CN113564182B

CN113564182B - Application of iris japonica SVP-like gene and method for obtaining iris japonica gene silencing or plant knockout

Info

Publication number: CN113564182B
Application number: CN202111025324.7A
Authority: CN
Inventors: 李丹青; 邵灵梅; 张亮生; 许曈; 王秀云; 张佳平; 夏宜平
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2023-04-07
Anticipated expiration: 2041-09-02
Also published as: CN113564182A

Abstract

The invention discloses application of an iris japonica SVP-like gene and a method for obtaining an iris japonica gene silencing or plant knockout. The research of the invention discovers that the germination rate and the growth speed of the plants of the mutants with the IjSVP1-like or IjSVP2-like gene silencing are obviously improved compared with the wild type under the dormancy induction condition; and the growth of the iris japonica can be promoted under the low-temperature condition in winter by reducing the expression quantity of the ABA synthetic gene IjnCED1, the discovery not only enriches the understanding of SVP gene functions, but also confirms that the functions of the iris japonica are different from those of the existing dicotyledons and provide a theoretical basis for cultivating evergreen florescent ground cover new varieties.

Description

Application of iris japonica SVP-like gene and method for obtaining iris japonica gene silencing or plant knockout

Technical Field

The invention relates to the technical field of biology, in particular to application of an iris japonica SVP-like gene and a method for obtaining an iris japonica gene silencing or knocking out a plant.

Background

Iris japonica (Iris japonica L.) is a perennial herbaceous plant of Iris of Iridaceae, has peculiar flower type, elegant flower color and evergreen color in four seasons, and is an important evergreen flower-viewing ground cover plant. It is warm and exposed to the sun or slightly shaded, late frost and winter cold are avoided, and it can be displayed as evergreen in winter under the condition of proper environment, however, its growth is severely limited by the low temperature in winter or early spring, and further its winter green display and ornamental effect are affected. Digging winter growth and dormancy control factors of the butterflies, and constructing germplasm resources which are resistant to low temperature in winter and continuously grow through gene directed mutation, thereby having important significance for obtaining new varieties of evergreen florescence ground covers.

Plant hibernation is a complex biological process that can be physiologically divided into physiological hibernation, which is a stop in growth caused by the surrounding environment or by endogenous signals, and restoration of growth within a certain time when given suitable circumstances, and ecological hibernation, which is a growth point restored to its growth ability but a growth arrest due to environmental condition restrictions (Lang GA, early JD, martin GC, darnell RL.1987.Endo-, para-, and ecodomanancyclics: physical science and classification for biological research. Hortscience.). At present, researches on plant overwintering dormancy are mostly concentrated in dicotyledonous perennial root plants, the plants generally have typical physiological dormancy and ecological dormancy processes, and after key genes for controlling the physiological dormancy are silenced or knocked out, the plants cannot directly recover growth due to the action of the ecological dormancy. However, in monocotyledons, only ecological dormancy exists in the winter dormancy of a perennial root plant represented by a rhizome iris, and the growth of the perennial root plant can be rapidly restored when the environment is suitable, and the plant shows dormancy characteristics different from those of the dormant mode plants (Li D, zhang J, zhang J, li K, xia Y.2017.Green period characteristics and folar colour in 12 iris species and cubars in the Yangtze Delta, china.Horttechnology 27, 399-407.) and the growth of the perennial root plant can be restored after key genes for controlling the ecological dormancy are silenced or knocked out, so that a mechanism for forming and releasing special overwintering dormancy characters is very necessary.

