Application of tadsk2a protein in regulating wheat resistance to stripe rust
A protein and rust technology, applied in the application field of TaDSK2a protein in the regulation of wheat stripe rust resistance, can solve the problems of no reports on the function of TaUbiL and its regulation method
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Embodiment 1
[0075] , Gene Cloning cDNA sequence TaDSK2a Example 1
[0076] 1, the wheat cDNA sequence
[0077] Total RNA (1) Wheat
[0078] Using RNA extraction kit original total RNA was extracted 11 of common wheat. RNA extraction kit is PEXBIO Products (Product Number: A010400), the specific extraction procedures and conditions required according to the product instructions.
[0079] (2) reverse transcription of RNA
[0080] Of the above step (1) obtained in wheat leaf total RNA was reverse transcribed to obtain cDNA: (RR047A Num) using the product of Takara reverse transcription kit. Specific steps and conditions required in accordance with product instructions.
[0081] 2, PCR amplification and sequencing of TaDSK2a cDNA sequence
[0082] cDNA was obtained in the above step 1 as template using primer pair DSKCDS-F and DSKCDS-R1, R2, wheat varieties raw water 11 (anti stripe rust strains CYR17) cDNA as template for PCR amplification.
[0083] DSKCDS-F: 5'-ATGGGCGGAGCAGGCGACG-3 ';
[0084]...
Embodiment 2
[0090] Example 2, experiments using BSMV-VIGS stripe rust resistance gene functional TaDSK2a
[0091] First, the wheat gene silencing TaDSK2a get
[0092] 1, induce gene down-regulation TaDSK2a BSMV-VIGS construct vector system
[0093] (1) According to Example 1 TaDSK2a-1D gene sequence (SEQ ID No.6), selected two different positions silence segments, a segment location for the particular + 984bp ~ + 1115bp, 132-bp long, silencing sequences named TaDSK2a- 1, using the primer pair V-TaDSK-F1 and V-TaDSK.2-R1 for PCR amplification. Another fragment starting at + 1279bp, end to + 1366bp long 88bp, silencing sequences named TaDSK2a-2, using the primer pair V-TaDSK-F2 and V-TaDSK-R2 for PCR amplification.
[0094] V-TaDSK-F1: 5'-CTA GCTAGC GCAAGGAACAACACGGTCA-3 ';
[0095] V-TaDSK-R1: 5'-CTA GCTAGC GCAGCACCAGTTCTTGCAT-3 '.
[0096] V-TaDSK-F2: 5'-CTA GCTAGC CCAAATGCACGTAGCCTGA-3 ';
[0097] V-TaDSK-R2: 5'-CTA GCTAGC CTGGGGATGCCATCTGG-3 '.
[0098] Underlined sequence is the restrictio...
Embodiment 3
[0129] Example 3, using Agrobacterium-mediated gene transfer methods to verify stripe rust resistance gene functional TaDSK2a
[0130] A, TaDSK2a overexpression plants obtained
[0131] 1, TaDSK2a gene over-expression vector construct vectors and primer design
[0132] To verify the anti-TaDSK2a wheat gene function in strip rust, the present invention in maize ubiquitin promoter (Ubil) drive (pLGY-02 comes with a promoter), the gene construct TaDSK2a-1D to pLGY-02 vector. Expression vectors designed OETaDSK2a-F primer and OETaDSK2a-R.
[0133] OETaDSK2a-F: 5'-CTAGAGGATCCCCG GGTACC -ATGGGCGGAGCAGGCGACGGCGAGG-3 ';
[0134] OETaDSK2a-R: 5'-GCTCTCTAGA Actagt -CTACTGGCCAAAGTTCCCGAGAAG-3 '.
[0135] PCR reaction system: the sequence SEQ ID No.6 embodiment product DNA 0.5μL (<0.5μg), dNTP (2.5mM) 4μl, OETaDSK2a-F (10μM) 1μl, OETaDSK2a-R (10μM) 1μl obtained in Example 1, 5 × TransStartFastPfuBuffer 10μl, TransStartFastPfu DNA Polymerase 1μl (2.5U), washed with water and finally made up to...
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