Culture method for increasing yield of microalgae Triglyceride (TAG) and application thereof
阅读说明：本技术 一种提升微藻甘油三酯(tag)产量的培养方法及其应用 (Culture method for increasing yield of microalgae Triglyceride (TAG) and application thereof ) 是由 张鹏 辛一 徐健 于 2020-05-27 设计创作，主要内容包括：本发明属于生物技术领域。本发明涉及一种影响甘油三酯(TAG)合成的功能基因和一种可高效提升微藻TAG产量的培养方法,及其在高效提升微藻TAG产量中的结合应用。影响甘油三酯(TAG)合成的基因NobZIP77,1)具有SEQ ID NO 1所示的碱基序列；或,2)与序列表中序列1所限定的核酸序列具有95％以上同源性、且编码相同生物学功能蛋白质的DNA序列。本发明获得的基因及氨基酸序列为微藻中的首次报道,敲除上述基因能够显著提高微拟球藻合成TAG的能力,此外,通过结合上述基因工程与培养方法可提高微拟球藻的TAG产量,因此该方法在生物能源及保健品行业均具有应用潜力。(The invention belongs to the field of biotechnology. The invention relates to a functional gene influencing Triglyceride (TAG) synthesis, a culture method capable of efficiently increasing microalgae TAG yield, and a combined application thereof in efficiently increasing microalgae TAG yield. A gene NobZIP77 affecting Triglyceride (TAG) synthesis, 1) having the base sequence shown in SEQ ID NO 1; or, 2) DNA sequence which has more than 95 percent of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function. The gene and the amino acid sequence obtained by the invention are reported in microalgae for the first time, the ability of the nannochloropsis to synthesize TAG can be obviously improved by knocking out the gene, and in addition, the TAG yield of the nannochloropsis can be improved by combining the genetic engineering and the culture method, so the method has application potential in biological energy and health care product industries.)
1. A gene, NobZIP77, affecting Triglyceride (TAG) synthesis, characterized by:
1) has a base sequence shown as SEQ ID NO 1;
or the like, or, alternatively,
2) DNA sequence which has more than 95% of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function.
2. A protein encoded by the gene of claim 1, wherein: the protein coded by the gene has an amino acid sequence with one of the following conditions:
1) has an amino acid sequence shown as SEQ ID NO 2;
2) an amino acid sequence of a derivative protein produced by substituting, deleting or adding one or more amino acid residues to the amino acid residue sequence of SEQ ID NO2, the derivative protein having the same biological function as the protein of SEQ ID NO 2.
3. A carrier for improving microalgae Triglyceride (TAG) yield, which is characterized in that: the vector contains a base sequence which causes silencing of the NobZIP77 gene.
4. The vector of claim 3, wherein: the vector has a base sequence shown by SEQ ID NO 3.
5. A nannochloropsis oculata gene engineering strain is characterized in that: the genome of the engineered strain is silenced with the NobZIP77 gene containing the vector of claim 3.
6. A culture method capable of improving the yield of microalgae TAGs is characterized in that: culturing the engineered bacterium of claim 5 in a culture medium and applying 30-50 μmol of phosns m-2s-1Blue ofCulturing for 3-4 days with light (445 nm).
7. A method for improving microalgae TAG yield is characterized by comprising the following steps:
1) using the nannochloropsis oculata genetically engineered strain of claim 5;
and the number of the first and second electrodes,
2) the culture method comprises the following steps: culturing the engineering strain to contain NaNO3In the culture solution with concentration of 2-4g/L, 30-50 μmol phosns m is applied-2 s-1For 9-12 days, followed by application of 30-50. mu. mol of phosns m-2 s-1Blue light (445nm), cultured for 3-4 days.
Technical Field
The invention belongs to the field of biotechnology. The invention relates to a functional gene influencing Triglyceride (TAG) synthesis, a culture method capable of efficiently increasing microalgae TAG yield, and a combined application thereof in efficiently increasing microalgae TAG yield.
Background
Under the guidance of the current concept of energy conservation and emission reduction, the development of renewable energy becomes a popular research direction. Biofuel plays an important role in the development of renewable energy sources, wherein biodiesel is more suitable for the current internal combustion engine and has a plurality of characteristics better than petroleum, such as reduction of carbon monoxide emission, improvement of combustion efficiency and the like. Unlike bioethanol and natural gas, biodiesel can be efficiently distributed by using the currently mature petroleum transportation system, which creates favorable conditions for large-scale popularization. Biodiesel is mainly derived from Triglycerides (TAG) in plants. Theoretically, if oil crops are produced on a large scale, the current energy demand must be met, however, the measure also causes a plurality of problems, including competition for food and land with people, improvement of net carbon emission and the like. In this context, oleaginous microalgae are an important concern for the biodiesel industry. Most microalgae have higher TAG unit yield than terrestrial plants, and some microalgae are reported to have TAG content of more than 75% of dry weight, and in addition, microalgae culture does not occupy arable land and has potential of water culture, marine algae avoid hidden danger of competing for fresh water with people, so that microalgae oil production has very optimistic prospect.
