RNA helicase mutant, mutant gene and application of RNA helicase mutant in preparation of vitamin B2
A technology of RNA helicase and mutants, applied in the biological field, can solve problems such as RNA helicase mutant genes, and achieve the effect of improving ability
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Embodiment 1
[0035] CshB validation and verification of gene mutation: Example 1
[0036] Since cshB Gene in Bacillus subtilis is not only responsible for maturation of ribosomal 70s, further responsible for encoding RNA helicase, i.e., the control of mRNA degradation, thus selected for site-directed mutagenesis cshB gene.
[0037] The inventors of the gene encoding for the RNA helicase cshB, analyzed using software Jalview highly conserved amino acid sequence regions. While using Swiss-Model tool cshB RNA encoded by the gene for protein helicase simulation modeling, using the active center of the enzyme HotSpot Wizard 2.0 predict. Found genes encoding RNA helicase CSHB 236 amino acid sequence of the enzyme, near the active site and a highly conserved region, subjected to site-directed mutagenesis is likely to lead to decreased RNA helicase activities.
[0038] The present inventors have pre-screened out a high yield of vitamin B 2 Bacillus subtilis (CGMCC NO.4018), may be fermentative product...
Embodiment 2
[0041] Example 2: Construction cshB revertant strain 18-cB
[0042] In Bacillus subtilis 168 chromosome as a template, with primers UPcshB-F, UPcshB-R (including DR) amplification of the wild-type UPcshB with connectors (including DR) fragments, spread with primers araR-F, araR-R (including DR) fragment increased with araR linker (containing the DR); in pC194 plasmid as a template, with primers cat-F, cat-R to amplify cat fragment with a linker; Bacillus subtilis Bacillus subtilis chromosome of 168 as a template, with primers DNcshB- F, DNcshB-R fragment was amplified downstream homology arm DNcshB, wherein the nucleotide sequence of the gene cshB amino acid sequence shown in SEQ ID, encoded by SEQ IDNo.3 No.1 as shown.
[0043] In UPcshB fragment, cat fragment, ARAR fragments and DNcshB fragments as template, with primers UPcshB-F, DNcshB-R fusion the PCR, to obtain assembled fragment UCR-cshB, by nucleic acid electrophoresis correct, gel recovered to obtain a purified UCR-cshB f...
Embodiment 3
[0049] Example 3: Construction of strain 19-cB mutants containing cshB
[0050] In synthetic RNA helicase mutant (L236R) encoding gene as template, with primers UPcshB-F, UPcshB-R (including DR) amplification UPcshB (including DR) and with a point mutation with connector clips, with primer araR-F, araR-R (including DR) amplification ARAR with connectors (including DR) fragments; in pC194 plasmid as a template, with primers cat-F, cat-R to amplify cat fragment with a linker; to Bacillus coli Bacillus subtilis 168 chromosome as a template, with primers DNcshB-F, DNcshB-R fragment was amplified downstream homology arm DNcshB. Wherein said RNA helicase nucleotide sequence (L236R) of mutations such as amino acid sequence shown in SEQ ID No.2 coded as shown in SEQ ID No.4.
[0051] In UPcshB fragment, cat fragment, ARAR fragments and DNcshB fragments as template, with primers UPcshB-F, DNcshB-R fusion the PCR, to obtain assembled fragment UCR-cshB, by nucleic acid electrophoresis correc...
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