阅读说明：本技术 一种暗脉obv基因及其应用 (Dark pulse obv gene and application thereof ) 是由 刘磊 鹿京华 李君明 李鑫 杜永臣 国艳梅 黄泽军 王孝宣 舒金帅 于 2021-07-22 设计创作，主要内容包括：本发明提供了一种新的暗脉基因obv,该暗脉基因为编码C2H2L结构域的锌指类转录因子,该基因主要在叶脉中的维管束和周围的栅栏组织里表达,并定位于细胞核。本发明的暗脉基因obv能够提高叶脉叶绿素含量、光合速率、气孔导度和增加叶绿体数量。本发明的obv基因参与调节番茄叶片的叶绿体发育和光合作用的调控,可为高光效番茄遗传改良提供了理论依据和技术支撑。本发明的obv基因可提高番茄的产量和可溶性固形物含量,对番茄的优质高产具有重要意义,可应用于番茄杂种优势新品种的培育,具有巨大的开发利用价值,市场应用前景广阔。(The invention provides a novel dark vein gene obv, which is a zinc finger transcription factor encoding C2H2L structural domain, is mainly expressed in vascular bundles in veins and surrounding palisade tissues and is positioned in cell nucleus. The dark vein gene obv can improve the chlorophyll content of veins, the photosynthetic rate, the stomatal conductance and increase the chloroplast quantity. The obv gene of the invention participates in the regulation and control of chloroplast development and photosynthesis of tomato leaves, and can provide theoretical basis and technical support for genetic improvement of high-photosynthetic-efficiency tomatoes. The obv gene can improve the yield and the soluble solid content of the tomato, has important significance for the high quality and the high yield of the tomato, can be applied to the cultivation of new species of the heterosis of the tomato, has great development and utilization values and has wide market application prospect.)

1. The dark vein gene obv is characterized in that the nucleotide sequence of the dark vein gene obv is shown as SEQ ID No: 1 is shown.

2. The dark vein gene obv of claim 1, wherein the coding sequence of the dark vein gene obv is as set forth in SEQ ID No: 2, respectively.

3. The dark vein gene obv of claim 1, wherein the amino acid sequence of the dark vein gene obv is as set forth in SEQ ID No: 3, respectively.

4. The use of the dark vein gene obv of claim 1 or 2 for regulating the light and dark veins in a plant.

5. Use of the dark vein gene obv of claim 1 or 2 for regulating plant photosynthetic rate.

6. Use of the dark vein gene obv of claim 1 or 2 in genetic improvement and breeding of plants.

7. Use according to any one of claims 3 to 5, wherein the plant is selected from at least one of tomato, pepper, eggplant and cucumber.

8. Use according to claim 6, wherein the plant is tomato.

Technical Field

The invention relates to a dark pulse obv gene and application thereof. The invention adopts a forward genetics method, the tomato dark vein controlling gene obv is cloned, and obv gene is knocked out by a CRISPR/Cas9 knocking-out technology, so that dark veins appear on the tomato leaves with bright veins; and the overexpression of obv gene can make the dark-vein tomato leaf appear bright vein, thus confirming the function of the gene. Also relates to a vector containing the gene and homologous genes of other species, and relates to the regulation and control of plant vein change and chlorophyll content by using the gene or functional analogues thereof.

Background

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

Tomatoes are native to Andes mountain, Ecuador coast and Peru in south America, and become one of the most important vegetables in the world after centuries of domestication and improvement, and are widely planted in the world. Tomato leaves are compound leaves, and a lot of variation is formed in the evolution process, but the variation of veins is very limited. A naturally mutated dark vein obv (obscuravenosa) gene is found in production, under the drought and high-light intensity environment conditions, such as California of America and Xinjiang in China, the photosynthetic efficiency and stomatal conductance of leaves can be effectively improved, the fruit yield is increased, and high-light intensity plasticity is shown, so that the naturally mutated dark vein obv (obscuravenosa) gene is well fixed in a processed tomato variety in the breeding process.