However, even though oleaginous microalgae do not accumulate TAG under good culture conditions, they need to be stress-cultured. At present, the most common stress method is nitrogen deficiency culture, which is divided into natural nitrogen deficiency and two-step nitrogen deficiency methods, however, the natural nitrogen deficiency requires long-term culture and the time cost is high, the two-step method requires artificial nitrogen deficiency, and although the time cost can be obviously reduced, the labor cost and the material cost are high. In summary, there is a need to develop a technology for efficiently increasing the yield of microalgae TAG at low cost, so as to promote the development of the microalgae biodiesel industry. The current common methods comprise a process control method and a metabolic engineering method, wherein the process control method focuses on external factors and has the characteristics of blindness and quick response; the metabolic engineering method focuses on internal factors, has the characteristics of rationality but not obvious effect, and in recent years, transcription factors are used for replacing key enzymes to solve the effect problem to a great extent, so that the utilization of the transcription factors to efficiently improve the yield of microalgae TAGs becomes the development direction of the industry.
Nannochloropsis is an outstanding representative of industrial oil-producing microalgae, and has the advantages of high photosynthetic efficiency, strong carbon-fixing capacity, high TAG content and the like, so that the nannochloropsis is an ideal species for applying the method. Earlier researches show that the lipid content of nannochloropsis oculata can be increased by 26.9% and 39.4% respectively (Sung, 2018) compared with a control group by irradiating red light and blue light for 10 days, which indicates that the light quality can possibly become an environmental factor for efficiently increasing the yield of nannochloropsis oculata TAG. In addition, the prediction and modification of the transcription factor of nannochloropsis has been reported (Hu, 2014; Kang, 2015), which lays an objective foundation for the development of the technology.
Disclosure of Invention
The invention aims to provide a gene with a Triglyceride (TAG) synthesis regulation function, a culture method capable of efficiently increasing the yield of microalgae TAG, and application of the gene in efficiently increasing the yield of microalgae TAG.
In order to achieve the purpose, the invention adopts the technical scheme that:
a gene affecting the synthesis of Triglyceride (TAG), NobZIP77,
1) has a base sequence shown as SEQ ID NO 1;
or the like, or, alternatively,
2) DNA sequence which has more than 95% of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function.
The protein coded by the gene is an amino acid sequence with one of the following conditions:
1) has an amino acid sequence shown as SEQ ID NO 2;
2) an amino acid sequence of a derivative protein produced by substituting, deleting or adding one or more amino acid residues to the amino acid residue sequence of SEQ ID NO2, the derivative protein having the same biological function as the protein of SEQ ID NO 2.
A vector for increasing microalgae Triglyceride (TAG) yield, wherein the vector contains a base sequence causing silencing of NobZIP77 gene.
The vector is combined with a double-stranded fragment with a BspQI cohesive end formed by annealing a single-stranded primer with a ribozyme gene and a NobZIP77 gRNA target sequence by taking a vector with a BspQI cohesive end as a framework, and the vector is the vector.
The vector has a base sequence shown by SEQ ID NO 3.
A nannochloropsis oculata gene engineering strain, in the genome of which the NobZIP77 gene containing said carrier has been silenced. The host bacterium is an industrial alga strain IMET1 of nannochloropsis oculata.
A culture method for increasing the yield of microalgae TAG comprises culturing the engineering bacteria in culture solution, and adding 30-50 μmol of photons m-2s-1Blue light (445nm), cultured for 3-4 days.
Or;
1) using the nannochloropsis oculata gene engineering strain;
and the number of the first and second electrodes,
2) the culture method comprises the following steps: culturing the engineering strain to contain NaNO3In the culture solution with concentration of 2-4g/L, 30-50 μmol phosns m is applied-2s-1For 9-12 days, followed by application of 30-50. mu. mol of phosns m-2s-1Blue light (445nm), cultured for 3-4 days.
The culture solution is f/2 liquid culture medium.
Drawings
FIG. 1 shows the gene structure of NobZIP77 used in the present invention.
FIG. 2 is a knock-out vector containing a partial sequence of NobZIP77 for use in the present invention.
FIG. 3 shows the alignment result of the genome sequence of the knockout strain NobZIP77ko-1 and the wild IMET1 obtained by the invention.
FIG. 4 shows the comparison of TAG production of wild IMET1 under artificial nitrogen deficiency conditions under blue and white light irradiation in the present invention, with asterisks indicating that p is less than or equal to 0.05 in t-test.
FIG. 5 shows the comparison of the TAG yields of the knockout strain NobZIP77ko-1 and the wild IMET1 under normal culture conditions under white light irradiation in the present invention, and asterisks indicate that p is less than or equal to 0.05 in t-test.
FIG. 6 shows the comparison of TAG yields of white light treated wild IMET1 and blue light treated knockout strain NobZIP77ko-1 under artificial nitrogen deficiency conditions in the present invention, with asterisks indicating that p is less than or equal to 0.05 in t-test.
FIG. 7 shows the comparison of TAG yields of white and blue light rational treatment knockout strain NobZIP77ko-1 and white light treatment wild IMET1 under normal conditions in the present invention, and asterisks indicate that p in t-test is less than or equal to 0.05.
