CN107384979B

CN107384979B - Application of high osmotic pressure glycerol protein kinase gene RKHog1

Info

Publication number: CN107384979B
Application number: CN201710454495.9A
Authority: CN
Inventors: 张琦; 李珊; 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: 2017-06-16
Filing date: 2017-06-16
Publication date: 2020-11-17
Anticipated expiration: 2037-06-16
Also published as: CN107384979A

Abstract

The invention discloses a high osmotic pressure glycerol protein kinase geneRKHog1The novel use of, i.e., a high osmotic pressure glycerol protein kinase geneRKHog1At low temperatureThe polyunsaturated fatty acid is produced at low temperature by using the gene, the content of the unsaturated fatty acid LA and ALA is improved by modifying microorganisms by means of genetic engineering, good application prospect and economic benefit are provided for the industrial production of PUFAs, and a foundation is laid for the large-scale commercial production of PUFAs.

Description

High osmotic pressureGlycerol protein kinase geneRKHog1Use of

Technical Field

The invention belongs to the field of biotechnology and genetic engineering, and relates to a high osmotic pressure glycerol protein kinase geneRKHog1The new application of (1).

Background

Polyunsaturated Fatty Acids (PUFAs) refer to Fatty Acids containing two or more unsaturated double bond structures, also known as polyenoic Fatty Acids. According to the position of the first unsaturated bond, PUFAs can be classified into omega-3, omega-6, omega-7, omega-9 and other series (i.e. omega numbering system, also called n numbering system). Many fatty acids in the omega-3 series and the omega-6 series are essential fatty acids for the human body, such as Linoleic Acid (LA), alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), Arachidonic Acid (AA), and the like. LA, ALA, DHA, and AA are not synthesized in humans and need to be ingested from food, known as Essential Fatty Acids (EFAs) in humans. The omega-3 and omega-6 PUFAs have important biological significance and are closely related to human health, and in many cases, the two PUFAs coordinate and restrict each other in function to jointly regulate the life activities of organisms.

Polyunsaturated fatty acids have more effects than saturated fatty acids, and can lower blood cholesterol and triglyceride, regulate cardiac function, lower blood viscosity, improve blood microcirculation, improve brain cell activity, enhance memory and thinking ability, enhance body defense system, and remove excessive waste. Therefore, the potential medical and medicinal value of the compound is widely concerned by the world, and the compound draws high attention in the industries of food, medicine, even cosmetics and the like.

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, the production of PUFAs by microbial technology has been sought as a new source of 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.

The high osmotic pressure glycerol protein kinase gene Hog1 is a core molecule of HOG-MAPK pathway and plays an important role in signal transduction. The previous researches suggest that Hog1 is mainly involved in the response of osmotic stress, but the recent researches show that Hog1 is also involved in the response of low-temperature stress, and related documents report that Hog1 or homologous proteins thereof are all found in mammals, insects, fission yeast and saccharomyces cerevisiae to be involved in the regulation under the low-temperature stress. Although the relationship between Hog1 and low-temperature stress in fungi has been researched, the specific action mechanism is not clear, and no report is found about the Hog1 and the low-temperature adaptability mechanism thereof and the synthesis mechanism of polyunsaturated fatty acid at low temperature in the oleaginous fungi, so that the research on the low-temperature adaptability of Hog1 and the oleaginous fungi provides a new theoretical basis for the research on oleaginous microorganisms. Provides reference for the research of elucidating the low-temperature adaptation mechanism of the fungus, and the knowledge of the regulation mechanism of the synthesis of the PUFAs under the low-temperature condition is helpful to lay a foundation for the research and the application of the large-scale production of the PUFAs in the future by utilizing the relevant synthesis regulation process.

Disclosure of Invention

The invention aims to provide a high osmotic pressure glycerol protein kinase geneRKHog1The novel use of, i.e., a high osmotic pressure glycerol protein kinase geneRKHog1Use of polyunsaturated fatty acids produced at low temperatures.

High osmotic pressure glycerol protein kinase geneRKHog1From 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 1080bp (basic group), and the amino acid sequence coded by the gene is shown as SEQ ID NO: 2 or a fragment thereof.

