CN113564178B - Blind vein obv gene and application thereof - Google Patents

Abstract

The invention provides a novel dark vein gene obv, which is a zinc finger transcription factor encoding a C2H2L structural domain, is mainly expressed in vascular bundles in veins and surrounding palisade tissues and is positioned in cell nuclei. The dark vein gene obv can improve the chlorophyll content, photosynthetic rate and stomatal conductance of veins and increase the quantity of chloroplasts. The obv gene of the invention participates in regulation and control of chloroplast development and photosynthesis of tomato leaves, and can provide theoretical basis and technical support for genetic improvement of tomatoes with high light efficiency. The obv gene of the invention can improve the yield and the content of soluble solids of tomatoes, has important significance on the high quality and the high yield of tomatoes, can be applied to the cultivation of new varieties of tomato heterosis, has huge development and utilization values and has wide market application prospect.

Description

Blind vein obv gene and application thereof

Technical Field

The invention relates to a dark vein obv gene and application thereof. The invention adopts a forward genetics method, clones to control tomato dark vein gene obv, knocks out obv gene by CRISPR/Cas9 knockout technology, and causes dark vein to appear in the tomato leaves with clear veins; and through over-expression of obv gene, the vein of the dark vein tomato leaves appears, thus proving the function of the gene. Also relates to a vector containing the gene and homologous genes of other species, and to the use of the gene or functional analogues thereof for regulating plant vein changes and chlorophyll content.

Background

Leaf blade is the main organ for photosynthesis and respiration of plant, photosynthesis is the basis of plant growth and development, and is also the main determinant of yield and quality, and leaf blade related character is one of the key characters for genetic improvement of crops.

Tomatoes are grown in the andes mountain, the ecuador coast and the peru of south america, and after several hundred years of domestication and improvement, tomatoes become one of the most important vegetables in the world and are widely planted worldwide. Tomato leaves are complex leaves, and many variations are formed in the evolution process, but the variation of the veins is very limited. A naturally mutated dark vein obv (Obscuravenosa) gene is found in the production, and under drought and high light intensity environment conditions, such as areas of California and Xinjiang in China, the photosynthetic efficiency and stomatal conductance of leaves can be effectively improved, the fruit yield is increased, and stronger plasticity is shown on high light, so that the gene is well fixed in tomato varieties for processing in the breeding process.

The wild tomato leaf veins are all transparent leaf veins, namely, the bright vein, the dark vein obv gene mutation can be derived from a variety Earliana in the twentieth of the nineteenth century, the character is single recessive gene control, is caused by the increase of the chlorophyll content of the leaf veins, has obvious gain on the leaf vein gas exchange related characters, and can obviously improve the water utilization rate and the yield. The brightness of tomato veins is caused by the high and low chlorophyll content in the veins, and the periphery of the lower epidermis cells and mesophyll cells of the tomatoes usually have no chloroplasts, but the dark vein tomato materials are opposite. Second, the tomato dark vein phenotype is caused by the lack of vascular bundle sheath extension (Bundle Sheath Extensions, BSEs) in the leaves, the dark vein leaf palisade tissue has continuity in the upper epidermis, while the palisade tissue of the bright vein is discontinuous.

Tomatoes are important worldwide vegetable crops, china is the first world production country, the cultivation area is about 1600 tens of thousands of mu, and annual output value is 1800 hundred million. The application of the tomato dark vein gene obv has important significance for genetic improvement of tomato varieties with high photosynthetic efficiency and improvement of tomato yield and quality. However, the gene is not cloned at present, the function of the gene is not clear, and the further application of the gene is limited. The research of the gene has great significance for genetic improvement of high-light-efficiency tomatoes, and has great promotion effect on the differentiation, formation and development of tomato vascular bundle sheaths, and the research of photosynthetic regulation mechanisms and photosynthetic systems of C3 crop tomatoes.

Disclosure of Invention

The invention aims at providing a dark vein gene obv and application thereof.

According to one aspect of the present application, the present invention provides a dark vein gene obv, wherein the nucleotide sequence of the dark vein gene obv is as shown in SEQ ID No: 1.

As a specific embodiment of the present application, the coding sequence of the dark vein gene obv is shown in SEQ ID No: 2.

As a specific embodiment of the present application, the amino acid sequence of the dark vein gene obv is as shown in SEQ ID No: 3.

According to another aspect of the present application, the present invention also provides the application of the dark vein gene obv in regulating the darkness of plants She Maiming.

According to another aspect of the application, the invention also provides the application of the dark vein gene obv in regulating and controlling the photosynthetic rate of plants.

According to another aspect of the application, the invention also provides the application of the dark vein gene obv in plant genetic breeding.

As a preferred embodiment of the present application, the plant is selected from at least one of tomato, capsicum, eggplant and cucumber.

As a specific embodiment of the present application, the plant is tomato. The green content of leaves in tomato leaves, veins and vascular bundle sheaths is controlled by controlling the expression quantity of obv genes or homologous genes thereof in tomatoes, so that the photosynthetic efficiency is enhanced, and the purposes of yield increase and high quality are achieved.

The technology for realizing the invention is as follows:

1. identification of clear tomato leaf vein as dark vein

Phenotype identification of tomato She Maiming veins and dark veins (FIG. 1), paraffin sections of tomato leaf veins were identified (FIG. 2).

