
A technology that encodes genes and proteins, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of slow progress, large chance, unstable varieties, etc., and achieve the effect of improving drought resistance
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Embodiment 1
[0034] Example 1. Acquisition of the gene encoding the drought resistance-related protein OsQR1 in upland rice
[0035] 1. Take the leaves of upland rice IRAT109 seedlings cultivated under normal conditions, extract total RNA by Trizol method, and reverse transcribe with M-MLV reverse transcriptase to obtain cDNA.
[0036] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with primer F1 and primer R1 to obtain a PCR amplification product.
[0037] The primer sequences are as follows:
[0038] F1: 5'-G TTAATTAA CAATGGCGGTGAAGGTCT-3' (SEQ ID No. 1)
[0039] (The underlined sequence is the restriction endonuclease PacI restriction endonuclease recognition site)
[0040] R1: 5'-C GAGCTC TCAAGCGGACCCCTTGA-3' (SEQ ID No. 2)
[0041] (The underlined sequence is the restriction endonuclease SacI restriction endonuclease recognition site)
[0042] 3. Perform agarose gel electrophoresis on the PCR amplification product, recover and purify the 629bp ...
Embodiment 2
[0043] Embodiment 2, real-time fluorescent quantitative PCR analysis endogenous OsQR1 gene expression
[0044] 1. Coercion treatment
[0045] The rice variety Nipponbare and the upland rice variety IRAT109 seedlings that are 4 weeks old in hydroponics are subjected to any of the following (1) to (4) treatments respectively:
[0046] (1) ABA treatment: soak the roots of the seedlings in an aqueous solution of 100 μM ABA, and cultivate them under light for 1 hour, 4 hours, 6 hours, 8 hours, 9 hours, 16 hours, and 24 hours, then take the leaves and freeze them with liquid nitrogen, Store at 80°C for later use.
[0047] (two) H 2 o 2 Treatment: Soak the roots of the seedlings in 1mM H 2 o 2 After being placed in the aqueous solution for 1 hour, 3 hours, 6 hours, 8 hours, 9 hours, 16 hours, and 24 hours, the leaves were taken out, quickly frozen with liquid nitrogen, and stored at -80°C for later use.
[0048] (3) PEG (drought) treatment: Soak the roots of the seedlings in 20...
Embodiment 3
[0064] Embodiment 3, the construction of recombinant expression vector
[0065] PacI and SacI double-digest the PCR amplification product obtained in step 2 of Example 1 to obtain the gene fragment; PacI and SacI double-enzyme-digest the vector pMDC83 to obtain the large vector fragment; connect the gene fragment to the large vector fragment to obtain the recombinant vector 35S ::OsQR1, the recombinant vector 35S::OsQR1 is inserted between the PacI and SacI sites of the vector pMDC83 (ie downstream of the double tobacco mosaic virus promoter 35S) in the 79th position from the 5' end of SEQ ID No.3 35S::OsQR1 was sent for sequencing to the DNA fragment indicated by the 690th nucleotide, and the result was correct.
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