Detailed description of the invention
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The invention knocks out the gene NobZIP77 for coding the negative regulation transcription factor of the nuclear genome of an industrial Nannochloropsis oceanica strain IMET1 (namely, Nannochloropsis oceanica IMET1 which is given by the university of Maryland and can be used for releasing the strain in the research direction of the public), wherein the gene and the amino acid sequence are first reported in microalgae, and the purpose of efficiently and obviously improving the yield of the TAG of the Nannochloropsis oceanica can be realized by combining blue light irradiation. The full-length cDNA sequence of the NobZIP77 gene is separated, and the NobZIP77 gene is subjected to endogenous knockout by using a gene transformation system of nannochloropsis, and experiments prove that the knockout of NobZIP77 can obviously improve the TAG yield of nannochloropsis. The disclosed full-length gene and amino acid sequence are reported for the first time in nannochloropsis, and the gene knockout can obviously improve the TAG synthesis capability of nannochloropsis, so that the application value of the gene in the aspect of improving the TAG production of organisms by using a genetic engineering means is proved; the light quality regulation and control method is reported in microalgae for the first time, can efficiently improve the TAG yield of nannochloropsis, and proves the application value of the method in the aspect of improving the industrial oil-producing microalgae culture process; in addition, TAG production of nannochloropsis is carried out by reasonably combining the genetic engineering and the culture method, and the remarkably accelerated improvement of the TAG yield can be obtained compared with that of the two methods when the two methods are used independently, so that the method has application potential in the biological energy and health care product industries.
Experimental methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional barsMolecular Cloning (Molecular Cloning: A Laboratory Manual, 3)rded.) or according to the manufacturer's recommendations.
Example 1: cloning and analysis of NobZIP77 Gene
Cloning NobZIP77 gene from cDNA of nannochloropsis oculata IMET1 by PCR technology, designing used primers, and handing to Shanghai to synthesize:
1)NobZIP77-for：
5’ATGGAAGGGCTAGGACAGC 3’；
2)NobZIP77-rev:
5’CTATCCCTTCAAATGCATCC 3’。
the PCR instrument used was a MasterCycler from Eppendorf, 50. mu.L of a reaction system including 4. mu.L of dNTP (2.5mM each of each, TAKARA), 2. mu.L (10. mu.M) of forward and reverse primers, and 5. mu.L of 10 XBuffer (Mg)2+plus, TAKARA), 0.4. mu.L rTaq enzyme (5U/. mu.L, TAKARA), 1. mu.L wild IMET1cDNA template (50 ng/. mu.L), and 35.6. mu.L ultrapure water. The reaction system is as follows: initial 94 ℃ pre-denaturation for 3min, then 94 ℃ denaturation for 30sec, 55 ℃ annealing for 30sec, 72 ℃ extension for 1min, 30 cycles, and finally 72 ℃ reaction for 7 min.
After the reaction, 5. mu.L of the PCR product was mixed with 1. mu.L of 6 × loading buffer (TAKARA), spotted on 1% (w/V) agarose (BIOWEST) gel, electrophoresed at 120V for 25min on an electrophoresis system manufactured by six instruments of Beijing, and then observed and photographed by using a UV gel imager BioChemiHR of UVP. The desired fragment was purified and recovered from the PCR product using the Cycle-Pure Kit or Gel Extraction Kit of Omega, the operation of which was completely performed according to the instructions.
The obtained purified fragment is connected into a pMD18-T vector of TAKARA company, and is transferred into Escherichia coli competent cells Trans 5 alpha of the whole gold company by using a heat shock transformation mode, and the positive clone is sent to Invitrogen company for sequencing, so that the full-length coding region sequence of the NobZIP77 gene is finally obtained, and is shown in a sequence table SEQ ID NO 1. In addition, the gene structure of NobZIP77 was obtained by alignment with the genome sequence, as shown in fig. 1, and the 5 'end and 3' end of the gene each had a flanking sequence, and 1 intron was present inside the gene.
Example 2: construction of knockout (primary) knockout vector of NobZIP77 in marine nannochloropsis oceanica IMET1
See fig. 2. The method for constructing the vector reference Poliner et al is as follows: designing a single-chain primer containing a ribozyme gene and a NobZIP77 gRNA target sequence, wherein the sequence is as follows:
1)g77-Cas9-for：
5’CGACACCGTCTGATGAGTCCGTGAGGACGAAACGAGTA AGCTCGTCACGGTGGTGAACAATGGAGGG 3’；
2)g77-Cas9-rev:
5’AAACCCTCCATTGTTCACCACCGTGACGAGCTTACTCGT TTCGTCCTCACGGACTCATCAGACGGTG 3’。
the above two primers were combined into a double-stranded fragment having a BspQI cohesive end by gradient dip annealing (95 ℃ to 25 ℃ C., 0.1 ℃ C. per cycle, holding for 1 second) and ligated to a pNOC-ARS-CRISPR-v2 vector backbone also having a BspQI cohesive end, which was awarded by Poliner, Michigan State university of Michigan, USA, to obtain a recombinant vector pXJ 630.
