CN107287222B

CN107287222B - Application of Hisk2301 gene of histidine kinase

Info

Publication number: CN107287222B
Application number: CN201710593680.6A
Authority: CN
Inventors: 张琦; 肖虎; 魏云林; 林连兵; 季秀玲
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2017-07-20
Filing date: 2017-07-20
Publication date: 2020-07-10
Anticipated expiration: 2037-07-20
Also published as: CN107287222A

Abstract

The invention discloses a histidine kinase geneHisk2301The use of, i.e., histidine kinasesHisk2301The gene is used for producing the polyunsaturated fatty acid at low temperature, the content of the polyunsaturated fatty acid L A and A L A is improved by modifying microorganisms through a genetic engineering means, good application prospect and economic benefit are provided for the industrial production of the polyunsaturated fatty acid, and a foundation is laid for the large-scale commercial production of the polyunsaturated fatty acid.

Description

Application of Hisk2301 gene of histidine kinase

Technical Field

The invention belongs to the field of biotechnology and genetic engineering, and relates to a histidine kinase geneHisk2301 and its application.

Background

The oleaginous yeast cells are rich in polyunsaturated fatty acids (PUFAs), and the PUFAs have a wide range of physiological functions in human bodies, and synthetic changes of the PUFAs can cause various diseases such as cardiovascular diseases, obesity, non-insulin-dependent diabetes mellitus, hypertension, neurological diseases, cancers and the like. PUFAs also have important functions in the aspect of cell membrane biology, and the relative content of PUFAs in the membrane lipid of the cell membrane has important influence on the physical properties of most biological membranes, such as the fluidity of the cell membrane, and the stable fluidity has very important function for maintaining the normal functions of the cell membrane. The problems of the traditional PUFAs sources in the aspects of oil and fat in the square cells such as yield, quality, production cost, safety and the like cannot meet the large demands of the market, and research and development of new PUFAs resources become hot spots. The oil-producing yeast has similar components to common vegetable oil, and the oil in the oil-producing yeast has unique and definite biochemical and physicochemical properties, so that the oil-producing yeast has important potential application value in industry as a substitute of vegetable edible oil in life and a raw material for producing biodiesel. In addition to obtaining high-yield oil through fermentation technology research, research on improving the activity or gene expression level of fatty acid dehydrogenase under different conditions to improve the composition and content of PUFAs in cells has also become a research hotspot in the field.

The extraction of PUFAs from animal and vegetable fats and oils has been studied for a long time, but the growth of animals and plants is changed with the influence of seasons, geographical locations, etc., so that the content and composition of PUFAs are changed, and the extraction of PUFAs from animals and plants is costly, long in cycle, and not suitable for the market. In addition, the oil content of animal and vegetable oil resources, the type and the proportion of unsaturated fatty acid are limited to a certain extent. Therefore, in recent years, new sources of PUFAs have been explored-namely, microbial technology for producing PUFAs; the microorganism has the advantages of short fermentation period, high oil content, low culture cost, no raw material control, strong environmental adaptability, high biological conversion rate and the like.

Histidine Kinases (HK) are transmembrane transferase-like proteins that act as cellular receptors for signaling molecules during signal transduction. HK generally transmits signals as a component of Two-component signal transduction systems (TCSs), which are commonly found in prokaryotes and eukaryotes and mainly consist of an HK and a response regulator protein (RR). The two-component signal transduction system participates in regulation of various mechanisms such as cell growth, stress resistance, secondary metabolite regulation, cell virulence maintenance and the like. Therefore, HK plays an important regulatory role in many cell biological processes, but no research on the promotion of polyunsaturated fatty acid synthesis by HK has been reported so far.

Disclosure of Invention

The purpose of the invention is to provide a histidine kinase geneHisk2301The novel use of histidine kinase geneHisk2301Use in the production of polyunsaturated fatty acids at low temperatures.