SVP (SHORT VEGETTING PHASE) is a class of MADS-box transcription factor genes, first found in non-dormancy mutants of peach (Prunus persica), the first candidate gene identified in association with physiological dormancy control (Bielenberg DG, wang Y, li Z, zhebengyayayayayayyeva T, fan S, reighard GL, scorza R, abbott AG.2008.Sequencing and mutation of the evolving and mutation in peaches [ Prunus persica (L.) Batsch ] genes, culture of MADS-box transcription factors for differentiation of terminal bud transformation. Genetics & gt, genes & 495-507, poplar-507, and grape vine all of which are identified in the same species: the expression change of SVP homologous gene is related to the formation, maintenance and release of plant physiological dormancy. However, at present, perennial plants generally have no homologous gene function verification system constructed yet, and can only depend on mode plants such as arabidopsis thaliana, and the like, and the arabidopsis thaliana is annual plants without an overwintering dormancy process, so that the related gene function research is obviously delayed. The genetic constitution and dormancy characteristics of the iris japonica, which is a perennial monocotyledon, are greatly different from those of the dicotyledonous dormant plant, and at present, whether the SVP plays a function in the dormancy of monocotyledon buds is not reported. Virus Induced Gene Silencing (VIGS) can silence and analyze a target gene in the current generation of infected plants, can avoid plant transformation, overcome function repetition, can play a role in different genetic backgrounds, and can analyze the gene function more thoroughly according to phenotypic variation (Lijie, rongjiang macro, yankwan. The research progress of the application of virus induced gene silencing on vegetable crops [ J ] Chinese agricultural science, 2021, 54 (10): 2154-2166.). Therefore, the method has wide application prospect in the gene function verification of plants, particularly perennial plants.

Disclosure of Invention

The invention provides a new application of an SVP-like gene of Iris japonica as a negative regulatory factor in promoting low-temperature growth of plants, and provides a theoretical basis for cultivating new varieties of evergreen florists.

The application of the Iris laevigata SVP-like gene as a negative regulatory factor in promoting low-temperature growth of Iris laevigata, wherein the Iris laevigata SVP-like gene is IjSVP1-like gene or IjSVP2-like gene, the gene sequence of the IjSVP1-like gene is shown in SEQ ID No.1, and the gene sequence of the IjSVP2-like gene is shown in SEQ ID No. 2.

The invention also provides a method for obtaining the Iris gene silencing or knockout plant, which comprises the steps of carrying out gene silencing or gene knockout on the tissue or plant organ from the Iris, wherein the silenced or knocked-out gene is at least one of IjSVP1-like gene and IjSVP2-like gene, then obtaining the plant,

wherein, the gene sequence of the IjSVP1-like gene is shown as SEQ ID No.1, and the gene sequence of the IjSVP2-like gene is shown as SEQ ID No. 2.

Gene silencing or knockout uses VIGS technology, T-DNA insertion, CRISPR/Cas9 gene editing technology, EMS mutagenesis, or RNA interference.

When the gene is silenced or knocked out, after a recombinant plasmid for gene silencing or knocking out is constructed, the recombinant plasmid is introduced into tissues or plant organs of the butterfly flower source by an agrobacterium transformation method. The plasmid backbone used in the construction of the recombinant plasmid is the pTRV2 vector, and when used, the recombinant plasmid is used in combination with the pTRV1 plasmid. Respectively introducing the recombinant plasmid and the pTRV1 plasmid into agrobacterium, and then, mixing the two kinds of agrobacterium according to the volume ratio of 1:1 mixed and used for transfection. The agrobacterium is GV3101.

The fragment for gene silencing IjSVP1-like gene used in the construction of recombinant plasmid is shown as SEQ ID No.5, and the fragment for gene silencing IjSVP2-like gene is shown as SEQ ID No. 6.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention proves the effect of SVP-like gene on the growth and ecological dormancy of low-temperature plants by a method of homologous gene silencing in monocotyledon perennial plants for the first time, and confirms that the gene is positioned in cell nucleus, mainly plays a function in stem tip meristem of butterfly flower and has the highest expression level in the low-temperature period in winter; in the prior art, SVP genes mainly play a role in inducing, maintaining and inhibiting physiological dormancy release in dicotyledonous dormant plants, when the SVP genes are silenced or knocked out, the plants are still in an ecological dormant state and cannot recover normal growth, but the iris japonica only has ecological dormancy in the wintering period, the SVP-like genes promote the plants to enter the ecological dormancy, and when the SVP-like genes of the iris japonica are silenced or knocked out, the plants can be prevented from entering the ecological dormant state, so that the plants can continue to grow under the low-temperature condition in winter.