(II) electroporation method for introducing the vector pXJ630 into Nannochloropsis
1h before transformation, the concentration is about 1-3X 107cells/mL Nannochloropsis oculata solution in logarithmic growth phase, centrifuging at 4000g for 5min, discarding the supernatant, rinsing with 375mM sorbitol for 2 times, and adjusting the cell concentration to 2 × 10 with sorbitol8cells/mL. The concentrated algae are divided into 200 μ l small portions, pXJ015 empty carrier is selected as a control group, 3 μ g pXJ630 carrier and 1 μ l salmon sperm DNA (15 μ g/mL) denatured at 95 ℃ for 1min are added into each portion, and the mixture is evenly mixed and then is kept for 10min on ice. The mixture was transferred into a 2mm cuvette and shocked at 2200V (HV), 50 μ F, and immediately after shocking, the algae were transferred to 5mL of fresh F/2 medium. After being thawed in a shaker at 25 ℃ at 100rpm under low light for 48h, the mixture was spread on an f/2 plate containing zeocin at a concentration of 5. mu.g/mL and incubated at 25 ℃ and a concentration of 50. mu. mol m-2s-1And (5) culturing in light until the clone grows out.
Molecular identification of nannochloropsis NobZIP77 knockout strain
The transformed clones were picked up in f/2 medium containing 5. mu.g/mL zeocin for activation and total DNA extracted for PCR identification using the following primer sequences:
1)g77-Cas9-DNA-for：
5’ACGATGATGATGCCTACGGG 3’；
2)g77-Cas9–DNA-rev:
5’GAACTTCTTCCTCACACGGGA 3’。
the PCR instrument used was a MasterCycler from Eppendorf, 50. mu.L of a reaction system including 4. mu.L of dNTP (2.5mM each of each, TAKARA), 2. mu.L (10. mu.M) of forward and reverse primers, and 5. mu.L of 10 XBuffer (Mg)2+plus, TAKARA), 0.4. mu.L of rTaq enzyme (5U/. mu.L, TAKARA), 1. mu.L of cDNA template of the transformant (50 ng/. mu.L), and 35.6. mu.L of ultrapure water. The reaction system is as follows: initial 94 ℃ pre-denaturation for 3min, then 94 ℃ denaturation for 30sec, 55 ℃ annealing for 30sec, 72 ℃ extension for 1min, 30 cycles, and finally 72 ℃ reaction for 7 min.
After the reaction, 5. mu.L of the PCR product was mixed with 1. mu.L of 6 × loading buffer (TAKARA), spotted on 1% (w/V) agarose (BIOWEST) gel, electrophoresed at 120V for 25min on an electrophoresis system manufactured by six instruments of Beijing, and then observed and photographed by using a UV gel imager BioChemiHR of UVP. The desired fragment was purified and recovered from the PCR product using the Cycle-Pure Kit or Gel Extraction Kit of Omega, the operation of which was completely performed according to the instructions. The recovered product is sent to Invitrogen company for sequencing, the sequencing result is compared with the genome sequence of NobZIP77, 1 strain of NobZIP77ko-1 is finally screened, the comparison result with the sequencing result of wild IMET1 is shown in figure 3, the sequence ACGGTGGTGAACAATGGAGG at 501-19 bp 521bp of NobZIP77 gene in the wild type of the screened strain and the Nannochloropsis sp industrial strain IMET1 is mutated into ACGGTGGTGAACAATG-AGG, so that the code shift mutation of the subsequent sequence is caused, and the NobZIP77 gene in the algae strain is silenced.
(IV) Photoplasmic culture of Nannochloropsis oculata strain and TAG yield determination
1) Photoplasmic culture and TAG yield determination of Nannochloropsis sp:
the nannochloropsis wild IMET1 and the knockout strain NobZIP77ko-1 are firstly respectively at 25 ℃ and the light intensity of 50 mu mol phosns m-2s-1Introducing sterile air into f/2 liquid culture medium under white light to culture to OD750Centrifuging at room temperature 3500g for 5min, discardingAfter the supernatant, the pellet was resuspended in 150ml of a nitrogen-containing or nitrogen-free f/2 liquid medium (containing NaNO or nitrogen-free f/2 liquid medium)3Adding into f/2 liquid culture medium with nitrogen content of 2g/L), resuspending, and respectively adding into a light intensity of 50 μmol photons m-2s-1Under the irradiation of white light or blue light (445nm), sterile air is introduced at 25 ℃ for culture, and samples are taken at different time points for subsequent detection.
The TAG yield was measured by Raman spectroscopy (He, 2017) using 1ml each of the above azoospermum parvum samples cultured in the absence of light, and 3 biological replicates were set for each strain. Centrifugation at 3000g for 5min at room temperature collected algal bodies, washing 3 times with 200 μ l deionized water and resuspending the cells, aspiration into quartz capillaries (length 50mm × width 1mm × height 0.1mm, Camlab, UK) followed by bleaching to remove pigments, 20 cells per sample were randomly selected, each cell was irradiated with 532nm laser light source for 1 sec to obtain raman spectra between 393.8cm and 3341.3cm, followed by baseline normalization with Labspec 5(HORIBA JobinYvon ltd., UK), and then raman raw data was converted into TAG yield with PLSR model (Almeida, 2010).
