Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene

A technology of phenylalanine ammonia lyase and honeysuckle, applied in the field of biological and medicinal plant genetic engineering

Inactive Publication Date: 2013-10-30
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the present invention was published, there was no disclosure or report of the phenylalanin...

Method used

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  • Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene
  • Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene
  • Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene

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Experimental program
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Embodiment 1

[0018] Embodiment 1, the construction of honeysuckle full-length cDNA library

[0019] 1. Isolation and detection of honeysuckle total RNA

[0020] Take 2 g of honeysuckle (Lonicera japonica Thunb) flowers, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer to 10 mL of extraction buffer (CTAB (W / V) 2%, Tris-HCl ( pH8.0) 100mmol L -1 , EDTA 25mmol·L -1 , NaCl 2.0mol·L -1 , PVP40 2%, spermidine 0.5g / L, mercaptoethanol 2%), fully shake and mix; extract twice with equal volume of chloroform, and centrifuge at 7500g for 15 minutes. Add 1 / 4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, and use 500 μL SSTE (SDS 0.5%, NaCl 1mol L -1 , Tris-HCl (pH8.0) 10mmol L -1 , EDTA 1mmol·L -1 , dissolved at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13,000g for 5 minutes; add 2 times the volume of absolute ethanol to the supernatant, and ...

Embodiment 2

[0023] Embodiment 2: Cloning of honeysuckle-related genes

[0024] Randomly pick 5000 single clones for colony PCR identification. Take an appropriate amount of PCR thin-walled tubes, place them on ice, and add 17.3ul of sterilized water to each tube. Use a sterilized 10ul small tip to pick up the monoclonal white spot into sterilized water, shake and mix. Add in sequence: Taq buffer 2.5μL, MgCl 2 (25mM) 1.8μL, dNTP (2.5mM) 1μL, M13+ primer (10pmol) 1μL, M13-primer (10pmol) 1μL, Taq enzyme 0.4μL. The PCR reaction conditions were 5 minutes of pre-denaturation at 94°C, 40 seconds at 94°C, 40 seconds at 54°C, 4 minutes at 72°C, 35 cycles of extension at 72°C for 10 minutes, and storage at 4°C. After the PCR reaction enters 4°C, remove the thin-walled PCR tube, take 7ul of the PCR product and add 3ul of Bromine Finland to run electrophoresis, take a picture half an hour later, observe the gel map, and roughly identify the size of the inserted fragment and the small fragment rat...

Embodiment 3

[0025] Embodiment 3, the bioinformatics analysis of LJPAL gene

[0026] The length of the full-length cDNA of the honeysuckle phenylalanine ammonia-lyase gene involved in the present invention is 2145bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 1-2145bp. The full-length cDNA sequence of Lonicerae japonica was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR databases using BLAST program. The gene has high homology with PAL in other species at the amino acid level, and has a typical PAL-HAL domain. Such as figure 1 .

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Abstract

The invention discloses a lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as a protease coded by the same and application of the gene. The LJPAL gene has nucleotide sequences shown in SEQ ID NO.1 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of nucleotides or nucleotide sequences derived from alleles of the gene and the gene. A protein coded by the gene has amino acid sequences shown in SEQ ID NO.2 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of amino acids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to the use of a full-length cDNA library to clone the phenylalanine ammonia-lyase gene and its encoded product and its application, especially to the biosynthesis of organic acids and flavonoid enzyme genes with pharmacologically active ingredients and their encoding The product and application belong to the field of medicinal plant genetic engineering. Background technique [0002] The formation of medicinal plant active ingredients is the product of a unique group of genes in plant secondary metabolic pathways. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to secondary metabolism synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. It provides a theoretical basis for the biosynthetic pathway and its regulation mechanism and explains the formati...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N1/21C12R1/19
Inventor 黄璐琦袁媛汪周勇蒋超
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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