(2) Obtaining a mutant with IjSVP1-like or IjSVP2-like gene silencing from Iris japonica by using VIGS technology, and finding that the germination rate and the growth speed of the mutant are obviously improved compared with the wild type under the condition of dormancy induction; and the growth of the iris japonica can be promoted under the low-temperature condition in winter by reducing the expression quantity of the ABA synthetic gene IjnCED1, the discovery not only enriches the understanding of the SVP gene function, but also provides a theoretical basis for cultivating a new variety of evergreen florescence by making clear that the function of the iris japonica is to promote the monocotyledon perennial plants to enter ecological dormancy and is different from the function of the existing dicotyledons.

Drawings

FIG. 1 is a diagram showing the results of the SVP gene phylogeny analysis of Iris japonica, and monocotyledonous and dicotyledonous representatives.

FIG. 2 is a graph showing the results of the subcellular localization detection of IjSVP1-like and IjSVP2-like genes in example 1.

FIG. 3 is a graph showing the results of examining the expression of IjSVP1-like and IjSVP2-like genes in different organ tissues of Iris japonica in example 2.

FIG. 4 is a graph showing the results of examining the expression of IjSVP1-like and IjSVP2-like genes in the shoot apical meristem of butterfly flower in different months in winter in example 3.

FIG. 5 is an electrophoretogram of recombinant plasmid fragments detected by PCR in Iris japonica silenced IjSVP1-like or IjSVP2-like gene obtained by VIGS technique in example 4.

FIG. 6 is a table of growth patterns in wild-type Iris lactea under the conditions of dormancy induction and IjSVP1-like or IjSVP2-like gene-silenced Iris lactea obtained by VIGS technique in example 5.

FIG. 7 is a graph showing the results of detecting the changes in expression levels of IjSVP1-like, ijSVP2-like and ABA-synthesizing gene IjNCED1 in wild-type Iris japonica and IjSVP1-like or IjSVP2-like genes in Iris japonica silenced under the dormancy induction conditions in example 6.

Detailed Description

The following detailed description will be made on embodiments of the present invention and the accompanying drawings, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following embodiments. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Because of the great difference of the monogenus and the dicotyledonous plants in the heredity, the SVP-like in the monocotyledonous plants is SVP-like homologous genes (figure 1), wherein the nucleotide sequences of protein coding regions of IjSVP1-like genes and IjSVP2-like genes in the iris are respectively shown as SEQ ID No.1 and SEQ ID No.2, the protein sequences are obtained by sequence comparison and conservative domain analysis of the overwintering dormancy transcriptome sequence of the iris of the subject group and the SVP of the model species, the lengths of the protein coding regions are respectively 702bp and 675bp, and the corresponding amino acid sequences are respectively shown as SEQ ID No.3 and SEQ ID No. 4.

The primer sequences referred to in the examples are shown in Table 1.

TABLE 1

Example 1: subcellular localization of IjSVP1-like and IjSVP2-like genes

And carrying out subcellular localization of IjSVP1-like and IjSVP2-like genes by using tobacco leaves.

The pCAMBIA 1-GFP plant green fluorescent expression vector was used for subcellular localization. According to the nucleotide sequences of coding regions of IjSVP1-like and IjSVP2-like genes, specific primers with homology arms are designed for amplifying full-length coding sequences except for stop codons, so that IjSVP1-like full-length cloning primers are IjSVP1-like-GFP-F and IjSVP1-like-GFP-R, and IjSVP2-like full-length cloning primers are IjSVP2-like-GFP-F and IjSVP2-like-GFP-R.

