阅读说明：本技术 一个表观遗传操作植物表型可塑性性状的方法 (Method for epigenetic manipulation of plant phenotypic plasticity traits ) 是由 强胜 解洪杰 于 2018-03-07 设计创作，主要内容包括：提供一个表观遗传操作植物表型可塑性性状的方法,通过简并密码子定量替换目的基因的可甲基化位点,通过设计合成基因,进行转基因操作；或利用CRISPR/Cas9技术构建植物表达载体进行原位在体基因简并密码子定量替换可甲基化位点,定量调控外源或原位在体目的基因甲基化程度和基因表达水平,达到定量调控目标植物的可塑性性状。通过定量调节ICE1基因的甲基化程度培育不同抗寒能力植物,为通过表观遗传技术培育抗寒作物提供了新的选择,为降低低温伤害对作物产量和品质等的影响提供了技术方法。(Providing a method for epigenetic manipulation of plant phenotypic plasticity traits, quantitatively replacing methylatable sites of a target gene by degenerate codons, and carrying out transgenic manipulation by designing a synthetic gene; or a plant expression vector is constructed by using the CRISPR/Cas9 technology to carry out in-situ in-vivo gene degenerate codon quantitative replacement on the methylatable site, and the methylation degree and the gene expression level of the exogenous or in-situ in-vivo target gene are quantitatively regulated, so that the quantitative regulation of the plasticity of the target plant is achieved. Plants with different cold resistance abilities are cultivated by quantitatively regulating the methylation degree of the ICE1 gene, a new choice is provided for cultivating cold-resistant crops by an epigenetic technique, and a technical method is provided for reducing the influence of low-temperature injury on the yield, quality and the like of the crops.)

A method for epigenetic manipulation of plant phenotypic plasticity traits, characterized in that degenerate codons are used to quantitatively replace methylatable sites of a target gene, synthetic genes are designed, and transgenic manipulation is performed; or a plant expression vector is constructed by using a CRISPR/Cas9 editing technology to quantitatively regulate the methylation degree and the gene expression level of an exogenous or in-vivo target gene in a receptor after in-situ in-vivo gene degenerate codon quantitative replacement methylatable sites, so that the quantitative regulation of the plasticity of the plant is achieved.

The method of claim 1, wherein the synthetic gene is designed after the methylation cytosine site of the target gene is quantitatively replaced by the degenerate codon, and transgenic genetic manipulation is performed to quantitatively regulate the methylation degree and gene expression level of the exogenous target gene in a receptor, so as to achieve quantitative regulation of the plasticity of the plant.

The method according to claim 1, characterized in that after the plant expression vector is constructed by using CRISPR/Cas9 technology to carry out in-situ quantitative substitution of methylated cytosine sites by in-vivo gene degenerate codons, the methylation degree and the gene expression level of in-vivo target genes are quantitatively regulated, so as to achieve quantitative regulation of the plasticity traits of the plant.

The method according to any one of claims 1 to 3, wherein the plastic trait is cold tolerance.

The method of claim 4, wherein the gene of interest is the ICE1 gene.