2. Whole genome association analysis (GWAS) of tomato natural populations

The widely collected 299 parts of processed tomato germplasm material is used as a natural population, and through carrying out phenotypic observation record and combining whole genome re-sequencing data, whole genome association analysis is completed. It was confirmed that the obv gene was located at the end of the long arm of chromosome 5 and linked to SP5G (fig. 3).

3. Genetic localization of tomato light and dark vein gene obv

F2 isolated population is constructed by using tomato vein material and vein material, and fine localization of obv gene is completed by constructing linkage map. According to the genotyping and phenotypic identification results of the recombinant single plant, the obv gene is finally positioned between the No. 5 chromosome molecular markers SNP20 and SNP24, and the interval size is about 24.141kb (FIG. 4). The primer nucleotide sequences of the molecular markers SNP20 and SNP24 are respectively shown in SEQ ID NO:3 and SEQ ID NO: 4.

4. Determining mutation site to obtain candidate gene

Using SGN website (https:// solgenomics. Net), gene prediction was performed within the located 24.141kb interval, and combined with sequence variation analysis, it was found that there was a mutation of 1 base G substitution to A on the third exon of Solyc05G054030, located at the 404 th base in CDS region, solyc05G054030 was finally determined as a candidate gene belonging to the C2H2 type zinc finger structural transcription factor, comprising 4 exons, CDS region length 1,149bp, translation 381 amino acids, and base G mutation to A at the 404 th base in CDS region, resulting in arginine (R) to histidine (H).

Functional verification of the obv Gene

The Crispr/Cas9 knockout experiments verify obv gene function. The method comprises the steps of constructing a CRISPR/Cas9 vector by using a pMGET (pKSE 401-S) vector as a skeleton vector and adopting a T4 connection method, and obtaining a transgenic plant by using the Mingmai tomato Micro-Tom as a material. Compared to the vein of wild-type Micro-Tom, which was Ming vein, the veins of the transgenic lines were all dark veins (FIG. 6). At the same time, the chlorophyll content of leaf veins of transgenic plants is obviously increased, and the number of chloroplasts in She Maiwei tube bundles is obviously increased (figure 7).

The function of the overexpression obv gene is verified. Cloning the full-length coding sequence (CDS) of obv into a pBI121 binary vector, and transforming the overexpression vector into a dark vein tomato M82 by adopting an agrobacterium GV3101 mediated genetic transformation method to obtain a transgenic plant. Both the main and collateral vessels of the leaves of the transgenic plants exhibited a Ming pulse phenotype compared to wild-type M82 (FIG. 8). Obv has the function of regulating and controlling the brightness of veins.

Application of obv Gene

The brightness of veins can be changed by combining agrobacterium-mediated genetic transformation methods such as Crispr/Cas9 gene editing or overexpression, and the like, so that the chlorophyll content of tomato leaves and vascular bundle sheaths can be changed. The brightness of the veins can be changed according to the requirements of different tomato varieties, so that the high efficiency is formed, and the purposes of increasing yield and improving quality are achieved. The tomato dark vein control gene obv can be applied to transgenic tomatoes and transgenic tomato seeds to carry out genetic improvement of varieties.

The beneficial effects of the invention are as follows:

(1) The invention provides a novel dark vein gene obv, which is a zinc finger transcription factor encoding a C2H2L structural domain, is mainly expressed in vascular bundles in veins and surrounding palisade tissues and is positioned in cell nuclei.

(2) The dark vein gene obv can improve the chlorophyll content, photosynthetic rate, stomatal conductance and chloroplast quantity of veins. The obv gene of the invention participates in regulation and control of chloroplast development and photosynthesis of tomato leaves, can provide theoretical basis and technical support for genetic improvement of tomatoes with high light efficiency, can be applied to cultivation of new varieties of tomato heterosis, has great development and utilization values and wide market application prospect.

Drawings

FIG. 1 tomato leaf vein phenotype observation;

FIG. 2 tomato leaf vein paraffin section;

FIG. 3 Whole genome association analysis of tomato leaf vein obv trait;

FIG. 4 Fine localization of tomato leaf vein obv trait

FIG. 5 tomato light and dark leaf photosynthesis index;

FIG. 6 tomato Micro-tom and obv gene editing plant leaf vein phenotype and paraffin sections;

FIG. 7 tomato Micro-tom and obv Gene-edited plant, leaf vein chlorophyll content and chloroplast number in vascular bundles;

FIG. 8 tomato M82 and obv overexpressing plant leaf vein.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

1. Identification of clear and dark veins of tomato leaves.

Cotyledons of tomato material of both genotypes already show different veins when budding, making it easier to distinguish between bright and dark vein phenotypes as true leaves develop. The difference of the size, thickness and shape of the leaf is not large, but the difference of the vein brightness and darkness phenotype is obvious, and the obvious difference can be observed by naked eyes under direct sunlight, as shown in figure 1.

In order to further observe the difference of the light and dark vein materials, paraffin sections are adopted to observe the cross sections of the material light veins and dark vein leaf veins, and it is obvious that the extension of the vascular bundle sheath is absent in the light vein material She Maizhong, and the palisade tissue becomes discontinuous; while in the presence of the vascular bundle sheath extension of the vein material She Maizhong, the palisade tissue remains in a continuous state with the veins, as shown in fig. 2.