Extracting the total RNA of the stem tip of the iris japonica, performing reverse transcription on the total RNA of the iris japonica into cDNA by using a TAKARA first strand cDNA synthesis kit (Cat # 6210A), and performing PCR amplification by using high fidelity enzyme to obtain the full-length coding sequences of IjSVP1-like and IjSVP2-like genes. Performing agarose gel electrophoresis, cutting gel and recovering to obtain a PCR product, obtaining a target fragment and a linearized pCAMBIA 1-GFP vector recombinant product by using a Clonexpress II one step cloning kit (Novozan), transforming escherichia coli DH5 alpha competence, performing dark culture at 37 ℃ overnight, picking out a single clone, performing PCR identification by using a vector universal primer to obtain a positive clone, and sequencing to confirm the correctness of the PCR product to respectively obtain a PCR product containing 35S: ijSVP1-like-GFP and 35S: ijSVP2-1ike-GFP in E.coli.

Plasmids were extracted using E.coli shakers of which sequencing was correct, and the objective plasmids (35S. Centrifuging at 5000rpm for 5min to collect thallus, and collecting thallus with infection solution (containing 10mM MgCl) ₂ MES of 100mM ph =5.6 and 100 μ M acetosyringone) were resuspended and injected onto the back of leaves of transgenic nicotiana benthamiana containing mCherry nuclear localization signal. After three days of light culture, fluorescence was observed under a laser confocal microscope (Olympus FV3000, japan) and photographed.

The results are shown in FIG. 2, and indicate that the IjSVP1-like-GFP and IjSVP2-1ike-GFP proteins are localized in the nucleus, while the free GFP is distributed throughout the cell.

Example 2: qRT-PCR analysis of expression of IjSVP1-like and IjSVP2-like genes in different tissues and organs of iris japonica

qRT-PCR was used to study the expression patterns of IjSVP1-like and IjSVP2-like genes in Iris florida roots, rhizomes, shoot apical meristems, flowers, leaves and fruits.

The specific method comprises the following steps:

(1) When the plant is in the full-bloom stage in the last March, taking young leaves, buds, white young root systems, rhizomes and shoot apical meristems, and taking young fruits in the last May, extracting RNA and carrying out reverse transcription to obtain cDNA;

(2) The expression level was detected by a fluorescent quantitative PCR instrument, and the details of the reaction system are shown in TAKARA SYBR Premix EX Taq (Peffect Real time, DRR 041A). Specific primers for amplifying IjSVP1-like are IjSVP1-like-qpcr-F and IjSVP1-like-qpcr-R, and specific primers for amplifying IjSVP2-like are IjSVP2-ilke-qpcr-F and IjSVP2-like-qpcr-R.

By use of 2 ^-ΔΔCT The relative expression level of the gene was calculated.

As shown in FIG. 3, it was found that IjSVP1-like and IjSVP2-like genes are expressed in different tissues, and the expression level in the root, rhizome and shoot apical meristem of the underground tissue is significantly higher than that in the flowers, leaves and fruits of the above ground tissue, wherein the shoot apical meristem has the highest expression level and has tissue specificity.

Example 3: qRT-PCR analysis of expression of IjSVP1-like and IjSVP2-like genes in meristems of butterfly flower stem tips in different winter months

The specific method comprises the following steps:

(1) Taking healthy plant shoot apical meristem samples in one year in 15 days per month in the key winter months, extracting RNA and carrying out reverse transcription to obtain cDNA;

(2) The expression of IjSVP1-like and IjSVP2-like genes in the shoot apical meristem of the butterfly flower in different months was determined according to the method and primer sequences described in example 2.

As shown in FIG. 4, the expression levels of IjSVP1-like and IjSVP2-like genes are gradually increased in the induction process of overwintering dormancy, and reach the highest level in 1 month of dormancy period, and then gradually decrease along with dormancy release; wherein the up-regulation multiple of the expression quantity of IjSVP2-like in the 1 month of the dormancy stage is obviously higher than that of IjSVP1-like in the 10 months.

Example 4: mutant obtained by VIGS technology silencing IjSVP1-like or IjSVP2-like gene of Iris japonica and molecular detection

In order to determine the influence of IjSVP1-like and IjSVP2-like gene deletion on the overwintering growth of the Iris laevigata plants, the IjSVP1-like or IjSVP2-like gene is subjected to homologous silencing by a VIGS technology to obtain mutants.