The method according to claim 4, wherein the ICE1 gene is selected from the group consisting of Eupatorium adenophorum AaICE1 gene, Manihot esculenta MeICE1 gene, Kiwi AcICE1 gene, wild banana MaICE1 gene, Japanese banana MabICE1 gene, cocoa TcICE1 gene, Hovenia CtICE1 gene, grape VvICE1 gene, apple MdICE1 gene, mustard CbICE1 gene, eggplant ScICE1 gene, tomato SlICE1 gene, peach PpICE1 gene, maize ZmICE1 gene, cotton GhICE1 gene, peanut AhICE1 gene, mustard BjICE1 gene, BrICE1 gene, chrysanthemum CdICE1 gene, small salt mustard tuber 1 gene, wild carrot DcICE1 gene, soybean GmICE1 gene, radish Rs3672 gene, wheat TavICE 1 gene, Bradychii Tsai gene, Japanese pepper Haematococcus 1 gene, Japanese rice red cabbage Hance 1 gene, rice Isaria 36363672 gene, European red cabbage ICE 363672 gene, European red cabbage ICE 36363672 gene, European red cabbage ICE 36363636363636363672 gene, European red cabbage ICE 36363672 gene, European red cabbage ICE 3636363636363672 gene, European ICE 363672 gene, European ICE arctic 3636363636363672 gene, sweet poplar PsICE1 gene, poplar PtICE1 gene, potamogeton paniculata PopICE1 gene and potamogeton paniculata PwICE1 gene; wherein, the nucleotide sequence of the eupatorium adenophorum AaICE1 gene is SEQ ID NO: 1, the cassava MeICE1 gene has a nucleotide sequence of SEQ ID NO: 9, the nucleotide sequence of the kiwi fruit AcICE1 gene is SEQ ID NO: 10, the nucleotide sequence of the wild banana MaICE1 gene is SEQ ID NO: 11, the nucleotide sequence of the banana MabICE1 gene is SEQ ID NO: 12, the nucleotide sequence of cocoa TcICE1 gene is SEQ ID NO: 13, the nucleotide sequence of the trifoliate orange CtICE1 gene is SEQ ID NO: 14, the nucleotide sequence of the grape vvICE1 gene is SEQ ID NO: 15, the nucleotide sequence of the apple MdICE1 gene is SEQ ID NO: 16, the nucleotide sequence of the mustard CbICE1 gene is SEQ ID NO: 17, the nucleotide sequence of the eggplant ScICE1 gene is SEQ ID NO: 18, the nucleotide sequence of the tomato SlICE1 gene is SEQ ID NO: 19, the nucleotide sequence of the peach PpICE1 gene is SEQ ID NO: 20, the nucleotide sequence of the corn ZmICE1 gene is SEQ ID NO: 21, the nucleotide sequence of the cotton GhICE1 gene is SEQ ID NO: 22, the nucleotide sequence of the AhICE1 gene of the peanut is SEQ ID NO: 23, the nucleotide sequence of the mustard BjICE1 gene is SEQ ID NO: 24, the nucleotide sequence of the rape BrICE1 gene is SEQ ID NO: 25, the nucleotide sequence of the chrysanthemum CdICE1 gene is SEQ ID NO: 26, the nucleotide sequence of theice 1 gene of thellungiella halophila is SEQ ID NO: 27, the nucleotide sequence of the wild carrot DcICE1 gene is SEQ ID NO: 28, the nucleotide sequence of the soybean GmICE1 gene is SEQ ID NO: 29, the nucleotide sequence of the radish RsICE1 gene is SEQ ID NO: 30, the nucleotide sequence of the wheat TaICE1 gene is SEQ ID NO: 31, the nucleotide sequence of the barley HvICE1 gene is SEQ ID NO: 32, the nucleotide sequence of the rubber tree HbICE1 gene is SEQ ID NO: 33, the nucleotide sequence of the walnut JrICE1 gene is SEQ ID NO: 34, the lettuce LsICE1 gene has the nucleotide sequence of SEQ ID NO: 35, the nucleotide sequence of the rice OsICE1 gene is SEQ ID NO: 36, the nucleotide sequence of the phalaenopsis PaICE1 gene is SEQ ID NO: 37, the nucleotide sequence of the plum blossom PmICE1 gene is SEQ ID NO: 38, the nucleotide sequence of the tea CsICE1 gene is SEQ ID NO: 39, the nucleotide sequence of the arabidopsis AtICE1 gene is SEQ ID NO: 40, the nucleotide sequence of the turnip BrrICE1 gene is SEQ ID NO: 41, the nucleotide sequence of the Isatis tinctoria ItICE1 gene is SEQ ID NO: 42, the nucleotide sequence of the eucalyptus globulus EcICE1 gene is SEQ ID NO: 43, the nucleotide sequence of jatropha curcas JcICE1 gene is SEQ ID NO: 44, the nucleotide sequence of the PicaICE 1 gene of the European spruce is SEQ ID NO: 45, the nucleotide sequence of the populus tremuloides PsICE1 gene is SEQ ID NO: 46, the nucleotide sequence of the popptICE 1 gene is SEQ ID NO: 47, the potamogeton perforatum PopICE1 gene has the nucleotide sequence of SEQ ID NO: 48, the nucleotide sequence of the potamogeton gracilis PwICE1 gene is SEQ ID NO: 49.

