A carrier for inhibiting the expression of cytochrome p450 gene and its application
A technology of gene expression and cell inhibition, applied in the field of agricultural biology, can solve problems such as death, inability to degrade bendazolone in vivo, plant death, etc., and achieve the effect of preventing transgene escape
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Embodiment 1
[0032] Embodiment 1 Agrobacterium transformed rice and identification of transgenic plants
[0033] The pTCK303-P450i-3 recombinant plasmid was introduced into the Agrobacterium EHA105 strain, and the callus of japonica rice Zhonghua 11 was transformed. After hygromycin resistance screening, differentiation, and rooting, 25 regenerated transgenic lines were obtained, and the transgenic plants were identified by PCR. The hygromycin resistance gene was introduced, and the results showed that 20 PCR positive plants ( figure 1 ), the positive plants were transplanted into the soil, and 17 survived.
Embodiment 2
[0034] Example 2 Obtaining of Rice Bentasone Sensitive and Resistant Transgenic Lines
[0035]In order to detect the effect of the designed interference carrier fragment in the transgenic plants, 0.25g / L bentazone solution was prepared with 48% bentazone mother solution (Changzhou Precision Biotechnology Co., Ltd.), and the pTCK303-P450i-3 transgenic T0 generation three Leaf stage seedlings, according to 100ml / m 2 Calculated, the spraying amount is 0.01-0.05g / m 2 , observed continuously after spraying. 4 days after spraying, some lines of P450i-3 transgenic T0 seedlings had curled and yellowed leaf tips; 7 days after spraying, 10 of the 17 P450i-3 transgenic lines died irreversibly, which belonged to highly sensitive lines There are 3 strains with severe leaf withering before but gradually recovering growth, which belong to moderately sensitive strains; there are 4 strains without significant changes before and after spraying, which belong to resistant strains ( figure 2 )...
Embodiment 3
[0036] Example 3 Detection of Cytochrome P450 Gene CYP81A6 Expression
[0037] In order to further confirm that the herbicide dominant sensitivity caused by the interference carrier fragment is due to its inhibition of the cytochrome P450 gene CYP81A6, we detected the expression level of the CYP81A6 gene. Two plants from the leaves of the transgenic sensitive line, moderately sensitive line, and resistant line were taken respectively, and Zhonghua 11 was used as a negative control, total RNA was extracted, and cDNA was obtained by reverse transcription. In the Thermo PikoReal96 real-time fluorescent quantitative PCR system, the Actin gene was used as an internal reference to detect the expression of CYP81A6 gene. The reaction system and procedure are as follows:
[0038] RT-PCR primer sequences:
[0039]
[0040] Fluorescent quantitative PCR reaction system and procedures are as follows:
[0041] Program: pre-denaturation at 94°C for 7min, denaturation at 94°C for 15s, a...
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