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Cane sugar non-fermentation type protein kinase regulatory subunit and application thereof

A protein kinase and regulatory subunit technology, applied in the fields of genetic engineering and microbial engineering, can solve the problem of less functional analysis of the SNF1 complex and achieve the effect of reducing production costs

Active Publication Date: 2020-01-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of the regulatory subunit in the SNF1 complex, that is, the γ subunit, especially the mediating role between the response to nutritional signals and the regulation of intracellular metabolism, has not been revealed, and in filamentous fungi, especially oleaginous silk In fungi, there are few studies on the SNF1 complex and its functional analysis

Method used

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  • Cane sugar non-fermentation type protein kinase regulatory subunit and application thereof
  • Cane sugar non-fermentation type protein kinase regulatory subunit and application thereof
  • Cane sugar non-fermentation type protein kinase regulatory subunit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Screening of genes encoding regulatory subunits of sucrose nonfermenting protein kinases

[0048] According to the gene sequence of the sucrose non-fermenting protein kinase regulatory subunit whose function has been identified in NCBI as a template, perform BLAST comparison in the gene bank of the sequenced M.alpina ATCC 32222 strain to obtain an alternative target gene; then Alternative target gene is carried out secondary alignment screening in NCBI storehouse, the target protein that finally obtains is named as Masnf4 (amino acid sequence is as shown in SEQ ID No.1), and its coding gene is named as Masnf4 (nucleotide sequence As shown in SEQ ID No.2).

[0049] The full length of Masnf4 corresponding cDNA is 1596bp, encoding 531 amino acids. In order to further determine whether the screened Masnf4 belongs to the regulatory subunit of sucrose non-fermenting protein kinase, the amino acid homology and conserved structure analysis was carried out with the A...

Embodiment 2

[0057] Example 2: Cloning of Masnf4

[0058] The total RNA of Mortierella alpina (Mortierella alpina) ATCC 32222 was extracted using the Trizol method, and cDNA was obtained by reverse transcription according to the instructions of the Takara reverse transcription kit. The reaction amplifies Masnf4, and the primers used for amplifying Masnf4 are listed in Table 2.

[0059] The PCR instrument used is BIO-RAD T100 Thermal Cycler, using KOD plus high-fidelity DNA polymerase, the reaction system is 50 μL, and the content of the system is carried out according to the instructions of the DNA polymerase; the reaction process is as follows: pre-denaturation at 95°C for 5 minutes, then denaturation at 95°C 30s, annealing at 58°C for 30s, extension at 68°C for 1.5min, repeat the above three steps 30 times, then fully extend at 68°C for 7min, and finally drop to 4°C for 10min and stop.

[0060] After the reaction is completed, the amplified product is obtained, and after the amplified p...

Embodiment 3

[0064] Example 3: Expression and RNA interference of Masnf4 in Mortierella alpina

[0065] (1) Construction of Mortierella alpina expression vector

[0066] Masnf4 obtained in Example 2 and the expression vector pBIG2-ura5s-ITs were digested using restriction endonucleases HindⅢ and SmaI, and then T 4 Ligase ligated the digested and purified DNA to obtain a ligation product. The specific enzyme digestion system (20 μL) is shown in Table 3.

[0067] Table 3 enzyme digestion system

[0068] Reagent Dosage 10×cutmart buffer 2μL restriction endonuclease 1μL PCR product or vector 200ng or 1μg wxya 2 o

Make up to 20μL

[0069] After connecting the obtained ligation product overnight at 4-16°C, transform it into Escherichia coli DH5α competent cells. The transformation method is as follows: take 100 μL of competent cells in a sterile state, add 5-8 μL of the ligation product, and mix by pipetting; Transfer the mixed competent cells i...

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Abstract

The invention discloses a cane sugar non-fermentation type protein kinase regulatory subunit and an application thereof, and belongs to the technical fields of genetic engineering and microorganism engineering. The cane sugar non-fermentation type protein kinase regulatory subunit of which the amino acid sequence is shown as SEQID No.1 has the function of responding extracellular nutrient level and adjusting and controlling lipid metabolism, and participates in response of thalli to extracellular glucose level. The gene has the negative regulation functions on growth and lipid accumulation, and can supply energy for the thalli by restraining growth of mountain mortierella and strengthening catabolism of fatty acids under the action of limitation by glucose.

Description

technical field [0001] The invention relates to a sucrose non-fermenting protein kinase regulatory subunit and its application, especially the application in responding to extracellular nutrient levels and regulating lipid metabolism, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Mortierella alpina is an oleaginous filamentous fungus with strong lipid accumulation ability, which can synthesize a variety of biologically active polyunsaturated fatty acids (PUFAs), which has good industrial development value. The level of carbon and nitrogen sources is an important factor affecting the lipid accumulation of Mortierella alpina. High sugar or nitrogen limitation can promote its lipid accumulation. This phenomenon exists widely in oleaginous microorganisms. Previous studies on oleaginous microorganisms mostly focused on the optimization of fermentation conditions, or the genetic modification of genes related to li...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N1/15C12P7/64C12R1/645
CPCC12N9/12C12P7/64C12Y207/11011
Inventor 陈海琴唐鑫常璐璐赵建新张灏陈卫
Owner JIANGNAN UNIV
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