Histidine kinase geneHisk2301From Rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) YM25235, the nucleotide sequence of the gene is shown in SEQ ID NO:1 or a fragment of the nucleotide sequence shown in SEQ ID NO:1, the length of the gene sequence is 3888bp (basic group), and the amino acid sequence coded by the gene is shown as SEQ ID NO: 2 or a fragment thereof.

The present invention relates to histidine kinase geneHisk2301Directly connecting with different expression vectors (plasmids, viruses or vectors) to construct a recombinant vector, and promoting the rhodosporidium toruloides YM25235 to produce polyunsaturated fatty acids at low temperature.

In particular to a method for preparing a compound from rhodosporidium toruloidesRhodosporidium kratochvilovae) Histidine kinase gene cloned in YM25235Hisk2301Inserting pRH2034 plasmid to construct recombinant expression plasmid pRHHisk2301, transferring the recombinant expression plasmid pRHHisk2301 into Rhodosporidium toruloides YM25235 for overexpression to obtain transgenic strain YM25235/pRHHisk2301, and researching the expression characteristics of the transgenic strain YM25235/pRHHisk2301 to lay a foundation for clarifying Hisk2301 gene and low-temperature adaptability mechanism thereof and synthesis mechanism of polyunsaturated fatty acid at low temperature.

The invention has the beneficial effects that: the invention relates to a method for preparing a red wintergreen spore yeast (Rhodosporidium toruloides)Rhodosporidium kratochvilovae) Gene of core molecule histidine kinase of two-component system separated from chromosome genome of YM25235Hisk2301. The full length of the gene is 3888bp, and Hisk2301 is considered to be mainly involved in response to osmotic stress in previous researches. The research shows that the gene Hisk2301 not only has the function of osmotic stress resistance, but also is related to salt pressure resistance and low-temperature synthesis of PUFAs in the rhodosporidium toruloides, and the gene can improve delta for catalyzing oleic acid to be converted into linoleic acid and linolenic acid in the rhodosporidium toruloides YM25235 at low temperature^12/15This study will help elucidate the signal transduction pathway regulating the synthesis of linoleic acid and α -linolenic acid in Rhodosporidium toruloides YM25235 at low temperature, reveal the regulatory mechanism of the synthesis of linoleic acid and α -linolenic acid in YM25235 strain at low temperature, and will help further elucidate the regulatory mechanism of polyunsaturated fatty acid synthesis in fungi, and provide reference for the study of elucidating the mechanism of adaptation to fungi at low temperature, and explore the mechanism of synthesis of polyunsaturated fatty acids at low temperatureThe formed regulating mechanism is beneficial to utilizing the relevant synthesis regulating process, and lays a foundation for the research and application of large-scale production of polyunsaturated fatty acid in the future.

Drawings

FIG. 1 is a PCR amplification map of Hisk2301 gene of Rhodosporidium toruloides YM25235 of the present invention;

FIG. 2 is a plasmid map of recombinant plasmid pRHHisk 2301;

FIG. 3 shows restriction analysis of recombinant plasmid pRHHisk 2301; wherein: 1. of empty plasmid pRH2304BamHⅠ、EcoRV, double enzyme digestion; 2. of recombinant plasmid pRHHisk2301BamHⅠ、EcoRV, double enzyme digestion, 3, PCR products of Hisk2301 gene, 4, DNA molecular weight mark D L15000;

FIG. 4 shows gas chromatography analysis of fatty acids in Hisk2301 transformed Rhodosporidium toruloides YM25235 cells under low temperature conditions A YM25235/pRH2304 at 15 ℃; YM25235/pRHRKHisk2301 at 15 ℃.

Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and conventional methods.