From the analysis of the results shown in fig. 4 to 6, the following conclusions were made: firstly, the TAG yield irradiated by blue light is improved by 3.6-48.3% compared with that of white light (figure 4) between 6h and 264h of artificial nitrogen deficiency, which shows that the blue light has better TAG yield promotion effect than the white light; secondly, the TAG yield is improved by 60.9-184.5% compared with that of the wild type between 24h and 264h after the white light normal culture, and reaches 184.5% of the peak value at the 9 th (216h) (figure 5), which indicates that the knockout strain has better TAG yield promotion effect compared with the wild type; thirdly, at 72h and 96h of artificial nitrogen deficiency, the TAG yield of the blue light treated knockout strain is respectively improved by 35.3% and 21.1% compared with that of the white light treated wild type, and reaches 35.3% of the peak value at 3 rd day (72h) (figure 6), which shows that the blue light treated knockout strain has better TAG yield promotion effect than that of the white light treated wild type.
2) Photoplasmic culture and TAG yield determination of Nannochloropsis sp:
according to the research results, the experiment is designed: the nannochloropsis wild IMET1 and the knockout strain NobZIP77ko-1 are firstly respectively at 25 ℃ and the light intensity of 50 mu mol phosns m-2s-1Introducing sterile air into f/2 liquid culture medium under white light to culture to OD750Centrifuging at room temperature of 3500g for 5min, discarding supernatant, and resuspending the precipitate in 150ml of nitrogen-containing f/2 liquid culture medium (containing NaNO or nitrogen-free f/2 liquid culture medium)3Adding into f/2 liquid culture medium with nitrogen content of 2g/L), resuspending, and respectively adding into a light intensity of 50 μmol photons m-2s-1Under white light irradiation, culturing in sterile air at 25 deg.C for 9 days, and culturing at light intensity of 50 μmol photons m-2s-1Under blue light irradiation, 3 days at 25 ℃ in sterile air, samples were taken at different time points for subsequent detection (see FIG. 7).
As can be seen in FIG. 7, TAG production was improved by 62.9% for the knockout compared to the 12-day white-cultured wild type. In conclusion, the invention provides a technical method capable of efficiently increasing the yield of microalgae TAG, and provides a feasible idea for improving the industrial oil-producing microalgae culture process.
While specific examples of the invention have been described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.
SEQ ID NO1
1401
DNA
Nannochloropsis oceanica IMET1
SEQ ID NO：2
466
PRT
Nannochloropsis oceanica IMET1
SEQ ID NO3
12282
DNA
pXJ630
Sequence listing
<110> institute of bioenergy and Process in Qingdao, China academy of sciences
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<213> Artificial Sequence (Artificial Sequence)
<400> 3
accggtttct tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga 60
tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt 120
agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt 180
ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga 240
ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt 300
gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagta 360
caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac 420
aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta tatatttata 480
ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac 540
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 600
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 660
tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 720
tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 780
acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 840
cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 900
ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 960
ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 1020
atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 1080
cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 1140
tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 1200
gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 1260
ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 1320
ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 1380
tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 1440
cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 1500
ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 1560
tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 1620
gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 1680
caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 1740
accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 1800
ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 1860
aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 1920
accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 1980
gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2040
ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2100
gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2160
gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2220
ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2280
aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2340
gttctttcct gcgttatccc ctgattctgt ggtttaaacc aggtcactgg attttggttt 2400
taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt 2460
tcttatatgc tcaacacatg agcgaaaccc tataagaacc ctaattccct tatctgggaa 2520
ctactcacac attattctgg agaaaaatag agagagatag atttgtagag agagactggt 2580
gatttttgcg gactccggtc ggcatctact actagcctat tcctttgccc tcggacgagt 2640
gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc 2700
cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc 2760
gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata 2820
gagttggtca agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc 2880
cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca 2940
gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat 3000
gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac 3060
gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc 3120
gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc 3180
aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg 3240
gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac 3300
cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc 3360
acggcgggag atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg 3420
aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc 3480
ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg 3540
agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag 3600
aaacttctcg acagacgtcg cggtgagttc aggctttttc atatcttatt gccccccggg 3660
gccctcgact gtgttgatgc gggctgagat tggtggtggt ctatcacgaa tatgtgtgag 3720
gggtaagtgc ggtgttttgc gtgagatttt agaatattgc cccgccccgg ggcaggccgg 3780
cgtggcggaa caaccaggca cacgagcgcg aatggtgata ccgacggagt caaaactttg 3840
tgacaagtag ctgcaccatg ggcagtggtg agctttcaga cgtggtatca ctgtccacta 3900
gttcacacac agaatgcgtg tccaaaaggt ctagagccgt ctcgcttgcg tctctccgtc 3960
gaagaacagt gaagaggctc gtcacgtcga ccagacgacg ggaggctggt caccatcgca 4020
gatgtctccc acaaagcagc acggcaactc ctactccttc acacaatgga agaaaaggtg 4080
gtctgatggt tctcagtgga aaagaacgat atcaggctga aaaaaatgat ctgcaggctc 4140
cagattcctg aatcacgtcg actgtgacga agcaaaccgc gtcgaacaac atcggtcatg 4200
ccaacgggtc tcgtctctcg agcccttttg gcggcgacta agaagtatga agcttcaggc 4260
cgcaacgcgc gacacagcgt tttgtgtggt gggcctcggc attgctcttt gcatggccca 4320
gcgtgattag tgcgtggatt ttaagcccga gaccgaagga ttgcgacatg tgcctggctg 4380
tataactcac gcttgctgct acgctcgcct cctcctccat ccactccatc gcggccgcca 4440
tggcctctag tggatcagct tgcatgcctg caggagacga tatcacctct tctgtttcca 4500
cgataaaaat agactgctca tttcttcgtc gtcttcatcg tctgcttttt ctgcttcgcc 4560
tctgtctggg gtctgaaacc actacacaca cacacaacac tcgtactccc actttcacaa 4620
aagcgtaagc tcaccggctt ttcttacacg tacattttag tggatcccat cacgccacta 4680
ccacgcccgc gggggatgga acggagggga gagagagaag ggggaagcat ggatgaatga 4740
gacattgagg gaaaggaggg gagggagcag tccatcaggg cgctacctct cttgtccccc 4800
aaaccctgtt gagccgttca acatgtttca tgtttcctcc ctcccccctt ccctccctgg 4860
cctttccgcg gagccattca agtgacgtct ggaccgcacc gtaacaaaat cgtttctatg 4920
gggggtttgt ttgacaacca cgtcttcagc gtttttaaaa aaaaaagcgg gcaagccctc 4980
tcaccctcac tcatgcccat cctcctcctc tcctgcggaa cattcttaca aaaggcgtaa 5040
ctcgacgaca actcaaagaa cgacaaacat caatcccaaa aaaaaaatct ctactcgtct 5100
ctcttggatc tttccaattg tcagaccttc ctcttctttt tcgggctagc caccatggac 5160
