CN112280797B

CN112280797B - Can improve coenzyme Q in tomato 10 Content combined vector and construction method and application thereof

Info

Publication number: CN112280797B
Application number: CN202011182841.0A
Authority: CN
Inventors: 许晶晶; 范航; 陈晓亚; 杨蕾
Original assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Current assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2022-08-05
Anticipated expiration: 2040-10-29
Also published as: CN112280797A

Abstract

The invention relates to a method for improving coenzyme Q in tomato ₁₀ The method for constructing the combined vector comprises the following steps: obtaining an E8 promoter sequence, a tobacco Coq2 gene coding sequence, a DPS gene coding sequence, an HMGR2 gene coding sequence, a Ubic gene coding sequence and an hsp terminator sequence; constructing level 0 vectors of the six sequences; constructing a level 1 vector of each of the four genes; and constructing a level 2 vector. The invention also relates to a method for improving coenzyme Q ₁₀ The production method of the content tomato comprises the steps of transforming agrobacterium GV3101 by the combined vector obtained by the construction method through heat shock, and transforming wild tomato; after obtaining independent resistant callus, further differentiating and regenerating to obtain high coenzyme Q ₁₀ Content of tomato lines. Through cultivation and screening, the T3 generation fruit coenzyme Q of the tomato strain HUCD provided by the invention ₁₀ The yield can reach more than 7 times (about 0.151mg/g) of wild tomato fruits at most, and the wild tomato fruits can be stably inherited without difference in phenotype; namely coenzyme Q in the tomato lines HUCD of the invention ₁₀ The content is obviously improved.

Description

Can improve coenzyme Q in tomato 10 Content combined vector and construction method and application thereof

Technical Field

The invention relates to the technical field of plants, in particular to a method for improving coenzyme Q in tomato ₁₀ The combined vector of the content and the construction method and the application thereof.

Background

Coenzyme Q ₁₀ (Coenzyme Q ₁₀ Also known as CoQ ₁₀ ) The molecular structure of (a) consists of a benzoquinone parent group and a quinone with a decaisopentene side chain. Coenzyme Q ₁₀ In the human body, it is mainly used as an electron transporter for oxidizing complex II/III in the respiratory chain, and helps the cell to produce ATP. In addition, it is alsoAn antioxidant substance on a membrane is used as a cofactor for a number of enzymes. In the human body, coenzyme Q is deficient ₁₀ It causes various severe neurological diseases such as mitochondrial encephalomyopathy, cerebellar ataxia or Leigh's encephalopathy, etc., and affects the functions of various organs such as skeletal muscle, heart and kidney. In general, coenzyme Q ₁₀ The level decreases with the age of the person and coenzyme Q is present in certain drugs (e.g. statins) and in the case of cardiovascular diseases ₁₀ The content is abnormally decreased.

The present coenzyme Q ₁₀ Production is mainly by microbial (yeast, etc.) synthesis. Coenzyme Q extracted from engineered yeast or bacterial cells in view of food safety ₁₀ Is not completely suitable for food addition, and coenzyme Q is extracted from plant ₁₀ This problem can be effectively avoided. But due to coenzyme Q in plants ₁₀ The content is too low, and the coenzyme Q in the crops needs to be improved by a certain molecular biological method ₁₀ And (4) content. Because of the coenzyme Q in the current plants ₁₀ The synthetic route has not been completely resolved, and the improvement of coenzyme Q in crops has not been successful ₁₀ The report of (1).

To obtain exogenous coenzyme Q from the diet ₁₀ For maintenance of coenzyme Q in vivo ₁₀ The horizontal stability is of great significance. Meat, fish and oil are generally rich in coenzyme Q in our dietary structure ₁₀ (about 0.12mg/g), but coenzyme Q in vegetables and fruits ₁₀ The content is relatively low (less than 0.02 mg/g). Under the concept of advocating increasing the ratio of the dietary structures of vegetables and fruits in the society at present, coenzyme Q in food is maintained ₁₀ The stable intake of (2) becomes extremely difficult. In order to meet the requirement of daily nutrient intake, people have to be rich in coenzyme Q ₁₀ The vegetable food of (b) has great demand. Thus increasing coenzyme Q in crops ₁₀ The invention has practical production value and definite transformation prospect.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for improving coenzyme Q in tomatoes ₁₀ Combined vector of contents and its structureA construction method and application.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the present invention provides a method for increasing coenzyme Q in tomato ₁₀ The method for constructing the combined vector comprises the following steps:

s1, extracting tomato genome DNA, and obtaining an E8 promoter sequence through PCR amplification; obtaining a tobacco Coq2 gene coding sequence through total synthesis, as shown in SEQ ID NO. 1; extracting tobacco genome RNA, obtaining cDNA through reverse transcription, and obtaining a DPS gene coding sequence through PCR amplification, wherein the DPS gene coding sequence is shown as SEQ ID NO. 2; obtaining an arabidopsis HMGR2 gene coding sequence through total synthesis, wherein the coding sequence is shown as SEQ ID NO. 3; obtaining an escherichia coli UbicC gene coding sequence with the N end fused with a tomato chloroplast signal peptide through total synthesis, wherein the sequence is shown as SEQ ID NO. 4; extracting arabidopsis genome DNA, and obtaining an hsp terminator sequence through PCR amplification;

s2, connecting the E8 promoter sequence into a vector pUAP1 by a Golden Gate cloning method to obtain a level 0 vector of the E8 promoter; the Coq2 gene coding sequence was ligated into another vector pUAP1 by the Golden Gate cloning method to obtain a level 0 vector for Coq2 gene; connecting the DPS gene coding sequence into another vector pUAP1 by a Golden Gate cloning method to obtain a level 0 vector of the DPS gene; the HMGR2 gene coding sequence is connected into another vector pUAP1 by a Golden Gate cloning method to obtain a level 0 vector of the HMGR2 gene; connecting the coding sequence of the Ubic gene into another vector pUAP1 by a Golden Gate cloning method to obtain a level 0 vector of the Ubic gene; ligating the hsp terminator sequence into another vector pUAP1 by the Golden Gate cloning method to obtain a level 0 vector for the hsp terminator;

s3, sequentially connecting the E8 promoter sequence, the Coq2 gene coding sequence and the hsp terminator sequence into a vector pICH47742 through a Golden Gate cloning method to obtain a level 1 vector of Coq2 genes; sequentially connecting the E8 promoter sequence, the DPS gene coding sequence and the hsp terminator sequence into a vector pICH47751 by a Golden Gate cloning method to obtain a level 1 vector of the DPS gene; sequentially connecting the E8 promoter sequence, the HMGR2 gene coding sequence and the hsp terminator sequence into a vector pICH47761 by a Golden Gate cloning method to obtain a level 1 vector of the HMGR2 gene; sequentially connecting the E8 promoter sequence, the ubiC gene coding sequence and the hsp terminator sequence into a vector pICH47772 by a Golden Gate cloning method to obtain a level 1 vector of the ubiC gene;

s4, connecting a 35S promoter sequence, a hygromycin resistance gene coding sequence and a 35S terminator sequence, the E8 promoter sequence, the Coq2 gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the DPS gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the HMGR2 gene coding sequence and the hsp terminator sequence in sequence by a Golden Gate cloning method, and connecting the E8 promoter sequence, the Ubic gene coding sequence and the hsp terminator sequence into a vector pICSL4723-P1 in sequence to obtain a level 2 vector, namely the combined vector, as shown in SEQ ID NO. 5.

Preferably, each of the PCR amplification systems described in step S1 is: a template, i.e., 500ng of said genomic DNA or 50ng of said cDNA; 0.2. mu.M forward primer; 0.2. mu.M reverse primer; 10. mu.L Fastpfu buffer; 0.2mM dNTPs; 2.5units Fastpfu DNA polymerase; h ₂ O is complemented to 50 mu L;

the PCR amplification procedures were all as follows: at 95 ℃ for 2 min; circulating for 33 times at 95 deg.C, 20s, 58 deg.C, 20s, 72 deg.C, 2 min; 72 ℃ for 5 min.

Preferably, the PCR amplification primers of tomato genomic DNA are shown as SEQ ID NO.6 and SEQ ID NO. 7; PCR amplification primers of tobacco cDNA are shown as SEQ ID NO.8 and SEQ ID NO. 9; PCR amplification primers of arabidopsis genomic DNA are shown as SEQ ID NO.10 and SEQ ID NO. 11.

Preferably, when the corresponding gene coding sequence is obtained by the total synthesis, some of the bases therein are modified to remove BsaI and BpiI cleavage sites.

