Compound for improving crop resistance and application of compound
A compound and wheat technology, applied in the field of plant cultivation, can solve the problems of pollution, burden on farmers, easy loss of proline, etc., and achieve the effects of low fermentation cost, reduced burden, and easy popularization.
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Embodiment 1
[0034] Embodiment 1 The expression construct of the gene of cold resistance protein
[0035] According to our design, Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize the gene encoding the cold resistance protein. The nucleotide sequence is shown in SEQ ID No: 2 of the sequence list, and the amino acid sequence of the encoded protein is shown in SEQ ID No: 2. ID No:1 is shown. Then, according to the "Molecular Cloning Experiment Guide" and the operating guide of the commercial reagents used to construct the secretory expressed yeast, in short, the gene encoding the xylanase is used as a forward primer such as SEQ ID No: 3 of the sequence listing As shown (the EcoR I endonuclease site was introduced), the reverse primer was shown in SEQ ID No: 4 of the sequence table (the Not I endonuclease site was introduced) and EcoR I and Not I double enzymes were used after amplification and ligated with the pPIC3.5 plasmid digested by these two endonuclea...
Embodiment 2
[0037] Example 2 Expression of anti-cold protein
[0038] Inoculate the positive yeast transformant obtained in Example 1 into 50ml BMGY liquid medium (100mM potassium phosphate buffer (pH6.0), containing 1% yeast extract, 2% peptone, 1.34% yeast nitrogenous base, biological Prime 4*10 -5 %, glucose 2%, glycerol 1%), cultured at 30°C, 200rpm until OD600 reached 5.0.
[0039] Transfer the above culture to 1L BMGY liquid medium, cultivate at 30°C, 200rpm for 24 hours, add 5ml of methanol, and then continue to cultivate at 30°C, 200rpm for 8 days, during which 5ml of methanol was added every 24 hours, and the solution was controlled by ventilation. Oxygen (DO) is greater than 20%, and the pH is controlled at 6.0 (adjusted with ammonia water). After the cultivation is completed, filter with a 0.22 μm filter membrane and retain the supernatant. The supernatant is detected by SDS-PAGE corresponding to the 8kDa cold resistance protein (proline content accounts for about 61%), and...
Embodiment 3
[0041] Example 3 Wheat Anti-Lodging Spring Cold Test
[0042] The natural phenomenon of late spring cold is difficult to predict, and there is no suitable field simulation test standard at present. Therefore, we sowed the newly cultivated cold-resistant wheat variety Xinmai No. 19 on October 19, and randomly divided the plot into 6 groups including positive control, negative control, proline 1, proline 2, protein 1 and protein 2 groups. Among them, the cultivation conditions (fertilizer application, etc.) of the positive control group were all referred to Dunhuang Seed Industry’s instructions for “Xinmai No. 19”, that is, field cultivation, without a greenhouse, and waited until the wheat began to turn green (February 27, 2011). day) normal water and fertilizer management; the other 5 groups set up greenhouse supports after sowing, but did not cover the plastic film until December 6th, and the temperature in the shed was controlled at 8-16°C. When the daily wheat began to ...
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