CN117721148A

CN117721148A - Application of TaRVR1 protein or biological material in regulation and control of wheat traits

Info

Publication number: CN117721148A
Application number: CN202311644038.8A
Authority: CN
Inventors: 何跃辉; 牛德
Original assignee: Institute Of Modern Agriculture Peking University
Current assignee: Institute Of Modern Agriculture Peking University
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-03-19

Abstract

The invention provides an application of TaRVR1 protein or biological material in regulating and controlling wheat traits. Wherein the TaRVR1 protein comprises the sequence of SEQ ID NOs:1-3, a TaRVR1-A protein, a TaRVR1-B protein or a TaRVR1-D protein; or the TaRVR1 protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues, or has at least 75% of identity, or is obtained by N-terminal or/and C-terminal connexin labels, and has the same function; regulating the wheat trait includes regulating the growth cycle of the wheat, regulating the effective tillering capacity of the wheat, or regulating the yield of the wheat. The invention is suitable for the biotechnology field, and can further improve the yield of wheat.

Description

Application of TaRVR1 protein or biological material in regulation and control of wheat traits

Technical Field

The invention relates to the field of biotechnology, in particular to application of TaRVR1 protein or biological material in regulation and control of wheat traits.

Background

Wheat (Triticum aestivum, AABBDD) is one of the most important food crops, providing 20% of calories and proteins to the world population. Wheat is divided into winter wheat and spring wheat according to the need of overwintering, wherein winter wheat can bloom and fruit in spring and summer in the next year only after being subjected to low temperature in winter (vernalization treatment). Spring wheat is planted in spring without being subjected to winter low temperature. Compared with winter wheat, the winter wheat variety has higher tillering and spike number and higher single plant yield than spring wheat variety, and the winter wheat with unit area yield is also obviously increased. Winter wheat varieties are commonly planted in northern areas of China, and the yield of the winter wheat varieties accounts for about 95% of the total yield of wheat.

In the first to tenth months of winter wheat, the beginning of the fifth year is the beginning of the flowering period, the last ten days of june starts to harvest successively, and the fifth and sixth months are the frequent periods of severe weather, so that the ineffective tillering number of wheat is increased, the weight of seeds and grains in effective tillers is reduced, and serious yield reduction is often caused. In addition, autumn grain crops are usually planted in time after wheat is harvested, so that the growth cycle of the wheat is properly shortened, the effective tillering number of the wheat is increased, the influence of extremely uncomfortable weather on flowering and fruiting of the wheat can be reduced, and the yield of the wheat is improved.

TaRVR1 encodes a protein comprising a BAH domain that mediates protein-protein interactions in complexes that modify or "read" chromatin states, but little is currently known about the function of the TaRVR1 gene, which is reported whether it is involved in the regulation of wheat vernalization and the regulation of effective tillering numbers.

Disclosure of Invention

The main purpose of the invention is to provide an application of TaRVR1 protein or biological material in regulating and controlling the characteristics of wheat, so as to solve the problem of low wheat yield in the prior art and further improve the wheat yield.

In order to achieve the above object, according to a first aspect of the present invention, there is provided the use of a TaRVR1 protein or biomaterial for regulating a wheat trait, the TaRVR1 protein comprising any one of the following a 1) -a 4): a1 SEQ ID NO:1, the TaRVR1-A protein shown in SEQ ID NO:2 or the TaRVR1-B protein shown in SEQ ID NO:3, any one or more of the TaRVR1-D proteins shown in figure 3; or a 2) a TaRVR1 protein having the same function by substituting and/or deleting and/or adding one or more amino acid residues to the TaRVR1 protein in a 1); or a 3) a TaRVR1 protein having at least 75% identity and the same function as the TaRVR1 protein in a 1); or a 4) a TaRVR1 protein having the same function obtained by linking protein tags at the N-terminal or/and C-terminal of the TaRVR1 protein in a 1), a 2) or a 3); the biomaterial comprises any one of the following b 1) -b 7): b1 A nucleic acid molecule encoding a TaRVR1 protein according to any of a 1) to a 4); b2 An expression cassette comprising a nucleic acid molecule; b3 A recombinant vector comprising a nucleic acid molecule or an expression cassette; b4 A recombinant microorganism comprising a nucleic acid molecule, an expression cassette or a recombinant vector; b5 A transgenic wheat cell line containing a nucleic acid molecule or expression cassette; b6 Transgenic wheat tissue containing a nucleic acid molecule or expression cassette; b7 A transgenic wheat organ comprising a nucleic acid molecule or expression cassette; regulating a wheat trait includes one or more of regulating a growth cycle of the wheat, regulating an effective tillering capacity of the wheat, or regulating a yield of the wheat.