As shown in FIG. 5, the results of FIG. 5 show that the photosynthetic rate, stomatal conductance and transpiration rate of the dark pulse tomato leaf are 1.98 times, 2.64 times and 2.94 times that of the bright pulse tomato leaf, respectively, indicating that the dark pulse gene can improve the photosynthetic efficiency of the tomato leaf.

1. Whole genome association analysis (GWAS) of tomato natural populations

The 299 parts of widely collected processed tomato germplasm material is used as a natural population, wherein the natural population comprises 129 parts of vein material, 163 parts of vein material, 7 parts of data deletion, and specific phenotype investigation results are shown in annex 1. By phenotyping it, whole genome association analysis was completed in combination with whole genome re-sequencing data, as shown in fig. 3. The obv gene is located at the end of the long arm of chromosome 5 with a confidence interval of SL2.50chr05:63,049,460 bp, section size 963,238bp, and linked to SP 5G.

2. Genetic localization of tomato light and dark vein gene obv

F2 isolated population of 1500 strains is constructed by using tomato vein material 05-62 and vein material 05-49, phenotype identification accords with 3:1 isolation rule, and the vein characters are controlled by recessive single genes. According to the GWAS result, selecting SNPs locus to develop into KASP mark, combining with phenotype identification result, and constructing linkage map to finish fine positioning of obv gene. According to the genotyping and phenotype identification results of the recombinant single plant, the obv gene is finally positioned between the No. 5 chromosome molecular markers SNP20 and SNP24, and the interval size is about 24.141kb, as shown in FIG. 4. The primer nucleotide sequences of the molecular markers SNP20 and SNP24 are shown in Table 1, respectively.

TABLE 1

3. Determining mutation site to obtain candidate gene

Using SGN website (https:// solgenomics. Net), gene predictions were made within a targeted 24.141kb interval, finding a total of 3 open reading frames within this region. We examined the expression of the three genes using Ensemblplants website (https:// plants. Ensembl. Org/Solanum_lycopersicum/Info/Index) and found that Solyc05g054030 and Solyc05g054040 were expressed in leaves, and sequencing analysis of the full length of the 2 genes indicated: a mutation in the third exon of Solyc05G054030, wherein 1 base G is replaced by A, and the mutation is positioned at the 404 th base of CDS region and codes for the 135 th amino acid; the method comprises the steps of carrying out a first treatment on the surface of the Solyc05g054040 coding region sequence was not different; solyc05g054030 was finally identified as a candidate gene belonging to the C2H2 type of zinc finger structural transcription factor. The nucleotide sequence of the gene is shown below.