Preferably, in step S2, each of the level 0 vector construction systems is: 100ng of pUAP1 vector; the molar ratio of the corresponding sequence fragment obtained in the step S1 to the pUAP1 vector is 2: 1; 5units BpiI endonuclease; 200units T4 ligase;1.5 μ L T4 ligase buffer; 1.5. mu.L BSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

the reaction procedures are as follows: circulating for 26 times at 37 deg.C, 3min, 16 deg.C, 4 min; 5min at 80 ℃;

after the reaction is finished, taking the reaction solution to transform the heat shock competence of escherichia coli DH10B, and growing on an LB solid culture medium containing chloramphenicol; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing and obtaining the corresponding correct level 0 vector.

Preferably, in step S3, each of the level 1 vector constructs is: corresponding level 1 vector, 100 ng; the molar ratio of the level 0 vector of the E8 promoter obtained in the step S2 to the corresponding level 1 vector is 2: 1; the molar ratio of the level 0 vector of the corresponding gene obtained in the step S2 to the corresponding level 1 vector is 2: 1; the molar ratio of the level 0 vector of the hsp terminator obtained in the step S2 to the corresponding level 1 vector is 2: 1; 5units BsaI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

after the reaction is finished, taking reaction liquid to transform escherichia coli DH10B heat shock competence, and growing on an LB solid culture medium containing ampicillin; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing and obtaining the corresponding correct level 1 vector.

Preferably, the level 2 vector construction systems in step S4 are all: 100ng pICSL4723-P1 vector; the molar ratio of the pICSL11059 vector to the pICSL4723-P1 vector is 2: 1; the molar ratio of the Coq2 gene level 1 vector obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the level 1 vector of the DPS gene obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the level 1 vector of the HMGR2 gene obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the Ubic gene level 1 vector obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the PICH41800 linker vector is described,the molar ratio of the pICSL4723-P1 vector to the pICSL4723-P1 vector is 2: 1; 5units BsaI endonuclease; 5units BpiI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

the reaction procedure is as follows: circulating for 26 times at 37 deg.C, 3min, 16 deg.C, 4 min; 5min at 80 ℃;

after the reaction is finished, taking reaction liquid to transform escherichia coli DH10B heat shock competence, and growing on an LB solid culture medium containing kanamycin; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing and obtaining correct level 2 vector.

A second aspect of the present invention is to provide a combination vector obtained by the above-mentioned combination vector construction method.

In a third aspect of the present invention, there is provided a method for increasing coenzyme Q ₁₀ The production method of the content tomato comprises the steps of transforming agrobacterium GV3101 by the combined vector obtained by the construction method through heat shock, and transforming wild tomato; after obtaining independent resistant callus, further differentiating and regenerating to obtain high coenzyme Q ₁₀ Content of tomato lines.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

coenzyme Q in wild type tomato fruit ₁₀ The dry weight content is far below 0.02 mg/g. Through cultivation and screening, the T3 generation fruit coenzyme Q of the tomato strain HUCD provided by the invention ₁₀ The yield can reach more than 7 times (about 0.151mg/g) of wild tomato fruits at most, and the wild tomato fruits can be stably inherited without difference in phenotype; namely coenzyme Q in the tomato lines HUCD of the invention ₁₀ The content is obviously improved.

Drawings

FIG. 1 is a diagram of the design concept of the combination vector of the present invention;

FIG. 2 is a schematic structural view of a combination vector of the present invention;

FIG. 3 shows coenzyme Q in fruits of different lines of HUCD according to the example of detection of the present invention ₁₀ Content results plot; wherein the letter a indicates that there is a significant difference between HUCD-5 and other strains; letter b denotes HUCD-4 and HUCD-2 and lower strainsSignificant differences between lines; letter c indicates significant differences between HUCD-2 and HUCD-1 and lower lines; letter d indicates a significant difference between HUCD-1 and WT; the letter bc indicates no significant difference between HUCD-3 and HUCD-4 and HUCD-2, respectively (n-5, P-value)<0.05)。

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.

Example 1

As shown in FIG. 1, coenzyme Q ₁₀ The biosynthetic pathway of (a) mainly comprises four processes: (1) formation of a quinone ring precursor aromatic ring structure; (2) isoprene pyrophosphate polymerizes into chains; (3) the combination of an aromatic ring structure with an isoamylene side chain; (4) modification at the quinone ring after conjugation. The first three processes directly determine the downstream coenzyme Q ₁₀ The yield of (2). Therefore, it is the main goal of our technical scheme to facilitate the first three steps of reaction process:

(1) promoting the formation of the quinone ring precursor aromatic ring structure: coenzyme Q ₁₀ The precursor of the quinone ring structure is p-hydroxybenzoic acid (4-hydroxybenzoic acid, 4-HB). In prokaryotes, 4-HB is cleaved by chorismate lyases (Ubic) catalyzed by chorismate lyases. In eukaryotes, 4-HB is converted from phenylalanine or tyrosine, but the specific formation process is not yet clear. We therefore used overexpressed Ubics to catalyze the replacement of chorismate for phenylalanine or tyrosine to complement 4-HB synthesis.

(2) Promoting isoprene pyrophosphate polymerization into chains: isoprene pyrophosphate (IPP) is mainly synthesized in plants by Mevalonate (MVA) pathway and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which is used for coenzyme Q ₁₀ The synthesized IPP is mainly derived from the MVA pathway. In the MVA pathway, HMG-CoA reductase (HMGR-CoA reductase), which regulates the conversion of hydroxymethylglutaryl-CoA (HMG-CoA) to mevalonate, is a key rate-limiting enzyme, and its expression directly affects the yield of IPP downstream. For coenzyme Q ₁₀ In particular, IPP is transported to mitochondria via the cytoplasm, and then polymerized into a decaisopentenyl side chain in the mitochondria under the catalysis of decaprenyl-diphosphate synthase (DPS). Therefore I increase IPP accumulation by increasing HMGR expression. Further IPP is then converted to the decaisopentenyl side chain by up-regulating the expression of DPS.

(3) Promote the bonding of the aromatic ring structure to the decaprenyl side chain: in mitochondria, the decaisopentenyl side chain is catalyzed by isopentenyl p-hydroxybenzoate transferase (4-hydroxybenzoate polyprenyltransferase, Coq2) to be combined with the C-3 position of 4-HB to form coenzyme Q ₁₀ Structural precursor, reaction of which is believed to confine coenzyme Q ₁₀ The last step of the synthesis is the key enzyme. Therefore, we promoted the upstream accumulated 4-HB and isopentenyl polymeric side chain to the downstream coenzyme Q by increasing the expression of Coq2 ₁₀ And (4) converting the direction.

Thus, this example provides a method for increasing coenzyme Q in tomato ₁₀ The method for constructing the combined vector comprises the following steps:

s1, extracting tomato genome DNA, and obtaining an E8 promoter sequence through PCR amplification; obtaining a tobacco Coq2 gene (XM _016628012.1) coding sequence through total synthesis, which is shown as SEQ ID NO. 1; extracting genome RNA of tobacco (Nicotiana tabacum), obtaining cDNA through reverse transcription, and obtaining a DPS gene (NM-001325324.1) coding sequence through PCR amplification, wherein the sequence is shown as SEQ ID NO. 2; obtaining an arabidopsis HMGR2 gene (AY143930.1, 99 amino acids of N segment are removed) coding sequence through total synthesis, and the coding sequence is shown as SEQ ID NO. 3; obtaining an Escherichia coli Ubic gene coding sequence with the N end fused with tomato chloroplast signal peptide (NM-001308944.1, the first 67 amino acids of Solanum lycopersicum tribute carboxylase small chain 2A) through total synthesis, wherein the coding sequence is shown as SEQ ID NO. 4; extracting arabidopsis genome DNA, and obtaining an hsp terminator sequence through PCR amplification;

wherein, each PCR amplification system is as follows: a template (i.e., 500ng of the genomic DNA or 50ng of the cDNA); 0.2. mu.M forward primer; 0.2. mu.M reverse primer; 10. mu.L Fastpfu buffer; 0.2mM dNTPs; 2.5units Fastpfu DNA polymerase; h ₂ O is complemented to 50 mu L;

the PCR amplification procedures were all as follows:

wherein, PCR amplification primers of tomato genome DNA are shown as SEQ ID NO.6 and SEQ ID NO. 7; PCR amplification primers of tobacco cDNA are shown as SEQ ID NO.8 and SEQ ID NO. 9; PCR amplification primers of arabidopsis genomic DNA are shown as SEQ ID NO.10 and SEQ ID NO. 11.