Firstly, respectively obtaining the conserved region sequences of IjSVP1-like and IjSVP2-like through sequence comparison, designing conserved region sequence primers by using NCBI websites, wherein the length of products is 200-300bp, and respectively adding recognition bases and protection base sequences GCTCTAGA and CGAGCTC of restriction enzymes Xba I and Sac I at the 5' ends of forward primers and reverse primers, so that IjSVP1-like fragment cloning primers are IjSVP 1-like-viss-F and IjSVP 1-like-viss-R, and IjSVP2-like fragment cloning primers are IjSVP 2-like-viss-F and IjSVP 2-like-viss-R.

Subsequently, total RNA of the stem tip of the iris japonica was extracted, the total RNA of the iris japonica was reverse-transcribed into cDNA using a TAKARA first strand cDNA synthesis kit (Cat # 6210A), and IjSVP1-like and IjSVP2-like gene fragments containing the cleavage site 7 and the protected base were obtained by PCR amplification using high fidelity enzyme. Obtaining a PCR product by agarose gel electrophoresis and gel cutting recovery, connecting a 5min TA/Blunt-Zero (Novozan) vector, transferring the connection product into Escherichia coli DH5a, screening and picking single clones on a resistance culture medium, carrying out PCR identification on positive clones by using the vector with a universal primer M13, and sequencing to confirm the correctness of the PCR product.

The vector plasmid containing the IjSVP1-like or IjSVP2-like fragment with the correct sequence was extracted, and the vector plasmid and pTRV2 plasmid were digested with two restriction enzymes Xba I and Sac I at 37 ℃ for 30 minutes. The IjSVP1 (SEQ ID No. 5) with the fragment size of 216bp, the IjSVP2 (SEQ ID No. 6) with the fragment size of 208bp and the linearized pTRV2 were recovered by electrophoresis and gel cutting, and the IjSVP1-like and IjSVP2-like fragments were ligated with the pTRV2 linearized vector fragment at 16 ℃ for 3 hours, respectively, using T4 DNA ligase.

And transforming the ligation product into escherichia coli DH5a competence, screening and selecting a single clone on a resistance culture medium, designing detection primers pTRV2-F and pTRV2-R according to sequences on two sides of the restriction enzyme site of the pTRV2 vector, carrying out bacteria liquid PCR, identifying positive clone and sequencing. The positive clone with correct sequence is selected to extract the plasmid, the agrobacterium GV3101 is transformed by a freeze-thaw method, the monoclonal is selected after two days of dark culture at 28 ℃, and the PCR detection of bacterial liquid is carried out by using primers pTRV2-F and pTRV 2-R.

Finally, 50. Mu.L of Agrobacterium GV3101 containing pTRV1, pTRV2 and IjSVP1-pTRV2, ijSVP2-pTRV2 was added to 5mL LB medium containing 50mg/L kanamycin and 50mg/L rifampicin, shaken at 28 ℃ at 200rpm for 16 hours, transferred to 200mL LB medium containing 50mg/L kanamycin, 50mg/L rifampicin, 10mM MES and 20. Mu.M AS, and shaken at 28 ℃ at 200rpm for 16 hours, respectively; OD of bacterial liquid ₆₀₀ When the concentration was about 2.0, the cells were centrifuged at 5000rpm and 25 ℃ for 10 minutes to collect the cells. With a solution containing 10mM MgCl ₂ 10mM MES, 200. Mu.M AS 200mL of the invader solution was used to resuspend the cells. Mixing the following bacterial liquid components in each group according to a volume ratio of 1:1, standing and activating for 3 hours at 25 ℃ in a dark place, and injecting by using a 1mL injector1 year-old iris plants with consistent growth had leaves: the Agrobacterium GV3101 strain containing pTRV1 and pTRV2, the Agrobacterium GV3101 strain containing pTRV1 and IjSVP1-like-pTRV2, and the Agrobacterium GV3101 strain containing pTRV1 and IjSVP2-like-pTRV 2. The plants were subsequently cultured in the dark at 21 ℃ for 3 days, then placed under conditions of 25 ℃/14 hours in light, 2000lxs in light intensity, 20 ℃/10 hours in the dark, and cultured for 2 weeks.