The leaf veins of the wild species tomato are all shown as transparent leaf veins, namely, bright veins, the gene mutation of the dark vein obv can be derived from a variety Earliana in the twentieth decade of nineteen-century, the character is single recessive gene control, the character is caused by the increase of the chlorophyll content of the leaf veins, the leaf gas exchange related character is obviously gained, and the water utilization rate and the yield can be obviously improved. The light and shade of the tomato veins are caused by the high and low content of chlorophyll in the veins, the periphery of epidermal cells and mesophyll cells of the tomato generally has no chloroplast, and the dark vein tomato materials are opposite. Second, the tomato dark vein phenotype is due to the lack of vascular Bundle Sheath Extensions (BSEs) in the leaves, with the dark vein leaf palisade tissue having continuity in the epidermis and the bright vein palisade tissue being discontinuous.

Tomatoes are important worldwide vegetable crops, China is the first major producing country in the world, the cultivation area is about 1600 mu or more and ten thousand mu, and the annual output value is 1800 hundred million. The application of the tomato dark vein gene obv has important significance for genetic improvement of tomato high photosynthetic efficiency varieties and improvement of tomato yield and quality. However, the gene is not cloned at present, and the function of the gene is not clear, so that the further application of the gene is limited. The research of the gene has important significance for genetic improvement of high-photosynthetic-efficiency tomatoes, and also has great promotion effect on revealing the differentiation, formation and development of the tomato vascular bundle sheath and the evolution research of the photosynthetic regulation mechanism and the photosynthetic system of the C3 crop tomatoes.

Disclosure of Invention

The invention aims to provide a dark pulse 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 shown in SEQ ID No: 1 is shown.

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

As a specific embodiment of the present application, the amino acid sequence of the venation dulcis gene obv is shown in SEQ ID No: 3, respectively.

According to another aspect of the application, the invention also provides application of the dark vein gene obv in regulating and controlling the light and dark veins of the plant leaves.

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

According to another aspect of the application, the invention also provides 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, pepper, eggplant and cucumber.

As a specific embodiment of the present application, the plant is a tomato. The green content of leaves in tomato leaves, veins and vascular bundle sheaths is controlled by controlling the expression level of obv gene or its homologous gene in tomato, so as to enhance photosynthetic efficiency and achieve the purpose of increasing yield and quality.

The technology for realizing the invention is concretely as follows:

1. identification for determining tomato leaf vein as dark vein

The phenotype identification of the bright and dark veins of tomato leaves (figure 1) and the paraffin section identification of the veins of tomato leaves (figure 2).

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

299 parts of processed tomato germplasm materials which are widely collected are used as natural populations, phenotype observation and recording are carried out on the natural populations, and whole genome re-sequencing data are combined to complete whole genome association analysis. 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 vein-light gene obv

F2 segregation population is constructed by utilizing tomato bright vein material and tomato dark vein material, and the fine positioning of obv gene is completed by constructing linkage map. According to the genotyping and phenotyping results of the recombinant individuals, the obv gene was finally mapped between the 5 th chromosomal molecular marker SNP20 and SNP24, with an interval size of about 24.141kb (FIG. 4). The primer nucleotide sequences of the molecular markers SNP20 and SNP24 are shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

4. Determining mutation sites to obtain candidate genes

By using SGN website (https:// solgenomics. net), gene prediction is carried out in a located 24.141kb interval, and through combination of sequence variation analysis, a mutation of replacing 1 base G with A exists on the third exon of Solyc05G054030, the mutation is located at 404 th base of a CDS region, and finally Solyc05G054030 is determined as a candidate gene, the gene belongs to a zinc finger structural transcription factor of C2H2 type, and comprises 4 exons, the CDS region is 1,149bp in length, 381 amino acids are translated, and the base G existing at the 404 th base of the CDS region is mutated into A, so that arginine (R) is changed into histidine (H).

Functional verification of the obv Gene

The criprpr/Cas 9 knock-out experiment verified obv gene function. The method comprises the steps of taking a pMGET (pKSE401-S) vector as a skeleton vector, constructing a CRISPR/Cas9 vector by adopting a T4 connection method, and taking the Mingmai tomato Micro-Tom as a material to obtain a transgenic plant. The veins of the transgenic lines were all dark, compared to the wild type Micro-Tom, where the veins were bright (FIG. 6). Meanwhile, the chlorophyll content of the leaf veins of the transgenic plants is obviously increased, and the number of chloroplasts in vascular bundles of the leaf veins is obviously increased (figure 7).

