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, Cry3Bb and CP4EPSPS genes were comprehensively optimized to remove AT-rich sequences such as ATTTA, AATTAA and other sequences that affect mRNA stability, and to increase the GC content in the coding region. Add the sequence that improves the translation efficiency, so that it can be expressed and translated efficiently in maize. According to the codon optimized Cry1Ab, Cry3Bb and CP4EPSPS gene sequences in maize, they are respectively named as mCry1Ab, mCry3Bb and mCP4EPSPS, and the nucleotide sequences are as shown in sequence 1-3 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 located ou...
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, mCry3Bb, mCP4EPSPS in maize transformant BBL2 were analyzed by PCR technique.
[0070] The results showed that the target genes mCry1Ab, mCry3Bb, 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 BBL2 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.
[007...
Embodiment 3
[0081] Example 3: BBL2 flanking sequence analysis and specific PCR detection
[0082] The 30 transgenic corn events obtained were first identified for glyphosate resistance, and 13 events had a resistance of more than 4 times. The insect resistance was identified for each of them, and 5 events were screened out. High resistance, further using the whole genome resequencing method to preliminarily determine the integration of the target gene in the maize genome. One of the event expression frames was missing, 2 were inserted inside the maize gene, and 2 were inserted in the intergenic region. Further research found that the BBL2-2 event had no effect on the growth and development of maize, and the expression frame was complete and the gene expression was high. , genetically stable.
[0083] According to the maize genome sequence next to the obtained BBL2-2 insertion fragment, and further PCR amplification and sequencing method was used to obtain the maize genome specific sequen...
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