Wherein, when the corresponding gene coding sequence is obtained through the total synthesis, partial bases in the corresponding gene coding sequence are modified to remove BsaI and BpiI enzyme cutting sites.

wherein, the stepsIn step S2, each level 0 vector construction system is: 100ng of pUAP1 vector; the corresponding sequence fragment obtained in step S1 (in a molar ratio of 2:1 to the pUAP1 vector); 5units BpiI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA (1 mg/mL); h ₂ O is complemented to 20 mu L;

the reaction procedures are as follows:

after the reaction is finished, 10 mu L of reaction liquid is taken to transform the heat shock competence of escherichia coli DH10B, and the escherichia coli is grown on an LB solid culture medium containing chloramphenicol; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing and obtaining the corresponding correct level 0 vector.

wherein, in the step S3, each level 1 vector construction system is: the corresponding level 1 vector (100 ng); a level 0 vector of the E8 promoter obtained in the step S2 (the molar ratio of the E8 promoter to the corresponding level 1 vector is 2: 1); level 0 vector of the corresponding Gene obtained in step S2 (corresponding to the aboveThe molar ratio of the level 1 carrier of (a) is 2: 1); level 0 vector of hsp terminator obtained in step S2 (molar ratio to the corresponding level 1 vector is 2: 1); 5units BsaI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA (1 mg/mL); h ₂ O is complemented to 20 mu L;

the reaction procedures are as follows:

after the reaction is finished, 10 mu L of reaction liquid is taken to transform the heat shock competence of escherichia coli DH10B, and the escherichia coli is grown on an LB solid culture medium containing ampicillin; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing and obtaining the corresponding correct level 1 vector.

S4, connecting a 35S promoter sequence, a hygromycin resistance gene coding sequence and a 35S terminator sequence, the E8 promoter sequence, the Coq2 gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the DPS gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the HMGR2 gene coding sequence and the hsp terminator sequence in sequence by a Golden Gate cloning method, wherein the E8 promoter sequence, the Ubic gene coding sequence and the hsp terminator sequence are connected into a vector pICSL4723-P1 in sequence, and the obtained level 2 vector is the combined vector, and is shown as SEQ ID NO. 5;

wherein, the level 2 vector construction systems in the step S4 are all as follows: 100ng pICSL4723-P1 vector; pICSL11059 vector (and the pICSL4723-P1 vector molar ratio is 2: 1); level 1 vector of Coq2 gene obtained in step S3 (molar ratio to pICSL4723-P1 vector is 2: 1); the level 1 vector of the DPS gene obtained in step S3 (the molar ratio to the pICSL4723-P1 vector is 2: 1); the level 1 vector of the HMGR2 gene obtained in step S3 (in a molar ratio of 2:1 to the pICSL4723-P1 vector); the level 1 vector of the Ubic gene obtained in the step S3 (the molar ratio to the pICSL4723-P1 vector is 2: 1); PICH41800 linker vector (2: 1 molar ratio to the pICSL4723-P1 vector); 5units BsaI endonuclease; 5units BpiI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA (1 mg/mL); h ₂ O is complemented to 20 mu L;

the reaction procedure is as follows:

after the reaction is finished, 10 mu L of reaction liquid is taken to transform escherichia coli DH10B heat shock competence and grow on an LB solid culture medium containing kanamycin; selecting positive clones, culturing in LB liquid culture medium, extracting plasmid, sequencing to obtain correct level 2 vector, as shown in FIG. 2.

Example 2

This example provides a method for increasing coenzyme Q ₁₀ The production method of the tomato with the content comprises the steps of transforming agrobacterium GV3101 by the level 2 vector obtained in the example 1 through heat shock, and transforming wild tomato MicroTom; and after independent resistant callus is obtained, further differentiating and regenerating to obtain a tomato strain HUCD.

Detection examples

Determination of tomato coenzyme Q ₁₀ The content of (A): collecting and freeze-drying the fruits at the same time after the color changing period, adding isopropanol according to the proportion of 1:20, ultrasonically extracting for 1h at low temperature in a dark place, and then injecting the filtered samples into a liquid phase-mass spectrometry combination for analysis. The liquid phase conditions were as follows: HPLC-DAD-MS (1260Infinity II-6460, Agilent) is matched with a ZORBAX Eclipse XDB C18 column (3.5 mu m, 2.1 multiplied by 50mm), isopropanol is taken as a mobile phase (A), and (B) 87.5% acetonitrile water solution (containing 10mmol/L ammonium acetate) is taken as a mobile phase, and the conditions of the mobile phase are 0-10min and 70-15% B; 10.5-20min, 15-70% B, and flow rate of 0.25 mL/min. The mass spectrometry conditions were as follows: adopting an ESI ion source, setting the capillary voltage to be 4.0kV, the neutral voltage to be 15psi, high-purity nitrogen as carrier gas, and the flow rate to be 11L/min; coenzyme Q10 (parent ion m/z 863.69, daughter ion m/z 197.1) was measured in a positive ion mode under conditions of a dissociation voltage of 230V and a collision energy of 42V.

And (3) detection results: as shown in FIG. 3, generation T3 (T3 ge)neration) coenzyme Q of each strain ₁₀ The content is obviously higher than that of a wild type strain, wherein the content of the HUCD-5 strain is the highest, about 0.151mg/g and higher than that of the wild type strain (C: (A))<0.02mg/g) 7 times or more.

In summary, coenzyme Q is present in wild-type tomato fruit ₁₀ The dry weight content is far below 0.02 mg/g. Through cultivation and screening, the T3 generation fruit coenzyme Q of the tomato strain HUCD of the invention ₁₀ The yield can reach more than 7 times (about 0.151mg/g) of wild tomato fruits at most, and the wild tomato fruits can be stably inherited without difference in phenotype; namely coenzyme Q in the tomato lines HUCD of the invention ₁₀ The content is obviously improved.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Sequence listing

<110> Shanghai mountain plant garden

120 combined vector capable of improving coenzyme Q10 content in tomato and construction method and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1206

