CN113430215A - Acetyl CoA synthetase gene RKACS1 and application thereof - Google Patents

Abstract

The invention discloses an acetyl CoA synthetase geneRKACS1The nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2; the gene is red winter cell yeast (Rhodosporidium kratochvilovae) The acetyl CoA synthetase gene separated from YM25235 is transformed into Rhodosporidium toruloides YM25235, and the experimental result shows that the acetyl CoA synthetase geneRKACS1Overexpression of (A) can cause the recombination of Rhodosporidium toruloides YM25235The invention improves the microorganism by genetic engineering means to improve the yield of carotenoid and grease in the microorganism, and lays a foundation for large-scale commercial production of carotenoid and grease.

Description

Acetyl CoA synthetase geneRKACS1And uses thereof

Technical Field

The invention belongs to the technical field of biological and genetic engineering, and relates to an acetyl CoA synthetase geneRKACS1In particular to a method for preparing a compound from rhodosporidium toruloidesRhodosporidium kratochvilovae) Acetyl CoA synthetase Gene cloned in YM25235RKACS1And connecting the gene with a vector, transferring the gene into a yeast cell to improve the expression level of the gene and promote the synthesis of carotenoid and grease.

Background

Carotenoids (carotenoids) are a kind of natural pigments widely existing in nature, generally presenting yellow, red or orange-red color, and at present, more than 800 kinds of natural carotenoids found in organisms such as higher plants, animals, fungi and the like exist, and carotenoids rich in common vegetables and fruits such as oranges, mangoes, pumpkins and the like exist. Most carotenoids are a C group consisting of 8 isoprenoids connected end to end₄₀Terpenoids and derivatives thereof, some carotenoids containing C₄₅Or C₅₀The skeleton, known as high carotenoids, also has a small fraction of carbon skeleton less than C₄₀The carotenoids of (a), are called apocarotenoids; all carotenoids contain a polyisoprene structure.

Carotenoids can be divided into two major classes, carotene (carotene) and lutein (xanthophyll), according to the chemical structure of the carotenoids, wherein the carotenes are alpha-carotene, beta-carotene, gamma-carotene, lycopene and the like containing only C, H two elements; xanthophyll is an oxidized carotene, contains C, H, O three major elements, and can form oxygen-containing functional groups such as hydroxyl, keto, carboxyl, methoxyl, etc., such as xanthophyll, zeaxanthin, astaxanthin, etc.; the oxygen-containing group makes the molecular structure of the carotenoid generate complex and various changes, and the polarity change makes the carotenoid easily combined with body fatty acid, sugar, protein and the like to form active molecules with various functions. Carotenoids have strong absorptivity, and usually have an absorption peak at a wavelength of 430-570 nm, so that the carotenoids in a sample are qualitatively and quantitatively analyzed by adopting a reversed-phase high performance liquid chromatography and matching with an ultraviolet-visible light detector, a mass spectrum detector, a nuclear magnetic resonance detector or a diode array detector.

Carotenoids play a very important role in the fields of human nutrition and health, and alpha-carotene, beta-carotene, gamma-carotene, beta-cryptoxanthin and the like can form vitamin A under the action of dioxygenase, and therefore, the carotenoids are called provitamin A active substances and are the main sources of the vitamin A. As a micronutrient essential to the human body, carotenoids have a variety of functions such as anti-oxidation, inhibition and elimination of free radicals in the body, slowing down aging, etc. Medical research shows that besides the functions, the carotenoid also has the functions of resisting cancers, preventing fundus macular degeneration and cataract, preventing cardiovascular diseases, preventing non-alcoholic fatty liver, strengthening the immunity of the organism and the like. Besides, the carotenoid can be combined with animal protein to make organism show body color, and has certain protection effect. At present, carotenoid is determined as A-type nutrient by international organizations such as Food and Agricultural Organization (FAO) and World Health Organization (WHO) of the United nations, is approved as a food additive with double functions of nutrition and pigment in more than 50 countries, and is widely applied to industries such as medicine, food, health care, beauty treatment and the like. The production processes of carotenoids reported so far include natural extraction, chemical synthesis and microbial fermentation, wherein microbial fermentation for producing carotenoids has the advantages of high biological activity, easy processing of the production process, safe product, short production period and the like, and has attracted much attention in recent years. Although many strategies have been used to promote the synthesis of carotenoids in microorganisms, there is still a lack of corresponding strategies in terms of industrial production for the production of various carotenoids using conventional microbial fermentation techniques due to the limitations of technical problems in terms of cost-effectiveness, pigment production and isolation and extraction. Therefore, we hope to obtain high-content raw materials by using gene technology, species discovery, hybrid culture and other technologies from the perspective of bioengineering, thereby alleviating the production and development limitations of the current natural carotenoids. In recent years, metabolic engineering to optimize the original metabolic pathways and regulatory networks of strains or to assemble heterologous metabolic pathways in combination with classical genetic and modern molecular biological methods to make microorganisms highly beneficial and cost effective for the production of carotenoids has provided a potential alternative.