Example 1: rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) YM25235 histidine kinase GeneHisk2301Cloning of genes

The OMEGA Kit E.Z.N.A Fungal RNA Kit is adopted to extract the total RNA of the Rhodosporidium toruloides YM25235, and the reverse transcription Kit Takara First Strand cDNA Synthesis Kit is adopted to synthesize cDNA. Designing specific primers (a primer 1 and a primer 2) according to a transcriptome sequence of the rhodosporidium toruloides YM25235 for PCR amplification; the primers, components and amplification conditions used in the reaction were as follows:

primer 1: hisk 2301-F5' -CACACCGCGGATCCATGCCGCAGACGCC -3’（SEQ ID NO:3）

Primer 2: hisk 2301-R5' -CTGACCGCGATATCTCACTGCGGGTCGTT -3’（SEQ ID NO:4）

（GGATCCIs composed ofBamHIII, enzyme cutting sites are used,GATATCis composed ofEcoRV enzyme cleavage site);

the PCR amplification system was as follows (50. mu. L):

PCR amplification conditions, pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 s, annealing at 62 ℃ for 15 s, extension at 72 ℃ for 2min30 s for 30 cycles, and finally complete extension at 72 ℃ for 10 min, taking a product 2 mu L after reaction, performing electrophoresis analysis in agarose gel with the concentration of 1%, wherein the result is shown in figure 1, after the correct size of the fragment is confirmed by imaging of a gel imaging system, recovering a target fragment by using a multifunctional DNA purification and recovery kit from Battach Biotechnology Ltd, connecting a target gene obtained by PCR amplification to pMD-18T, transforming the connected product to escherichia coli DH5 α competent cells, and using a plasmid containing Ampicillin (AMP)⁺) The L B solid plate is screened, transformants on the plate are selected to carry out colony PCR screening positive clones, and then the Shanghai worker is sent to carry out sequencing, the sequencing result shows that a 3888bp long sequence is obtained, which is named Hisk2301, and the sequence composition is the nucleotide sequence shown as SEQ ID NO. 1.

Example 2: construction of Hisk2301 overexpression vector pRHHisk2301

Using reverse transcribed YM25235 cDNA as a template, using Hisk2301-F and Hisk2301-R as primers to amplify the Hisk2301 coding sequence to obtain Hisk2301 fragment with the size of about 3888bp, and subjecting the Hisk2301 fragment obtained by amplification to reverse transcriptionBamHⅠ、EcoRV two restriction enzymes are cut and connected to an expression vector pRH2304 to obtain a recombinant plasmid pRHHisk2301 (figure 2). The obtained recombinant plasmid is transferred into escherichia coli DH5 α for amplification, and the recombinant plasmid is extracted after colony PCR verification and is usedBamHⅠ、EcoRV, double digestion verification is carried out on pRHHisk 2301. The results showed that the recombinant plasmid pRHHisk2301 produced two bands of about 3.8 kb and 10 kb after double digestion (FIG. 3, lane 2), which were identical in size to the Hisk2301 fragment and the pRH2304 vector after double digestion, and the construction of the recombinant plasmid pRHHisk2301 was preliminarily demonstratedAnd (4) working. Sequencing by using a sequencing primer, and sending out the plasmid with correct enzyme digestion verification for further verification. The sequencing result shows that the sequence obtained by sequencing is completely consistent with the target sequence, and no base mutation, deletion and the like occur.

Example 3: analysis of relationship between Hisk2301 gene and low-temperature synthesized polyunsaturated fatty acid

1. Recombinant plasmid pRH2034-Hisk2301 transformed Rhodosporidium toruloides YM25235 strain

The recombinant plasmid pRH2034-Hisk2301 is transformed into an Rhodosporidium toruloides YM25235 strain by a lithium acetate transformation method, transformants are screened by a YPD culture medium containing hygromycin B (hygromycin B) with the final concentration of 150 mug/m L, and the transformants are extracted by genomic DNA PCR verification.