<212> DNA

<213> Artificial Sequence

<400> 1

atggcattgt accgccgtct ttctcgaact tctcgtgtcc tccggcggca ccctcgcccc 60

tctatcgctg ccctctgctc ttctcccgat caaacaaacc ctagcacaac tgctcctcat 120

tctcagttaa tcaacccctt tcaatcttct tcttcttctg atcagtatat atatataccc 180

aactccaatt ttaagaaatc tttgacatat tattactctc cacattcttc aagaattgaa 240

cttttagggt accgtcaagt tcttcacttt tcaaccttag ctaattcaga agaggagaag 300

aagaatgagc aacaaaaaca gccttcttgg attgatacgt atttgcccca aagaattaga 360

ccctacgctc atcttgcccg acttgataag cctattggca cttggttact cgcttggccc 420

tgcatgtggt cgattgcttt ggctgctacg ccagggagcc ttcctgatgt gaaaatgatg 480

acactatttg gttgcggggc tttgcttttg cgaggtgctg gttgtacagt taacgatctt 540

cttgatcgag atattgacac taaggtggaa agaacaaggt cgaggccagt cgcgagtggt 600

gtattgacac cctttcaagg gctctgtttc cttgggttcc agttgctatt aggtcttggg 660

attcttttgc aattaaacaa ttttagccgc attttgggtg cttcatccct gttgctggtg 720

ttctcatacc ccctcatgaa gaggttgaca ttttggcctc aagcctatct tggtctaact 780

ttcaactggg gagctttgtt aggttgggct gctattaaag gaagtattga tcctgcagtt 840

gtgcttccat tgtacgcctc tggggtgttt tggacgctcg tgtatgacac aatatatgca 900

catcaggata aagaggacga tctcaaagtg ggtgtcaagt ctacagcctt gagatttgga 960

gattccacaa aagaatggat cagtggtttt ggattagcat gcatcagcag tcttgctctt 1020

tccggagtta atgctgatat tggatggcca tactatgcat ttctgacagc tgcttctggc 1080

cagttggcct ggcaaatttg gaccgttgac ttatcatctc gtgcagattg caacagaaaa 1140

tttgtgtcca acaaatggtt tggtgctttt gtctttggcg gggttctatt tggtagacta 1200

ttgtga 1206

<210> 2

<211> 1248

<212> DNA

<213> Artificial Sequence

<400> 2

atgctatttg caaggggttt gtctcagatt tcgagacgca gtatcaatag atatcaatgg 60

ctatgtcccc agcaacaaca atttcaccat tccaatcact ttccctctcc catcgatgct 120

tctcataagg ttttgggttg cagagtaatt cattcatggg tttctaatgc tcttagtggt 180

attggtcaac aaattcatca tcaaagcact gctgtagcag aggagcaagt ggacccattt 240

tcccttgttg ctgatgaatt atcccttctg acaaacaggc tgagatcaat ggtagttgcc 300

gaggtcccaa agctggcttc agctgctgag tatttcttca aaatgggagt agaagggaag 360

aggtttcgac ccacagttct gctgttgatg gcaacggcac tgaacgcacc ggtacttaga 420

ccccaggtgg atgttgattc tttgtccagg gatttgcgta cgaggcagca gtgtatagct 480

gaaatcacgg agatgatcca tgttgccagc ctactccatg atgatgtact agatgatgct 540

gacacacgac gtgggatagg ttctttaaac tttgtgatgg gaaataagtt agctgtacta 600

gctggagact ttctgctttc ccgagcgtgt gtggcacttg cctctttgaa gaatacagag 660

gttgtatccc ttctggcaac tgttgtggaa catcttgtta ctggagagac aatgcaaatg 720

acgacttctt ctgatgaacg ttgtagcatg gattattaca tgcagaaaac atattacaag 780

actgcatcat taatttcaaa cagttgcaaa gcaatagcgc tacttgctgg gcatactgct 840

gaagtctcca tgttagcttt tgactatggg aaaaatttgg gcttggcatt tcaattaata 900

gatgatgttc ttgatttcac aggcacatct gccaccctcg gcaagggttc attgtctgat 960

attcgtcatg ggattgtaac tgccccaata ttgtatgcca tggaggaatt tcctcaactg 1020

cgaacggtgg tggaccgggg attcgatgat cctgtcaatg tggagattgc tctggactac 1080

cttggtaaga gccgagggat acagagaaca agagaacttg caagaaaaca tgctaacctt 1140

gcctcagcgg ctgttgactc tcttcctgag agcgatgacg aggatgttca gagatcaaga 1200

cgggcacttg tggaacttac tcaaagagtc atcacaagaa caaaatag 1248

<210> 3

<211> 1395

<212> DNA

<213> Artificial Sequence

<400> 3

atgttccgat ccagctctga cgacgacgtg tgggtgaacg acgggatgat cccctgcaac 60

cagagtttgg actgccgaga ggtgctccca atcaagccca acagtgtcga cccaccccga 120

gagagcgagc tagactctgt cgaggacgag gagatcgtga agctcgtgat cgacggtacc 180

atcccctctt acagcctcga gactaagctc ggcgactgca agagggccgc cgcaatcagg 240

cgagaggccg tgcagcgaat caccgggaag tccttgaccg ggctccccct cgagggcttc 300

gactacaact ctatcttagg gcagtgttgc gagatgcccg tgggatacgt gcagatcccc 360

gtgggtatcg ccggaccact gctcctggat ggagtcgagt acagcgtgcc catggccacc 420

actgagggtt gcctcgtggc atcaactaac cgaggcttca aggccatcca cttgagcggc 480

ggagccttct ctgtgctcgt gaaggatgca atgacacgag ctcccgtggt gaggttccca 540

agcgcccgta gggctgcttt ggtgatgttc tacctccagg acccctcaaa cttcgagcga 600

ctctcactca tcttcaacaa gtcaagcagg ttcgccaggt tgcagtctat cacttgcacc 660

atcgccggta ggaaccttta ccccaggttc gcctgcagca ctggcgacgc catgggtatg 720

aacatggtgt caaagggtgt gcagaacgtg cttgattttg tgaagtccga attccctgac 780

atggacgtca tcggcatcag cggaaactac tgcagcgaca agaaggcctc cgccgtgaac 840

tggatcgagg ggaggggaaa gcacgtggtg tgcgaggctt tcatcaaggc agagatcgtg 900

gagaaggtcc tcaagacttc cgtcgaggct ctggtggagt tgaacaccct caagaacctc 960

gtcgggtctg ccatggccgg gagcctgggg ggtttcaacg cacactccag caacatcgtg 1020

agtgcagtgt tcatcgcaac aggccaggac cctgctcaga acgtcgaatc ttcccactgc 1080

atgaccatga tcctccctga cggtgacgac ttgcatatca gtgtgtccat gccctgcatc 1140

gaggtcggga ccgtgggtgg ggggacccag ctcgcctccc aggccgcctg cctgaacctg 1200

ctgggggtga agggctccaa caacgagaag cctggctcca acgcccagca gttggcaaga 1260

atagtggctg gttcggttct tgcaggagag ctttcactaa tgtctgctat tgcagctgga 1320

cagcttgtga agagtcacat gaaatacaac agatccagca gagacattgg cccttcgtct 1380

caagtcaaca ggtga 1395

<210> 4

<211> 699

<212> DNA

<213> Artificial Sequence

<400> 4

atggcttcct ctgtcatttc ttcagcagct gttgccacaa ggtctaacgt gacccaggcc 60

tccatggtgg cccctttcac cgggctcaag tcctccgcta ccttccctgt gaccaagaag 120

cagaacctcg acatcacatc tatcgcatcc aacggcggcc gtgtgagctg cgccgtgcca 180

ctctgcaacg gggagttcgg gatgagtcac cctgctttga cacagctgcg tgctctgcga 240

tactgcaagg agatccccgc cctcgaccct cagcttctcg actggctcct ccttgaggac 300

tccatgacaa agaggttcga gcagcaggga aagaccgtgt ccgtgaccat gatccgtgag 360

gggttcgtgg agcagaacga gatccctgag gagcttcccc tgctccccaa ggagtctagg 420

tactggctca gggagatcct gttgtgcgca gacggggagc cctggctcgc aggaaggacc 480

gtcgtccccg tgtccaccct ctcaggccct gagctcgccc tccagaagct cggtaagacc 540

cccttgggac gatacttgtt cacctccagc accctcaccc gagacttcat cgagatcggg 600

cgagacgctg gactctgggg taggaggtcc aggctcaggc tctccgggaa gccactcctc 660

ctcaccgagc tcttcctgcc agcctcccct ctctactga 699

<210> 5

<211> 22105

<212> DNA

<213> Artificial Sequence

<400> 5

ggaggaattc caatcccaca aaaatctgag cttaacagca cagttgctcc tctcagagca 60

gaatcgggta ttcaacaccc tcatatcaac tactacgttg tgtataacgg tccacatgcc 120

ggtatatacg atgactgggg ttgtacaaag gcggcaacaa acggcgttcc cggagttgca 180

cacaagaaat ttgccactat tacagaggca agagcagcag ctgacgcgta cacaacaagt 240

cagcaaacag acaggttgaa cttcatcccc aaaggagaag ctcaactcaa gcccaagagc 300

tttgctaagg ccctaacaag cccaccaaag caaaaagccc actggctcac gctaggaacc 360

aaaaggccca gcagtgatcc agccccaaaa gagatctcct ttgccccgga gattacaatg 420

gacgatttcc tctatcttta cgatctagga aggaagttcg aaggtgaagg tgacgacact 480

atgttcacca ctgataatga gaaggttagc ctcttcaatt tcagaaagaa tgctgaccca 540

cagatggtta gagaggccta cgcagcaagt ctcatcaaga cgatctaccc gagtaacaat 600

ctccaggaga tcaaatacct tcccaagaag gttaaagatg cagtcaaaag attcaggact 660

aattgcatca agaacacaga gaaagacata tttctcaaga tcagaagtac tattccagta 720

tggacgattc aaggcttgct tcataaacca aggcaagtaa tagagattgg agtctctaaa 780

aaggtagttc ctactgaatc taaggccatg catggagtct aagattcaaa tcgaggatct 840

aacagaactc gccgtcaaga ctggcgaaca gttcatacag agtcttttac gactcaatga 900

caagaagaaa atcttcgtca acatggtgga gcacgacact ctggtctact ccaaaaatgt 960

caaagataca gtctcagaag atcaaagggc tattgagact tttcaacaaa ggataatttc 1020

gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcgaaa ggacagtaga 1080

aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga 1140

tctctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 1200

agaagaggtt ccaaccacgt ctacaaagca agtggattga tgtgacatct ccactgacgt 1260

aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc 1320

atttcatttg gagaggacac gctcgagtat aagagctcat ttttacaaca attaccaaca 1380