SEQ ID No：1

Solyc05g054030 gene sequence

>SL4.0ch05 SL4.0ch05:63395462..63398588(+strand)length＝3127

ATGCTAACTAGCAACTCTTTCTTGTTTGGTGCTCCTTCTAATTATTCTGATCCATTTTCTTCCCCAGAAAATGGTTTTATTATCAAAAGAAAAAGAAGACCTGCTGGTACTCCAGGTATATATATATATTTTTAATTAATTAATTAGTATATTTTTAAAAAAAAATTAATTTACATAAATATATGAAGAAAATGGTACTTTTTTTGATAATTATGTGAAAAAACACTTGAGTTTTAGCTCTTGTGTGTCTATTATATTTCTAAATTGATCAACATGTTCAGTCAGTGACGAAAACAGAATTTTCATCAGAGGATTCATGAGGATGTAACGAAAAGAATTCAGATGAACCTCCTTTTGGCTTTTTCTATCTCCGACCTTGTGTTTTTGAATTCAGAATTTAAACGTTATAGATGAGAAAGTTGAATTATGATTTAACCTTATCTTTATAGTCAAGGGCGGAGCTATAGGTAACAAAGATTGTTTGGTTGATACAACCCCTTTCGTCAGAAAATTATATTTTTATATATTTATTTTTTAAAAAAAATTCTTAACCTAATAGATTTAATTTTTTAAAATTTTCTTAACCTAATAAATTTAGATGTGAAAATTATATTTGAATTACTGGCTCCGCTACTATTGCTAACACACATATGTTTAGGGTTATTCGACTGGTAAGAATGCTATTGAATTCTGTTGAACTCGTAATAATTAAATTTACGAATTTGCACAGATCCCGATGCACAAGTTGTATATCTTACAGCTGAGATGTTAATGGAATCTGATCGTTACGTTTGTGAAATCTGCAACCTTAGCTTTCAAAGAGAGCAAAATCTACAAATGCATCGTCGTCGCCATAAGGTTCCATGGAAGTTGAAGAAGAAGGTAGTTTAATTTATGTATAATTACGTCATCAATATATCGTCTCATCTAAAATCTTAAACTGTTCGATAGAACACAAGTTCTTCATTCGTTCAATAGGGAGTGAGTCTTCCCCTTTTTGAAAAATGAGTTAATATCATGTGTAGACGGAGAATTCATATATCTGATAAGAACAGATGTTACACTTGATCTTAGCCACAAGACCGAGAAAGATATTGATGAGAACTATACAATTTTTATTTACTAAATTATACTTTATATTTCAACACATCTCCTCACGTGCAAGTCATGAAGTTCTTCTTCTTCTTTTTTATTACGAGAACGATACATTTTAATATTTAGAATTTCTCTGTTTATTCTTACTGAAATGATTTATAATAATCACACAAATTGCTAAGGCTTAGTTTTCGACAATAATTTTCAAAAGTCTTTCAATTCTAGACGTCACTCCCCAGTTAAATATAGTCACATAAATTGTAACTGACATATTAGATTATATGATTAGATATGTTAATTTTTTTAATTAAATATAAATATAATTTCATTTACTTGATTATATTTTCAACGTGATCATCAGGAAGAAGAGAAAAATGAGATGGATCAAGTTATTAAGAAGAGAGTATATGTGTGTCCAGAGCCAAGTTGTGTGCACCATGATCCATGTCATGCATTAGGTGATCTTGTTGGAATCAAAAAACATTTTAGAAGAAAACGTAGCAATTACAAACAATGGATTTGTCAAAAATGCAACAAAGGTTATGCTGTTCAATCAGATTATAAAGCTCACATCAAAACTTGTGGTACTAGAGGCCATTCTTGTGATTGTGGAAGAGTTTTCTCTAGGTAAATTCATCTTCTTAATTATATATCTGTGTTCTGTTTTACTTGAGTCGAGAATCTATAAGAAAAAATAGAATCTATTTATCCTCATAGGAGTAAGGTTACAACGTCCTATTCAGATTCCACTAAATATGTTATTGTTATAGTAATTTTTATCATCAGCGTATCTTTATTATCTAGGTTATATTAAATATACTACTAAAAAACGTTAAAGAATTAGCTATGAAATTCGTAGCTGGTTAATTTATAACTAAATAGTCTATATCTACTAAGATTGTCTCATTATAAAATGTCATTTCTATGTAGTCAAATAGAATTAGGTTTAATTCATTGTTTAGTGATATAAATTAAATTTATAAAAATCTTTTAAGTGACTTAATAGCGTAAAAAGTAAATTTACACTATCTTATATATAAAAATTATACACATATATCAAGATGAGATTACCACATGTTACTTGAATTGGTAACATCCTTTAGGTCTAAAACCTAATGTATATATATGTCTTGTAAATGTACAAACATATTTTGTGTGCTCACATTTGAAAATTTCTTCCTTATCTATATGATTATAAAAATCACTATCTTTTTAGTTAAAAACATGAATATTATTATCAGAAAATCACTAATTTTCGACGATATTATATGAGTCAAATTCTGATAGATTTGTTGGAAATATTTTTAATTAAAAATTAGCGATTTTCTGATAGTAAATTTGAGTTATATATAGTATGTTTCTTCTAATTAATCTACTTTTTTTTTCCTCCCATTTTTATTGTGTTTTTTTTTTCAGAGTTGAAACATTTATTGAGCATCAAGATTCATGCAAACCACAAAGTACAACTACTAAAGAATGTCATGATATGCAAATACCAAAACCAATTTTCTTGCCTACTACTACAACTCATATCCCACCACATGATCAATATTCAAAAATATTGCCTAATCTTGATCTTGAGCTTTTCACTTCTCCAAATTATTTCAACCAAAACACACACAATTTTTCATCATTTGTTGATCAAAGTGATCATCATCATCATAATAATAATTACATAGTCCAAAACAATGATATTGAAGTCAAAGAAATTATTGAAGAGGCAACAACACAAGTAACAAGATTGAAAAGTGAAGCAAATGAAATACTCAAAATAGCAATGGAAGAAAAGGCAATGGCTATAGAGAAGAGACAAGAAGCAAAGTGTTTGATTGAATTAGCCAACCTTGAAATGGCAAAAGCAATGGAAATTAGACAAAGTGTTTGTGCTTCATCATCATCATCATCACATGTCATGAAGATAATAAAATGTAGTTCTTGTAATAATAAACAATTTCAAAGTGTGTCATCATCAAAAGATGCTACTTTGACTAATAATTATTATTTGTCATCTTCTATTTATAGAAGATGATGA

SEQ ID No：2

Coding sequence (CDS)

ATGCTAACTAGCAACTCTTTCTTGTTTGGTGCTCCTTCTAATTATTCTGATCCATTTTCTTCCCCAGAAAATGGTTTTATTATCAAAAGAAAAAGAAGACCTGCTGGTACTCCAGATCCCGATGCACAAGTTGTATATCTTACAGCTGAGATGTTAATGGAATCTGATCGTTACGTTTGTGAAATCTGCAACCTTAGCTTTCAAAGAGAGCAAAATCTACAAATGCATCGTCGTCGCCATAAGGTTCCATGGAAGTTGAAGAAGAAGGAAGAAGAGAAAAATGAGATGGATCAAGTTATTAAGAAGAGAGTATATGTGTGTCCAGAGCCAAGTTGTGTGCACCATGATCCATGTCATGCATTAGGTGATCTTGTTGGAATCAAAAAACATTTTAGAAGAAAACGTAGCAATTACAAACAATGGATTTGTCAAAAATGCAACAAAGGTTATGCTGTTCAATCAGATTATAAAGCTCACATCAAAACTTGTGGTACTAGAGGCCATTCTTGTGATTGTGGAAGAGTTTTCTCTAGAGTTGAAACATTTATTGAGCATCAAGATTCATGCAAACCACAAAGTACAACTACTAAAGAATGTCATGATATGCAAATACCAAAACCAATTTTCTTGCCTACTACTACAACTCATATCCCACCACATGATCAATATTCAAAAATATTGCCTAATCTTGATCTTGAGCTTTTCACTTCTCCAAATTATTTCAACCAAAACACACACAATTTTTCATCATTTGTTGATCAAAGTGATCATCATCATCATAATAATAATTACATAGTCCAAAACAATGATATTGAAGTCAAAGAAATTATTGAAGAGGCAACAACACAAGTAACAAGATTGAAAAGTGAAGCAAATGAAATACTCAAAATAGCAATGGAAGAAAAGGCAATGGCTATAGAGAAGAGACAAGAAGCAAAGTGTTTGATTGAATTAGCCAACCTTGAAATGGCAAAAGCAATGGAAATTAGACAAAGTGTTTGTGCTTCATCATCATCATCATCACATGTCATGAAGATAATAAAATGTAGTTCTTGTAATAATAAACAATTTCAAAGTGTGTCATCATCAAAAGATGCTACTTTGACTAATAATTATTATTTGTCATCTTCTATTTATAGAAGATGA