Fats and oils (lipids) are key substances in the biological activities of various living things, and common fats and oils include functional fats and oils such as Linoleic Acid (LA), α -linolenic acid (ALA), γ -linolenic acid (GLA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Research shows that the omega-3 fatty acids such as ALA, DHA, EPA and the like can effectively promote the growth and development of human bodies as important life active substances of the human bodies, have the physiological functions of preventing diabetes, cardiovascular and cerebrovascular diseases, resisting cancer, resisting inflammation, reducing blood fat, enhancing the immunity of the organisms and the like, and lack of the omega-3 fatty acids and the omega-6 fatty acids for a long time easily causes hereditary obesity and metabolic disorder of the organisms, thereby influencing the health of the human bodies; in addition, LA, ARA, DHA, etc. can be used as medicinal additives to lower blood sugar and blood pressure, prevent pregnancy, relieve respiratory tract epidemic disease, and prevent arteriosclerosis and digestive system ulcer. Most functional oil is necessary for human body and cannot be synthesized by human body, ALA can be obtained from partial vegetable oil, DHA and EPA are mainly derived from fish oil and microbial oil. However, fish oil has high cost, is easy to be polluted, has unstable quality and has output which is difficult to meet the global demand.

The oil-producing microorganism has high oil content, and the fatty acid composition of the microorganism is similar to that of vegetable oil. Compared with vegetable oil, the oil-producing microorganism has the advantages of no land competition with grains, no influence of climate and environment, short growth period, easy realization of large-scale production and the like, so that the oil production by utilizing the microbial fermentation is more and more concerned. Currently, more than 30 yeasts have been identified as oleaginous yeasts, which are most capable of increasing the intracellular accumulation of oil by culture or genetic modification under specific conditions. Therefore, the strain is hoped to be purposefully transformed by utilizing technologies such as gene technology, synthetic biology and the like from the perspective of bioengineering, and the oil conversion rate of the existing strain is improved, so that the pressure of the shortage of the oil resources at present is relieved. In recent years, the combination of classical genetics and modern molecular biology methods to optimize the original metabolic pathway and the regulation network of the strain or assemble heterologous metabolic pathways provides a potential alternative pathway for microbial metabolic engineering for producing grease with high benefit and low cost.

Disclosure of Invention

The invention aims to provide acetyl CoA synthetaseGeneRKACS1The gene is derived from Rhodosporidium toruloides (Rhodosporidium kratochvilovae) YM25235, its nucleotide sequence is shown in SEQ ID NO:1 or is the fragment of the nucleotide sequence, or the nucleotide sequence complementary to SEQ ID NO:1, the gene sequence is 1977bp (base), the coded amino acid sequence is polypeptide or fragment shown in SEQ ID NO: 2.

Another object of the present invention is to provide an acetyl-CoA synthetase geneRKACS1The recombinant expression vector of (1) is constructed by directly connecting the gene shown in SEQ ID NO. 1 with different expression vectors (plasmids, viruses or carriers). Construction of acetyl-CoA synthetase genes can be accomplished by methods well known to those skilled in the artRKACS1An expression vector of (a) and a suitable transcription/translation regulatory element; these methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like; the acetyl CoA synthetase geneRKACS1Can be operably linked to an appropriate promoter of an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; the PL promoter of lambda phage; eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling the expression of genes in prokaryotic cells or eukaryotic cells or viruses thereof; the expression vector also comprises a ribosome binding site for translation initiation, a transcription terminator and the like; the transcription of the vector in higher eukaryotic cells is enhanced by inserting an enhancer sequence into the vector; enhancers are cis-acting elements of DNA expression, usually about 10-300bp, that act on a promoter to enhance gene transcription, such as adenovirus enhancers.

Another object of the present invention is to provide a gene containing acetyl CoA synthetaseRKACS1Or a host cell of the above recombinant expression vector.

