Protein TaMYB85 related to stress resistance, and coding gene and application thereof
A coding and protein technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as quality deterioration and yield decline
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Embodiment 1
[0078] Embodiment 1, the cloning of the coding gene of protein TaMYB85
[0079] 1. Take the seedlings of TAM107 cultured in water for 10 days, place them in a 40°C incubator, and cultivate them under light (light intensity is 120-150 μmol / m 2 ·s -1 ) for 1 h; then quick-frozen in liquid nitrogen and stored at -80°C as experimental materials.
[0080] 2. Take the experimental materials obtained in step 1, extract total RNA with TRIZOL, and then use primer T15 and M-MLV reverse transcriptase to carry out reverse transcription to obtain cDNA of wheat. For wheat cDNA, the cDNA concentration was 100 ng / μL.
[0081] 3. Using the cDNA of wheat as a template, PCR amplification was carried out with TaMYB85-L: 5'-CCAATCAACTCACAACGCAAG-3' and TaMYB85-R: 5'-AGCCTACACAAATCC-3' as primers, and a PCR amplification product of about 1077 bp was obtained.
[0082] Reaction conditions: pre-denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min; exten...
Embodiment 2
[0084] Embodiment 2, expression pattern analysis
[0085] 1. Expression pattern of TaMYB85 gene in different tissues and organs of Chinese spring
[0086] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:
[0087] 1. Get 3 leaves (hereinafter referred to as seedling stage leaves) of Chinese Spring normally grown to seedling stage in field, extract total RNA with TRIZOL, then adopt primer T15 and M-MLV reverse transcriptase to carry out reverse transcription, obtain cDNA of seedling leaves. In the cDNA of leaves at the seedling stage, the cDNA concentration was 100 ng / μL.
[0088]According to the above-mentioned method, the leaves at the seedling stage are replaced with the root (hereinafter referred to as the root of the seedling stage) of Chinese spring normally grown to the seedling stage in the field, the root of the Chinese spring normally grown to the flowering stage in the field (hereinafter referred to as the ...
Embodiment 3
[0126] Example 3, subcellular localization
[0127] 1. Using the PCR amplification product obtained in Example 1 as a template, using TaMYB85-HindIII-L:
[0128]5'-AGCCCAAGCTTATGGGGAGGGCGCC-3' (underlined is the recognition sequence of restriction endonuclease HindIII) and TaMYB85-XbaI-R: 5'-GC TCTAGA CATGTCATACAC-3' (underlined is the recognition sequence of restriction endonuclease XbaI) was used as a primer for PCR amplification, and a PCR amplification product of about 970bp was obtained.
[0129] 2. Digest the PCR amplified product obtained in step 1 with restriction endonucleases HindIII and XbaI, and recover a digested product of about 960 bp.
[0130] 3. Digest the vector pJIT163hGFP with restriction endonucleases HindIII and XbaI, and recover the vector backbone of about 4.5kb.
[0131] 4. Ligate the digested product with the vector backbone to obtain the recombinant plasmid pJIT163hGFP-TaMYB85.
[0132] The recombinant plasmid pJIT163hGFP-TaMYB85 was sequenced. ...
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