2. Analysis of changes in fatty acid content of Hisk2301 gene and Rhodosporidium toruloides

Two transgenic Rhodosporidium ymbiorum YM25235 transformed into empty plasmid pRH2304 and recombinant plasmid pRHHisK2301 were cultured at 30 ℃ for 24 hours, respectively, immediately after the culture was further cultured at 15 ℃ for 24 hours for cold treatment, and after the cold treatment, fatty acids of the two transgenic Rhodosporidium ymbiorum YM were extracted and methyl-esterified, and the control Rhodosporidium toruloides strain transformed into empty plasmid pRH2304 was used as a control, and gas chromatography analysis was performed on the changes in fatty acid content of the two plasmid transformed strains under the same treatment conditions, it can be seen from the graphs in A and B in FIG. 4 that the control strain transformed into empty plasmid pRH2304 and the overexpression strain transformed into recombinant plasmid pRHHisK2301 had a significantly increased linoleic acid content at 13.3min (C18: 2L A) (indicated by gray arrows) and a 29.53% of the total fatty acid content (Table 1) and a significantly increased linolenic acid content at 29.53% of 13.3min in the control strain transformed empty plasmid pRHHisK 2304 (Table 1), while the total linoleic acid content of the control strain transformed empty plasmid pRHHisK 2304 had a significantly increased linolenic acid content (Table 1) and a significantly increased linoleic acid content of the control strain expressed at black arrows 3947.18-35% of the total fatty acid content of the fatty acid (total fatty acid) and a of the control strain of the fatty acid expressed by black fatty acid strain treated fatty acid (PRHHisK 19. 12.17. the same fatty acid) and a-19. the fatty acid content of the control strain indicated by black fatty acid content of the fatty acid (total fatty acid content of the fatty acid strain of the same fatty acid strain, thus the control strain, 9-19. the fatty acid content of the.

Table 1: at 15 deg.CHisk2301Fatty acid content in transgenic Rhodosporidium toruloides YM25235 cells