Further, the nucleic acid molecule in b 1) comprises: c1 A gene encoding a TaRVR1-A protein, a TaRVR1-B protein or a TaRVR1-D protein; the coding gene of the TaRVR1-A protein comprises SEQ ID NO: 4; the coding gene of the TaRVR1-B protein comprises SEQ ID NO:5, a nucleic acid shown in FIG. 5; the coding gene of the TaRVR1-D protein comprises SEQ ID NO: 6; or c 2) a nucleic acid molecule which has at least 75% identity to the coding gene in c 1) and which codes for any one of the TaRVR1 proteins from a 1) to a 4); or c 3) a nucleic acid molecule which hybridizes under stringent conditions to the coding gene of c 1) and which codes for any one of the TaRVR1 proteins from a 1) to a 4).

Further, wheat includes diploid wheat, tetraploid wheat or hexaploid wheat; preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat; preferably, the commonly cultivated hexaploid wheat comprises Kenong wheat; preferably, regulating the growth cycle of wheat comprises shortening the vernalization time of the wheat; preferably, the ability to regulate effective tillering of wheat comprises increasing the number of effective tillers of wheat; preferably, the effective tillering number is the number of tillers containing a heading ear obtained after wheat tillering; preferably, regulating the yield of wheat comprises increasing the yield of wheat; preferably, increasing the yield of wheat comprises increasing the yield of wheat kernels.

Further, the TaRVR1 protein in a 3) has at least 85% identity and the same function as the TaRVR1 protein in a 1); preferably, at least 95% identity, more preferably 99%, still more preferably 99.9%; preferably, the TaRVR1 protein in a 3) is derived from wheat.

Further, the application includes any one or more of the following: down-regulating in wheat the expression level of the TaRVR1 protein in a 1), replacing the TaRVR1-A protein expressed in wheat with the sequence of SEQ ID NO:7 or SEQ ID NO:8 or a TaRVR1-A-1 or TaRVR1-A-2 protein expressed in wheat by replacing the TaRVR1-B protein expressed in wheat with the sequence of SEQ ID NO:9 or replacing the TaRVR1-B-1 protein expressed in wheat with the sequence shown in SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, taRVR1-D-1, taRVR1-D-2 or TaRVR1-D-3 protein.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for breeding wheat, which comprises decreasing the expression level of a TaRVR1 protein in a target wheat, decreasing the activity of the TaRVR1 protein or decreasing the expression level of a nucleic acid molecule encoding the TaRVR1 protein in the target wheat, to obtain a wheat with improved traits; wherein the TaRVR1 protein is a TaRVR1 protein according to any one of claims 1 to 5 and the nucleic acid molecule encoding the TaRVR1 protein is a nucleic acid molecule according to any one of claims 1 to 5; trait improvement includes one or more of a shortened growth cycle, an increased effective tillering capacity, or an increased yield.

Further, a method comprising gene editing or RNAi is employed to reduce the expression level of the TaRVR1 protein, the activity of the TaRVR1 protein or the expression level of the nucleic acid molecule in the wheat of interest; preferably, the gene editing comprises cleavage of the nucleic acid molecule, and/or insertion, deletion or substitution of bases on the nucleic acid molecule; preferably, the reduction in growth cycle comprises a reduction in vernalization time; preferably, the increase in effective tillering capacity includes an increase in the number of tillers that heading; preferably, the yield increase comprises a yield increase of the kernel.

