Siderophilic Synthetic Gene Cluster and Its Application
A technology for synthesizing genes and siderophils, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as no reports of siderophil biosynthesis gene clusters.
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Embodiment 1
[0079] Example 1 Extraction of Amycolatopsis orientalis CGMCC No.6023 Total DNA
[0080] 1) Take fresh slant spores and inoculate them in 20ml of YMB liquid medium, culture at 28°C and shake at 220rpm for 36hrs.
[0081] 2) Collect the mycelia by centrifugation (3000g, 15min), and wash once with sterile water.
[0082] 3) The mycelia were suspended in 3ml TE and dispersed by vortexing.
[0083] 4) Add 300ul lysozyme (10mg / ml) and 30ul RNase (10mg / ml), bathe in water at 37°C for 90min, and shake continuously during this period.
[0084] 5) Add 210 ul of 20% SDS and 30 ul of proteinase K (10 mg / ml), and bathe in water at 55° C. for 2 hr.
[0085] 6) Add 600ul of 5M NaCl and 480ul of 10% CTAB, and bathe in water at 65°C for 30min.
[0086] 7) After cooling to room temperature, add an equal volume of chloroform:phenol:isoamyl alcohol (25:24:1), mix well, and centrifuge (8900g, 15min).
[0087] 8) Take the supernatant, add an equal volume of chloroform, mix well, and centrifuge...
Embodiment 2
[0090] Example 2 High-throughput sequencing
[0091] 1) Fragmenting the DNA obtained in Example 1 to construct a sequencing library;
[0092] 2) High-throughput sequencing was performed using the GS FLX sequencer of Roche 454 Company: 561,423 reads (reads) were obtained. After splicing with the assembly software that comes with 454 Company, the number of contigs was 56 (>500bp), and the coverage 25.6 times that of the whole genome.
[0093] 3) Carry out further splicing and assembly to complete the gap closing work: A total of 548 specific PCRs and shotgun sequencing of 2 fosmid clones were designed, and the complete genome sequence of Amycolatopsis orientalis CGMCC No.6023 was finally assembled. Among them, the error rate per 100,000 bases is less than 0.5, exceeding the international requirement that the error rate per 100,000 bases be less than 1. Whole genome assembly uses the phred / phrap / consed data package.
Embodiment 3
[0094] Example 3 Genome Functional Annotation
[0095] 1) Determination of the open reading frame (ORF): firstly predict it with Glimmer3.02, and then use Genemark and Z-curve to correct it.
[0096] 2) Protein function annotation: For the predicted CDSs, preliminary gene annotation was performed by comparing the KEGG and NR databases through BLASTP, and all annotation results were manually corrected; while the orthologous genes (clusters of orthologous groups, COGs) and protein conservation The prediction of the structural domain is obtained by comparing the CDD database of NCBI, and the confirmation parameters of COGs are identity≥30%, and length diversity≤20%.
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