SEQ ID No：3

Amino acid sequence

MLTSNSFLFGAPSNYSDPFSSPENGFIIKRKRRPAGTPDPDAQVVYLTAEMLMESDRYVCEICNLSFQREQNLQMHRRRHKVPWKLKKKEEEKNEMDQVIKKRVYVCPEPSCVHHDPCHALGDLVGIKKHFRRKRSNYKQWICQKCNKGYAVQSDYKAHIKTCGTRGHSCDCGRVFSRVETFIEHQDSCKPQSTTTKECHDMQIPKPIFLPTTTTHIPPHDQYSKILPNLDLELFTSPNYFNQNTHNFSSFVDQSDHHHHNNNYIVQNNDIEVKEIIEEATTQVTRLKSEANEILKIAMEEKAMAIEKRQEAKCLIELANLEMAKAMEIRQSVCASSSSSSHVMKIIKCSSCNNKQFQSVSSSKDATLTNNYYLSSSIYRR*

4. Gene knockout test of obv Gene

To further confirm that the phenotype of tomato vein as dark vein is due to the variation of the gene Solyc05g054030, obv gene function was verified by Crispr/Cas9 knockout experiments in the context of wild type Micro-Tom. The method comprises the steps of constructing a CRISPR/Cas9 vector by using a pMGET (pKSE 401-S) vector as a skeleton vector and adopting a T4 connection method, and obtaining a transgenic plant by using the Mingmai tomato Micro-Tom as a material. Finally, 12 transgenic positive lines are obtained, three of the positive plants (Cris-1, cris-3 and Cris-24) are finally selected for further experiments, and compared with the leaf veins of the wild type Micro-Tom, the leaf veins of the three transgenic lines are all dark veins, as shown in figure 6.

Results of paraffin section experiments observing the cross section of the knockdown mutant and the wild type vein show that the palisade tissue in the Cris-24 vein of the transgenic plant is continuously arranged on the upper epidermis, while the palisade tissue in the wild type Micro-Tom vein shows discontinuous arrangement, as shown in FIG. 6, which is completely consistent with the previous paraffin section results.

Meanwhile, the chlorophyll content in the veins of the material is detected, and as shown in the figure 7, the content of Chla and Chlb in the veins of Cris-obv is about 1.4 times of that of WT, which is obviously higher than that in the wild type, as shown in the figure 7; the number of chloroplasts in the She Maiwei tube bundle was significantly increased.

5. overexpression test of obv Gene

To further verify obv gene function we performed over-expression of obv gene. Cloning the full-length coding sequence (CDS) of obv into a pBI121 binary vector, and transforming the overexpression vector into a dark vein tomato M82 by adopting an agrobacterium GV3101 mediated genetic transformation method to obtain a transgenic plant. Compared with the wild type M82, the main pulse and the side pulse of the leaf of the transgenic plant show a bright pulse phenotype, as shown in FIG. 8. According to the result of the comprehensive knockout and overexpression experiment, we can determine that Solyc05g054030 is the target gene for regulating and controlling the formation of the dark veins of tomato leaves, and the gene has the function of regulating and controlling the brightness of the veins.

The invention provides a dark vein gene obv and application thereof. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

<110> institute of vegetable and flower at national academy of agricultural sciences

<120> a dark vein obv gene and application thereof

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tatatatatt tttaattaat taattagtat atttttaaaa aaaaattaat ttacataaat 180

atatgaagaa aatggtactt tttttgataa ttatgtgaaa aaacacttga gttttagctc 240

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tttcatcaga ggattcatga ggatgtaacg aaaagaattc agatgaacct ccttttggct 360

ttttctatct ccgaccttgt gtttttgaat tcagaattta aacgttatag atgagaaagt 420

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gtgaaaatta tatttgaatt actggctccg ctactattgc taacacacat atgtttaggg 660

