A Wheat Scab Resistance Gene taxax1 and its application
A technology for head blight and wheat, applied in the field of molecular breeding, can solve the problems of declining resistance to various diseases
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Embodiment 1
[0016] Embodiment 1, gene TaXAX1 screening
[0017] Using the leaves of the wheat variety "Wangshuibai" as the material, DON (Deoxynivalenol, deoxynivalenol, sigma company) toxin standard solution was diluted to 100PPM, and then injected into the epidermis of wheat seedling leaves with a disposable syringe. Bacterial water was injected in the same way as the control. After culturing for 12 and 24 hours, samples were taken for future use.
[0018] The 2 pieces of Wangshuibai leaf tissue samples treated with DON were mixed equally as the experimental sample, and the 2 pieces of Wangshuibai leaf tissue samples treated with sterile water were mixed in equal amounts as the control sample. The total RNA ( Figure 5 A). After mRNA was isolated, double-stranded cDNA was synthesized according to the instruction of BD sMART PCR cDNA synthesis kit (Clontech Company). After 1.4% agarose gel electrophoresis, the cDNA appeared as a smear of 0.3-2 kb ( Figure 5 B), in terms of size, i...
Embodiment 2
[0021] Embodiment 2, gene TaXAX1 cDNA sequence acquisition
[0022] Using Wangshuibai leaf cDNA as a template, primers TaXAX1 F (5'-TATATGCGGCCGCATGGCGTCCACGGCGTACG-3', SEQ ID NO: 3), TaXAX1 R (5'-TATATGGCGCGCCCCATCCTGCAGCTGGTCGAG-3', SEQ ID NO: 4) PCR amplified gene TaXAX1 . Use ultra-fidelity DNA polymerase (Phanta Max Super-Fidelity DNA polymerase, Nanjing Novozyme):
[0023] PCR reaction (50 μL):
[0024] 2 × Phanta Max Buffer 25 μL
[0025] dNTP Mix (2.5 mM) 4 μL
[0026] Primer 1 (10 μM) 2 μL
[0027] Primer 2 (10 μM) 2 μL
[0028] Template x μL (approximately 100 ng)
[0029] Phanta Max Super-Fidelity
[0030] DNA polymerase (1U / μL) 1 μL
[0031] ddH2O to 50 μL
[0032] PCR reaction program: Denaturation at 95°C for 3min, denaturation at 94°C for 30s, annealing at 68°C for 30s, extension at 72°C (1min / 1Kb), a total of 30~40 cycles, followed by final extension at 72°C for 5min, and storage at 16°C. PCR products were analyzed by electrophoresis on 1% agarose ...
Embodiment 3
[0034] Will TaXAX1 The gene is connected to the downstream of the expression vector PC611 strong promoter Ubi to obtain the overexpression vector PC611- TaXAX1. After verification, the built PC611- TaXAX1 The carrier was transformed into the susceptible material Fielder by the Wheat Genetic Transformation Platform of the Chinese Academy of Agricultural Sciences.
[0035] After the transformation is completed, transfer the differentiated seedlings to the medium for rooting and strong seedlings. When the seedlings grow to about 10 cm, they are hardened and transplanted. Plant leaf DNA was extracted for PCR verification, and the verification primers were TaXAX1 F / R. The verification result is as image 3 shown.
[0036] The positive lines were planted in the greenhouse of Shandong Agricultural University, and the negative transgenic lines and wild-type Fielder were planted as controls. During the flowering period, Fusarium graminearum ( F. graminearum ) wild-type strain ...
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