Further, the method comprises the steps of: introducing genetic material expressing nuclease into cells or tissues of target wheat, and culturing the introduced cells or tissues into complete plants to obtain wheat with improved properties; preferably, the nuclease is capable of specifically cleaving a functional segment of the nucleic acid molecule; genetic material includes DNA circular plasmids, or linear fragments of DNA, or RNA transcribed in vitro; preferably, the genetic material is a recombinant vector capable of transcribing the guide RNA and expressing the Cas protein; the guide RNA is RNA with palindromic structure formed by combining crRNA and tracrRNA through partial base pairing; crRNA includes RNA fragments capable of complementary binding to functional segments; preferably, the method of introducing genetic material into the wheat cell or tissue of interest comprises a gene gun method, an agrobacterium infection method, a PEG-induced protoplast method, an electrode method, a silicon carbide fiber-mediated method, or a vacuum infiltration method; preferably, the cells include any cell that can serve as an infusion recipient and can be regenerated into a whole plant by tissue culture; the tissue is any tissue which can be used as an introduction receptor and can be regenerated into a complete plant through tissue culture; preferably, the cells comprise protoplast cells or suspension cells; tissues include callus, immature embryo, mature embryo, leaf, stem tip, young ear or hypocotyl.

Further, the wheat of interest includes diploid wheat, tetraploid wheat or hexaploid wheat; preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat; preferably, the commonly cultivated hexaploid wheat comprises Kenong wheat.

In order to achieve the above object, according to a third aspect of the present invention, there is provided a TaRVR1 protein or biomaterial for use in any one of the above applications, in any one of the above methods of wheat breeding.

By applying the technical scheme of the invention, the application discovers the TaRVR1 gene and protein related to wheat tillering, and the expression level of the TaRVR1 protein or biological material in wheat is regulated and controlled to be reduced, so that the change of the wheat characteristics including the growth cycle, the effective tillering capacity and/or the yield of the wheat can be realized, and the characteristics can stabilize inherited new wheat varieties and further improve the yield of the wheat.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

fig. 1 shows different culture forms and statistics of the Kenong 9204 wheat which is not vernalized and is vernalized for 6 weeks according to example 1 of the present invention; in fig. 1, a is a morphological diagram of a plant 9204 which is cultivated to bloom and fruit after being not vernalized and vernalized for 6 weeks in the plant 9204; FIG. 1B is a statistical plot of days before flowering of non-vernalized Kenong 9204 wheat and vernalized 6-week Kenong 9204 wheat; figure 1C is a morphology of ears and kernels after harvest of non-vernalized crop 9204 wheat and 6 week vernalized crop 9204 wheat; in FIG. 1, D is a statistical plot of the tillering numbers of non-vernalized Kenong 9204 wheat and 6 week vernalized Kenong 9204 wheat cultivated to flowering and fruiting.

A schematic diagram of the structure of the alignment of TaRVR1 with AtAIPP3 and BdRVR1 according to embodiment 2 of the present invention is shown in fig. 2; wherein, A in FIG. 2 is a schematic diagram of the gene structures of three TaRVR1 genes (TaRVR 1A, taRVR B and TaRVR 1D) AtAIPP3 gene and BdRVR1 gene; FIG. 2B is a schematic diagram of an amino acid sequence alignment of three TaRVR1 proteins (TaRVR 1A, taRVR B and TaRVR 1D) with AtAIPP3 and BdRVR1 proteins; FIG. 2C shows the prediction of the BAH protein structures of AtAIPP3 and TaRVR 1.

A schematic diagram of gene editing using CRISPR/Cas9 technology of three TaRVR1 genes according to example 3 of the present invention is shown in fig. 3; wherein, a in fig. 3 is a schematic diagram of the TaRVR1 gene structure and the setting of a target point by using CRISPR/Cas9 technology; FIG. 3B is a schematic diagram of the gene structure of the binding of sgRNA1 and sgRNA2 to three TaRVR1 genes; in fig. 3, C is a schematic diagram of amino acid sequences of wild-type control acceptor wheat and three genetically engineered TaRVR1 homozygous mutant wheat.

A schematic representation of the phenotypic identification of wheat TaRVR1 gene editing mutants according to example 4 of the present invention is shown in fig. 4; wherein, in fig. 4, a is a schematic diagram of growth morphology of wild-type acceptor control group wheat and three TaRVR1 homozygous mutant wheat in the same period; FIG. 4B is a schematic diagram of the morphology of the wild type receptor control wheat during the maturation period; FIG. 4C is a statistical plot of days before flowering of the target wheat and three TaRVR1 homozygous mutant wheat.