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atttgcacag atcccgatgc acaagttgta tatcttacag ctgagatgtt aatggaatct 780

gatcgttacg tttgtgaaat ctgcaacctt agctttcaaa gagagcaaaa tctacaaatg 840

catcgtcgtc gccataaggt tccatggaag ttgaagaaga aggtagttta atttatgtat 900

aattacgtca tcaatatatc gtctcatcta aaatcttaaa ctgttcgata gaacacaagt 960

tcttcattcg ttcaataggg agtgagtctt cccctttttg aaaaatgagt taatatcatg 1020

tgtagacgga gaattcatat atctgataag aacagatgtt acacttgatc ttagccacaa 1080

gaccgagaaa gatattgatg agaactatac aatttttatt tactaaatta tactttatat 1140

ttcaacacat ctcctcacgt gcaagtcatg aagttcttct tcttcttttt tattacgaga 1200

acgatacatt ttaatattta gaatttctct gtttattctt actgaaatga tttataataa 1260

tcacacaaat tgctaaggct tagttttcga caataatttt caaaagtctt tcaattctag 1320

acgtcactcc ccagttaaat atagtcacat aaattgtaac tgacatatta gattatatga 1380

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aaaatgcaac aaaggttatg ctgttcaatc agattataaa gctcacatca aaacttgtgg 1680

