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Phenylalanine ammonia lyase gene ThPAL derived from radix tetrastigme and application thereof

A technology of phenylalanine ammonia lyase and gene, which is applied in the field of phenylalanine ammonia lyase gene ThPAL and its application, can solve the problem of relatively little research at the molecular level

Active Publication Date: 2021-02-12
杭州鑫旺途科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on Clover mainly focuses on cultivation, seedling cultivation, extraction and separation of chemical components and pharmacological effects, etc., and there are few studies on its molecular level.

Method used

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  • Phenylalanine ammonia lyase gene ThPAL derived from radix tetrastigme and application thereof
  • Phenylalanine ammonia lyase gene ThPAL derived from radix tetrastigme and application thereof
  • Phenylalanine ammonia lyase gene ThPAL derived from radix tetrastigme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0025] Example 1 Obtaining the full-length sequence of the ThPAL gene cDNA of Sanyeqingqing

[0026] The leaves of the fresh plants of cloverleaf were taken, wrapped in tin foil, and quickly frozen with liquid nitrogen, and total RNA was extracted and reverse transcribed into cDNA. Total RNA was extracted according to the instructions of TIANGEN RNAprep Pure Plant Total RNA Extraction Kit (DP441), and its integrity and concentration were detected by 1.0% agarose gel electrophoresis and nucleic acid concentration detector.

[0027] Reverse transcription of total RNA according to Takara PrimeScript TM II 1st Strand cDNA Synthesis Kit instructions.

[0028] Based on the existing transcriptome data and the PAL gene sequences of the same family and genus in NCBI, BLAST analysis was performed, and the sequence with the highest similarity was selected as the target gene sequence, and multiple pairs of primers were designed based on the open reading frame sequence of this sequence. ...

Embodiment 2

[0033] Example 2 Secondary structure and tertiary structure prediction of ThPAL and phylogenetic tree analysis

[0034] Using the online software SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html) to predict the secondary structure of ThPAL protein in the biosynthetic pathway of resveratrol, the results like figure 2 As shown, the protein consists of 54.07% Alpha helix, 8.43% Extended strand, 5.34% Beta turn and 32.16% Random coil, It shows that the α-helical structure is the backbone of the secondary structure of protein PAL.

[0035]The online software SWISS-MODEL (http: / / swissmodel.expasy.org / ) was used to predict the three-dimensional structure of ThPAL protein in the biosynthetic pathway of resveratrol. The method was X-ray, and the resolution was The result is as image 3 shown. The template number used is 1w27.1.A, the sequence identity (Seq Identity) is 85.19%, the oligo-state (Oligo-state) is Homo-tetramer (homotetramer), and the seq...

Embodiment 3

[0044] Example 3 Functional verification of ThPAL gene

[0045] The cDNA sequence of ThPAL gene and the distribution of restriction sites on the plasmid vector pCMBIA1301 were analyzed, and PCR primers with SmaI and XbaI restriction sites (upstream primer: TCCCCCGGGATGGAAGCAAAGAACTGCAATGG; downstream primer: GCTCTAGATTAGCAGATCGGGAGT GGAGCA) were designed for Construction of overexpression vectors.

[0046] The cloverleaf cDNA was used as a template for PCR amplification. The reaction system was the same as above. After the amplification product was subjected to 1.0% agarose gel electrophoresis, the DNA fragment consistent with the target gene was purified and recovered by a kit. The purified and recovered product was double digested with plasmid pCMBIA1301 at 37°C, and then purified and recovered by agarose gel electrophoresis. The purified and recovered digested products were ligated with T4 DNA ligase and incubated at 16°C overnight. The ligation system was: 2 μL plasmid ve...

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Abstract

The invention discloses a phenylalanine ammonia lyase gene ThPAL derived from radix tetrastigme and an application thereof. The nucleotide sequence of the phenylalanine ammonia lyase gene ThPAL is shown as SEQ ID NO.1. According to the invention, radix tetrastigme is used as a biological source to design a primer to amplify the cDNA sequence of radix tetrastigme to obtain the phenylalanine ammonialyase gene ThPAL, and the gene is used as one of key enzymes in a phenylpropane metabolic pathway and can be used for producing resveratrol.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and mainly relates to a phenylalanine ammonia lyase gene ThPAL derived from cloverleaf and application thereof. Background technique [0002] As the state attaches great importance to traditional Chinese medicine in recent years, the government's strong support has driven the rapid development of the traditional Chinese medicine industry and expanded the market demand for Tetrastigma hemsleyanum Diels et Gilg. The continuous in-depth research on the anti-tumor effect of cloverleaf has led to a rapid increase in the market price of cloverleaf in recent years, and its market demand is also increasing. [0003] In recent years, the gene regulation of the synthesis of secondary metabolites in medicinal plants has become a very active frontier research field in molecular biology. The amount and composition of metabolites mainly depend on the key enzymes of biosynthesis and their expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/88C12Y403/01024C12N15/70C12P7/22
Inventor 夏鹏国张宇胡婉莹杨东风陈享梁宗锁
Owner 杭州鑫旺途科技有限公司
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