Optimization of host cells with the nucleotide sequences described herein or recombinant vectors containing the nucleotide sequences can be performed using methods well known to those skilled in the art.

Another object of the present invention is to provide the above-mentioned acetyl-CoA synthetase geneRKACS1It is applied in producing carotenoid.

Another object of the present invention is to provide the above-mentioned acetyl-CoA synthetase geneRKACS1The method is applied to oil production.

The invention relates to a method for preparing a red wintergreen spore yeast (Rhodosporidium toruloides)Rhodosporidium kratochvilovae) Separating the total RNA of YM25235 to obtain the acetyl CoA synthetase geneRKACS1The total length of the gene is 1977 bp; in Rhodosporidium toruloides YM25235RKACS1The over-expression of the gene can cause the transcription level of the gene in the cell to be improved to a certain extent, which indicates that the exogenous gene is transcribed in the thallus and then translated into corresponding protein to cause the expression quantity of enzymes related to the synthesis of the carotenoid or the grease in the cell to be improved.

Drawings

FIG. 1 shows a scheme for producing Rhodosporidium toruloides YM25235 of the present inventionRKACS1PCR amplification of the gene; DNA molecular weight marker DL 2000; 2. negative control; 3.RKACS1a cDNA fragment of (1);

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

FIG. 3 is a PCR-verified electrophoretogram of colonies; DNA molecular weight marker DL 2000; 2. RKACS1a cDNA fragment of (1); 3-7 is a transformant;

FIG. 4 shows the verification of positive clones of recombinant plasmid pRHRKACS1 transformed Rhodosporidium toruloides YM 25235; DNA molecular weight marker DL 2000; 2. a wild type strain specific gene band; 3.RKACS1; 4. verifying a transformant;

FIG. 5 comparison of carotenoid content of overexpression strain YM25235/pRHRKACS1 with control strain YM 25235;

FIG. 6 shows the comparison of oil and fat contents between the overexpression strain YM25235/pRHRKACS1 and the control strain YM 25235.

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 methods are used.

Example 1: from Rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) Isolation of acetyl CoA synthetase Gene from YM25235RKACS1Nucleotide sequence of (A)

Extracting total RNA of Rhodosporidium toruloides YM25235 by using UNlQ-10 column type Trizol total RNA extraction kit (product number: SK 1321) of bio-engineering (Shanghai) Co., Ltd, performing reverse transcription according to PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) of TaKaRa Co., Ltd to synthesize cDNA, performing polymerase chain reaction by using 1 mu L cDNA as a template, and sequencing according to the transcription set to obtain the cDNARKACS1Designing specific primers RKACS1-F and RKACS1-R (primer 1 and primer 2), carrying out PCR amplification on the cDNA template obtained by the method on a PCR instrument (BIOER company), wherein the primers, components and amplification conditions used in the reaction are as follows:

primer 1: RKACS 1-F: 5' -GATCACTCACCATGGTGACCGAACACACCTACGAC-3' (SEQ ID NO: 3) (upstream vector end homology sequence underlined)

Primer 2: RKACS 1-R: 5' -CCGGTCGGCATCTACCTACGCCTTGGCGAACTT-3' (SEQ ID NO: 4) (the downstream vector end homology is underlined);

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

Template cDNA	1µL
		Foward Primer (RKACS1-F)	2µL
Reverse Primer (RKACS1-R)	2µL
		dNTPs Mix (10 mM)	1µL
2×phanta Max Buffer	25µL
		Phanta Max Super-Fidelity Polymerase	1µL
ddH2O	adding to 50 mu L

Amplification conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 2min for 30 cycles, and final extension at 72 deg.C for 10 min; after the reaction, 2. mu.L of the product was taken and subjected to electrophoresis analysis in 1% agarose gel, and the results are shown in FIG. 1; amplifying to obtain a fragment with the size of about 2000bp, and naming the fragmentRKACS1(ii) a pRH2034 throughBamHⅠ、EcoRV, carrying out double enzyme digestion by two restriction enzymes; the two fragments were recovered with agarose gel DNA recovery Kit (Beijing Solebao technologies, Ltd.), and the two recovered fragments were ligated to obtain recombinant plasmid pRHRKACS1 (FIG. 2) in a Clonexpress II One Step Cloning Kit (20. mu.L):

Exnase ™ II	2µL
		RKACS1fragments	79ng
Linearized pRH2034	200ng
		5×CEIIBuffer	4µL
ddH2O	Adding to 20 mu L

Gently blowing and beating the mixture by using a pipettor, mixing the mixture evenly, centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube, and then reacting the reaction liquid for 30min at 37 ℃; cooled to 4 ℃ or immediately placed on ice to cool.