FIG. 5 shows graphs of the results of qPCR of the relative expression amounts of TaVRN1 genes of the acceptor control group wheat according to example 5 of the present invention at different periods of time from three TaRVR1 homozygous mutant wheat.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

Term interpretation:

sequence identity: refers to sequence similarity between two polynucleotide sequences. When a position in both comparison sequences is occupied by the same base, for example if each position of both DNA molecules is occupied by adenine, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100.

Tillering (Tiller): the term "adventitious bud" refers to a meristematic stem (branch) that grows from a plant such as Gramineae, which is a plant that grows from a root below the ground or from a node with a dense stem base near the ground.

Effective tillering: tillers capable of heading and having a grain size of 10 or more are called effective tillers.

Ineffective tillering: tillers that cannot heading or set less than 10 grains are called ineffective tillers.

Vernalization: plants must undergo a prolonged period of sustained low temperature to grow from the vegetative to the reproductive stage.

As mentioned in the background art, the yield of wheat needs to be further improved in the prior art, and thus, the inventors have tried to explore genes and proteins related to wheat tillering in the present application, and have proposed a series of protection schemes of the present application according to the research results.

In a first exemplary embodiment of the present application, there is provided the use of a TaRVR1 protein or biomaterial for modulating a wheat trait, the TaRVR1 protein comprising any one of the following a 1) -a 4): a1 SEQ ID NO:1, the TaRVR1-A protein shown in SEQ ID NO:2 or the TaRVR1-B protein shown in SEQ ID NO:3, any one or more of the TaRVR1-D proteins shown in figure 3; or a 2) a TaRVR1 protein having the same function by substituting and/or deleting and/or adding one or more amino acid residues to the TaRVR1 protein in a 1); or a 3) a TaRVR1 protein having at least 75% identity and the same function as the TaRVR1 protein in a 1); or a 4) a TaRVR1 protein having the same function obtained by linking protein tags at the N-terminal or/and C-terminal of the TaRVR1 protein in a 1), a 2) or a 3); the biological material comprises any one of the following b 1) -b 7): b1 A nucleic acid molecule encoding a TaRVR1 protein according to any of a 1) to a 4); b2 An expression cassette comprising a nucleic acid molecule; b3 A recombinant vector comprising a nucleic acid molecule or an expression cassette; b4 A recombinant microorganism comprising a nucleic acid molecule, an expression cassette or a recombinant vector; b5 A transgenic wheat cell line containing a nucleic acid molecule or expression cassette; b6 Transgenic wheat tissue containing a nucleic acid molecule or expression cassette; b7 A transgenic wheat organ comprising a nucleic acid molecule or expression cassette; regulating the wheat trait includes regulating the growth cycle of the wheat, regulating the effective tillering capacity of the wheat, or regulating the yield of the wheat.

The TaRVR1 protein comprises proteins in wheat and endogenous homologous proteins in other wheat varieties; also included are exogenous TaRVR1 protein-related biomaterials transformed in wheat that does not contain such homologous proteins, such that exogenous TaRVR1 proteins are expressed in the wheat. The application can regulate and control the expression level or activity of the TaRVR1 protein in the wheat, can realize the regulation and control of the vernalization time, the growth cycle and the effective tillering capacity of the wheat, and can further regulate and control the yield of the wheat. The same functions described above include the ability to regulate the vernalization time as well as the growth cycle and effective tillering of wheat containing the TaRVR1 protein. The wheat containing no TaRVR1 protein or less expression of the TaRVR1 protein needs short vernalization time or no vernalization, the growth cycle is reduced, and the effective tillering capability is strong.

The winter wheat flowering phase is mainly regulated and controlled by vernalization, the TaVRN1 gene is induced and expressed by vernalization, and is a direct target gene of vernalization, and activation of a TaVRN1-TaVRN2-TaVRN3 regulation network promotes wheat to enter a flowering conversion phase, wherein TaRVR1 in the winter wheat flowering phase has BAH domain protein and a TFS2M structure (the TFS2M structure participates in gene transcription regulation through interaction with RNA polymerase II), and is presumed to participate in regulating and controlling expression of the TaVRN1 so as to influence vernalization time of the wheat, further lead flowering time of the wheat to be advanced, realize avoidance of severe weather in a normal period and further improve yield. The effective tillering capacity is improved, so that the effective tillering number in the single wheat is increased, the number of seeds is increased, and the yield is improved.