the method of claim 6, wherein the nucleotide sequences of ICE1 gene after the total replacement of methylated cytosine by degenerate codons are Eupatorium adenophorum: SEQ ID NO: 3, cassava: SEQ ID NO: 50, kiwi fruit: SEQ ID NO: 51, wild banana: SEQ ID NO: 52, banana: SEQ ID NO: 53, cocoa: SEQ ID NO: 54, Zhi: SEQ ID NO: 55, grape: SEQ ID NO: 56, apple: SEQ ID NO: 57, mustard: SEQ ID NO: 58, eggplant: SEQ ID NO: 59, tomato: SEQ ID NO: 60, peach: SEQ ID NO: 61, corn: SEQ ID NO: 62, cotton: SEQ ID NO: 63, peanut: SEQ ID NO: 64, mustard: SEQ ID NO: 65, rape: SEQ ID NO: 66, chrysanthemum: SEQ ID NO: 67, Thellungiella halophila: SEQ ID NO: 68, wild carrot: SEQ ID NO: 69, soybean: SEQ ID NO: 70, radish: SEQ ID NO: 71, wheat: SEQ ID NO: 72, barley: SEQ ID NO: 73, rubber tree: SEQ ID NO: 74, walnut: SEQ ID NO: 75, lettuce: SEQ ID NO: 76, rice: SEQ ID NO: 77, moth orchid: SEQ ID NO: 78, plum blossom: SEQ ID NO: 79, tea: SEQ ID NO: 80, Arabidopsis thaliana: SEQ ID NO: 81, turnip: SEQ ID NO: 82, isatis indigotica fort: SEQ ID NO: 83, eucalyptus globulus rubra: SEQ ID NO: 84, jatropha curcas: SEQ ID NO: 85, European spruce: SEQ ID NO: 86, sweet poplar: SEQ ID NO: 87, poplar: SEQ ID NO: 88, potamogeton perfoliatus: SEQ ID NO: 89, herba phyllanthi: SEQ ID NO: 90, respectively; the nucleotide sequences of the ICE1 gene after partial replacement of methylated cytosine by degenerate codons are eupatorium adenophorum: SEQ ID NO: 4, cassava: SEQ ID NO: 91, kiwi fruit: SEQ ID NO: 92, wild banana: SEQ ID NO: 93, plantain: SEQ ID NO: 94, cocoa: SEQ ID NO: 95, Zhi: SEQ ID NO: 96, grape: SEQ ID NO: 97, apple: SEQ ID NO: 98, mustard: SEQ ID NO: 99, eggplant: SEQ ID NO: 100, tomato: SEQ ID NO: 101, peach: SEQ ID NO: 102, corn: SEQ ID NO: 103, cotton: SEQ ID NO: 104, peanut: SEQ ID NO: 105, mustard: SEQ ID NO: 106, rape: SEQ ID NO: 107, chrysanthemum: SEQ ID NO: 108, Thellungiella halophila: SEQ ID NO: 109, wild carrot: SEQ ID NO: 110, soybean: SEQ ID NO: 111, radish: SEQ ID NO: 112, wheat: SEQ ID NO: 113, barley: SEQ ID NO: 114, rubber tree: SEQ ID NO: 115, walnut: SEQ ID NO: 116, lettuce: SEQ ID NO: 117, rice: SEQ ID NO: 118, moth orchid: SEQ ID NO: 119, plum blossom: SEQ ID NO: 120, tea: SEQ ID NO: 121, arabidopsis thaliana: SEQ ID NO: 122, turnip: SEQ ID NO: 123, woad: SEQ ID NO: 124, eucalyptus globulus rubra: SEQ ID NO: 125, jatropha curcas: SEQ ID NO: 126, european spruce: SEQ ID NO: 127, sweet poplar: SEQ ID NO: 128, young: SEQ ID NO: 129, perforales paniculata: SEQ ID NO: 130, herba phyllanthi: SEQ ID NO: 131.