。

Sequence listing

<110> university of Kunming science

<120> application of Hisk2301 gene of histidine kinase

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30

Ser Gly Ser Ser Ala Ser Ser Ala Ala Val Ser Thr Pro Ala Thr Ser Val Phe Ser Tyr

50

Ser Thr Ser Pro Ala Ser Ser Phe Val Asp MET Thr Ala Cys Pro Thr Pro Ala Pro Ala

70

Leu Ala Leu Ala Gln Glu Ala Gly Ala Val Gly Leu Ser Pro Glu Pro Leu Ser Pro Asp

90

Asn MET Ser Arg Pro Leu Ala Ser Ala Gln Pro Leu His Thr Phe Pro Thr Phe Leu Leu

110

Ser Leu Leu Thr Ala Leu Glu Ser Asp Pro Ala Ser Val Arg Ser His Leu Ala Asn Leu

130

Ala Ala Ser His Ala Ala Pro Ala Thr Phe Ser Pro Thr Pro Glu Gly Lys Pro Val Glu

150

Thr Asp Ala Ile Ile Ala Cys Ala Ser Arg Ile Ala Asp Arg Leu Gln Ala Ala Glu Arg

170

Pro Gln Asp Glu Pro Val Gln Pro Ala Ser Glu Pro Ala Ala Glu Glu Glu Ala Val Pro

190

Pro Pro Val Val Ala Pro Ala Val Ser Arg Thr Thr Ser Asp Asp Ser Val Pro Pro Lys

210

Lys Arg Pro Val Val Pro Gly Asp Thr Val Glu Glu Val Arg Arg Asn Cys Glu Asp Gln

230

Ile Gln Ala Leu Lys Val Leu His Ala Glu Glu Leu His Arg Ala Gln Leu Ser His Asp

250

Asn Glu Val Arg Ser Ile Ala Val Val Thr Ser Ala Ile Ala Arg Gly Asp Leu Thr Lys

270

Thr Ile Asp Ala Glu Val Glu Gly Glu MET Ala Thr Leu Lys Glu Thr Ile Asn Asp MET

290

Val Tyr Lys Leu Arg Leu Phe Ser Ser Glu Val Thr Arg Val Ser Leu Asp Val Gly Thr

310

MET Gly Gln Leu Gly Gly Gln Ala Val Val Thr Gly Val Glu Gly Thr Trp Lys Thr Leu

330

Thr Glu Thr Val Asn Thr MET Ala Thr Gln Leu Thr Val Gln Ile Arg Ser Ile Ala Ile

350

Val Thr Ser Ala Ile Ala Arg Gly Asp Leu Ser Lys Thr Ile Asp Ala Glu Val Gln Gly

370

Glu MET Ala Thr Leu Lys Glu Thr Val Asn Ser MET Val Thr Arg Leu Arg Val Phe Ser

390

Ser Glu Val Thr Arg Val Ala Arg Glu Val Gly Thr Asp Gly Gln Leu Gly Gly Gln Ala

410

Val Val His Gly Val Glu Gly Thr Trp Lys Glu Leu Thr Asp Ser Val Asn Arg MET Ala

430

Gln Asn Leu Thr Leu Gln Val Arg Glu Ile Ala Ser Val Thr Lys Ala Val Ala Asn Gly

450

Asp Leu Ser Lys Thr Val Asp Ile Gly Ala Ser Gly Glu Ile Arg Glu Leu Lys Thr Thr

470

Val Asn Ser MET Val Ala Gln Leu Arg Arg Phe Ala Ala Glu Val Thr Arg Val Ala Leu

490

Glu Val Gly Thr Glu Gly Gln Leu Gly Gly Thr Ala Asn Val Glu Gly Val Gln Gly Glu

510

Trp Lys Ser Leu Val Ser Ser Val Asn Thr MET Ala MET Asn Leu Thr Gln Gln Val Arg

530

Ser Ile Ala Asp Val Thr Arg Ala Val Ala Glu Gly Asp Leu Ser Arg Lys Ile Asp Val

550

Lys Val Lys Gly Glu MET Leu Asp Leu Lys Leu Thr Val Asn Ser MET Val Asp Ser Leu

570

Arg Leu Phe Ala Ala Glu Val Thr Arg Val Ala Lys Glu Val Gly Thr Asp Gly Gln Leu

590

Gly Gly Gln Ala Tyr Val Ile Asn Val Ser Gly Glu Trp Lys Ser Leu Val Asp Ser Val

610

Asn Gln MET Cys Gly Asn Leu Thr Asp Gln Val Arg Ser Ile Ala Lys Ala Thr Thr Ala

630

Val Ala Arg Gly Asp Leu Thr Gln Lys Val MET Ile Glu Ala Asn Gly Glu Val Leu Gln

650

Leu Val Val Thr Ile Asn Glu MET Val Asp Arg Leu Ala Thr Phe Ala Ser Glu Val Thr

670

Arg Val Ala His Glu Val Gly Thr Lys Gly Asn Leu Gly Val Thr Ala Lys Val Asp Asn

690

Ile Glu Gly Thr Trp Gln Glu Ile Thr Asn Asn Val Asn Thr MET Ala Thr Asn Leu Thr

710

Ser Gln Val Arg Ala Phe Ala Gln Ile Ser Ala Ala Ala Thr Glu Gly Asp Phe Ser Ser

730

Phe Val Thr Val Glu Ala Ser Gly Glu MET Asp Ser Leu Lys Thr Lys Ile Asn Lys MET