The invention is highOsmolyte glycerol protein kinase geneRKHog1Directly 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) High osmotic pressure glyceroprotein kinase gene cloned in YM25235RKHog1The pRH2304 plasmid is inserted to construct a recombinant expression plasmid pRHRKHog1, the recombinant expression plasmid is transferred into Rhodosporidium toruloides YM25235 to be overexpressed, a transgenic strain YM25235/pRHRKHog1 is obtained, and the expression characteristics of the transgenic strain YM25235/pRHRKHog1 are researched, so that a foundation is laid for the elucidation of the gene of the RKHoRKHog 1, the low-temperature adaptability mechanism of the gene and the synthesis mechanism of polyunsaturated fatty acids 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 Hog1p of HOG pathway isolated from chromosome genome of YM25235RKHog1. The gene has a total length of 1080bp, and the previous researches suggest that Hog1 is mainly involved in the response of osmotic stress. This study showed that the geneRKHog1The gene not only has the function of osmotic stress resistance, but also is related to the low-temperature synthesis of PUFAs in the rhodosporidium toruloides, and the gene can improve the delta of catalyzing oleic acid to be converted into linoleic acid and linolenic acid in the rhodosporidium toruloides YM25235 at low temperature^12/15-an increase in the mRNA transcription level of the fatty acid dehydrogenase gene RKD12 gene, thereby causing a significant increase in the amount of PUFAs synthesized in the cell lipids; the research result is helpful for clarifying the signal transduction pathway and synthesis regulation mechanism of low-temperature synthesis of PUFAs in oleaginous yeast-Rhodosporidium toruloides YM25235, provides reference for disclosing the mechanism of low-temperature adaptability of microorganisms, is helpful for improving the content of unsaturated fatty acids LA and ALA by modifying the PUFAs through a genetic engineering means, provides good application prospect and economic benefit for industrial production of PUFAs, and lays a foundation for large-scale commercial production of PUFAs.

Drawings

FIG. 1 is a PCR amplification map of Rhodosporidium toruloides YM25235 RKHog1 gene;

FIG. 2 is a plasmid map of recombinant plasmid pRHRKHog 1;

FIG. 3 shows restriction analysis of the recombinant plasmid pRHRKHog 1; wherein: 1 is DNA molecular weight marker DL 10000; 2 is empty plasmid pRH2304NcoⅠ、EcoRV, double enzyme digestion; 3 is recombinant plasmid pRHRKHog1NcoⅠ、EcoRV, double enzyme digestion; 4 is the PCR product of RKHog1 gene;

FIG. 4 is a gas chromatography analysis of fatty acids in cells of Rhodosporidium toruloides YM25235 transformed with RKHog1 gene under low temperature conditions; a, YM25235/pRH 230430 ℃ cultured at 30 ℃; YM25235/pRHRKHog1 cultured at 30 ℃;

FIG. 5 is a gas chromatography analysis of fatty acids in cells of Rhodosporidium toruloides YM25235 transformed with RKHog1 gene under low temperature conditions; a is YM25235/pRH2304 cultured at 15 ℃; YM25235/pRHRKHog1 cultured 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 RKHog1 Gene cloning

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: RKHog 1-F5' -CGCAAGCTTATGGCCGACTTCGTGAAG -3’（SEQ ID NO:3）

Primer 2: RKHog 1-R5' -TAATGGATCCTTACTGCTGCGGCGCG -3’（SEQ ID NO:4）

（AAGCTTIs composed ofHindIII, enzyme cutting sites are used,GGATCCis composed ofBamH i cleavage site);

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

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min, denaturation at 95 deg.C for 30 s, annealing at 63 deg.C for 15 s, and extension at 72 deg.C for 1 min for 30 cycles, and final extension at 72 deg.C for 10 min. After completion of the reaction, 2. mu.L of the product was collected and subjected to electrophoresis analysis on a 1% agarose gel, and the results are shown in FIG. 1. After confirming the correct size of the fragment by imaging with a gel imaging system, recovering the target fragment with a multifunctional DNA purification recovery kit from Betach Biotechnology Ltd, ligating the target gene obtained by PCR amplification to pMD-18T, transforming the ligation product into E.coli DH5 alpha competent cells, and using a DNA fragment containing Ampicillin (AMP)⁺) The LB solid plate is screened, transformants on the plate are selected to carry out colony PCR screening positive clone, and then the colony PCR screening positive clone is sent to Shanghai worker for sequencing. The sequencing result showed that a 1080bp long sequence was obtained, designated RKHog1, consisting of the nucleotide sequence shown in SEQ ID NO. 1.