tactagaggc cattcttgtg attgtggaag agttttctct aggtaaattc atcttcttaa 1740

ttatatatct gtgttctgtt ttacttgagt cgagaatcta taagaaaaaa tagaatctat 1800

ttatcctcat aggagtaagg ttacaacgtc ctattcagat tccactaaat atgttattgt 1860

tatagtaatt tttatcatca gcgtatcttt attatctagg ttatattaaa tatactacta 1920

aaaaacgtta aagaattagc tatgaaattc gtagctggtt aatttataac taaatagtct 1980

atatctacta agattgtctc attataaaat gtcatttcta tgtagtcaaa tagaattagg 2040

tttaattcat tgtttagtga tataaattaa atttataaaa atcttttaag tgacttaata 2100

gcgtaaaaag taaatttaca ctatcttata tataaaaatt atacacatat atcaagatga 2160

gattaccaca tgttacttga attggtaaca tcctttaggt ctaaaaccta atgtatatat 2220

atgtcttgta aatgtacaaa catattttgt gtgctcacat ttgaaaattt cttccttatc 2280

tatatgatta taaaaatcac tatcttttta gttaaaaaca tgaatattat tatcagaaaa 2340

tcactaattt tcgacgatat tatatgagtc aaattctgat agatttgttg gaaatatttt 2400

taattaaaaa ttagcgattt tctgatagta aatttgagtt atatatagta tgtttcttct 2460

aattaatcta cttttttttt cctcccattt ttattgtgtt ttttttttca gagttgaaac 2520

atttattgag catcaagatt catgcaaacc acaaagtaca actactaaag aatgtcatga 2580

tatgcaaata ccaaaaccaa ttttcttgcc tactactaca actcatatcc caccacatga 2640

tcaatattca aaaatattgc ctaatcttga tcttgagctt ttcacttctc caaattattt 2700

caaccaaaac acacacaatt tttcatcatt tgttgatcaa agtgatcatc atcatcataa 2760

taataattac atagtccaaa acaatgatat tgaagtcaaa gaaattattg aagaggcaac 2820

aacacaagta acaagattga aaagtgaagc aaatgaaata ctcaaaatag caatggaaga 2880

aaaggcaatg gctatagaga agagacaaga agcaaagtgt ttgattgaat tagccaacct 2940

tgaaatggca aaagcaatgg aaattagaca aagtgtttgt gcttcatcat catcatcatc 3000

acatgtcatg aagataataa aatgtagttc ttgtaataat aaacaatttc aaagtgtgtc 3060

atcatcaaaa gatgctactt tgactaataa ttattatttg tcatcttatg ctaactagca 3120

actctttctt gtttggtgct ccttctaatt attctgatcc attttcttcc ccagaaaatg 3180

gttttattat caaaagaaaa agaagacctg ctggtactcc aggtatatat atatattttt 3240

aattaattaa ttagtatatt tttaaaaaaa aattaattta cataaatata tgaagaaaat 3300

ggtacttttt ttgataatta tgtgaaaaaa cacttgagtt ttagctcttg tgtgtctatt 3360

atatttctaa attgatcaac atgttcagtc agtgacgaaa acagaatttt catcagagga 3420

ttcatgagga tgtaacgaaa agaattcaga tgaacctcct tttggctttt tctatctccg 3480

accttgtgtt tttgaattca gaatttaaac gttatagatg agaaagttga attatgattt 3540

aaccttatct ttatagtcaa gggcggagct ataggtaaca aagattgttt ggttgataca 3600

acccctttcg tcagaaaatt atatttttat atatttattt tttaaaaaaa attcttaacc 3660

taatagattt aattttttaa aattttctta acctaataaa tttagatgtg aaaattatat 3720

ttgaattact ggctccgcta ctattgctaa cacacatatg tttagggtta ttcgactggt 3780

aagaatgcta ttgaattctg ttgaactcgt aataattaaa tttacgaatt tgcacagatc 3840

ccgatgcaca agttgtatat cttacagctg agatgttaat ggaatctgat cgttacgttt 3900

gtgaaatctg caaccttagc tttcaaagag agcaaaatct acaaatgcat cgtcgtcgcc 3960

ataaggttcc atggaagttg aagaagaagg tagtttaatt tatgtataat tacgtcatca 4020

atatatcgtc tcatctaaaa tcttaaactg ttcgatagaa cacaagttct tcattcgttc 4080

aatagggagt gagtcttccc ctttttgaaa aatgagttaa tatcatgtgt agacggagaa 4140

ttcatatatc tgataagaac agatgttaca cttgatctta gccacaagac cgagaaagat 4200

attgatgaga actatacaat ttttatttac taaattatac tttatatttc aacacatctc 4260

ctcacgtgca agtcatgaag ttcttcttct tcttttttat tacgagaacg atacatttta 4320

atatttagaa tttctctgtt tattcttact gaaatgattt ataataatca cacaaattgc 4380

taaggcttag ttttcgacaa taattttcaa aagtctttca attctagacg tcactcccca 4440

gttaaatata gtcacataaa ttgtaactga catattagat tatatgatta gatatgttaa 4500

tttttttaat taaatataaa tataatttca tttacttgat tatattttca acgtgatcat 4560

caggaagaag agaaaaatga gatggatcaa gttattaaga agagagtata tgtgtgtcca 4620

gagccaagtt gtgtgcacca tgatccatgt catgcattag gtgatcttgt tggaatcaaa 4680

aaacatttta gaagaaaacg tagcaattac aaacaatgga tttgtcaaaa atgcaacaaa 4740

ggttatgctg ttcaatcaga ttataaagct cacatcaaaa cttgtggtac tagaggccat 4800

tcttgtgatt gtggaagagt tttctctagg taaattcatc ttcttaatta tatatctgtg 4860

ttctgtttta cttgagtcga gaatctataa gaaaaaatag aatctattta tcctcatagg 4920

agtaaggtta caacgtccta ttcagattcc actaaatatg ttattgttat agtaattttt 4980

atcatcagcg tatctttatt atctaggtta tattaaatat actactaaaa aacgttaaag 5040

aattagctat gaaattcgta gctggttaat ttataactaa atagtctata tctactaaga 5100

ttgtctcatt ataaaatgtc atttctatgt agtcaaatag aattaggttt aattcattgt 5160

ttagtgatat aaattaaatt tataaaaatc ttttaagtga cttaatagcg taaaaagtaa 5220

atttacacta tcttatatat aaaaattata cacatatatc aagatgagat taccacatgt 5280

tacttgaatt ggtaacatcc tttaggtcta aaacctaatg tatatatatg tcttgtaaat 5340

gtacaaacat attttgtgtg ctcacatttg aaaatttctt ccttatctat atgattataa 5400

aaatcactat ctttttagtt aaaaacatga atattattat cagaaaatca ctaattttcg 5460

acgatattat atgagtcaaa ttctgataga tttgttggaa atatttttaa ttaaaaatta 5520

gcgattttct gatagtaaat ttgagttata tatagtatgt ttcttctaat taatctactt 5580

tttttttcct cccattttta ttgtgttttt tttttcagag ttgaaacatt tattgagcat 5640

caagattcat gcaaaccaca aagtacaact actaaagaat gtcatgatat gcaaatacca 5700

aaaccaattt tcttgcctac tactacaact catatcccac cacatgatca atattcaaaa 5760

atattgccta atcttgatct tgagcttttc acttctccaa attatttcaa ccaaaacaca 5820

cacaattttt catcatttgt tgatcaaagt gatcatcatc atcataataa taattacata 5880

gtccaaaaca atgatattga agtcaaagaa attattgaag aggcaacaac acaagtaaca 5940

agattgaaaa gtgaagcaaa tgaaatactc aaaatagcaa tggaagaaaa ggcaatggct 6000

atagagaaga gacaagaagc aaagtgtttg attgaattag ccaaccttga aatggcaaaa 6060