gcgtagtcgg gcacgtcgta ggggtatccg gccaggatgc gctcgcacag gcgccagccg 5220
gttaccccgt tgatggtcac gcggaacaac agcgagccat ccgggttgat cagccgctcg 5280
tcaatgatct tattgccgtt ccacagggtg ccggtcaccg tgatcttctt gccgtcgaac 5340
acggcaatgc cctcgtaagg acggccgaag tagtcaatca tgttcggcgt cacaccgtcg 5400
atgaccagcg tcccgtagtg cagtatcacc ttgaagtggt gatcgtccac ggggtacaca 5460
accttgaaga tcttctcgat ctggcccatt tggtccccgc tcaggccctc gtagggaatg 5520
atgacgtgga tgtcgatctt caggccgttc tcgccgctca ggacgatgcg ctggattggg 5580
gtcacggaga cgcccaggtt ctggaaaagc gaggacacac cgccctgctc cagcacctgg 5640
tcgaggttat agccagctgt ctgccgccag tcgcccacga agtcctcgag agtaaacacc 5700
atgttaacgg acccgctgcc ggacccgtca gccctgctgt ctccaccgag ctgagagagg 5760
tcgattcttg tttcatagag ccccgtaatt gactgatgaa tcagtgtggc gtccaggacc 5820
tcctttgtag aggtgtaccg ctttctgtct atggtggtgt cgaagtactt gaaggctgca 5880
ggcgcgccca agttggtcag agtaaacaag tggataatgt tttctgcctg ctccctgatg 5940
ggcttatccc tgtgcttatt gtaagcagaa agcaccttat cgaggttagc gtcggcgagg 6000
atcactcttt tggagaattc gcttatttgc tcgatgatct catcaaggta gtgtttgtgt 6060
tgttccacga acagctgctt ctgctcatta tcttcgggag accctttgag cttttcatag 6120
tggctggcca gatacaagaa attaacgtat ttagagggca gtgccagctc gttacctttc 6180
tgcagctcgc ccgcactagc gagcattcgt ttccggccgt tttcaagctc aaagagagag 6240
tacttgggaa gcttaatgat gaggtctttt ttgacctctt tatatccttt cgcctcgaga 6300
aagtcgatgg ggtttttttc gaagcttgat cgctccatga ttgtgatgcc cagcagttcc 6360
ttgacgcttt tgagtttttt agacttccct ttctccactt tggccacaac cagtacactg 6420
taagcgactg taggagaatc gaatccgccg tatttcttgg ggtcccaatc ttttttgcgt 6480
gcgatcagct tgtcgctgtt ccttttcggg aggatacttt ccttggagaa gcctccggtc 6540
tgtacttcgg tctttttaac gatgttcacc tgcggcatgg acaggacctt ccggactgtc 6600
gcgaaatccc tacccttgtc ccacacgatt tctcctgttt ctccgtttgt ttcgataagt 6660
ggtcgcttcc gaatctctcc attggccagt gtaatctcgg tcttgaaaaa attcataata 6720
ttgctgtaaa agaagtactt agcggtggcc ttgcctattt cctgctcaga ctttgcgatc 6780
attttcctaa catcgtacac tttatagtct ccgtaaacaa attcagattc aagcttggga 6840
tattttttga taagtgcagt gcctaccact gcattcaggt aggcatcatg cgcatggtgg 6900
taattgttga tctctctcac cttataaaac tgaaagtcct ttctgaaatc tgagaccagc 6960
ttagacttca gagtaataac tttcacctct cgaatcagtt tgtcattttc atcgtacttg 7020
gtgttcatgc gtgaatcgag aatttgggcc acgtgcttgg tgatctggcg tgtctcaaca 7080
agctgccttt tgatgaagcc ggctttatcc aactcagaca ggccacctcg ttcagcctta 7140
gtcagattat cgaacttccg ttgtgtgatc agtttggcgt tcagcagctg ccgccaataa 7200
tttttcattt tcttgacaac ttcttctgag gggacgttat cactcttccc tctattttta 7260
tcggatcttg tcaacacttt attatcaata gaatcatctt tgagaaaaga ctggggcacg 7320
atatgatcca cgtcgtagtc ggagagccga ttgatgtcca gttcctgatc cacgtacatg 7380
tccctgccgt tctgcaggta gtacaggtag agcttctcat tctgaagctg ggtgttttca 7440
actgggtgtt ccttaaggat ttgggacccc agttctttta taccctcttc aatcctcttc 7500
atcctttccc tactgttctt ctgtcccttc tgggtagttt ggttctctcg ggccatctcg 7560
ataacgatat tctcgggctt atgccttccc attactttga cgagttcatc cacgacctta 7620
acggtctgca gtattccctt tttgatagct gggctacctg caagattagc gatgtgctcg 7680
tgaagactgt ccccctggcc agaaacttgt gctttctgga tgtcctcctt aaaggtgaga 7740
gagtcatcat ggatcaactg catgaagttc cggttggcaa atccatcgga cttaagaaaa 7800
tccaggattg tctttccact ctgcttgtct cggatcccat tgatcagttt tcttgacagc 7860
cgcccccatc ctgtatatcg gcgcctcttg agctgtttca tgactttgtc gtcgaagaga 7920
tgagcgtaag ttttcaagcg ttcttcaatc atctccctat cttcaaacaa cgtaagggtg 7980
aggacaatgt cctcaagaat gtcctcgttc tcctcattgt ccaggaagtc cttgtcttta 8040
atgattttca ggagatcgtg atacgttccc agggatgcgt tgaagcgatc ctccactccg 8100
ctgatttcaa cagagtcgaa acattcaatc tttttgaaat agtcttcttt gagctgtttc 8160
acggtaactt tccggttcgt cttgaagagg aggtccacga tagctttctt ctgctctcca 8220
gacaggaatg ctggctttct catcccttct gtgacgtatt tgaccttggt gagctcgtta 8280
taaactgtga agtactcgta cagcagagag tgtttaggaa gcaccttttc gttaggcaga 8340
tttttatcaa agttagtcat cctttcgatg aaggactggg cagaggcccc cttatccacg 8400
acttcctcga agttccaggg agtgatggtc tcttctgatt tgcgagtcat ccacgcgaat 8460
ctggaatttc cccgggcgag ggggcctaca tagtagggta tccgaaatgt gaggattttc 8520
tcaatctttt ccctgttatc tttcaaaaag gggtagaaat cctcttgccg cctgaggata 8580
gcgtgcagtt cgcccaggtg aatctggtgg gggatgcttc cattgtcgaa agtgcgctgt 8640
ttgcgcaaca gatcttctct gttaagcttt accagcagct cctcggtgcc gtccattttt 8700
tccaagatgg gcttaataaa tttgtaaaat tcctcctggc ttgctccgcc gtcaatgtat 8760