the method according to claim 6, characterized in that the synthetic or CRISPR/Cas9 in situ degenerate codon is performed to replace all or part of the methylated cytosine followed by transformation of the AaICE1 gene into rice, cassava, banana, tomato, cocoa, rubber, lemon, sweet orange, citrus, sugarcane, papaya, loquat, lychee, longan, mango, potato, pepper, sorghum, grape, apple, mustard, eggplant, kiwi, peach, maize, cotton, peanut, mustard, rape, chrysanthemum, carrot, soybean, radish, wheat, barley, walnut, lettuce, cymbidium, butterfly orchid, wintersweet, tea, eucalyptus plants to obtain transgenic plants with increased cold resistance; the sequence of the AaICE1 gene after all the methylated cytosines are replaced by the methylated cytosine is shown in SEQ ID NO: 3, the sequence of AaICE1 gene after partial replacement of methylated cytosine is SEQ ID NO: 4.

an hardiness gene selected from the group consisting of the ICE1 gene following the complete or partial replacement of a methylatable cytosine.

The gene according to claim 9, wherein the wild type ICE1 gene is selected from the group consisting of Eupatorium adenophorum AaICE1 gene, Manihot esculenta MeICE1 gene, Kiwi AcICE1 gene, wild banana MaICE1 gene, Japanese banana MabICE1 gene, cocoa TcICE1 gene, Hovenia CtICE1 gene, grape VvICE1 gene, apple MdICE1 gene, mustard CbICE1 gene, eggplant ScICE1 gene, tomato SlICE1 gene, peach PpICE1 gene, maize ZmICE1 gene, cotton GhICE1 gene, peanut AhICE1 gene, mustard BjICE1 gene, BrICE1 gene, chrysanthemum CdICE1 gene, small salt mustard tuber ThICE1 gene, wild carrot DcICE1 gene, soybean GmICE1 gene, radish Rs3672 gene, wheat TavICE 1 gene, Bradychira japonica Haemarginia tricuspidata Hance, rice Hance Fang 1 gene, rice Isaria strain Haemargia Hance 1 gene, rice strain Haematis 1 gene, rice strain Haemarginia indica 1 gene, rice strain 363672 gene, rice strain OsICE 36363672 gene, rice strain 363672 gene, rice strain, sweet poplar PsICE1 gene, poplar PtICE1 gene, potamogeton paniculata PopICE1 gene and potamogeton paniculata PwICE1 gene; wherein, the nucleotide sequence of the eupatorium adenophorum AaICE1 gene is SEQ ID NO: 1, the cassava MeICE1 gene has a nucleotide sequence of SEQ ID NO: 9, the nucleotide sequence of the kiwi fruit AcICE1 gene is SEQ ID NO: 10, the nucleotide sequence of the wild banana MaICE1 gene is SEQ ID NO: 11, the nucleotide sequence of the banana MabICE1 gene is SEQ ID NO: 12, the nucleotide sequence of cocoa TcICE1 gene is SEQ ID NO: 13, the nucleotide sequence of the trifoliate orange CtICE1 gene is SEQ ID NO: 14, the nucleotide sequence of the grape vvICE1 gene is SEQ ID NO: 15, the nucleotide sequence of the apple MdICE1 gene is SEQ ID NO: 16, the nucleotide sequence of the mustard CbICE1 gene is SEQ ID NO: 17, the nucleotide sequence of the eggplant ScICE1 gene is SEQ ID NO: 18, the nucleotide sequence of the tomato SlICE1 gene is SEQ ID NO: 19, the nucleotide sequence of the peach PpICE1 gene is SEQ ID NO: 20, the nucleotide sequence of the corn ZmICE1 gene is SEQ ID NO: 21, the nucleotide sequence of the cotton GhICE1 gene is SEQ ID NO: 22, the nucleotide sequence of the AhICE1 gene of the peanut