Plant heat resistance related protein TaXPD and coding gene and application thereof
A plant and coding technology, which is applied in the field of plant heat resistance-related protein TaXPD and its coding gene and application field, can solve the problems of harm, yield decline, quality reduction, etc., and achieve the effect of increasing heat resistance
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Embodiment 1
[0077] Cloning of the coding gene of embodiment 1, protein TaXPD
[0078] The gene encoding the protein TaXPD, ie the TaXPD gene, was cloned from wheat. Specific steps are as follows:
[0079] 1. Use TRNzol Total RNA Extraction Kit to extract total RNA from fresh wheat leaves, and then use M-MLV reverse transcriptase to reverse transcribe the first-strand cDNA.
[0080] 2. Artificially synthesized primers F: 5'-ATGAAGTTCGATCTTGAAGG-3' and R: 5'-TCACATCTCCATGGCGTCCC-3'.
[0081] 3. After completing steps 1 and 2, use the cDNA extracted in step 1 as a template and use F and R as primers for PCR amplification to obtain a double-stranded DNA molecule of about 2300 bp.
[0082] 4. Ligate the double-stranded DNA molecule with pEASY-Blunt Cloning Vector to obtain recombinant plasmid A.
[0083] According to the sequencing results, the recombinant plasmid A contains the DNA molecule shown in sequence 1 in the sequence listing (hereinafter referred to as TaXPD gene), and expresses t...
Embodiment 2
[0084] Embodiment 2. Real-time quantitative detection of the relative expression of TaXPD gene in wheat samples under the condition of high temperature treatment for different time
[0085] Three replicates were performed, and the steps for each replicate were as follows:
[0086] (1) Get samples
[0087] The 10-day-old wheat seedlings were treated at 22° C. for 3 hours, and then the leaves were removed and stored in liquid nitrogen to obtain sample 1 (as a control).
[0088] Wheat seedlings grown to 10 days were treated at 40°C for 1 hour, and then the leaves were taken out and stored in liquid nitrogen to obtain sample 2.
[0089] According to the above method, replace 1h with 2h, 3h, 6h, and 12h respectively, and keep other steps unchanged, and obtain sample 3, sample 4, sample 5, and sample 6 in sequence.
[0090] (2) Real-time quantitative detection of the relative expression of TaXPD gene in wheat samples processed for different times under high temperature conditions ...
Embodiment 3
[0096] Embodiment 3, the acquisition and identification of transgenic Arabidopsis
[0097] 1. Acquisition of recombinant plasmids pB2GW7-TaXPD and GV3101 / pB2GW7-TaXPD
[0098]1. Acquisition of recombinant plasmid pB2GW7-TaXPD
[0099] Using Gateway technology to construct an overexpression vector, the specific steps are as follows:
[0100] (1) Acquisition of the target gene
[0101] Primers were designed according to the open reading frame sequence of the TaXPD gene as follows:
[0102] TaXPD-TOPOF: 5'-CACCATGAAGTTCGATCTTGAAGG-3'
[0103] TaXPD-TOPOR: 5'-TCACATCTCCATGGCGTCCC-3'.
[0104] TRNzol Total RNA Extraction Kit was used to extract total RNA from fresh wheat leaves, and then M-MLV reverse transcriptase was used to reverse transcribe the first-strand cDNA to obtain a cDNA solution. The DNA concentration in the cDNA solution was 1000 ng / μL.
[0105] The cDNA solution is used as a template, and TaXPD-TOPOF and TaXPD-TOPOR are used as primers for PCR amplification to ...
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