Herbicide-resistant proteins and application thereof in plant breeding
A herbicide-resistant, herbicide-resistant technology applied in the field of plant protein
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Embodiment 1
[0019] Example 1: Screening of resistant plants from the M2 population of the sorghum EMS mutagenesis library
[0020] In order to carry out large-scale screening of the sorghum mutant library, we constructed the sorghum mutant library by chemical mutagenesis. The specific steps are as follows: Weigh about 1 kg of sorghum seeds, put them into 80-mesh mesh bags, and wash 2 times with tap water for a total of 10 parts. Sorghum seeds were then soaked in 0.3% (w / w) ethyl methanesulfonate (EMS) for 12 h, during which the seeds were shaken every 1 h. After 12 hours, the seeds were taken out, soaked in tap water for 5 times, and the EMS waste liquid and soaking waste liquid were collected and stored in special containers. Spread out the soaked seeds to air dry and arrange for sowing immediately thereafter. At the same time, the germination rate of the treated seeds was tested in the light incubator, and the germination rate was controlled at about 65%. After the plants are mature,...
Embodiment 2
[0021] Example 2: Molecular detection of screened herbicide-resistant sorghum
[0022]Molecular detection was carried out on the two suspected herbicide-resistant sorghum plants screened. The specific steps are as follows: Take 10-20 mg of leaves of plants that may have herbicide resistance, put them into a centrifuge tube, add steel balls, place them in liquid nitrogen for quick cooling, use a tissue grinder to fully shake and grind the leaves to powder, and use Sample DNA was extracted by SDS method, and then PCR sequencing was performed with specific primers. Sequencing company: Yingwei Jieji (Shanghai) Trading Co., Ltd. It was confirmed by sequencing that two mutation sites in the ALS gene were encoded in sorghum, one mutation site was the 503rd C-T base mutation, corresponding to the 168th P-L amino acid mutation; the other mutation site was The 1871st G-A base mutation corresponds to the 624th S-N amino acid mutation. The nucleic acid sequence of the wild-type plant i...
Embodiment 3
[0023] Example 3: Field Imidazolinone herbicide-resistant test of the screened herbicide-resistant plant progeny
[0024] The offspring of the identified mutant strains were harvested and planted in the field with a row spacing of 50×20 cm and a row length of 5 m. The wild type was used as a control. When the sorghum grows to the four-leaf stage, imazethapyr is sprayed at a dose of 48g ai / ha. After 14 and 21 days of spraying, the deadness of the plants was investigated, and it was found that the wild-type control almost stopped growing after spraying imazamox, and the leaves were yellow or dead, while the herbicide-resistant plants screened showed complete immunity to imazamox. For the tolerant phenotype, its normal growth and development were not affected, and eventually jointing and heading were normal. This indicates that the herbicide-resistant mutants we screened can stably inherit their resistance to imidazolinone herbicides (such as figure 1 and image 3 ).
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