Wheat Stress Resistance Regulatory Protein tacor58 and Its Encoding Gene and Application
A technology of stress resistance and protein, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear functions, achieve the effect of improving drought resistance and frost resistance, and improving stress resistance
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Embodiment 1
[0045] Example 1: Cloning of TaCOR58 protein coding gene
[0046] 1. Acquisition of cDNA
[0047] The root system of the one-leaf-one-heart wheat variety Bainong'ai Kang 58 seedlings was taken, and the total wheat RNA was extracted according to the steps indicated in the instructions of the Plant RNA Extraction Kit (TaKaRa). The resulting RNA was reverse-transcribed with polyT primers to obtain cDNA.
[0048] 2. PCR amplification
[0049] Using the cDNA obtained in step 1 as a template, the forward primer TaCOR58-F and the reverse primer TaCOR58-R were used for PCR amplification to obtain PCR amplification products. The primer sequences are as follows:
[0050] TaCOR58-F: 5'-ATGGACGCGCCGGCCCCCGACAG-3;
[0051] TaCOR58-R: 5'-TCACATCACATTCGTGTTGGTC-3'.
[0052] The above PCR amplification reaction conditions: 95°C for 1min; then 95°C for 15s, 60°C for 15s, 72°C for 2min, 30 cycles; finally 72°C for 5min.
[0053] 3. Electrophoresis and sequencing
[0054] The PCR amplifica...
Embodiment 2
[0055] Example 2: Expression analysis of TaCOR58 gene under different stress conditions
[0056] 1. Expression analysis of TaCOR58 gene under drought stress conditions:
[0057] (1) Experimental method:
[0058] Take the seeds of the wheat variety Bainong Ai Kang 58, and after accelerating germination at 25°C for three days, transfer the germinated seeds to Hogland nutrient solution. The normal growth temperature is 25 / 15°C, and the photoperiod is 16 / 8h (day / night). Two weeks later, the wheat seedlings were taken out from the nutrient solution, and the wheat root system was wrapped with filter paper and placed vertically on a support for direct water shortage and drought stress treatment. Quick-frozen in liquid nitrogen, with three biological replicates at each time point.
[0059] Grind the above-mentioned root material samples separately, extract total RNA, and perform reverse transcription to obtain cDNA. Using the cDNA as a template, the TaCOR58 gene fragment is amplifie...
Embodiment 3
[0087] Example 3: Obtaining Silenced TaCOR58 Wheat
[0088] 1. Obtaining gene silencing fragments:
[0089] Using the cDNA (SEQ ID NO.1) of the TaCOR58 gene as a template, BSMV-TaCOR58F and BSMV-TaCOR58R were amplified by PCR using primers to obtain a fragment of 379bp, which corresponds to the 5' of the TaCOR58 gene sequence SEQ ID NO.1 The 1st to 379th nucleotides at the end, which were recorded as TaCOR58 (VIGS), were used to construct the VIGS silencing vector.
[0090] Wherein, the nucleotide sequences of the above primers to BSMV-TaCOR58F and BSMV-TaCOR58R are as follows:
[0091] BSMV-TaCOR58F:5'-GCTAGCTGAGCGGCCGCCAAGGTCACCAAGCCGCAC-3';
[0092] BSMV-TaCOR58R:5'-AGCTAGCTGATTAATTAAATGGACGCGCCGGCCCCCGA-3'.
[0093] 2. Construction of gene silencing vector
[0094] According to conventional molecular biology methods, use Not I and Pac I to digest the BSMV-VIGS viral vector γ, and then reversely insert the silent fragment TaCOR58 (VIGS) obtained in the above step 1 into...
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