In early wheat planting, the field nutrition is sufficient, the wheat body is still in the primary growth period, more nutrients are supplied to the growth of tillers, the tillers can further spike and seed, namely effective tillers, if the effective tillers of the wheat are stronger, the obtained spike and seed grains are more, and the yield of the wheat is improved. However, as wheat grows, ineffective tillers, i.e., the middle tillers cannot shoot or the grains in the ears are less than 10 grains, may also be formed, which is a main cause of the yield reduction of wheat. While the main reasons for ineffective tillering include:

1) Insufficient illumination; 2) Too high or too low a temperature; 3) Insufficient or excessive moisture; 4) Undernutrition or overnutrition; 5) Attack by diseases and insect pests: 6) Genetic mutation or genetic defect. In addition, the excessive ineffective tillering number can also strive for the nutrition of effective tillers, so that the quality of seeds on the effective tillers is reduced. Therefore, the effective tillering number of the wheat is improved, the ineffective tillering number is reduced, the yield of the wheat can be directly increased, and the quality of wheat grains is further improved.

TaRVR1 proteins include any one or more of the TaRVR1-A proteins, the TaRVR1-B proteins or the TaRVR1-D proteins. Wherein the TaRVR1-A protein is wheat TaRVR1-A gene transcript, and the amino acid sequence is SEQ ID NO:1. the TaRVR1-B gene is expressed as SEQ ID NO:2, and a TaRVR1-B protein shown in the specification. The TaRVR1-D gene is expressed as SEQ ID NO:3, and a TaRVR1-D protein shown in the specification. The TaRVR1-A gene, the TaRVR1-B gene and the TaRVR1-D gene are three subgenoids of the wheat TaRVR1 gene, and the cDNA nucleic acid sequences of the three subgenoids are respectively shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: shown at 6. The expression quantity or activity of the corresponding TaRVR1 protein can be changed by adjusting any one or more of the plurality of nucleic acid molecules, so that the regulation and control of the effective tillering capacity or yield of the wheat in vernalization time, growth cycle and the like can be realized, and the method can be applied to wheat breeding or production.

The application of the TaRVR1 protein or the biological material in the regulation and control of the wheat properties can further improve the wheat yield, and has important application value in the improvement of wheat varieties and the agricultural production.

TaRVR1-A：SEQ ID NO：1：

MGRRRRFTQQPTSDDDDDDDDEAVPQPLRTTKPTAPLSSGAKKQQRHRADEDDLELEEEEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDSQEKKAESEEEEESREEDSTEAVPVGQPFKVTGKGKKQRRHYFSFEYEGNTFELEDPVLLTPEQQKEKPYVAIIKDITENDGSLSVTGQWFYRPEEADKKGGGNWTASDTRELFYSFHIDDVPAESVMHKCVVHFIPLNKQIPSRKEHPGFIVQKVYDTVAKKLWNLTDKDYEDNKQHEIDLLVKKTVDRIGQLPDREPIDVPADSTDQFSNKRGLRKRPPNPLDVSKDTTGKPEQFIKAETPGSDNLKHYAILAKYKAVTNATYRDKWLDKLVDTIPLTSKEGAEASHADAGSVAKISNSSSSARDTSSSDNENSYPPDVVVSIMASLERSTYDALHADFQKYNQKMRKLEFNIKNSPVLRRRLMNKELDPPVLLTMSPDELKAGLTPAEKTSEPEEARRLQMTDARCERCTEKRVGISDIIHAGHGDRYQLECISCGYTWFSSRDAISSLTVDTPSSGGNVGTAPWATAKFDVLQKQLVSPRDQPDNKASADALQKNTVASIPKLERQKSFIKPKPEEPSAPTLEKQKAFTKLKPEEPSAPTLEKQKAFTKPKPEEPSAPSASH。