The function of the obv gene is verified by overexpression. The full-length coding sequence (CDS) of obv is cloned into a pBI121 binary vector, and the overexpression vector is transformed into the dark-vein tomato M82 by adopting an agrobacterium GV3101 mediated genetic transformation method to obtain a transgenic plant. Compared with wild type M82, the main vein and the lateral vein of the leaf of the transgenic plant both showed a bright vein phenotype (FIG. 8). The specification obv has the function of regulating the vein brightness.

Application of 6, obv gene

By combining gene editing or overexpression of Crispr/Cas9 and the like with an agrobacterium-mediated genetic transformation method, the light and shade of veins can be changed, and further the chlorophyll content of tomato leaves and vascular bundle sheaths can be changed. Can change the light and shade of veins according to the requirements of different tomato varieties to form high efficiency and achieve the purpose of increasing production and high quality. The tomato dark vein control gene obv can be applied to transgenic tomatoes and also can be applied to transgenic tomato seeds for genetic improvement of varieties.

The invention has the beneficial effects that:

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

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

Drawings

FIG. 1 phenotypic observations of leaf veins in tomato leaves;

FIG. 2 Paraffin sections of leaf veins of tomato;

FIG. 3 genome-wide association analysis of the tomato vein obv trait;

FIG. 4 Fine localization of tomato vein obv trait

FIG. 5 shows the photosynthetic indexes of the leaves of the tomato in the light and dark veins;

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

FIG. 7 tomato Micro-tom and obv gene editing plants, leaf vein chlorophyll content and number of chloroplasts in vascular bundles;

FIG. 8 tomato M82 and obv overexpressing plant veins.

Detailed Description

The present invention is described in further detail below with reference to specific examples.

1. And (4) identifying the bright veins and the dark veins of the tomato leaves.

Cotyledons of the two genotype tomato materials already show different veins when budding, and the bright and dark vein phenotypes are easier to distinguish along with the development of true leaves. The size, thickness and shape of the leaves of the two plants are not greatly different, but the bright and dark phenotype difference of the veins is obvious, and the obvious difference can be observed by naked eyes under the direct sunlight, as shown in figure 1.

In order to further observe the difference of light and dark vein materials, paraffin sections are adopted to observe the cross sections of the bright vein and dark vein leaf veins of the materials, so that the defect of extension of a vascular bundle sheath in the bright vein material leaf veins can be obviously seen, and the fence tissues become discontinuous; while in the dark vein material, the presence of the extension of the vascular bundle sheath maintains the palisade tissue in a continuous state with the vein, as shown in fig. 2.

The photosynthetic indexes of the bright and dark pulse tomatoes are measured, 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 leaves are respectively 1.98 times, 2.64 times and 2.94 times of those of the bright pulse tomato leaves, which indicates that the dark pulse gene can improve the photosynthetic efficiency of the tomato leaves.

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

299 parts of processed tomato germplasm materials which are widely collected are used as natural populations, wherein the natural populations comprise 129 parts of bright vein materials, 163 parts of dark vein materials and 7 parts of data loss, and specific phenotype investigation results are shown in appendix 1. The whole genome association analysis is completed by performing phenotype observation recording on the genome and combining whole genome re-sequencing data, as shown in FIG. 3. It was confirmed that the obv gene was located at the end of the long arm of chromosome 5 with a confidence interval of SL2.50chr05: 63,049,462bp-64,012,700bp, interval size is 963,238bp, and is linked with SP 5G.

2. Genetic localization of tomato vein-light gene obv

The tomato bright vein material 05-62 and the tomato dark vein material 05-49 are utilized to construct an F2 segregation population of 1500 strains, the phenotype identification accords with the 3:1 segregation rule, and the light and dark vein characters are controlled by a recessive monogene. According to the GWAS result, SNPs sites are selected to be developed into KASP markers, and the fine positioning of obv genes is completed by combining a phenotype identification result and constructing a linkage map. According to the genotyping and phenotypic identification results of the recombinant single strains, the obv gene is finally positioned between the 5 th 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 sites to obtain candidate genes

Gene prediction was performed using the SGN website (https:// solgenomics. net) within the localized 24.141kb interval, and a total of 3 open reading frames were found in this region. We looked at the expression of the three genes using the EnsemblPlants website (https:// plants. ensemblel. org/Solanum _ lysopersicum/Info/Index) and found that Solyc05g054030 and Solyc05g054040 were expressed in leaves, sequencing-by-sequencing the full-length of the 2 genes indicated: a mutation of 1 base G to A exists on the third exon of Solyc05G054030, is positioned at the 404 th base of the CDS region and codes the 135 th amino acid; (ii) a The sequence of the Solyc05g054040 coding region is not different; solyc05g054030 was finally identified as a candidate gene belonging to the zinc finger transcription factor of C2H2 type. 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. obv Gene knockout test

