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Mevalonate kinase gene in synthetic route of ginkgo terpene lactones, encoded protein and application thereof

A technology of ginkgo mevalonate kinase and mevalonate kinase, which is applied in application, genetic engineering, plant gene improvement, etc., and can solve the problem that there is no mevalonate kinase gene

Inactive Publication Date: 2018-09-04
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an important gene in the terpene lactone synthesis pathway, there is no report of mevalonate kinase gene in Ginkgo biloba

Method used

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  • Mevalonate kinase gene in synthetic route of ginkgo terpene lactones, encoded protein and application thereof
  • Mevalonate kinase gene in synthetic route of ginkgo terpene lactones, encoded protein and application thereof
  • Mevalonate kinase gene in synthetic route of ginkgo terpene lactones, encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Obtaining of Ginkgo Mevalonate Kinase Gene

[0048] Extraction of total RNA from Ginkgo biloba leaves

[0049] Take 1 g of fresh ginkgo leaves, freeze and grind them into powder with liquid nitrogen, and use the MiniBEST Plant RNAExtraction Kit (TaKaRa Company) to extract ginkgo RNA. For specific methods, refer to the kit instructions.

[0050] The quality of sample RNA was detected by a nucleic acid analyzer.

[0051] Ginkgo RNA reverse transcription to obtain cDNA

[0052] The prepared ginkgo RNA was reverse-transcribed to obtain cDNA;

[0053] The first strand cDNA was synthesized using the PrimeScriptTM 1st strand cDNA synthesis Kit kit from TAKaRa Company, and the method was referred to the instruction manual.

[0054] Cloning of Ginkgo Mevalonate Kinase Gene

[0055] Ginkgo mevalonate kinase gene-specific primers Amplification-F and Amplification-R were designed according to Ginkgo transcriptome data, and RT-PCR amplification reaction was carried out...

Embodiment 2

[0058] Example 2 Ginkgo mevalonate kinase protein sequence analysis

[0059] Transmembrane structure analysis found that there are transmembrane structures at positions 113-135 and 148-170 of the protein ( figure 1 ), Ginkgo mevalonate kinase protein has 61%-63% similarity with other species of plant mevalonate kinase protein, such as plum (similarity 62%, accession number XP_008246488.1), Yali (similarity 63%, accession number XP_009349394.1), jatropha (62% similarity, accession number XP_012089078.1), jujube (62% similarity, accession number XP_015875214.1), rubber tree (63% similarity, accession number AIO11226. 1).

[0060] The results of multiple alignment of protein sequences showed that Ginkgo mevalonate kinase protein has the functional structure of mevalonate synthase, and the monomer structure of Ginkgo mevalonate kinase protein includes ribosomal protein S5 2-type, GHMP kinase N-terminal , GHMP kinase C-terminal structure. Isoleucine at position 146 to alanine at...

Embodiment 3

[0061] Phylogenetic Analysis of Example 3 Ginkgo Mevalonate Kinase

[0062] In order to study the evolutionary relationship between Ginkgo mevalonate kinase and other biological mevalonate kinases, this study screened the mevalonate kinase proteins of bacteria, fungi, gymnosperms and angiosperms respectively from the NCBI database. The phylogenetic tree of mevalonate kinase was constructed by NJ method (Neighbor-Joining), and the bootstrap value was set as 1000.

[0063] Such as image 3 As shown, the mevalonate kinases of different species also evolved from a common ancestor. The halophilic archaea and the thermophilic archaea constituted the bacterial clade, followed by the fungal clade composed of Gibberella fujikura and Candida dunili, and Ginkgo biloba. The valonate kinase gene constitutes an independent gymnosperm clade and appears earlier than the angiosperm clade.

[0064] Biological information shows that the Ginkgo mevalonate kinase protein has GHMP kinase activity...

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Abstract

The invention discloses a mevalonate kinase gene in the synthetic route of ginkgo terpene lactones, an encoded protein and application thereof. The invention discloses a nucleotide sequence of the gene and the gene encoded protein sequence. Tissue expression analysis shows that ginkgo biloba mevalonate kinase gene has the highest expression in leaves and significantly low expression in flowering organs. The yeast functional complementation of the ginkgo biloba mevalonate kinase gene verifies that the ginkgo biloba mevalonate kinase gene encoded protein has the enzymatic activity of mevalonatekinase and plays a catalytic role in the synthetic route of terpene lactones. Exogenous hormone methyl jasmonate and salicylic acid are employed to treat ginkgo biloba plants, the expression of ginkgobiloba mevalonate kinase gene and the content of terpene lactones are both increased. The ginkgo biloba mevalonate kinase gene provided by the invention is in positive correlation with the content ofterpene lactones in the synthetic route of ginkgo terpene lactones, and over-expression of the gene can improve the content of terpene lactones in ginkgo biloba.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the separation and cloning, functional verification and practical application of the mevalonate kinase gene of the ginkgolide lactone synthesis pathway. Background technique [0002] Ginkgo biloba L., also known as the Gongsun tree, can be traced back to the Carboniferous period 200 million years ago, and is the oldest relict plant in the world. At present, the medicinal value of ginkgo has received more and more attention. Studies have found that ginkgolides are important pharmaceutical ingredients for the treatment of cardiovascular and cerebrovascular diseases. However, the content of terpene lactones in ginkgo leaves grown under natural conditions is extremely low, and the ginkgo terpene lactones obtained by traditional methods are far from meeting the needs of the market. Therefore, new methods are needed to increase the content of ginkgo terpene lactones. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/82C12N1/19A01H5/00
CPCC12N9/1205C12N15/8243C12Y207/01036
Inventor 许锋宋启玲陈强文程水源廖咏玲叶家保周忠诚
Owner YANGTZE UNIVERSITY
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