is SEQ ID NO: 23, the nucleotide sequence of the mustard BjICE1 gene is SEQ ID NO: 24, the nucleotide sequence of the rape BrICE1 gene is SEQ ID NO: 25, the nucleotide sequence of the chrysanthemum CdICE1 gene is SEQ ID NO: 26, the nucleotide sequence of theice 1 gene of thellungiella halophila is SEQ ID NO: 27, the nucleotide sequence of the wild carrot DcICE1 gene is SEQ ID NO: 28, the nucleotide sequence of the soybean GmICE1 gene is SEQ ID NO: 29, the nucleotide sequence of the radish RsICE1 gene is SEQ ID NO: 30, the nucleotide sequence of the wheat TaICE1 gene is SEQ ID NO: 31, the nucleotide sequence of the barley HvICE1 gene is SEQ ID NO: 32, the nucleotide sequence of the rubber tree HbICE1 gene is SEQ ID NO: 33, the nucleotide sequence of the walnut JrICE1 gene is SEQ ID NO: 34, the lettuce LsICE1 gene has the nucleotide sequence of SEQ ID NO: 35, the nucleotide sequence of the rice OsICE1 gene is SEQ ID NO: 36, the nucleotide sequence of the phalaenopsis PaICE1 gene is SEQ ID NO: 37, the nucleotide sequence of the plum blossom PmICE1 gene is SEQ ID NO: 38, the nucleotide sequence of the tea CsICE1 gene is SEQ ID NO: 39, the nucleotide sequence of the arabidopsis AtICE1 gene is SEQ ID NO: 40, the nucleotide sequence of the turnip BrrICE1 gene is SEQ ID NO: 41, the nucleotide sequence of the Isatis tinctoria ItICE1 gene is SEQ ID NO: 42, the nucleotide sequence of the eucalyptus globulus EcICE1 gene is SEQ ID NO: 43, the nucleotide sequence of jatropha curcas JcICE1 gene is SEQ ID NO: 44, the nucleotide sequence of the PicaICE 1 gene of the European spruce is SEQ ID NO: 45, the nucleotide sequence of the populus tremuloides PsICE1 gene is SEQ ID NO: 46, the nucleotide sequence of the popptICE 1 gene is SEQ ID NO: 47, the potamogeton perforatum PopICE1 gene has the nucleotide sequence of SEQ ID NO: 48, the nucleotide sequence of the potamogeton gracilis PwICE1 gene is SEQ ID NO: 49;

the nucleotide sequences of the ICE1 gene after the methylation of cytosine is completely replaced by degenerate codons are eupatorium adenophorum: SEQ ID NO: 3, cassava: SEQ ID NO: 50, kiwi fruit: SEQ ID NO: 51, wild banana: SEQ ID NO: 52, banana: SEQ ID NO: 53, cocoa: SEQ ID NO: 54, Zhi: SEQ ID NO: 55, grape: SEQ ID NO: 56, apple: SEQ ID NO: 57, mustard: SEQ ID NO: 58, eggplant: SEQ ID NO: 59, tomato: SEQ ID NO: 60, peach: SEQ ID NO: 61, corn: SEQ ID NO: 62, cotton: SEQ ID NO: 63, peanut: SEQ ID NO: 64, mustard: SEQ ID NO: 65, rape: SEQ ID NO: 66, chrysanthemum: SEQ ID NO: 67, Thellungiella halophila: SEQ ID NO: 68, wild carrot: SEQ ID NO: 69, soybean: SEQ ID NO: 70, radish: SEQ ID NO: 71, wheat: SEQ ID NO: 72, barley: SEQ ID NO: 73, rubber tree: SEQ ID NO: 74, walnut: SEQ ID NO: 75, lettuce: SEQ ID NO: 76, rice: SEQ ID NO: 77, moth orchid: SEQ ID NO: 78, plum blossom: SEQ ID NO: 79, tea: SEQ ID NO: 80, Arabidopsis thaliana: SEQ ID NO: 81, turnip: SEQ ID NO: 82, isatis indigotica fort: SEQ ID NO: 83, eucalyptus globulus rubra: SEQ ID NO: 84, jatropha curcas: SEQ ID NO: 85, European spruce: SEQ ID NO: 86, sweet poplar: SEQ ID NO: 87, poplar: SEQ ID NO: 88, potamogeton perfoliatus: SEQ ID NO: 89, herba phyllanthi: SEQ ID NO: 90, respectively;