Transferring the obtained ligation product into escherichia coli DH5 alpha for amplification, culturing on an LB solid plate containing spectinomycin (100 mug/mL) for 12-16 h, selecting a white colony growing on the plate, verifying a positive clone through colony PCR (shown in figure 3), inoculating the positive clone into an LB liquid culture medium (containing 100 mug/mL spectinomycin) for culturing for 12-16 h, extracting a plasmid by using a high-purity plasmid miniprep kit (centrifugal column type) (Beijing Baitaike biotechnology limited), sequencing (Kunming technologies limited), and displaying that the amplified fragment has the size of 1977bp and is consistent with a transcriptome sequence and the nucleotide sequence is shown as SEQ ID NO: 1.

Example 2:RKACS1effect of Gene overexpression on Carotenoid Synthesis in Rhodosporidium toruloides YM25235

1. Agrobacterium mediated transformation of Rhodosporidium toruloides YM25235

Transforming the recombinant plasmid pRHRKACS1 into Rhodosporidium toruloides YM25235 by using an agrobacterium-mediated method, screening transformants by a YPD culture medium containing hygromycin B (hygromycin B) with the final concentration of 150 mug/mL, extracting genomic DNA of the yeast transformants according to the steps in the DNA extraction kit specification of Shanghai biological engineering GmbH, and then carrying out PCR verification, wherein the result is shown in FIG. 4;

2、RKACS1analysis of Carotenoid content in Gene-overexpressed Rhodosporidium toruloides YM25235

Culturing overexpression strain containing pRHRKACS1 at 28 deg.C, extracting carotenoid, and determining total carotenoid content (mg/g dry thallus) at 445nm with ultraviolet-visible spectrophotometer using wild type Rhodosporidium toruloides YM25235 strain as control, as shown in FIG. 5; as can be seen from the figure, the total carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACS1 is obviously improved compared with that of the wild type Rhodosporidium toruloides YM25235 strain, the carotenoid synthesis amount of the wild type Rhodosporidium toruloides YM25235 strain is 5.23 +/-0.25 mg/g, and the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACS1 is 9.57 +/-0.57 mg/g, namely the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACS1 is 1.83 times that of the control strain; the results showed that the acetyl CoA synthetase geneRKACS1The overexpression of (a) can cause the increase of the total carotenoid content in the rhodosporidium toruloides YM25235 strain,RKACS1the gene can promote the synthesis of total carotenoid.

3、RKACS1Analysis of oil content in Rhodosporidium toruloides YM25235 with Gene overexpression

Culturing overexpression strain containing pRHRKACS1 at 28 deg.C, extracting oil, and determining oil content (% dry thallus) with wild type Rhodosporidium toruloides YM25235 strain as control as shown in FIG. 6; as can be seen from the figure, the fat synthesis amount of the over-expression strain YM25235/pRHRKACS1 is obviously improved compared with that of the wild type Rhodosporidium toruloides YM25235 strain, the fat synthesis amount of the wild type Rhodosporidium toruloides YM25235 strain is 4.27 +/-0.51%, and the fat synthesis amount of the over-expression strain YM25235/pRHRKACS1 is 5.73 +/-0.24%, namely the fat synthesis amount of the over-expression strain YM25235/pRHRKACS1 is improved by 34.19% compared with that of the control strain; the results showed that the acetyl CoA synthetase geneRKACS1Can cause the increase of the oil content in the rhodosporidium toruloides YM25235 strain,RKACS1geneThe synthesis of grease can be promoted;

in summary, the acetyl CoA synthetase geneRKACS1Is related to the synthesis of carotenoid and grease in the rhodosporidium toruloides.

Sequence listing

<110> university of Kunming science

<120> acetyl CoA synthetase gene RKACS1 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1977

<212> DNA

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovaeYM25235)

