Glycosyl transferase participating in neoandrographolide biosynthesis as well as coding genes and application thereof
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A new technology of andrographolide and glycosyltransferase, applied in the field of glycosyltransferase, can solve the problem of low yield
Active Publication Date: 2018-11-02
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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Its active ingredients are mainly extracted by chemical methods, and the yield is low
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Embodiment 1
[0054] Example 1. Discovery of glycosyltransferases involved in neoandrographolide biosynthesis and their encoding genes
[0055]First, the inventors found that methyl jasmonate can induce the accumulation of andrographolide in Andrographis paniculata leaves. Further, the inventors found that methyl jasmonate can also induce the accumulation of neoandrographolide in Andrographis paniculata leaves.
[0056] Through high-throughput whole-genome sequencing, nine genes were initially screened, the expression of which was induced by methyl jasmonate. Further, prokaryotic expression and functional verification of nine genes were carried out, and a glycosyltransferase involved in the biosynthesis of neoandrographolide was found.
[0057] The glycosyltransferase involved in the biosynthesis of neoandrographolide is shown in sequence 1 of the sequence listing (the expected molecular weight is about 100KDa), and the coding region in its cDNA is shown in sequence 2 of the sequence listi...
Embodiment 2
[0058] Embodiment 2, function verification
[0059] 1. Construction of recombinant plasmids
[0060] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.
[0061] 2. Using the DNA molecule obtained in step 1 as a template, a primer pair composed of F1 and R1 is used for PCR amplification, and the PCR amplification product is recovered.
[0064] 3. Take the vector HIS-MBP-pET28a, perform double digestion with restriction endonucleases BamHI and SalI, and recover the linearized vector backbone.
[0065] 4. Take the PCR amplification product obtained in step 2, use the pEASY-Uni Seamless Cloning and Assembly Kit and operate according to the instructions, and clone it into the vector HIS-MBP-pET28a to obtain a recombinant plasmid.
[0066] After sequencing, the recombinant plasmid has an open reading frame shown in sequ...
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Abstract
The invention discloses glycosyl transferase participating in neoandrographolide biosynthesis as well as coding genes and application thereof. The protein provided by the invention is derived from herba andrographitis, and is (a1) or (a5) as follows: (a1) a protein composed of an amino acid sequence shown as a sequence 1 in a sequence table; and (a5) a protein composed of an amino acid sequence shown as a sequence 3 in the sequence table. The invention further discloses application of the protein serving as glycosyl transferase. The glycosyl transferase participates in plant growth and development and secondary metabolism processes. The discovery of the glycosyl transferase and coding genes thereof has important significance. The invention provides a basis for further describing biosynthesis of glycoside compounds in the herba andrographitis, contributes to analyzing a biosynthetic pathway of glycoside active ingredients in the herba andrographitis, and provides novel material basis and gene resources for new drug research and development.
Description
technical field [0001] The invention relates to a glycosyltransferase involved in the biosynthesis of neoandrographolide, its coding gene and application. Background technique [0002] Andrographis paniculata (Burm.f.) Nees is the dry aerial part of the plant Andrographis paniculata (Burm.f.) Nees. It has the functions of clearing heat and detoxifying, cooling blood and reducing swelling, and is widely used clinically. At present, Andrographis paniculata is not only used as a traditional Chinese medicine andrographis paniculata, but also made into a variety of modern dosage forms for clinical use, such as Chuanxinlian tablets, Chuanxinlian capsules, Chuanxinlian injection, Xiyanping injection and Chuanhuning injection, etc. Its active ingredients are mainly extracted by chemical methods, and the yield is low. Compared with chemical synthesis methods, biosynthesis avoids environmental pollution caused by the use of heavy metals, organic solvents, etc., and the substrate spec...
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