gcaatggaaa ttagacaaag tgtttgtgct tcatcatcat catcatcaca tgtcatgaag 6120

ataataaaat gtagttcttg taataataaa caatttcaaa gtgtgtcatc atcaaaagat 6180

gctactttga ctaataatta ttatttgtca tcttctattt atagaagatg atgactattt 6240

atagaagatg atga 6254

<210> 2

<211> 1146

<212> DNA

<213> Artificial Sequence

<400> 2

atgctaacta gcaactcttt cttgtttggt gctccttcta attattctga tccattttct 60

tccccagaaa atggttttat tatcaaaaga aaaagaagac ctgctggtac tccagatccc 120

gatgcacaag ttgtatatct tacagctgag atgttaatgg aatctgatcg ttacgtttgt 180

gaaatctgca accttagctt tcaaagagag caaaatctac aaatgcatcg tcgtcgccat 240

aaggttccat ggaagttgaa gaagaaggaa gaagagaaaa atgagatgga tcaagttatt 300

aagaagagag tatatgtgtg tccagagcca agttgtgtgc accatgatcc atgtcatgca 360

ttaggtgatc ttgttggaat caaaaaacat tttagaagaa aacgtagcaa ttacaaacaa 420

tggatttgtc aaaaatgcaa caaaggttat gctgttcaat cagattataa agctcacatc 480

aaaacttgtg gtactagagg ccattcttgt gattgtggaa gagttttctc tagagttgaa 540

acatttattg agcatcaaga ttcatgcaaa ccacaaagta caactactaa agaatgtcat 600

gatatgcaaa taccaaaacc aattttcttg cctactacta caactcatat cccaccacat 660

gatcaatatt caaaaatatt gcctaatctt gatcttgagc ttttcacttc tccaaattat 720

ttcaaccaaa acacacacaa tttttcatca tttgttgatc aaagtgatca tcatcatcat 780

aataataatt acatagtcca aaacaatgat attgaagtca aagaaattat tgaagaggca 840

acaacacaag taacaagatt gaaaagtgaa gcaaatgaaa tactcaaaat agcaatggaa 900

gaaaaggcaa tggctataga gaagagacaa gaagcaaagt gtttgattga attagccaac 960

cttgaaatgg caaaagcaat ggaaattaga caaagtgttt gtgcttcatc atcatcatca 1020

tcacatgtca tgaagataat aaaatgtagt tcttgtaata ataaacaatt tcaaagtgtg 1080

tcatcatcaa aagatgctac tttgactaat aattattatt tgtcatcttc tatttataga 1140

agatga 1146

<210> 3

<211> 381

<212> PRT

<213> Artificial Sequence

<400> 3

Met Leu Thr Ser Asn Ser Phe Leu Phe Gly Ala Pro Ser Asn Tyr Ser

1 5 10 15

Asp Pro Phe Ser Ser Pro Glu Asn Gly Phe Ile Ile Lys Arg Lys Arg

20 25 30

Arg Pro Ala Gly Thr Pro Asp Pro Asp Ala Gln Val Val Tyr Leu Thr

35 40 45

Ala Glu Met Leu Met Glu Ser Asp Arg Tyr Val Cys Glu Ile Cys Asn

50 55 60

Leu Ser Phe Gln Arg Glu Gln Asn Leu Gln Met His Arg Arg Arg His

65 70 75 80

Lys Val Pro Trp Lys Leu Lys Lys Lys Glu Glu Glu Lys Asn Glu Met

85 90 95

Asp Gln Val Ile Lys Lys Arg Val Tyr Val Cys Pro Glu Pro Ser Cys

100 105 110

Val His His Asp Pro Cys His Ala Leu Gly Asp Leu Val Gly Ile Lys

115 120 125

Lys His Phe Arg Arg Lys Arg Ser Asn Tyr Lys Gln Trp Ile Cys Gln

130 135 140

Lys Cys Asn Lys Gly Tyr Ala Val Gln Ser Asp Tyr Lys Ala His Ile

145 150 155 160

Lys Thr Cys Gly Thr Arg Gly His Ser Cys Asp Cys Gly Arg Val Phe

165 170 175

Ser Arg Val Glu Thr Phe Ile Glu His Gln Asp Ser Cys Lys Pro Gln

180 185 190

Ser Thr Thr Thr Lys Glu Cys His Asp Met Gln Ile Pro Lys Pro Ile

195 200 205

Phe Leu Pro Thr Thr Thr Thr His Ile Pro Pro His Asp Gln Tyr Ser

210 215 220

Lys Ile Leu Pro Asn Leu Asp Leu Glu Leu Phe Thr Ser Pro Asn Tyr

225 230 235 240

Phe Asn Gln Asn Thr His Asn Phe Ser Ser Phe Val Asp Gln Ser Asp

245 250 255

His His His His Asn Asn Asn Tyr Ile Val Gln Asn Asn Asp Ile Glu

260 265 270

Val Lys Glu Ile Ile Glu Glu Ala Thr Thr Gln Val Thr Arg Leu Lys

275 280 285

Ser Glu Ala Asn Glu Ile Leu Lys Ile Ala Met Glu Glu Lys Ala Met

290 295 300

Ala Ile Glu Lys Arg Gln Glu Ala Lys Cys Leu Ile Glu Leu Ala Asn

305 310 315 320

Leu Glu Met Ala Lys Ala Met Glu Ile Arg Gln Ser Val Cys Ala Ser

325 330 335

Ser Ser Ser Ser Ser His Val Met Lys Ile Ile Lys Cys Ser Ser Cys

340 345 350

Asn Asn Lys Gln Phe Gln Ser Val Ser Ser Ser Lys Asp Ala Thr Leu

355 360 365

Thr Asn Asn Tyr Tyr Leu Ser Ser Ser Ile Tyr Arg Arg

370 375 380

<210> 4

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 4

gaaggtgacc aagttcatgc tctacgtaca atcagagaaa ttacttcc 48

<210> 5

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 5

gaaggtcgga gtcaacggat tcctacgtac aatcagagaa attacttct 49

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 6

agcacggtat aaaaactgtt ataattaata tagaa 35

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 7

gaaggtgacc aagttcatgc tcctgcgagt caagagaata tcag 44

<210> 8

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 8

gaaggtcgga gtcaacggat taacctgcga gtcaagagaa tatcat 46

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 9

cataatatga aaatatatta tcatcaaatt tgtcagtac 39

Claims (4)

1. The application of the dark vein gene obv in regulating the darkness of tomatoes She Maiming is characterized in that the nucleotide sequence of the dark vein gene obv is shown as SEQ ID No:1 is shown in the specification;

wherein, knocking out the dark pulse gene obv, wherein the tomato leaf pulse is dark pulse; the dark pulse gene obv was overexpressed and the tomato She Maiwei pulse was clear.

2. The application of the dark vein gene obv in regulating and controlling the photosynthetic rate of tomatoes is characterized in that the nucleotide sequence of the dark vein gene obv is shown as SEQ ID No:1 is shown in the specification;

wherein, knocking out the dark vein gene obv increases the photosynthetic rate of tomatoes.

3. The use according to any one of claims 1-2, wherein the coding sequence of the dark vein gene obv is set forth in SEQ ID No: 2.

4. The use according to any one of claims 1-2, wherein the amino acid sequence of the dark vein gene obv is set forth in SEQ ID No: 3.