TaRVR1-B：SEQ ID NO：2：

QPLRTTKPTAPLFSGAKKQQRHRPDEDDLELEEEEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEEPEEKKAESEEEEESREEDSTEAVPVGEPLKVTGKGKKQRRHYFSFEYEGNTFELEDPVLLTPEQQKEKPYVAIIKDITENDGSLSVTGQWFYRPEEADKKGGGNWTASDTRELFYSFHIDDVPAESVMHKCVVHFIPLNKQIPSRKEHPGFIVQKVYDTVAKKLWNLTDKDYEDNKQHEIDLLVKKTVDRIGQLPDREPIDVPADSTDQFSNKRGLRKRPPNPLDVSRDTTGKPEQFIKAETPGSDNLKHYAILAKYKAVTNATYRDKWLDKLVDTIPLTSKEGAEASHADAGSVAKISNSSSSARDSSSSDNENSYPPDVVVSIMASLERSTYDALHADFQKYNQKMRKLEFNIKNSPVLRRRLMNKELDPPVLLTMSPDELKAGLTPAEKTSEPEEARRLQMTDARCGRCTEKRVGISDIIHAGHGDRYQLECISCGYTWFSSRDAISSLTVDTPSSGGNVGTAPWATAKFDALQKQLVSPRDQPDNKASADALQKNTVASIPKLERQKSLINPKPEEPSAPTLEKQKTFTKPKPEEPSAPSASH。

TaRVR1-D：SEQ ID NO：3：

MGRRRRFTQQPTSDDDDDDDEAVPQPLRTTKPTAPLSPGAKKQQRHRADEDDLELEEEEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDPEEKKAESEEEEESREEDSTEAVPVGEPLKVTGKGKKQRRHYLSFEYEGNTFELEDPVLLTPEQQKEKPYVAIIKDITENDGSLSVTGQWFYRPEEADKKGGGNWTASDTRELFYSFHIDDVPAESVMHKCVVHFIPLNKQIPSRKEHPGFIVQKVYDTVAKKLWNLTDKDYEDNKQHEIDLLVKKTVDRIGQLPDREPIDVPADSTDQFSNKRGLRKRPPNPLDVSRDTTGKPEQFIKAETPGSDNLKHYAILAKYKAVTNATYRDKWLDKLVDTIPLTSKEGAEASHADAGSVAKISNSSSSARDPSSSDNENSYPPDVVVSIMASLERSTYDALHADFQKYNQKMRKLEFNIKNSPVLRRRLMNKELDPPVLLTMSPDELKAGLTPAEKTSEPEEARRLQMTDARCERCTEKRVGISDIIHAGHGDRYQLECISCGYTWFSSRDAISSLTVDTPSSGGNVGTAPWATAKFDVLQKQLVSPRDQPDNKASADALQKNTVASIPKLERQKSFIKPKPEEPSAPTLEKQKAFTKPKPEEPSAPSASH。

In a preferred embodiment, the nucleic acid molecule of b 1) comprises: c1 A gene encoding a TaRVR1-A protein, a TaRVR1-B protein, or a TaRVR1-D protein; the coding gene of the TaRVR1-A protein comprises SEQ ID NO: 4; the coding gene of the TaRVR1 protein comprises SEQ ID NO:5, a nucleic acid shown in FIG. 5; the coding gene of the TaRVR1 protein comprises SEQ ID NO: 6; or c 2) a nucleic acid molecule which has at least 75% identity to the coding gene in c 1) and which codes for any one of the TaRVR1 proteins from a 1) to a 4); or c 3) a nucleic acid molecule which hybridizes under stringent conditions to the coding gene of c 1) and which codes for any one of the TaRVR1 proteins from a 1) to a 4).