<400> 1

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ggccaccctg tccacgccaa cacggacgag tacgtcgagc tgtacaagga gtcgatcgac 120

tcgcccaagc agttctggga ccgcatggcc aaggagcacc tctactggca ccgcccgtac 180

tcgaccgtca ccgccggctc tttcgaggca ggagacgtac agtggttccc cgagggcggc 240

ctgaacgtcg cgtacaactg cgtcgaccgc tgggcgtaca agcacccgaa caagacggcc 300

atcatctggg aggcggacga gcccggcgag catgtcgagc tcacgtacga gcagctgttc 360

caggaggtct gcaagactgc caacatcctc aagagctacg gcgtcaagaa gggcgacacc 420

gtcgccatct acctccccat ggtccccgag gcggcaatcg ccttcctcgc gtgcgcccgc 480

ctcggcgcga tccactcggt cgtcttcgcc ggcttctccg ccgagtcgct ccgcgaccgc 540

gtcaacgacg cccagtcgcg cgtcgtcatc accaccgacg agggcaagcg cggcggcaag 600

acgatcgcga ccaagtcgat cgtcgacgcg gcgctctccg agtgccccgt cgtcgagcat 660

gtcctcgtcc tcaagcgcac tggcggcgac gtcaagtgga ccgagggccg cgaccactgg 720

tggcacgagg agaaggagaa ggtccagccg tactgccccg tcgagatcgt ctcggccgag 780

gacccgctct tcatcctcta cacctcgggc tcgaccggca agcccaaggg tgtcgtccac 840

tcgtccgccg gctacctcct cggcgcgttc atgacgctca agtacgtctt cgacgtgcac 900

cccgaggacc gctacgcgtg catggcagac gtcggctgga tcaccggcca cacctacatc 960

gtttacggcc cgcttgcgaa cggcgtcacc accaccatct tcgagtcgac gccggtctac 1020

ccgaccccgt cgcgcttctg ggagacggtc gcgaagcaca agctcacgca gttctacacc 1080

gcgccgaccg ccatccgtct cctccgccgc ctcggcgagg agcacaccaa gggccacgac 1140

ctgtcgacgc tccgcaccat cggctcggtc ggcgagccga tcaaccccga ggcttgggag 1200

tggtactggg agcacgtcgg caagaaggag tgcgccgtcg tcgacacgta ctggcagacc 1260

gagaccggct ccatcatcat cacccccctt cccggcgcga ccaagaccaa gcccggcgcg 1320

gcgacgctgc cgttcttcgg catcgacccg gtcctcctcg acccgacgac gggcgaggag 1380

atcaagggca acgaggtcga gggcgtgctc tgcgtgcgca agccgtggcc gtcgatcgcg 1440

cgcaccgtct acggcgacca caagcggttc ctcgacacgt acatgaaccc gtacccgggc 1500

tactacttca ccggcgacgg cgccgggcgc gaccacgacg ggtactactg gatccgcggc 1560

cgcgtcgacg acgtgatcaa cgtctcgggc caccgcctct cgaccgccga aatcgagagc 1620

gccctcatcc accacaacgg cgtagccgag acggccgtcg tcggcatccc cgacgagctc 1680

accggccagg ccgtcgtcgc ctacgtcgcc ctcaagcccg agtttgcggc cgagaacccc 1740

gacgagccgg cgctgctgaa ggaattggtg ttgcaggtcc gcaagaccat cggtccgttc 1800

gcggccccga agaaactcgt gcttgtgggt gatctgccga agacccggag cggcaagatc 1860

gtccggcggg cactgcgcaa gatcgcgagc ggcgagggcg accagctcgg cgatctttcg 1920

acactggcgg agcctgccat catcgacgag atcaaggaga agttcgccaa ggcgtag 1977

<210> 2

<211> 658

<212> PRT

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovae YM25235)