750

Val Phe Ser Leu Arg Asp Ser Leu Gln Lys Asn Thr MET Ala Arg Glu Ala Ala Glu Leu

770

Ala Asn Arg Ser Lys Ser Glu Phe Leu Ala Asn MET Ser His Glu Ile Arg Thr Pro MET

790

Asn Gly Ile Ile Gly Leu Thr Gly Val Thr Leu Glu Thr Asp Leu Thr Arg Gln Gln Arg

810

Glu Asn Leu MET Ile Val Ser Asn Leu Ala Asn Ser Leu Leu Leu Ile Ile Asp Asp Ile

830

Leu Asp Ile Ser Lys Ile Glu Ala Gly Arg MET Thr Val Glu Gln Ile Pro Phe Ser Val

850

Arg Ser Ala Val Phe Gly Ile Leu Lys Thr Leu Ala Val Lys Ala Thr Gln Ser Arg Leu

870

Asp Leu MET Tyr Ser Val Glu Ser Asp Ile Pro Asp Leu Leu Val Gly Asp Pro Phe Arg

890

Leu Arg Gln Val Ile Thr Asn Leu Val Gly Asn Ala Ile Lys Phe Thr Gln Arg Gly Gln

910

Val Ala Leu Ser Cys Arg Leu Ala Ala Ala Asp MET Glu Glu Lys Thr Tyr Gln Leu Glu

930

Phe Cys Ile Ser Asp Thr Gly Ile Gly Ile Lys Pro Asp Lys Leu Asn Leu Ile Phe Asp

950

Thr Phe Ala Gln Ala Asp Gly Ser Thr Thr Arg Lys Tyr Gly Gly Thr Gly Leu Gly Leu

970

Thr Ile Ser Lys Arg Leu Val Gln Leu MET Gly Gly Glu Leu Trp Val Thr Ser Gln Phe

990

Gly Arg Gly Ser Gln Phe Tyr Phe Thr Ile Gln Cys Arg Ile Gly Glu Trp Asn Leu Asp

1010

Gln Val Arg Gln Lys Thr Leu Ile Pro His Pro Gly Arg Cys Ile Leu Phe Ile Asp Thr

1030

Leu His His Asp Pro Ser ValIle Asp Ser Val Glu Gln Leu Gly Leu Glu Ile Thr Val

1050

Val Asp Ser Leu Glu Glu Ala Cys MET Leu Asp His Ser Gln Asn Gly Tyr Phe Asp Thr

1070

Val Leu Val Asp Gln Leu Ser Ile Val Glu Arg Leu Arg Asp Val Glu His Leu Arg Tyr

1090

Ile Pro Leu Val Leu Ile Thr Pro Gln Ile Pro Gln Leu Asn Leu Lys Tyr Cys Leu Asp

1110

Phe Gly Ile Ala Asn Cys Val Glu Ser Pro Thr Asn Ala Gln Asp MET Cys Asn Ala Leu

1130

Leu Pro Ala Leu Glu Ala Ser Asn Arg Ile Pro Ser Glu Arg Gly Gly Asp Ala Ser Phe

1150

Lys Val Leu Leu Ala Glu Asp Asn Ile Val Asn Gln Lys Val Ala Leu Lys Phe Leu Glu

1170

Ser Ala Gly His Gln Ser Glu Val Val Glu Asn Gly Ala Leu Ala Leu Glu Ala Val Arg

1190

Lys Asn Tyr Tyr Asp Ile Val Leu MET Asp Leu Ser MET Pro Ile MET Gly Gly Gln Glu

1210

Ala Thr Gln Ile Ile Arg Gln Phe Glu Gln Asp Asn Gly Leu Glu Arg Leu Pro Ile Val

1230

Ala Leu Thr Ala His Ala MET Leu Gly Asp Arg Glu Lys Cys Ile Gln Ala Gly MET Asp

1250

Asp Tyr Leu Thr Lys Pro Leu Arg Lys Pro Asp Leu Leu Ala Thr Ile Asn Lys Ile Val

1270

Leu Ala Arg Arg Ala Gly Ser Ser Pro Thr Ala Leu Arg Phe Phe ***

1290 1295

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<211>28

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<213> Artificial sequence

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cacaccgcgg atccatgccg cagacgcc 28

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<212>DNA

<213> Artificial sequence

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ctgaccgcga tatctcactg cgggtcgtt 29

Claims

1. Histidine kinase geneHisk2301Use of histidine kinase gene for the production of polyunsaturated fatty acids at low temperaturesHisk2301The nucleotide sequence of (a) is shown as SEQ ID NO:1, the amino acid sequence coded by the gene is shown as SEQ ID NO: 2 is shown in the specification;

the application refers to the overexpression of histidine kinase genes in red wintergreen spore yeast strainsHisk2301It causes the content of two polyunsaturated fatty acids, linoleic acid and linolenic acid, to increase.