Example 2:RKHog1construction of Gene overexpression vector pRHRKHog1

Reverse transcription of YM25235 cDNA as template, RKHog1-F and RKHog1-R as primers to amplify RKHog1 coding sequence to obtain RKHog1 fragment of about 1080bp in size, and amplification of RKHog1 fragmentNcoⅠ、EcoRV two restriction enzymes were digested, and ligated to expression vector pRH2304 to obtain recombinant plasmid pRHRKHog1 (FIG. 2). Transferring the obtained recombinant plasmid into Escherichia coli DH5 alpha for amplification, performing colony PCR verification, extracting recombinant plasmid, and purifying withNcoⅠ、EcoRV double restriction enzyme validation of pRHRKHog 1. The result shows that the recombinant plasmid pRHRKHog1 generates two bands (shown in a 3 rd lane of a figure 3) of about 1.0 kb and 10 kb after double digestion, the sizes of the two bands are respectively consistent with the sizes of the RKHog1 fragment and the pRH2304 vector after double digestion, and the construction of the recombinant plasmid pRHRKHog1 is preliminarily shown to be successful; 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:RKHog1analysis of relationship between gene and low-temperature synthesized polyunsaturated fatty acid

1. Recombinant plasmid pRHRKHog1 transformed Rhodosporidium toruloides YM25235 strain

Transforming the recombinant plasmid pRHRKHog1 into an Rhodosporidium toruloides YM25235 strain by a lithium acetate transformation method, and screening transformants by a YPD culture medium containing hygromycin B (hygromycin B) at a final concentration of 150 mug/mL; genomic DNA was extracted and transformants verified by PCR.

2、RkHog1Analysis of Gene and fatty acid content variation in Rhodosporidium toruloides

Culturing the transgenic strain YM25235/pRHRKHog1 and the control strain YM25235/pRH2304 at 30 deg.C for 24 h, respectively, culturing one part at 30 deg.C for 24 h, and immediately transferring the other part to 15 deg.C for cold treatment and culturing for 24 h; respectively extracting total fatty acids in the cells of the 4 strains of bacteria subjected to cold treatment, and carrying out methyl esterification. And (3) carrying out gas chromatography analysis on the sample, and calculating the content of the fatty acid in the strain by using an area normalization method. The fatty acid gas chromatography chromatogram analysis chromatogram of the 4 strains is shown in fig. 4 and 5, wherein the retention time in the graph is about 11 min for saturated fatty acid C18:1 OA oleic acid, the retention time is about 12.5 min for unsaturated fatty acid C18:2 LA linoleic acid, and the retention time is about 15 min for unsaturated fatty acid C18:3 ALA linolenic acid. From FIGS. 4 and 5, it was found that the peak areas of OA, LA and ALA were substantially the same when the transgenic strain YM25235/pRHRKHog1 and the control strain YM25235/pRH2304 were at 30 ℃; when the transgenic strain YM25235/pRHRKHog1 and the control strain YM25235/pRH2304 were cultured at 15 ℃, the peak area size of oleic acid decreased, the peak area size of OA, LA and ALA increased, and the peak area size of the transgenic strain YM25235/pRHRKHog1LA and ALA was larger than that of the control strain YM25235/pRH2304 ALA (FIG. 4A: YM25235/pRH2304 cultured at 30 ℃, B: YM25235/pRHRKHog1 cultured at 30 ℃, FIG. 5A: YM25235/pRH2304 cultured at 15 ℃ and B: YM 25235/pRKHog 1 cultured at 15 ℃).

From the fatty acid content data in the table below, we can calculate that the content of linoleic acid in the transgenic strain YM25235/pRHRkHog1 after 15 ℃ culture was 37.76%, which was increased by 2.06-fold with respect to 17.83% of LA content in the same strain at 30 ℃ culture condition and by 1.30-fold with respect to 28.16% of LA content in the control strain under the same treatment condition. And under the culture condition of 15 ℃, the content of ALA in the transgenic strain YM25235/pRHRkHog1 is 21.11 percent, which is increased by 3.95 times relative to the content of ALA in the same strain under the culture condition of 30 ℃ which is 5.34 percent, and increased by 2.38 times relative to the content of ALA in the same strain under the same culture condition of a control strain which is 8.87 percent. Under the condition of 15 ℃, the content of OA is reduced by 1.58 times compared with the condition of 30 ℃ and reduced by 1.00 times compared with the control strain under the same culture condition. These results indicate that overexpression of the RkHog1 gene in Rhodosporidium toruloides YM25235 strain causes an increase in the contents of two polyunsaturated fatty acids, LA and ALA, and a concomitant decrease in the content of saturated fatty acid OA;

at 15 ℃ and 30 DEG CRkHog1Fatty acid content in transgenic Rhodosporidium toruloides YM25235 cells

。

Sequence listing

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Claims

1. High osmotic pressure glycerol protein kinase geneRKHog1The application of the polyunsaturated fatty acid produced at low temperature has the nucleotide sequence shown as SEQ ID NO:1 is shown in the specification;

the application refers to the overexpression of the high osmotic pressure glycerol protein kinase gene in the Rhodosporidium toruloides YM25235 strainRKHog1It can increase the content of two polyunsaturated fatty acids, linoleic acid and alpha-linolenic acid.