Taking 100mg of each of the control and infected plant leaves to extract total RNA, and carrying out reverse transcription to obtain cDNA; and (3) determining whether the recombinant plasmid enters the plant by using TRV1 and TRV2 vector detection primers.

As shown in FIG. 5, the TRV2 vector sequence is not amplified in the control sample, only the TRV2 vector sequence is present in the empty load sample, the length is about 281bp, the amplified band size in the silent IjSVP1-like plant sample is about 520bp, and the amplified band size in the silent IjSVP1-like plant sample is about 501bp. The marker used in the figure is 2000bp, with the 3 rd band from bottom to top being approximately 500bp in size, from which it can be seen that the recombinant plasmid has successfully entered the plant.

Example 5: growth condition observation of Iris laevigata IjSVP1-like or IjSVP2-like silent plants under dormancy induction condition

Transferring the Iris florida mutant containing the recombinant plasmid into an incubator with the conditions of illumination of 10 hours, illumination intensity of 4000lxs, 10 ℃/dark of 14 hours and 4 ℃ for cold acclimation and culture for 7 days, uniformly shearing the leaves to the height of 5cm away from the rhizome, uniformly putting the leaves into an artificial climate box under the conditions shown in the table 2 for dormancy induction and culture for 28 days, and recording the growth changes of wild plants and IjSVP1-like or IjSVP2-like silent plants.

TABLE 2 Iris japonica dormancy induction culture conditions

The plant height statistics takes the junction of the rhizome and the leaf as a starting point, and is measured to the highest position of the leaf expansion; the germination rate refers to the percentage of the number of plants with the height increased by more than 0.5cm in a certain treatment to the number of plants used in the treatment; the number of the blades is based on the number of the blades with the green part larger than 2/3; each treatment contained 6 biological replicates. As shown in figure 6, under the dormancy induction condition, the germination rate of IjSVP1-like or IjSVP2-like silenced plants of fringed iris is higher than that of a contrast wild type, and the plant height of the IjSVP2-like silenced plants of fringed iris is increased more quickly than that of the wild type and the IjSVP1-like silenced plants, so that the silenced SVP-like gene can effectively inhibit fringed iris from entering ecological dormancy and maintain the growth under the low-temperature condition.

Example 6: the expression quantity of IjSVP1-like, ijSVP2-like and ABA synthetic gene IjNCNCED 1 of Iris japonica IjSVP1-like or IjSVP2-like silent plants is changed under the condition of dormancy induction.

Taking 100mg of functional leaves of the Iris laevigata IjSVP1-like or IjSVP2-like silent plants subjected to 28-day dormancy induction treatment, extracting total RNA, and performing reverse transcription to obtain cDNA, and determining the expression condition of IjSVP1-like, ijSVP2-like and ABA synthesis related IjNCED1 genes in stem apical meristems of the Iris laevigata according to the method and primer sequences in the example 2, wherein specific primers of the IjNCED1 are IjNCED1-like-qpcr-F and IjNCED1-like-qpcr-R.

As shown in FIG. 7, expression levels of IjSVP1-like, ijSVP2-like and ABA synthetic gene IjNCED1 in IjSVP1-like or IjSVP2-like gene knockout mutants of Iris laevigata are all reduced, so that ABA content is reduced, and plant germination is promoted.

By combining the researches, the invention discovers that the SVP-like gene negatively regulates the overwintering ecological dormancy process of the iris japonica, and the silent mutant of the SVP-like gene can avoid entering an ecological dormancy state and promote the iris japonica to reduce ABA accumulation under the dormancy induction condition so as to promote growth.