acaacaaaca acaaacaaca ttacaattac atttacaatt atcgatacaa tgaaaaagcc 1440

tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga 1500

cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg 1560

tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta 1620

tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag 1680

cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc 1740

tgaaaccgaa ctgcccgctg ttctgcaggt aaatttctag tttttctcct tcattttctt 1800

ggttaggacc cttttctctt tttatttttt tgagctttga tctttcttta aactgatcta 1860

ttttttaatt gattggttat ggtgtaaata ttacatagct ttaactgata atctgattac 1920

tttatttcgt gtgtctatga tgatgatgat aactgcagcc ggtcgcggag gccatggatg 1980

cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa 2040

tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc 2100

actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc 2160

tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct 2220

ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga 2280

tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt 2340

gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc 2400

ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg 2460

gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag 2520

ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct 2580

gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg 2640

aataggcttc tctagctaga gtcgatcgac aagctcgagt ttctccataa taatgtgtga 2700

gtagttccca gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat 2760

aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt 2820

cctaaaacca aaatccagta ctaaaatcca gatcgctgca agaattcaag cttggagaag 2880

ctttccctaa tgatattgtt catgtaatta agttttgtgg aagtgagaga gtccaatttt 2940

gataagaaaa gagtcagaaa acgtaatatt ttaaaagtct aaatctttct acaaataaga 3000

gcaaatttat ttatttttta atccaataaa tattaatgga ggacaaattc aattcacttg 3060

gttgtaaaat aaacttaaac caataaccaa agaactaata aatcctgaag tggaattatt 3120

aaggataaat gtacatagac aatgaagaaa taataggttc gatgaattaa taataattaa 3180

ggatgttaca atcatcatgt gccaagtata tacacaatat tctatgggat ttataatttc 3240

gttacttcac ttaacttttg cgtaaataaa acgaattatc tgatatttta taataaaaca 3300

gttaattaag aaccatcatt tttaacaaca tagatatatt atttctaata gtttaatgat 3360

acttttaaat cttttaaatt ttatgtttct tttagaaaat aaaaattcaa aaaattaaat 3420

atatttacaa aaactacaat caaacacaac ttcatatatt aaaagcaaaa tatattttga 3480

aaatttcaag tgtcctaaca aataagacaa gaggaaaatg tacgatgaga gacataaaga 3540

gaactaataa ttgaggagtc ctataatata taataaagtt tattagtaaa cttaattatt 3600

aaggactcct aaaatatatg ataggagaaa atgaatggtg agagatattg gaaaacttaa 3660

taattaagga ttttaaaata tatggtaaaa gataggcaaa gtatccatta tcccctttta 3720

acttgaagtc tactaggcgc atgtgaaagt tgattttttg tcacgtcata tagctataac 3780

gtaaaaaaag aaagtaaaat ttttaatttt ttttaatata tgacatattt taaacgaaat 3840

ataggacaaa atgtaaatga atagtaaagg aaacaaagat taatacttac tttgtaagaa 3900

tttaagataa atttaaaatt taatagatca actttacgtc tagaaagacc ctatcttaga 3960

aggaatttca gaaatcggcc ctttttcaaa aataactttt aaataatgaa ttttaaattt 4020

taagaaataa tttccaatga ataaatgaca tgtagcattt tacctaaata tttcaactat 4080

tttaatccaa tattaatttg tttattccca acaatagaaa gtcttgtgca gacatttaat 4140

ctgacttttc cagtactaaa tattaatttt ctgaagattt tcgggtttag tccacaagtt 4200

ttagtgagaa gttttgctca aaattttagg tgagaaggtt tgatatttat cttttgttaa 4260

attaatttat ctaggtgact attatttatt taagtagaaa ttcatatcat tacttttgcc 4320

aacttgtagt cataatagga gtaggtgtat atgatgaagg aataaacaag ttcagtgaag 4380

tgattaaaat aaaatataat ttaggtgtac atcaaataaa aaccttaaag tttagaaagg 4440

caccgaataa ttttgcatag aagatattag taaatttata aaaataaaag aaatgtagtt 4500

gtcaagttgt gttctttttt ttggataaaa atagcagttg gcttatgtca ttcttttaca 4560

acctccatgc cacttgtcca attgttgaca cttaactaat tagtttgatt catgtatgaa 4620

tactaaataa ttttttagga ctgactcaaa tatttttata ttatcatagt aatatttatc 4680

taatttttag gaccacttat tactaaataa taaattaact actactatat tattgttgtg 4740

aaacaacaac gttttggttg ttatgatgaa acgtacacta tatcagtatg aaaaattcaa 4800

aacgattagt ataaattata ttgaaaattt gatatttttc tattcttaat cagacgtatt 4860

gggtttcata ttttaaaaag ggactaaact tagaagagaa gtttgtttga aactactttt 4920

gtctctttct tgttcccatt tctctcttag atttcaaaaa gtgaactact ttatctcttt 4980

ctttgttcac attttatttt attctattat aaatatggca tcctcatatt gagattttta 5040

gaaattattc taatcattca cagtgcaatg gcattgtacc gccgtctttc tcgaacttct 5100

cgtgtcctcc ggcggcaccc tcgcccctct atcgctgccc tctgctcttc tcccgatcaa 5160

acaaacccta gcacaactgc tcctcattct cagttaatca acccctttca atcttcttct 5220

tcttctgatc agtatatata tatacccaac tccaatttta agaaatcttt gacatattat 5280

tactctccac attcttcaag aattgaactt ttagggtacc gtcaagttct tcacttttca 5340

accttagcta attcagaaga ggagaagaag aatgagcaac aaaaacagcc ttcttggatt 5400

gatacgtatt tgccccaaag aattagaccc tacgctcatc ttgcccgact tgataagcct 5460

attggcactt ggttactcgc ttggccctgc atgtggtcga ttgctttggc tgctacgcca 5520

gggagccttc ctgatgtgaa aatgatgaca ctatttggtt gcggggcttt gcttttgcga 5580

ggtgctggtt gtacagttaa cgatcttctt gatcgagata ttgacactaa ggtggaaaga 5640

acaaggtcga ggccagtcgc gagtggtgta ttgacaccct ttcaagggct ctgtttcctt 5700

gggttccagt tgctattagg tcttgggatt cttttgcaat taaacaattt tagccgcatt 5760

ttgggtgctt catccctgtt gctggtgttc tcataccccc tcatgaagag gttgacattt 5820

tggcctcaag cctatcttgg tctaactttc aactggggag ctttgttagg ttgggctgct 5880

attaaaggaa gtattgatcc tgcagttgtg cttccattgt acgcctctgg ggtgttttgg 5940

acgctcgtgt atgacacaat atatgcacat caggataaag aggacgatct caaagtgggt 6000

gtcaagtcta cagccttgag atttggagat tccacaaaag aatggatcag tggttttgga 6060

ttagcatgca tcagcagtct tgctctttcc ggagttaatg ctgatattgg atggccatac 6120

tatgcatttc tgacagctgc ttctggccag ttggcctggc aaatttggac cgttgactta 6180

tcatctcgtg cagattgcaa cagaaaattt gtgtccaaca aatggtttgg tgcttttgtc 6240

tttggcgggg ttctatttgg tagactattg tgagcttata tgaagatgaa gatgaaatat 6300

ttggtgtgtc aaataaaaag cttgtgtgct taagtttgtg tttttttctt ggcttgttgt 6360

gttatgaatt tgtggctttt tctaatatta aatgaatgta agatctcatt ataatgaata 6420

aacaaatgtt tctataatcc attgtgaatg ttttgttgga tctcttctgc agcatataac 6480

tactgtatgt gctatggtat ggactatgga atatgattaa agataagcgc tactagaatt 6540

cgagctcgga gaagctttcc ctaatgatat tgttcatgta attaagtttt gtggaagtga 6600

gagagtccaa ttttgataag aaaagagtca gaaaacgtaa tattttaaaa gtctaaatct 6660

ttctacaaat aagagcaaat ttatttattt tttaatccaa taaatattaa tggaggacaa 6720

attcaattca cttggttgta aaataaactt aaaccaataa ccaaagaact aataaatcct 6780

gaagtggaat tattaaggat aaatgtacat agacaatgaa gaaataatag gttcgatgaa 6840

ttaataataa ttaaggatgt tacaatcatc atgtgccaag tatatacaca atattctatg 6900

ggatttataa tttcgttact tcacttaact tttgcgtaaa taaaacgaat tatctgatat 6960

tttataataa aacagttaat taagaaccat catttttaac aacatagata tattatttct 7020

aatagtttaa tgatactttt aaatctttta aattttatgt ttcttttaga aaataaaaat 7080

tcaaaaaatt aaatatattt acaaaaacta caatcaaaca caacttcata tattaaaagc 7140

aaaatatatt ttgaaaattt caagtgtcct aacaaataag acaagaggaa aatgtacgat 7200

gagagacata aagagaacta ataattgagg agtcctataa tatataataa agtttattag 7260

taaacttaat tattaaggac tcctaaaata tatgatagga gaaaatgaat ggtgagagat 7320

attggaaaac ttaataatta aggattttaa aatatatggt aaaagatagg caaagtatcc 7380

attatcccct tttaacttga agtctactag gcgcatgtga aagttgattt tttgtcacgt 7440

catatagcta taacgtaaaa aaagaaagta aaatttttaa ttttttttaa tatatgacat 7500

attttaaacg aaatatagga caaaatgtaa atgaatagta aaggaaacaa agattaatac 7560

ttactttgta agaatttaag ataaatttaa aatttaatag atcaacttta cgtctagaaa 7620