SEQ ID NO：4：

ATGGGGAGGCGCCGCCGGTTCACGCAGCAGCCCACCAGCGACGACGACGACGACGACGACGACGAAGCCGTCCCGCAGCCGCTCAGAACCACCAAGCCCACAGCCCCCCTCTCCTCGGGCGCCAAGAAGCAGCAGCGTCACCGTGCCGACGAGGACGACCTGGAACTCGAGGAAGAGGAAGAGGATGAGAAGGACCTGGCGGAGTTGAGGAAGAACGAGGAGGAGGAGAGGCGGGAGGAGACGCAGACCCGCCGCCGCAGGGGCCGCAAACCCAAGCGCCCCGCTGAGGAGAGCGACGAGGATTCGCAGGAGAAAAAGGCGGAATCAGAGGAGGAGGAGGAGTCCCGCGAGGAGGATAGCACGGAGGCGGTACCCGTCGGGCAACCTTTTAAGGTGACCGGCAAGGGGAAGAAGCAGCGGAGGCATTACTTCTCCTTTGAGTACGAGGGCAACACCTTCGAGCTTGAGGACCCTGTGCTGCTCACGCCCGAGCAGCAGAAAGAGAAGCCCTATGTCGCCATCATCAAGGATATAACAGAAAATGATGGGAGCTTATCGGTAACTGGTCAGTGGTTTTATAGGCCAGAAGAAGCTGACAAAAAGGGTGGTGGGAATTGGACAGCAAGTGACACAAGAGAGCTATTTTATAGTTTTCATATAGATGATGTGCCAGCGGAGTCAGTTATGCACAAATGTGTGGTCCATTTCATTCCACTGAACAAGCAGATTCCCAGTCGAAAGGAACACCCTGGATTCATTGTCCAAAAAGTTTACGACACAGTTGCGAAGAAATTGTGGAACTTAACAGATAAGGATTATGAGGATAACAAGCAACACGAAATCGATCTCCTCGTGAAGAAAACAGTGGACCGTATTGGACAGCTTCCTGACCGTGAACCTATAGATGTACCTGCTGACAGTACTGATCAGTTCTCAAATAAACGCGGTCTACGAAAGAGACCTCCGAACCCTTTAGACGTGTCGAAAGATACAACAGGCAAACCCGAGCAATTCATAAAAGCAGAGACACCTGGGAGTGATAATCTAAAGCATTATGCCATCCTCGCCAAATATAAAGCTGTCACTAACGCCACTTATCGTGATAAGTGGCTTGATAAACTAGTAGACACTATTCCATTAACATCAAAAGAAGGTGCTGAGGCTTCTCATGCCGATGCTGGCAGTGTGGCCAAAATTTCAAATAGCTCTTCATCTGCAAGGGATACTAGTTCAAGTGATAATGAAAATTCATATCCACCAGATGTGGTTGTTTCAATAATGGCCTCCTTAGAAAGGTCTACATATGACGCTCTTCATGCTGACTTCCAGAAGTATAATCAGAAAATGAGAAAATTAGAATTCAACATCAAGAATAGCCCTGTACTGCGCAGGCGACTGATGAACAAGGAGCTTGACCCTCCAGTTCTTTTAACCATGTCTCCAGATGAACTAAAGGCTGGGTTGACACCAGCAGAGAAAACGTCTGAACCAGAAGAAGCAAGGCGATTGCAGATGACTGACGCACGATGTGAAAGGTGCACAGAAAAGAGAGTAGGCATTTCTGATATTATTCATGCTGGTCATGGAGATCGATATCAGCTCGAATGTATTTCCTGTGGTTACACATGGTTTTCGTCAAGAGATGCCATCTCATCGCTGACGGTCGATACTCCAAGTTCTGGTGGGAATGTAGGCACCGCTCCCTGGGCAACAGCAAAGTTTGATGTCCTTCAGAAGCAGCTGGTGAGCCCTCGGGATCAGCCAGACAATAAGGCAAGTGCTGATGCTCTCCAGAAGAACACGGTGGCATCCATTCCTAAACTTGAGAGGCAGAAATCATTCATCAAACCGAAGCCAGAGGAGCCCTCTGCACCCACACTGGAGAAACAGAAGGCGTTCACCAAGCTAAAGCCGGAGGAACCCTCTGCACCCACACTGGAGAAACAGAAGGCGTTCACCAAGCCAAAGCCGGAGGAACCCTCTGCACCTTCGGCTAGTCATTAG。

