Cordyceps sinensis hirsutella-sinensis malic dehydrogenase B, encoding gene and application of two

A technology of malate dehydrogenase and Trichosporum sinensis, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of not being able to search and achieve high expression, great application prospects, and the effect of expanding biological applications

Inactive Publication Date: 2014-11-05
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] However, the NCBI database is currently unable to retrieve the gene-related information of malate dehydrogenase in Mortierella chinensis

Method used

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  • Cordyceps sinensis hirsutella-sinensis malic dehydrogenase B, encoding gene and application of two
  • Cordyceps sinensis hirsutella-sinensis malic dehydrogenase B, encoding gene and application of two
  • Cordyceps sinensis hirsutella-sinensis malic dehydrogenase B, encoding gene and application of two

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Cultivation of "Bailing" production fungus Cordyceps sinensis

[0041] Source of the strain: Firstly, the natural Cordyceps sinensis was collected from Qinghai, and brought back to Hangzhou for isolation and screening, and the L0106 strain was obtained, and the strain was identified as Hirsutella sinensis, which was preserved in a typical culture in China The collection center, the deposit number is CCTCC No: M2011278, which has been disclosed in the previously applied patent CN102373190A.

[0042] This bacterial classification is inoculated on the slant, and the culture medium formula (this is the liquid formula before solidification, makes the slant after being prepared according to the following ratio) is: glucose 2.0% (w / v, 1% means that 100mL culture medium contains 1g, the same below), corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm chrysalis powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydroge...

Embodiment 2

[0043] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0044] Extract total RNA with TRIzol reagent, the steps are as follows:

[0045] 1) Grinding with liquid nitrogen: Put 1 g of fresh bacteria into a mortar, add liquid nitrogen repeatedly to grind until powdery, dispense into pre-cooled 1.5 mL centrifuge tubes, add 1 mL of TRIzol reagent, mix well, and stand on ice. Set aside for 5 minutes to completely separate the nucleic acid-protein complex.

[0046] 2) RNA isolation: Add 0.2 mL of chloroform, shake vigorously for 15 s, let stand on ice for 2-3 min, centrifuge at 4°C, 12000 rpm for 15 min, separate the layers, and take the upper aqueous phase, about 600 μL.

[0047] 3) RNA precipitation: add 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, and discard the supernatant.

[0048] 4) RNA washing: add 1 mL of 75% (v / v) ethanol, suspend the precipitate, let stand on ice for 10 min, ...

Embodiment 3

[0050] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

[0051] After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments (200-700bp), use mRNA as a template, use six-base random primers (random hexamers) to synthesize the first cDNA strand, then synthesize the second cDNA strand, and then pass through QiaQuick PCR After the kit is purified and eluted with EB buffer, end repair is performed, polyA is added and sequencing adapters are connected, and then agarose gel electrophoresis is used for fragment size selection, and finally PCR amplification is performed. The built sequencing library is carried out with Illumina GA IIx sequencing. The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. The reads containing only the adapter sequence in the original sequ...

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Abstract

The invention relates to malic dehydrogenase A which comes from corbrin-capsule-producing strain cordyceps sinensis hirsutella sinensis, participates tricarboxylic acid cycle and is used for citric acid synthesis, a gene encoding the malic dehydrogenase B, and application of the above two. The amino acid sequence of the malic dehydrogenase B and protein shown as SEQ ID No. 1 have 90% or more of homology, and the nucleotide sequence of the encoding gene and a sequence shown as SEQ ID No. 2 have 90% or more of homology. A cloned DNA of the provided nucleotide sequence can be transferred into an engineering bacterium through a transduction, transformation or conjugal transferring method. By adjusting expression of the encoding gene corresponding to the enzyme catalyzing malic acid for preparing corresponding oxobutanedioic acid, a host is endowed with property of highly expressing malic dehydrogenase B, an effective approach is provided for expanding biological application of malic dehydrogenase A, and important application prospect is provided.

Description

(1) Technical field [0001] The invention relates to a malate dehydrogenase B (Malic dehydrogenase) from the "Bailing" production fungus Cordyceps sinensis which participates in the tricarboxylic acid cycle and is used in the synthesis of citric acid, the gene encoding the enzyme and its application. (2) Background technology [0002] Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is a complex of Cordyceps sinensis parasitizing on the larvae of Lepidoptera (Lepidoptera) bat moth (Hepialus armoricanus Oberthur) larvae and larval corpses (including subunits and worm bodies ). Cordyceps sinensis is a kind of precious traditional fungal medicinal resources, which has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention for its extensive and obvious medicinal effects, and is highly respected all over the world. Chinese med...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C12P7/46C12R1/19
CPCC12N9/0006C12P7/46C12Y101/01037C12Y101/01038C12Y101/01039C12Y101/0104
Inventor 柳志强郑裕国林善薛亚平吴晖李邦良许静许峰袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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