gaccctatct tagaaggaat ttcagaaatc ggcccttttt caaaaataac ttttaaataa 7680

tgaattttaa attttaagaa ataatttcca atgaataaat gacatgtagc attttaccta 7740

aatatttcaa ctattttaat ccaatattaa tttgtttatt cccaacaata gaaagtcttg 7800

tgcagacatt taatctgact tttccagtac taaatattaa ttttctgaag attttcgggt 7860

ttagtccaca agttttagtg agaagttttg ctcaaaattt taggtgagaa ggtttgatat 7920

ttatcttttg ttaaattaat ttatctaggt gactattatt tatttaagta gaaattcata 7980

tcattacttt tgccaacttg tagtcataat aggagtaggt gtatatgatg aaggaataaa 8040

caagttcagt gaagtgatta aaataaaata taatttaggt gtacatcaaa taaaaacctt 8100

aaagtttaga aaggcaccga ataattttgc atagaagata ttagtaaatt tataaaaata 8160

aaagaaatgt agttgtcaag ttgtgttctt ttttttggat aaaaatagca gttggcttat 8220

gtcattcttt tacaacctcc atgccacttg tccaattgtt gacacttaac taattagttt 8280

gattcatgta tgaatactaa ataatttttt aggactgact caaatatttt tatattatca 8340

tagtaatatt tatctaattt ttaggaccac ttattactaa ataataaatt aactactact 8400

atattattgt tgtgaaacaa caacgttttg gttgttatga tgaaacgtac actatatcag 8460

tatgaaaaat tcaaaacgat tagtataaat tatattgaaa atttgatatt tttctattct 8520

taatcagacg tattgggttt catattttaa aaagggacta aacttagaag agaagtttgt 8580

ttgaaactac ttttgtctct ttcttgttcc catttctctc ttagatttca aaaagtgaac 8640

tactttatct ctttctttgt tcacatttta ttttattcta ttataaatat ggcatcctca 8700

tattgagatt tttagaaatt attctaatca ttcacagtgc aatgctattt gcaaggggtt 8760

tgtctcagat ttcgagacgc agtatcaata gatatcaatg gctatgtccc cagcaacaac 8820

aatttcacca ttccaatcac tttccctctc ccatcgatgc ttctcataag gttttgggtt 8880

gcagagtaat tcattcatgg gtttctaatg ctcttagtgg tattggtcaa caaattcatc 8940

atcaaagcac tgctgtagca gaggagcaag tggacccatt ttcccttgtt gctgatgaat 9000

tatcccttct gacaaacagg ctgagatcaa tggtagttgc cgaggtccca aagctggctt 9060

cagctgctga gtatttcttc aaaatgggag tagaagggaa gaggtttcga cccacagttc 9120

tgctgttgat ggcaacggca ctgaacgcac cggtacttag accccaggtg gatgttgatt 9180

ctttgtccag ggatttgcgt acgaggcagc agtgtatagc tgaaatcacg gagatgatcc 9240

atgttgccag cctactccat gatgatgtac tagatgatgc tgacacacga cgtgggatag 9300

gttctttaaa ctttgtgatg ggaaataagt tagctgtact agctggagac tttctgcttt 9360

cccgagcgtg tgtggcactt gcctctttga agaatacaga ggttgtatcc cttctggcaa 9420

ctgttgtgga acatcttgtt actggagaga caatgcaaat gacgacttct tctgatgaac 9480

gttgtagcat ggattattac atgcagaaaa catattacaa gactgcatca ttaatttcaa 9540

acagttgcaa agcaatagcg ctacttgctg ggcatactgc tgaagtctcc atgttagctt 9600

ttgactatgg gaaaaatttg ggcttggcat ttcaattaat agatgatgtt cttgatttca 9660

caggcacatc tgccaccctc ggcaagggtt cattgtctga tattcgtcat gggattgtaa 9720

ctgccccaat attgtatgcc atggaggaat ttcctcaact gcgaacggtg gtggaccggg 9780

gattcgatga tcctgtcaat gtggagattg ctctggacta ccttggtaag agccgaggga 9840

tacagagaac aagagaactt gcaagaaaac atgctaacct tgcctcagcg gctgttgact 9900

ctcttcctga gagcgatgac gaggatgttc agagatcaag acgggcactt gtggaactta 9960

ctcaaagagt catcacaaga acaaaatagg cttatatgaa gatgaagatg aaatatttgg 10020

tgtgtcaaat aaaaagcttg tgtgcttaag tttgtgtttt tttcttggct tgttgtgtta 10080

tgaatttgtg gctttttcta atattaaatg aatgtaagat ctcattataa tgaataaaca 10140

aatgtttcta taatccattg tgaatgtttt gttggatctc ttctgcagca tataactact 10200

gtatgtgcta tggtatggac tatggaatat gattaaagat aagcgcttta cgaattccca 10260

tggggagaag ctttccctaa tgatattgtt catgtaatta agttttgtgg aagtgagaga 10320

gtccaatttt gataagaaaa gagtcagaaa acgtaatatt ttaaaagtct aaatctttct 10380

acaaataaga gcaaatttat ttatttttta atccaataaa tattaatgga ggacaaattc 10440

aattcacttg gttgtaaaat aaacttaaac caataaccaa agaactaata aatcctgaag 10500

tggaattatt aaggataaat gtacatagac aatgaagaaa taataggttc gatgaattaa 10560

taataattaa ggatgttaca atcatcatgt gccaagtata tacacaatat tctatgggat 10620

ttataatttc gttacttcac ttaacttttg cgtaaataaa acgaattatc tgatatttta 10680

taataaaaca gttaattaag aaccatcatt tttaacaaca tagatatatt atttctaata 10740

gtttaatgat acttttaaat cttttaaatt ttatgtttct tttagaaaat aaaaattcaa 10800

aaaattaaat atatttacaa aaactacaat caaacacaac ttcatatatt aaaagcaaaa 10860

tatattttga aaatttcaag tgtcctaaca aataagacaa gaggaaaatg tacgatgaga 10920

gacataaaga gaactaataa ttgaggagtc ctataatata taataaagtt tattagtaaa 10980

cttaattatt aaggactcct aaaatatatg ataggagaaa atgaatggtg agagatattg 11040

gaaaacttaa taattaagga ttttaaaata tatggtaaaa gataggcaaa gtatccatta 11100

tcccctttta acttgaagtc tactaggcgc atgtgaaagt tgattttttg tcacgtcata 11160

tagctataac gtaaaaaaag aaagtaaaat ttttaatttt ttttaatata tgacatattt 11220

taaacgaaat ataggacaaa atgtaaatga atagtaaagg aaacaaagat taatacttac 11280

tttgtaagaa tttaagataa atttaaaatt taatagatca actttacgtc tagaaagacc 11340

ctatcttaga aggaatttca gaaatcggcc ctttttcaaa aataactttt aaataatgaa 11400

ttttaaattt taagaaataa tttccaatga ataaatgaca tgtagcattt tacctaaata 11460

tttcaactat tttaatccaa tattaatttg tttattccca acaatagaaa gtcttgtgca 11520

gacatttaat ctgacttttc cagtactaaa tattaatttt ctgaagattt tcgggtttag 11580

tccacaagtt ttagtgagaa gttttgctca aaattttagg tgagaaggtt tgatatttat 11640

cttttgttaa attaatttat ctaggtgact attatttatt taagtagaaa ttcatatcat 11700

tacttttgcc aacttgtagt cataatagga gtaggtgtat atgatgaagg aataaacaag 11760

ttcagtgaag tgattaaaat aaaatataat ttaggtgtac atcaaataaa aaccttaaag 11820

tttagaaagg caccgaataa ttttgcatag aagatattag taaatttata aaaataaaag 11880

aaatgtagtt gtcaagttgt gttctttttt ttggataaaa atagcagttg gcttatgtca 11940

ttcttttaca acctccatgc cacttgtcca attgttgaca cttaactaat tagtttgatt 12000

catgtatgaa tactaaataa ttttttagga ctgactcaaa tatttttata ttatcatagt 12060

aatatttatc taatttttag gaccacttat tactaaataa taaattaact actactatat 12120

tattgttgtg aaacaacaac gttttggttg ttatgatgaa acgtacacta tatcagtatg 12180

aaaaattcaa aacgattagt ataaattata ttgaaaattt gatatttttc tattcttaat 12240

cagacgtatt gggtttcata ttttaaaaag ggactaaact tagaagagaa gtttgtttga 12300

aactactttt gtctctttct tgttcccatt tctctcttag atttcaaaaa gtgaactact 12360

ttatctcttt ctttgttcac attttatttt attctattat aaatatggca tcctcatatt 12420

gagattttta gaaattattc taatcattca cagtgcaatg ttccgatcca gctctgacga 12480

cgacgtgtgg gtgaacgacg ggatgatccc ctgcaaccag agtttggact gccgagaggt 12540

gctcccaatc aagcccaaca gtgtcgaccc accccgagag agcgagctag actctgtcga 12600

ggacgaggag atcgtgaagc tcgtgatcga cggtaccatc ccctcttaca gcctcgagac 12660

taagctcggc gactgcaaga gggccgccgc aatcaggcga gaggccgtgc agcgaatcac 12720

cgggaagtcc ttgaccgggc tccccctcga gggcttcgac tacaactcta tcttagggca 12780

gtgttgcgag atgcccgtgg gatacgtgca gatccccgtg ggtatcgccg gaccactgct 12840

cctggatgga gtcgagtaca gcgtgcccat ggccaccact gagggttgcc tcgtggcatc 12900

aactaaccga ggcttcaagg ccatccactt gagcggcgga gccttctctg tgctcgtgaa 12960

ggatgcaatg acacgagctc ccgtggtgag gttcccaagc gcccgtaggg ctgctttggt 13020

gatgttctac ctccaggacc cctcaaactt cgagcgactc tcactcatct tcaacaagtc 13080

aagcaggttc gccaggttgc agtctatcac ttgcaccatc gccggtagga acctttaccc 13140

caggttcgcc tgcagcactg gcgacgccat gggtatgaac atggtgtcaa agggtgtgca 13200

gaacgtgctt gattttgtga agtccgaatt ccctgacatg gacgtcatcg gcatcagcgg 13260

aaactactgc agcgacaaga aggcctccgc cgtgaactgg atcgagggga ggggaaagca 13320

cgtggtgtgc gaggctttca tcaaggcaga gatcgtggag aaggtcctca agacttccgt 13380

cgaggctctg gtggagttga acaccctcaa gaacctcgtc gggtctgcca tggccgggag 13440

cctggggggt ttcaacgcac actccagcaa catcgtgagt gcagtgttca tcgcaacagg 13500

ccaggaccct gctcagaacg tcgaatcttc ccactgcatg accatgatcc tccctgacgg 13560

tgacgacttg catatcagtg tgtccatgcc ctgcatcgag gtcgggaccg tgggtggggg 13620