<400> 2

Met Thr Glu His Thr Tyr Asp Thr Pro Ser His Pro Ile Ser Lys Arg

1 5 10 15

Asn Asp Gly Thr Gly His Pro Val His Ala Asn Thr Asp Glu Tyr Val

20 25 30

Glu Leu Tyr Lys Glu Ser Ile Asp Ser Pro Lys Gln Phe Trp Asp Arg

35 40 45

Met Ala Lys Glu His Leu Tyr Trp His Arg Pro Tyr Ser Thr Val Thr

50 55 60

Ala Gly Ser Phe Glu Ala Gly Asp Val Gln Trp Phe Pro Glu Gly Gly

65 70 75 80

Leu Asn Val Ala Tyr Asn Cys Val Asp Arg Trp Ala Tyr Lys His Pro

85 90 95

Asn Lys Thr Ala Ile Ile Trp Glu Ala Asp Glu Pro Gly Glu His Val

100 105 110

Glu Leu Thr Tyr Glu Gln Leu Phe Gln Glu Val Cys Lys Thr Ala Asn

115 120 125

Ile Leu Lys Ser Tyr Gly Val Lys Lys Gly Asp Thr Val Ala Ile Tyr

130 135 140

Leu Pro Met Val Pro Glu Ala Ala Ile Ala Phe Leu Ala Cys Ala Arg

145 150 155 160

Leu Gly Ala Ile His Ser Val Val Phe Ala Gly Phe Ser Ala Glu Ser

165 170 175

Leu Arg Asp Arg Val Asn Asp Ala Gln Ser Arg Val Val Ile Thr Thr

180 185 190

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

195 200 205

Asp Ala Ala Leu Ser Glu Cys Pro Val Val Glu His Val Leu Val Leu

210 215 220

Lys Arg Thr Gly Gly Asp Val Lys Trp Thr Glu Gly Arg Asp His Trp

225 230 235 240

Trp His Glu Glu Lys Glu Lys Val Gln Pro Tyr Cys Pro Val Glu Ile

245 250 255

Val Ser Ala Glu Asp Pro Leu Phe Ile Leu Tyr Thr Ser Gly Ser Thr

260 265 270

Gly Lys Pro Lys Gly Val Val His Ser Ser Ala Gly Tyr Leu Leu Gly

275 280 285

Ala Phe Met Thr Leu Lys Tyr Val Phe Asp Val His Pro Glu Asp Arg

290 295 300

Tyr Ala Cys Met Ala Asp Val Gly Trp Ile Thr Gly His Thr Tyr Ile

305 310 315 320

Val Tyr Gly Pro Leu Ala Asn Gly Val Thr Thr Thr Ile Phe Glu Ser

325 330 335

Thr Pro Val Tyr Pro Thr Pro Ser Arg Phe Trp Glu Thr Val Ala Lys

340 345 350

His Lys Leu Thr Gln Phe Tyr Thr Ala Pro Thr Ala Ile Arg Leu Leu

355 360 365

Arg Arg Leu Gly Glu Glu His Thr Lys Gly His Asp Leu Ser Thr Leu

370 375 380

Arg Thr Ile Gly Ser Val Gly Glu Pro Ile Asn Pro Glu Ala Trp Glu

385 390 395 400

Trp Tyr Trp Glu His Val Gly Lys Lys Glu Cys Ala Val Val Asp Thr

405 410 415

Tyr Trp Gln Thr Glu Thr Gly Ser Ile Ile Ile Thr Pro Leu Pro Gly

420 425 430

Ala Thr Lys Thr Lys Pro Gly Ala Ala Thr Leu Pro Phe Phe Gly Ile

435 440 445

Asp Pro Val Leu Leu Asp Pro Thr Thr Gly Glu Glu Ile Lys Gly Asn

450 455 460

Glu Val Glu Gly Val Leu Cys Val Arg Lys Pro Trp Pro Ser Ile Ala

465 470 475 480

Arg Thr Val Tyr Gly Asp His Lys Arg Phe Leu Asp Thr Tyr Met Asn

485 490 495

Pro Tyr Pro Gly Tyr Tyr Phe Thr Gly Asp Gly Ala Gly Arg Asp His

500 505 510

Asp Gly Tyr Tyr Trp Ile Arg Gly Arg Val Asp Asp Val Ile Asn Val

515 520 525

Ser Gly His Arg Leu Ser Thr Ala Glu Ile Glu Ser Ala Leu Ile His

530 535 540

His Asn Gly Val Ala Glu Thr Ala Val Val Gly Ile Pro Asp Glu Leu

545 550 555 560

Thr Gly Gln Ala Val Val Ala Tyr Val Ala Leu Lys Pro Glu Phe Ala

565 570 575

Ala Glu Asn Pro Asp Glu Pro Ala Leu Leu Lys Glu Leu Val Leu Gln

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Val Arg Lys Thr Ile Gly Pro Phe Ala Ala Pro Lys Lys Leu Val Leu

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

610 615 620

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

625 630 635 640

Thr Leu Ala Glu Pro Ala Ile Ile Asp Glu Ile Lys Glu Lys Phe Ala

645 650 655

Lys Ala

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

gatcactcac catggtgacc gaacacacct acgac 35

<210> 4

<211> 33

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

ccggtcggca tctacctacg ccttggcgaa ctt 33

Claims (3)

1. Acetyl CoA synthetase geneRKACS1The nucleotide sequence is shown in SEQ ID NO. 1.

2. The acetyl-CoA synthetase gene according to claim 1RKACS1Application in producing carotenoid and oil.

3. The acetyl-CoA synthetase gene according to claim 1RKACS1Application in oil production.

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