Herbicide-tolerant protein as well as coding gene and application thereof
A herbicide and protein technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve problems such as insufficient herbicide tolerance and environmental friendliness to provide HPPD inhibitors
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
no. 1 example
[0112] The first embodiment, mutation and screening of HP1 gene
[0113] 1. Synthesis of HP1 gene
[0114] The nucleotide sequence of the HP1 gene was synthesized, as shown in SEQ ID NO:2 in the sequence listing, which encodes the HP1 protein, as shown in SEQ ID NO:1 in the sequence listing. The HP1M nucleotide sequence encoding the amino acid sequence corresponding to the HP1 was obtained according to the preferred codons of Arabidopsis thaliana, as shown in SEQ ID NO:3 in the sequence listing.
[0115] 2. Construction of the HP1 gene mutation library
[0116] After the above-mentioned synthetic HP1 gene was amplified by PCR, it was cloned into the vector pUC118 according to the operation steps of the Takara company product pUC118 vector (Takara, CAT: 3318), and then the above-mentioned ligated product was introduced into Escherichia coli DH5α as a template, Error-prone PCR with forward and reverse primers resulted in mutations in the HP1 gene due to random base mismatches....
no. 2 example
[0134] The second embodiment, detection of the tolerance effect of herbicide tolerance proteins HP1-1 and HP1-2 to HPPD inhibitor herbicides
[0135] 1. Synthesize the nucleotide sequences of HP1, HP1-1 and HP1-2
[0136] 5' and 3 of the HP1 nucleotide sequence (SEQ ID NO:2), HP-1 nucleotide sequence (SEQ ID NO:7) and HP1-2 nucleotide sequence (SEQ ID NO:10) The 'ends were respectively connected with universal adapter primer 1:
[0137] 5' end universal linker primer 1: 5'-taagaaggagatatacatatg-3', as shown in SEQ ID NO:12 in the sequence listing;
[0138] 3' end universal adapter primer 1: 5'-gtggtggtggtggtgctcgag-3', as shown in SEQ ID NO: 13 in the sequence listing.
[0139] 2. Construction of prokaryotic recombinant expression vectors and acquisition of recombinant strains
[0140] The prokaryotic expression vector DBNBC-01 was subjected to a double digestion reaction with restriction endonucleases Nde I and Xho I, thereby linearizing the prokaryotic expression vector, ...
no. 3 example
[0151] The third embodiment, the acquisition and verification of transgenic Arabidopsis plants
[0152] 1. Construct recombinant expression vectors of Arabidopsis thaliana containing HP1, HP1-1 or HP1-2 genes respectively
[0153] The HP1M nucleotide sequence (SEQ ID NO:3) described in the above-mentioned first embodiment 1, the HP1-1M nucleotide sequence (SEQ ID NO:8) described in the above-mentioned first embodiment 4, and the HP1-2M The 5' and 3' ends of the nucleotide sequence (SEQ ID NO: 11) are respectively connected with universal linker primer 2:
[0154] 5' end universal linker primer 2: 5'-agtttttctgattaacagactagt-3', as shown in SEQ ID NO:14 in the sequence listing;
[0155] 3' end universal linker primer 2: 5'-caaatgtttgaacgatcggcgcgcc-3', as shown in SEQ ID NO: 15 in the sequence listing.
[0156] The plant expression vector DBNBC-02 was subjected to a double digestion reaction with restriction endonucleases Spe I and Asc I, thereby linearizing the plant express...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More