Sequence listing

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Lys Gln Val Thr Glu Ser Thr Glu Gln Leu Arg Lys Met Arg Gly Glu

100 105 110

Asp Leu Lys Gly Leu Ser Ile Glu Glu Leu Gln Gln Leu Glu Lys Thr

115 120 125

Leu Glu Ala Gly Leu Ser Arg Val Leu Asn Arg Lys Gly Glu His Ile

130 135 140

Met Glu Gln Ile Arg Gly Leu Glu Lys Lys Gly Leu Gln Leu Ile Glu

145 150 155 160

Glu Asn Thr Arg Leu Arg Glu Gln Val Val Asp Met Ser Arg Val Gly

165 170 175

Lys Gln Ile Val Thr Asp Ser Gly Asn Ala Ile Cys Glu Asp Gly Gln

180 185 190

Ser Ser Glu Pro Ala Thr Asn Thr Ser Gln Ser Gly Gly Pro Gln Asp

195 200 205

Tyr Asp Asp Ser Ser Asp Thr Ser Leu Lys Leu Gly Leu Leu Trp Lys

210 215 220

<210> 5

<211> 216

<212> DNA

<213> Iris japonica L.)

<400> 5

gggcctcaag attatgatga cagttttgac acctctctca agttagggct tccatggaag 60

gaatcgaagg ccacgatgat gcagtaaagc ttcatttttc ttttaatgtt tttgtaagat 120

atagatcagt aacaaacact tgtgagcaaa atgaactttc tgacataaaa tatgctataa 180

taaagacttg tttgtctagg cattggcatg gcttgc 216

<210> 6

<211> 208

<212> DNA

<213> Iris japonica L.)

<400> 6

agatgaaggg ctagctagtt tgtttaataa tactgatata tttcagtaaa cgtacttata 60

taagcgagag cataaatgaa ttttcagaca tgggtttgct aataaaaggc tcaattgggc 120

tcataatcta catgcatgta tgatgtacgt acttatgaat ttgtataaca atacttgata 180

atttcttggt gacatcgaga cctttctg 208

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agctcggtac ccggggatcc tacacgaccg accaattgcc 40

<210> 8

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttgcatgcct gcaggtcgac tgcatcatcg tggccttcg 39

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agctcggtac ccggggatcc gaaaggagga ggatggttag gg 42

<210> 10

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttgcatgcct gcaggtcgac ttccatagaa gccctagctt gag 43

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgacacctct ctcaagttag 20

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgccaatgc ctagacaa 18

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcagacatgg gtttgctaat 20

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtctcgatgt caccaagaa 19

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctctagagg gcctcaagat tatgatgaca g 31

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgagctcgca agccatgcca atgccta 27

<210> 17

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gctctagaag atgaagggct agctagtttg t 31

<210> 18

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cgagctccag aaaggtctcg atgtcacca 29

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgggagatga tacgctgtt 19

<210> 20

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cctaaaactt cagacacg 18

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cctgttgtgt ccttatcca 19

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gcgatgtcaa gaagatgtg 19

Claims

1. A method for inhibiting iris japonica from entering ecological dormancy is characterized in that tissue or plant organ from iris japonica is subjected to gene silencing or gene knockout, the silenced or knocked-out gene is at least one of IjSVP1-like gene and IjSVP2-like gene, then a plant is obtained,

2. The method of claim 1, wherein gene silencing is by VIGS technology.

3. The method according to claim 2, wherein the gene silencing is carried out by constructing a recombinant plasmid for gene silencing and then introducing the recombinant plasmid into a tissue or plant organ derived from a butterfly flower by an Agrobacterium transformation method.

4. The method of claim 3, wherein the plasmid backbone used in the construction of the recombinant plasmid is the pTRV2 vector, and wherein the recombinant plasmid is used in combination with the pTRV1 plasmid.

5. The method of claim 4, wherein the recombinant plasmid and the pTRV1 plasmid are introduced into Agrobacterium separately, and the two Agrobacterium are mixed at a volume ratio of 1:1 for transfection.

6. The method of claim 5, wherein the Agrobacterium is GV3101.

7. The method according to claim 3, wherein the fragment for gene silencing IjSVP1-like gene used in constructing the recombinant plasmid is represented by SEQ ID No.5, and the fragment for gene silencing IjSVP2-like gene is represented by SEQ ID No. 6.