ccggcgtagc catttttaga ctgatcgaag aaaatttcct tgtacttctc aggcagttgc 8820
tgtctgacaa gggccttcag caaagtcaag tcttggtggt gctcatcata gcgcttgatc 8880
atactagcgc tcagcggagc tttggtgatc tccgtgttca ctcgcagaat atcactcagc 8940
agaatggcgt ctgacaggtt ctttgccgcc aaaaaaaggt ctgcgtactg gtcgccgatc 9000
tgggccagca gattgtcgag atcatcatcg taggtgtctt tgctcagttg aagcttggca 9060
tcttcggcca ggtcgaagtt agatttaaag ttgggggtca gcccgagtga cagggcgata 9120
agattaccaa acaggccgtt cttcttctcc ccagggagct gtgcgatgag gttttcgagc 9180
cgccgggatt tggacagcct agcgctcagg attgctttgg cgtcaactcc ggatgcgttg 9240
atcgggttct cttcgaaaag ctgattgtaa gtctgaacca gttggataaa gagtttgtcg 9300
acatcgctgt tgtctgggtt caggtccccc tcgatgagga agtgtccccg aaatttgatc 9360
atatgcgcca gcgcgagata gatcaaccgc aagtcagcct tatcagtact gtctacaagc 9420
ttcttcctca gatgatatat ggttgggtac ttttcatggt acgccacctc gtccacgata 9480
ttgccaaaga ttgggtggcg ctcgtgcttt ttatcctcct ccaccaaaaa ggactcctcc 9540
agcctatgga agaaagagtc atccacctta gccatctcat tactaaagat ctcctgcagg 9600
tagcagatcc gattctttct gcgggtatat ctgcgccgtg ctgttctttt gagccgcgtg 9660
gcttcggccg tctccccgga gtcgaacagg agggcgccaa tgaggttctt ctttatgctg 9720
tggcgatcgg tattgcccag aactttgaat tttttgctcg gcaccttgta ctcgtccgta 9780
atgacggccc agccgacgct gtttgtgccg atatcgagcc caatggagta cttcttgtcc 9840
accttccgct ttttcttggg catgctcgag ggttgcgtgt gtatctgtgt gcagtggata 9900
ttgttaccga gtttggtgag cgtgagtccg ttagtgccct ggtggtggtg gattaggaga 9960
gtgggtgact cggtgtccat ggctttcttc gctcattata ggaggggaaa ggaatgaggg 10020
agggtgggga gaccgcgtct gttgttgacc accgatttac ttcttgcctc ccttcccctc 10080
cctcccctca atccgtacga cacaaatagt agccgagtgt ctgctgcaga gcgcatgatt 10140
agtgtggtag acaacgaggg agggaaggat gtacagggca tggcacggag aagcgatggt 10200
ggccaggaag aggagaggtc gcgagaacag gatgtgttgc gaatggataa aaacagaaag 10260
cgatggctct gggcttcgaa agcaggggac attaggacgt gtagaccatc tcgacggatc 10320
cctctgtatc tctgttgtgc gtgaatgttt tctgtgcacg tgtagtgtgt gagagtagaa 10380
cccgggaact cgaacagaga aaagcatggg tggctgtggt gtggaggctt cgttcccacc 10440
acatgccctt ctccttcgcc tcgcctctcc ctgccttctt ccacgcaccc ttgcgcccct 10500
cgttctcaat acctggctca cttccaccat tcaaacaacc atcacgatac aggcatttat 10560
ctatcgttga agacttcttc ctccggtaga tcttagccaa ggtaagaaga ggggcatgca 10620
gcaaggagaa agaaatgatg catgatgagg aatagaaggg gaggagggag ggatatgatg 10680
ggaagcgaaa gcgcatattc tggtggtctg ctgcctgatg gggacgcgtc tagctgtgac 10740
actgaggacg gtggctgctg gtggctgcgg gcgctgcctt ggtgatcaat gggagtaaag 10800
ggagggaagg aggtagcgtg aacggatgac gcggagaagt ttaggggtct ctttacgtat 10860
cgcccctgcc gcccgcctct ctgcgataaa tgtgcctgtt accctgcagc ctctattctt 10920
cactgtgttc ctgttttcca acagcctcta ttcttccctg tcttttgttg cagtggcgtc 10980
atcctctctt tgccccagtc gtcgttctct cgactcactc actccccccc tccttccctc 11040
cctccatcca cagaatcgag agtgactgat gagtccgtga ggacgaaacg agtaagctcg 11100
tctcactcca tcccaagcca ctgttttaga gctagaaata gcaagttaaa ataaggctag 11160
tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttggccggca tggtcccagc 11220
ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac tcctgggtac 11280
catgggaaag aaaggatgag aaaggagaaa ggacatctag ataaccggca tatcacggtg 11340
gtgtattagt gtagaatagt gaagagaaga cttgggaaaa tgtgtaggaa aggttgtttc 11400
tgtgtatgtg ggttgggatg ggtggctgtt tgagaaggaa cagcgggcag ggcgatgtag 11460
tgctgaacgg gcacggaacc actgagactg aaggaagtag ggagagagag gggcagggga 11520
cgtgcacttt aatctttgcc tcggtagagt atacccatgc aagagtatgt ggccacctgt 11580
ggtggctttt ggccaggtct ggtgcagtgc caatcatctc ccatcaataa tacaacttca 11640
gaacaacggc gcattgatgg ggagagagaa agtaaattta agtaaggggt acgtagtaga 11700
ggattcaact gaaatatttt cgaggagcgg ttgggaagtt gaccgattga aaggagaagg 11760
gaggggagca ggtgtgatag tcatgtgtaa agtaattctt ttttgccgtc gtcacacaat 11820
ccacatcaat gataaaatat gtttaaggat caatcacacg gagtcggtca taaggcaacc 11880
gcaaacgcaa tgcaaactag caagcaagca ggaccacaac aacaaccatt accatcacag 11940
gcgacagcag cagcagctgc agcagcagca aggcaagcaa aggccatctg cgcgtggact 12000
tttccaggca ccggctttaa gcgtaatttt caatgattgt tgtccgtgta tcctctcacc 12060
ctttcaccgc ttgcgcgcac gtgagcagca caactggtaa tgcccgagga caatagacga 12120
ggaaaaagaa gaagaaaaat gaagagggag tgatgaagaa gaagagaaga ataaaagaag 12180
acagaaaaga agataaaaaa gtcaaaagac gacaaagacg gaaaaaaaag aaacgcgcaa 12240
ttttaacacc agcaacgacg acgaaaaggg cgtcagcttt gc 12282