Transgenic insect-resistant herbicide-resistant corn and cultivation method thereof
A technology of transgenic corn and herbicide tolerance, applied in the field of plant biology, can solve the problem of insect resistance without joint tandem expression
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Embodiment 1
[0049] Example 1: Obtaining of transgenic materials
[0050] 1. Codon optimization
[0051] According to the codon usage frequency of maize, the Cry1Ab, Cry1F and CP4EPSPS genes were comprehensively optimized to remove AT-rich sequences such as ATTTA and AATTAA that affect mRNA stability, and to increase the GC content of the gene coding region. Sequences to improve translation efficiency were added before the codons to enable efficient expression and translation in maize. The Cry1Ab, Cry1F and CP4EPSPS gene sequences were named mCry1Ab, mCry1F and mCP4EPSPS respectively based on the comprehensive codon optimization of maize codons. The nucleotide sequences are as follows: Sequences 1-3 are shown.
[0052] 2. Expression vector construction
[0053] The basic vector is pCAMBIA1300, and its selection marker in bacteria is Kan gene, which encodes aminoglycoside phosphotransferase, and there is no report that its encoded product has toxic side effects on animals. Since it is lo...
Embodiment 2
[0068] Example 2: Detection of genetic stability of target gene in transgenic material
[0069] The integration and genetic stability of target genes mCry1Ab, mCry1F, mCP4EPSPS in maize transformant BFL5 were analyzed by PCR technique.
[0070] The results showed that the target genes mCry1Ab, mCry1F, and mCP4EPSPS could all be detected in the BC4F1, BC5F1, and BC6F1 generation test populations, and the size of the positive specific fragment was consistent with the expected fragment size, and there was only one band. Each gene was stably inherited in each generation of the transformant, and its segregation ratio conformed to the Mendelian single-site inheritance rule, indicating that the three target genes were only integrated at one site.
[0071] The PCR band map of BFL5 target gene is attached figure 2 shown.
[0072] The primers for the PCR of the target gene are shown in Table 4 to Table 6, and the PCR composition and reaction conditions are shown in Table 7.
[0073]...
Embodiment 3
[0081] Example 3: BFL5 flanking sequence analysis and specific PCR detection
[0082] The 25 transgenic corn events obtained were first identified for glyphosate resistance, and 10 events had a resistance of more than 4 times. The insect resistance of the 10 events was identified respectively, and 4 events were screened out to be effective against pests such as corn borer. It showed high resistance, and further used the whole genome resequencing method to preliminarily determine the integration of the target gene in the maize genome. Among them, 1 event expression box had a sequence loss, 2 were inserted into the maize gene, and 1 was integrated on the maize chromosome. The intergenic region of BFL5 (named BFL5), further research on BFL5 found that it has no effect on the growth and development of maize, and the expression frame is complete, the gene expression level is high, and the inheritance is stable.
[0083] According to the obtained maize genome sequence next to the BF...
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