Conch alkali synthase and application thereof
A technology for synthesizing enzymes and conchalkine, applied in the biological field, can solve the problems of complex process flow, high cost, unclear biosynthetic pathways of secondary metabolites, etc.
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Embodiment 1
[0033] Embodiment 1, the cloning of conch synthase (littorine synthase, LS) gene
[0034] (1) Extraction of total RNA from belladonna fibrous root
[0035] Take an appropriate amount of belladonna fibrous root tissue, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and the RNA concentration was determined on a spectrophotometer.
[0037] Using the extracted total RNA as a template, synthesize cDNA according to the instructions of the Tiangen FastKing cDNA First Strand Synthesis Kit; design gene-specific primers, and the specific primers are as follows:
[0038] F: 5'-gtattagtttctggcttgtaattca-3' (SEQ ID NO.7);
[0039]R: 5'-cctactctgttatccaagaaaaaga-3' (SEQ ID NO.8);
[0040] The LS gene was amplified from total cDNA by...
Embodiment 2
[0042] Example 2, using tobacco to verify the function of the LS gene
[0043] (1) LS expression vector construction
[0044] Since there is no commercial source of phenalactoylglucose required for conchine biosynthesis, tobacco was used to verify the function of LS. The LS transient expression vector pEAQ-LS was constructed, and pEAQ-YFP was used as a control. In vector construction, the restriction site of AgeI was introduced into the forward primer, and the restriction site of XhoI was introduced into the reverse primer. The specific primers are as follows:
[0045] pEAQ-LS-F: 5'-cgc accggt atgaagaaaacaattgtggttcca-3' (SEQ ID NO. 3);
[0046] pEAQ-LS-R:5'-cgc ctcgag ttatagaggttgataatatatcc-3' (SEQ ID NO. 4);
[0047] pEAQ-YFP-F:5'-cgc accggt atggtgagcaagggcgagga-3' (SEQ ID NO. 9);
[0048] pEAQ-YFP-R:5'-cgc ctcgag ttacttgtacagctcgtccatg-3' (SEQ ID NO. 10).
[0049] Then use the total cDNA of belladonna as a template, and the sequences shown in SEQ ID NO.3 and S...
Embodiment 3
[0060] Example 3, Agrobacterium rhizogenes-mediated LS overexpression vector genetically transforms belladonna to obtain transgenic belladonna hairy roots
[0061] (1) Construction of plant binary overexpression vector containing LS gene
[0062] In order to study the effect of LS gene on tropine alkaloids in belladonna, the LS overexpression vector pBI121-LS was constructed. In vector construction, a BamHI restriction site was introduced into the forward primer, and a SacI restriction site was introduced into the reverse primer. The primers are as follows:
[0063] pBI121-LS-F: 5'-cgc ggatcc atgcattataactcaacgtt-3' (SEQ ID NO.5)
[0064] pBI121-LS-R:5'-cgc gagctc ttagaatctagagagaaact-3' (SEQ ID NO.6)
[0065] Then take the belladonna total cDNA as template, and the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers to carry out PCR amplification, and the amplified product is digested with BamHI and SacI and connected to BamH I and Sac I of the pBI121 plasmid Res...
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