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Rhododendron phenylalanine ammonialyase PAL protein, coding gene and gene cloning method thereof

A technology of phenylalanine ammonia lyase and cloning method, applied in lyase, genetic engineering, carbon nitrogen lyase, etc., can solve the problems of complex biosynthesis process and unclear regulation mechanism, and achieve high application value

Pending Publication Date: 2021-07-23
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biosynthesis process of most floral aroma compounds is very complicated, and it is completed by multiple enzymes in different tissues. In the current study, the regulation mechanism of phenylalanine ammonia lyase on the downstream genes producing plant aroma substances is not yet clear

Method used

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  • Rhododendron phenylalanine ammonialyase PAL protein, coding gene and gene cloning method thereof
  • Rhododendron phenylalanine ammonialyase PAL protein, coding gene and gene cloning method thereof
  • Rhododendron phenylalanine ammonialyase PAL protein, coding gene and gene cloning method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the cloning of rhododendron PAL gene

[0035] 1. Acquisition of plant material

[0036] The plant material used in this experiment is Rhododendron yunnanensis, a high-quality germplasm resource of rhododendron. Yunjin Rhododendron is a rare aroma variety. The experimental materials were collected from Siming Mountain National Forest Park, Ningbo City, Zhejiang Province. Seedlings are raised, grown and flowered under natural conditions. The petals of Rhododendron in full bloom were collected for RNA and DNA extraction.

[0037] 2. Extraction of RNA and DNA

[0038] RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (the kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). After denaturation, 1% agarose gel electrophoresis was used to identify the integrity of RNA, and the purity and concentration of RNA were measured on a spectrophotometer (Thermo Scientific NanoDrop2000).

[00...

example 2

[0051] Example 2, the sequence information and homology analysis of Rhododendron PAL gene

[0052]The obtained PAL gene was predicted by ORF, 286-2430bp in the sequence was the ORF sequence, the length was 2145bp, and it was presumed that it encoded 715 amino acids. The molecular weight of the protein encoded by PAL is 77,58kDa, and the theoretical isoelectric point pI is 6.03. The genome of PAL contains 2 exons and 1 intron.

[0053] The open reading frame sequence of Rhododendron PAL and the amino acid sequence of its encoded protein were searched for nucleotide and protein homology using BLAST program on online analysis such as NCBI and EMBL.

example 3

[0054] Example 3, the expression difference of Rhododendron PAL gene in different tissues and the performance of petals in different developmental stages

[0055] 1. Acquisition of plant material

[0056] Petals at different developmental stages were collected, and leaves, petals, and male (gyne) pistils were collected during the flowering stage, and the samples were wrapped in ziplock bags, frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator.

[0057] 2. Extraction of RNA

[0058] RNA was extracted using the RNA prep Pure polysaccharide polyphenol plant total RNA extraction kit (the kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). After deformation, 1% agarose gel electrophoresis (1TAE; 120V, 20min) was used to identify the integrity of RNA, and the purity and concentration of RNA were determined on a spectrophotometer (Thermo Scientific NanoDrop2000).

[0059] 3. Acquisition of cDNA

[0060] Using 500ng of total R...

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Abstract

The invention discloses an rhododendron phenylalanine ammonialyase PAL protein and a coding gene thereof. The rhododendron phenylalanine ammonialyase PAL protein comprises an amino acid sequence in a sequence table and a coding gene thereof; the gene is expressed in leaves, stamens, pistils and petals, and the expression in the leaves is obviously higher than that in other tissues; and in different development stages, the expression quantity in the blooming stage is the maximum. The invention provides a theoretical basis for improving plant quality and obtaining plants with different flower fragrances by using a genetic engineering technology in the future, makes up the blank in the related fields of rhododendron research at present, and has application value.

Description

technical field [0001] The invention relates to a key enzyme in a rhododendron flower fragrance synthesis pathway and its coding gene, in particular to a rhododendron phenylalanine ammonia lyase (PAL) protein, a coding gene and a cloning method of the coding gene, and belongs to the field of biotechnology. Background technique [0002] Plant aroma substances are composed of many low molecular weight and volatile compounds, which are the products of secondary metabolism. The headspace solid-phase microextraction method was used to analyze the aroma composition in the petals of Rhododendron yunnanensis, and it was found that the relative content of benzene ring substances was the highest. Plant aroma substances phenylpropanoic acid / benzene ring compounds are mainly synthesized by phenylpropanoid metabolic pathway. Phenylalanine ammonia lyase is the key rate-limiting enzyme in the metabolic pathway of phenylpropanes, and is the most studied enzyme in plant secondary metabolism...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/10C12N15/70
CPCC12N9/88C12N15/1096C12N15/70C12Y403/01005C12Q2531/113
Inventor 吕思佳吴月燕贾永红谢晓鸿
Owner ZHEJIANG WANLI UNIV
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