To further determine that the tomato vein-dark phenotype is due to changes in the gene Solyc05g054030, obv gene function was verified by Crispr/Cas9 knock-out experiments in the context of wild type Micro-Tom. The method comprises the steps of taking a pMGET (pKSE401-S) vector as a skeleton vector, constructing a CRISPR/Cas9 vector by adopting a T4 connection method, and taking the Mingmai tomato Micro-Tom as a material to obtain a transgenic plant. Finally, 12 transgenic positive lines are obtained, and three positive plants (Cris-1, Cris-3 and Cris-24) are selected for further experiments, so that the veins of the three transgenic lines are dark veins compared with the veins of the wild type Micro-Tom which are bright veins, as shown in FIG. 6.

Results of paraffin section experiments observing cross sections of the knockout mutant and wild type veins show that fence tissues in veins of a transgenic plant Cris-24 are continuously arranged on the upper epidermis, and fence tissues in veins of wild type Micro-Tom are discontinuously arranged, as shown in figure 6, which is completely consistent with the results of previous paraffin sections.

Meanwhile, the chlorophyll content in the veins of the material is detected, and the result is shown in fig. 7, and as can be seen from fig. 7, the contents of Chl a and Chl b in the veins of Cris-obv are both about 1.4 times of that of WT, and are obviously higher than the chlorophyll content in wild type; the number of chloroplasts in the vascular bundle of the leaf vein is obviously increased.

5.obv Gene overexpression test

To further verify obv gene function, we performed overexpression of obv gene. The full-length coding sequence (CDS) of obv is cloned into a pBI121 binary vector, and the overexpression vector is transformed into the dark-vein tomato M82 by adopting an agrobacterium GV3101 mediated genetic transformation method to obtain a transgenic plant. Compared with the wild M82, the main vein and the lateral vein of the leaf of the transgenic plant show a bright vein phenotype, and the bright vein phenotype is shown in figure 8. By combining the results of knockout and overexpression experiments, the Solyc05g054030 can be determined to be a target gene for regulating and controlling the formation of dark veins of tomato leaves, and the gene has the function of regulating and controlling the light and shade of veins.

The dark pulse gene obv and the application thereof provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

<110> vegetable and flower institute of Chinese academy of agricultural sciences

<120> dark pulse obv gene and application thereof

<130> CNP210010

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6254

<212> DNA

<213> Artificial Sequence

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atgctaacta gcaactcttt cttgtttggt gctccttcta attattctga tccattttct 60

tccccagaaa atggttttat tatcaaaaga aaaagaagac ctgctggtac tccaggtata 120

tatatatatt tttaattaat taattagtat atttttaaaa aaaaattaat ttacataaat 180

atatgaagaa aatggtactt tttttgataa ttatgtgaaa aaacacttga gttttagctc 240

ttgtgtgtct attatatttc taaattgatc aacatgttca gtcagtgacg aaaacagaat 300

tttcatcaga ggattcatga ggatgtaacg aaaagaattc agatgaacct ccttttggct 360

ttttctatct ccgaccttgt gtttttgaat tcagaattta aacgttatag atgagaaagt 420

tgaattatga tttaacctta tctttatagt caagggcgga gctataggta acaaagattg 480

tttggttgat acaacccctt tcgtcagaaa attatatttt tatatattta ttttttaaaa 540

aaaattctta acctaataga tttaattttt taaaattttc ttaacctaat aaatttagat 600

gtgaaaatta tatttgaatt actggctccg ctactattgc taacacacat atgtttaggg 660

ttattcgact ggtaagaatg ctattgaatt ctgttgaact cgtaataatt aaatttacga 720

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

ttagatatgt taattttttt aattaaatat aaatataatt tcatttactt gattatattt 1440

tcaacgtgat catcaggaag aagagaaaaa tgagatggat caagttatta agaagagagt 1500

atatgtgtgt ccagagccaa gttgtgtgca ccatgatcca tgtcatgcat taggtgatct 1560

tgttggaatc aaaaaacatt ttagaagaaa acgtagcaat tacaaacaat ggatttgtca 1620

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