gacccagctc gcctcccagg ccgcctgcct gaacctgctg ggggtgaagg gctccaacaa 13680

cgagaagcct ggctccaacg cccagcagtt ggcaagaata gtggctggtt cggttcttgc 13740

aggagagctt tcactaatgt ctgctattgc agctggacag cttgtgaaga gtcacatgaa 13800

atacaacaga tccagcagag acattggccc ttcgtctcaa gtcaacaggt gagcttatat 13860

gaagatgaag atgaaatatt tggtgtgtca aataaaaagc ttgtgtgctt aagtttgtgt 13920

ttttttcttg gcttgttgtg ttatgaattt gtggcttttt ctaatattaa atgaatgtaa 13980

gatctcatta taatgaataa acaaatgttt ctataatcca ttgtgaatgt tttgttggat 14040

ctcttctgca gcatataact actgtatgtg ctatggtatg gactatggaa tatgattaaa 14100

gataagcgct cagagaattc gcatgcggag aagctttccc taatgatatt gttcatgtaa 14160

ttaagttttg tggaagtgag agagtccaat tttgataaga aaagagtcag aaaacgtaat 14220

attttaaaag tctaaatctt tctacaaata agagcaaatt tatttatttt ttaatccaat 14280

aaatattaat ggaggacaaa ttcaattcac ttggttgtaa aataaactta aaccaataac 14340

caaagaacta ataaatcctg aagtggaatt attaaggata aatgtacata gacaatgaag 14400

aaataatagg ttcgatgaat taataataat taaggatgtt acaatcatca tgtgccaagt 14460

atatacacaa tattctatgg gatttataat ttcgttactt cacttaactt ttgcgtaaat 14520

aaaacgaatt atctgatatt ttataataaa acagttaatt aagaaccatc atttttaaca 14580

acatagatat attatttcta atagtttaat gatactttta aatcttttaa attttatgtt 14640

tcttttagaa aataaaaatt caaaaaatta aatatattta caaaaactac aatcaaacac 14700

aacttcatat attaaaagca aaatatattt tgaaaatttc aagtgtccta acaaataaga 14760

caagaggaaa atgtacgatg agagacataa agagaactaa taattgagga gtcctataat 14820

atataataaa gtttattagt aaacttaatt attaaggact cctaaaatat atgataggag 14880

aaaatgaatg gtgagagata ttggaaaact taataattaa ggattttaaa atatatggta 14940

aaagataggc aaagtatcca ttatcccctt ttaacttgaa gtctactagg cgcatgtgaa 15000

agttgatttt ttgtcacgtc atatagctat aacgtaaaaa aagaaagtaa aatttttaat 15060

tttttttaat atatgacata ttttaaacga aatataggac aaaatgtaaa tgaatagtaa 15120

aggaaacaaa gattaatact tactttgtaa gaatttaaga taaatttaaa atttaataga 15180

tcaactttac gtctagaaag accctatctt agaaggaatt tcagaaatcg gccctttttc 15240

aaaaataact tttaaataat gaattttaaa ttttaagaaa taatttccaa tgaataaatg 15300

acatgtagca ttttacctaa atatttcaac tattttaatc caatattaat ttgtttattc 15360

ccaacaatag aaagtcttgt gcagacattt aatctgactt ttccagtact aaatattaat 15420

tttctgaaga ttttcgggtt tagtccacaa gttttagtga gaagttttgc tcaaaatttt 15480

aggtgagaag gtttgatatt tatcttttgt taaattaatt tatctaggtg actattattt 15540

atttaagtag aaattcatat cattactttt gccaacttgt agtcataata ggagtaggtg 15600

tatatgatga aggaataaac aagttcagtg aagtgattaa aataaaatat aatttaggtg 15660

tacatcaaat aaaaacctta aagtttagaa aggcaccgaa taattttgca tagaagatat 15720

tagtaaattt ataaaaataa aagaaatgta gttgtcaagt tgtgttcttt tttttggata 15780

aaaatagcag ttggcttatg tcattctttt acaacctcca tgccacttgt ccaattgttg 15840

acacttaact aattagtttg attcatgtat gaatactaaa taatttttta ggactgactc 15900

aaatattttt atattatcat agtaatattt atctaatttt taggaccact tattactaaa 15960

taataaatta actactacta tattattgtt gtgaaacaac aacgttttgg ttgttatgat 16020

gaaacgtaca ctatatcagt atgaaaaatt caaaacgatt agtataaatt atattgaaaa 16080

tttgatattt ttctattctt aatcagacgt attgggtttc atattttaaa aagggactaa 16140

acttagaaga gaagtttgtt tgaaactact tttgtctctt tcttgttccc atttctctct 16200

tagatttcaa aaagtgaact actttatctc tttctttgtt cacattttat tttattctat 16260

tataaatatg gcatcctcat attgagattt ttagaaatta ttctaatcat tcacagtgca 16320

atggatctag acaatatggc ttcctctgtc atttcttcag cagctgttgc cacaaggtct 16380

aacgtgaccc aggcctccat ggtggcccct ttcaccgggc tcaagtcctc cgctaccttc 16440

cctgtgacca agaagcagaa cctcgacatc acatctatcg catccaacgg cggccgtgtg 16500

agctgcgccg tgccactctg caacggggag ttcgggatga gtcaccctgc tttgacacag 16560

ctgcgtgctc tgcgatactg caaggagatc cccgccctcg accctcagct tctcgactgg 16620

ctcctccttg aggactccat gacaaagagg ttcgagcagc agggaaagac cgtgtccgtg 16680

accatgatcc gtgaggggtt cgtggagcag aacgagatcc ctgaggagct tcccctgctc 16740

cccaaggagt ctaggtactg gctcagggag atcctgttgt gcgcagacgg ggagccctgg 16800

ctcgcaggaa ggaccgtcgt ccccgtgtcc accctctcag gccctgagct cgccctccag 16860

aagctcggta agaccccctt gggacgatac ttgttcacct ccagcaccct cacccgagac 16920

ttcatcgaga tcgggcgaga cgctggactc tggggtagga ggtccaggct caggctctcc 16980

gggaagccac tcctcctcac cgagctcttc ctgccagcct cccctctcta ctgagaggaa 17040

aaaaatatgg agtggagtct gacgcagaat ggggatcctc tagagtcgag cttatatgaa 17100

gatgaagatg aaatatttgg tgtgtcaaat aaaaagcttg tgtgcttaag tttgtgtttt 17160

tttcttggct tgttgtgtta tgaatttgtg gctttttcta atattaaatg aatgtaagat 17220

ctcattataa tgaataaaca aatgtttcta taatccattg tgaatgtttt gttggatctc 17280

ttctgcagca tataactact gtatgtgcta tggtatggac tatggaatat gattaaagat 17340

aagcgcttgt ggaggatgca catgtgaccg agggacacga agtgatccgt ttaaactatc 17400

agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt attagaataa 17460

tcggatattt aaaagggcgt gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca 17520

accacagggt tcccctcggg agtcagccgt gcggctgcat gaaatcctgg ccggtttgtc 17580

tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg 17640

tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat 17700

gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt 17760

aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa 17820

ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg 17880

gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac 17940

cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg 18000

gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca 18060

agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag 18120

gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc 18180

atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt 18240

atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca 18300

gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa 18360

ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg 18420

gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag 18480

ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg 18540

cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag 18600

agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg 18660

gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc 18720

ggttggccag gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc 18780

aagcccgagg aatcggcgtg agcggtcgca aaccatccgg cccggtacaa atcggcgcgg 18840

cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 18900

tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 18960

aatcccggca accgccggca gccggtgcgt cgtcgattag gaagccgccc aagggcgacg 19020

agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 19080

tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 19140

gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 19200

tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 19260

accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 19320

tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 19380

ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 19440

tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 19500

ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 19560

aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 19620

tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 19680

tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 19740

ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 19800

ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 19860

ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 19920

gaaaaagctt ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 19980

accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 20040

tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 20100

aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaacagc 20160

tgcaaaaagc gcccacatca aggctccgag tgcgcggaac ccctatttgt ttatttttct 20220

aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 20280

attgaaaaag gaagagtatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg 20340

aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg 20400

tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct 20460

atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa 20520

aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg 20580

gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt 20640

atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata 20700

gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg 20760

attgcgaaaa ctgggaagag gacactccat ttaaagatcc gcgcgagctg tatgattttt 20820

taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca 20880

acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg 20940

cggacaagtg gtatgacatt gccttctgcg tccggtcgct cagggaggat atcggggaag 21000

aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa 21060

aatattatat tttactggat gaattgtttt agctgtcaga ccaagtttac tcatatatac 21120

tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 21180

ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 21240

tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 21300

aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 21360

tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 21420

agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 21480

taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 21540

caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 21600

agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 21660

aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 21720

gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 21780

tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 21840

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctgctcgga tctgttggac 21900

cggacagtag tcatggttga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc 21960

cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg 22020

cttagacaac ttaataacac attgcggacg tttttaatgt actggggttg aacactctgt 22080

ggaagacaat gccgaattcg gatcc 22105

<210> 6

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 6

ccgaagacgg ctcaggagca tccctaatga tattgttcac gtaa 44

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 7

ccgaagacgg ctcgaatggc actgtgaatg attagaataa tttc 44

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 8

ccgaagacgg ctcaaatgct atttgcaagg ggtttg 36

<210> 9

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 9

ccgaagacgg ctcgaagcct attttgttct tgtgatgact 40

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 10

ccgaagacgg ctcagcttat atgaagatga agatgaaata 40

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 11

ccgaagacgg ctcgagcgct tatctttaat catattccat 40

Claims

1. Coenzyme Q in tomato can be improved ₁₀ The method for constructing the combined vector is characterized by comprising the following steps:

s4, connecting a 35S promoter sequence, a hygromycin resistance gene coding sequence and a 35S terminator sequence, the E8 promoter sequence, the Coq2 gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the DPS gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the HMGR2 gene coding sequence and the hsp terminator sequence, the E8 promoter sequence, the Ubic gene coding sequence and the hsp terminator sequence into a vector pICSL4723-P1 in sequence by a Golden Gate cloning method, wherein the obtained level 2 vector is the combined vector, and the gene sequence of the combined vector is shown in SEQ ID No. 5.

2. The method for constructing a combined vector according to claim 1, wherein each of the PCR amplification systems in step S1 is: a template, i.e., 500ng of the genomic DNA or 50ng of the cDNA; 0.2. mu.M forward primer; 0.2. mu.M reverse primer; 10. mu.L Fastpfu buffer; 0.2mMdNTPs; 2.5units Fastpfu DNA polymerase; h ₂ O is complemented to 50 mu L;

3. The method for constructing a combined vector according to claim 2, wherein the nucleotide sequence of the PCR amplification primer of tomato genomic DNA is shown as SEQ ID No.6 and SEQ ID No. 7; the nucleotide sequence of the PCR amplification primer of the tobacco cDNA is shown as SEQ ID NO.8 and SEQ ID NO. 9; the nucleotide sequence of the PCR amplification primer of the arabidopsis genomic DNA is shown as SEQ ID NO.10 and SEQ ID NO. 11.

4. The method for constructing a combined vector according to claim 1, wherein when the coding sequence of the corresponding gene is obtained by the total synthesis, the bases of a part of the coding sequence are modified to remove BsaI and BpiI cleavage sites.

5. The method for constructing a combined vector according to claim 1, wherein each of the level 0 vector constructs in step S2 is: 100ng of pUAP1 vector; the molar ratio of the corresponding sequence fragment obtained in the step S1 to the pUAP1 vector is 2: 1; 5units BpiI endonuclease; 200units T ₄ A ligase; 1.5 μ L T4 ligase buffer; 1.5 μ LBSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

6. The method for constructing a combined vector according to claim 1, wherein in step S3, each level 1 vector construction system is: corresponding level 1 vector, 100 ng; e8 start-up obtained in step S2The molar ratio of the level 0 carrier of the rotor to the corresponding level 1 carrier is 2: 1; the molar ratio of the level 0 vector of the corresponding gene obtained in the step S2 to the corresponding level 1 vector is 2: 1; the molar ratio of the level 0 vector of the hsp terminator obtained in the step S2 to the corresponding level 1 vector is 2: 1; 5units BsaI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

7. The combined vector construction method according to claim 1, wherein the level 2 vector construction system in step S4 is: 100ng pICSL4723-P1 vector; the molar ratio of the pICSL11059 vector to the pICSL4723-P1 vector is 2: 1; the molar ratio of the Coq2 gene level 1 vector obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the level 1 vector of the DPS gene obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the level 1 vector of the HMGR gene obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the molar ratio of the Ubic gene level 1 vector obtained in the step S3 to the pICSL4723-P1 vector is 2: 1; the PICH41800 linker vector has a molar ratio of 2:1 to the pICSL4723-P1 vector; 5units BsaI endonuclease; 5units BpiI endonuclease; 200units T4 ligase; 1.5 μ L T4 ligase buffer; 1.5. mu.L BSA, 1 mg/mL; h ₂ O is complemented to 20 mu L;

8. A combined vector obtainable by the method of constructing a combined vector according to any one of claims 1 to 7.

9. A method for producing tomato with increased coenzyme Q10 content, characterized in that the combined vector obtained by the construction method according to any one of claims 1-7 is transformed into Agrobacterium GV3101 by heat shock and transformed into wild type tomato; after obtaining the independent resistant callus, further differentiating and regenerating to obtain the tomato strain with high coenzyme Q10 content.