Coding gene of peptidyl proacyl cis-trans isomerase
A peptidyl prolyl cis and isomerase technology, applied in the directions of isomerase, genetic engineering, plant genetic improvement, etc., can solve problems such as aggravating environmental pollution, drug residues, and increasing agricultural costs.
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Embodiment 1
[0026] Embodiment 1. Construction of Xanthomonas campestris mutant library and identification of XC2964 gene
[0027] Tn5gusA5 was introduced into Xcc wild-type strain 8004 using the shuttle plasmid pLAFR1 between E. coli and Xcc as a vector, then pLAFR1 was driven out by introducing the incompatible plasmid pPH1JI, and Xcc::Tn5gusA5 insertion was screened by an antibiotic (kanamycin) resistance marker Mutants, combined with Xcc 8004 whole genome sequence and TAIL-PCR (Thermal Asymmetric Interlaced-PCR) technology (Liu YG et al.1995 Efficient isolation and mapping of Arabidopsisthaiiana T-DNA insert junctions by thermal asymmetric interlacedPCR.Plant J.8: 457 -63.), determine the insertion position of Tn5gusA5 on the genome (specific method: first, amplify the DNA sequence downstream of the insertion of Tn5gusA5 by TAIL-PCR, then sequence the sequence, and then compare the sequencing sequence with the whole genome sequence of Xcc8004 Yes, get the insert location information). ...
Embodiment 2
[0029] Cloning and sequence determination of embodiment 2.XC2964 gene (peptidyl prolyl cis-trans isomerase gene)
[0030] According to the gene sequence of XC2964, primers were designed (see XC2964-F and XC2964-R), and the total DNA of Xanthomonas campestris was used as a template to amplify the full-length sequence of the gene by PCR (amplification conditions: 95°C (1min ); 35 cycles of 94°C (30s), 55°C (30s), 72°C (1min)]), and cloned it into the cloning vector pGEM3Zf(+) between the EcoRI and BamHI sites, and obtained the gene containing Recombinant plasmid pXC2964. The plasmid was digested with EcoRI+BamHI, and besides a 3.1kb vector band, there was also a 1.1kb insert (see figure 1 ). The DNA nucleotide sequence was determined on an ABI 377 DNA automatic sequencer (purchased from PE Company, USA) by the dideoxynucleotide method.
Embodiment 3
[0031] Construction and verification of embodiment 3.XC2964 gene deletion mutant
[0032] Using pGEM-3Zf(+) as a carrier, the kanamycin resistance gene (Kan gene, which is a gene commonly used in molecular biology methods) amplified by PCR method was cloned into the carrier. Design PCR primers (see instructions) to amplify the flanking sequence of XC2964 [amplification conditions: 95°C (1min); 30 cycles of 94°C (5s), 53°C (1min), 72°C (2min)] to obtain The left and right flanking sequences were named 2964L and 2964R, respectively. 2964L and 2964R were respectively cloned on both sides of the Kan gene on the vector (refer to Note 1 for the restriction sites), and the recombinant plasmid pGK2964 with the Kan gene and XC2964 flanking sequences was constructed. The insert in pGK2964 was cloned into the cosmid pLAFR3 with a wide host range to construct plasmid pLGK2964, and pLGK2964 was introduced into the wild-type 8004 strain through three-parent conjugation for homologous doubl...
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