the nucleotide sequences of the ICE1 gene after partial replacement of methylated cytosine by degenerate codons are eupatorium adenophorum: SEQ ID NO: 4, cassava: SEQ ID NO: 91, kiwi fruit: SEQ ID NO: 92, wild banana: SEQ ID NO: 93, plantain: SEQ ID NO: 94, cocoa: SEQ ID NO: 95, Zhi: SEQ ID NO: 96, grape: SEQ ID NO: 97, apple: SEQ ID NO: 98, mustard: SEQ ID NO: 99, eggplant: SEQ ID NO: 100, tomato: SEQ ID NO: 101, peach: SEQ ID NO: 102, corn: SEQ ID NO: 103, cotton: SEQ ID NO: 104, peanut: SEQ ID NO: 105, mustard: SEQ ID NO: 106, rape: SEQ ID NO: 107, chrysanthemum: SEQ ID NO: 108, Thellungiella halophila: SEQ ID NO: 109, wild carrot: SEQ ID NO: 110, soybean: SEQ ID NO: 111, radish: SEQ ID NO: 112, wheat: SEQ ID NO: 113, barley: SEQ ID NO: 114, rubber tree: SEQ ID NO: 115, walnut: SEQ ID NO: 116, lettuce: SEQ ID NO: 117, rice: SEQ ID NO: 118, moth orchid: SEQ ID NO: 119, plum blossom: SEQ ID NO: 120, tea: SEQ ID NO: 121, arabidopsis thaliana: SEQ ID NO: 122, turnip: SEQ ID NO: 123, woad: SEQ ID NO: 124, eucalyptus globulus rubra: SEQ ID NO: 125, jatropha curcas: SEQ ID NO: 126, european spruce: SEQ ID NO: 127, sweet poplar: SEQ ID NO: 128, young: SEQ ID NO: 129, perforales paniculata: SEQ ID NO: 130, herba phyllanthi: SEQ ID NO: 131.

an expression vector comprising the ICE1 gene of claim 10 following the replacement, in whole or in part, of a methylatable cytosine.

Use of the ICE1 gene of claim 10 to breed hardy plants.

Use according to claim 12, characterized in that: the method comprises the following steps:

(1) constructing a plant transformation plasmid containing the ICE1 gene by using a DNA recombination technology;

(2) introducing the plant transformation plasmid constructed in the step (1) into plant tissues by a gene gun or an agrobacterium infection method or a pollen guide method or the like, or carrying out in-situ replacement on a receptor plant by a CRISPR/Cas9 technology to obtain transgenic or gene edited plants containing ICE1 genes with different methylation degrees.

Use according to claim 12 or 13, characterized in that the plant is selected from rice, cassava, banana, tomato, cocoa, rubber, lemon, sweet orange, citrus, sugarcane, papaya, loquat, lychee, longan, mango, potato, capsicum, sorghum, grape, apple, mustard, eggplant, kiwi, peach, corn, cotton, peanut, mustard, rape, chrysanthemum, carrot, soybean, radish, wheat, barley, walnut, lettuce, cymbidium, phalaenopsis, wintersweet, tea, eucalyptus globulus.