SEQ ID NO：5：

CAGCTTCTCAATCGGACATTAGGAGAGATGGGGAGGCGCCGCCGGTTCACGCAGCAGCCCACCAGCGACGACGACGACGACGACGACAAACCCGTCCCGCAGCCGCTCAGAACGACCAAGCCCACAGCCCCCCTCTTCTCGGGCGCCAAGAAGCAGCAGCGTCACCGTCCCGACGAGGACGACCTGGAACTCGAGGAAGAGGAAGAGGATGAGAAGGACCTGGCGGAGTTGAGGAAGAACGAGGAGGAGGAGAGGCGGGAGGAGACGCAGACCCGCCGCCGCAGGGGCCGCAAACCCAAGCGCCCCGCCGAGGAGAGCGACGAGGAGCCGGAGGAGAAAAAGGCGGAATCAGAGGAGGAGGAGGAGTCCCGCGAGGAGGATAGCACGGAGGCGGTACCCGTCGGGGAGCCTTTGAAGGTGACCGGCAAGGGGAAGAAGCAGCGGAGGCATTACTTCTCCTTTGAGTACGAGGGCAACACCTTCGAGCTTGAGGACCCGGTGCTGCTCACGCCCGAGCAGCAGAAAGAGAAGCCCTATGTCGCCATCATCAAGGATATAACAGAAAATGATGGGAGCTTATCGGTAACTGGTCAGTGGTTTTATAGGCCAGAAGAAGCTGACAAAAAGGGTGGTGGGAATTGGACAGCAAGTGACACAAGAGAGCTATTTTATAGTTTTCATATAGATGATGTGCCAGCGGAGTCAGTTATGCACAAATGTGTGGTCCATTTCATTCCACTGAACAAGCAGATTCCCAGTCGAAAGGAACACCCTGGGTTCATTGTCCAAAAAGTTTACGACACAGTTGCGAAGAAATTGTGGAACTTAACAGATAAGGATTATGAGGATAACAAGCAACACGAAATCGATCTCCTCGTGAAGAAAACAGTGGACCGTATTGGACAGCTTCCTGACCGTGAACCTATAGATGTACCTGCTGACAGTACTGATCAGTTCTCAAATAAACGCGGTCTACGAAAGAGACCTCCAAACCCTTTAGACGTGTCGAGAGATACAACAGGCAAACCCGAGCAATTCATAAAAGCAGAGACACCTGGGAGTGATAATCTAAAGCATTATGCCATCCTCGCCAAATATAAAGCTGTCACCAACGCCACTTATCGCGATAAGTGGCTTGATAAACTAGTAGACACTATTCCATTAACATCAAAAGAAGGCGCTGAGGCTTCTCATGCCGATGCTGGCAGTGTGGCCAAAATTTCAAATAGCTCTTCATCTGCAAGGGATAGTAGTTCAAGTGATAATGAAAATTCATATCCACCAGATGTGGTTGTTTCAATAATGGCCTCCTTAGAAAGGTCTACATATGACGCTCTTCATGCTGACTTCCAGAAGTATAATCAGAAAATGAGAAAATTAGAATTCAACATCAAGAATAGCCCTGTACTGCGCAGGCGACTGATGAACAAGGAGCTTGACCCTCCAGTTCTTTTAACCATGTCTCCAGATGAACTAAAGGCTGGGTTGACACCAGCAGAGAAAACGTCTGAACCAGAAGAAGCAAGGCGATTGCAGATGACTGACGCACGATGTGGAAGGTGCACAGAAAAGAGAGTAGGCATTTCTGATATTATTCATGCTGGTCATGGGGATCGATATCAGCTCGAATGTATTTCCTGTGGTTACACATGGTTTTCGTCAAGAGATGCCATCTCATCGCTGACGGTCGATACTCCAAGTTCTGGTGGGAATGTAGGCACCGCTCCCTGGGCGACAGCAAAGTTTGATGCCCTTCAGAAGCAGCTGGTGAGCCCTCGGGATCAGCCAGACAACAAGGCAAGTGCTGATGCTCTCCAGAAGAACACGGTGGCATCCATTCCTAAACTTGAGAGGCAGAAATCACTAATCAATCCGAAGCCAGAGGAGCCCTCTGCACCCACACTGGAGAAGCAGAAGACGTTCACCAAGCCAAAGCCGGAGGAACCCTCTGCACCTTCAGCTAGTCATTAG。

SEQ ID NO：6：

ATGGGGAGGCGCCGCCGGTTCACGCAGCAGCCCACCAGCGACGACGACGACGACG

ACGACGAAGCCGTCCCGCAGCCGCTCAGAACCACCAAGCCCACAGCCCCCCTCTCCCCG

GGCGCCAAGAAGCAGCAGCGTCACCGTGCCGACGAGGACGACCTGGAACTCGAGGAA

GAGGAAGAGGATGAGAAGGACCTGGCGGAGTTGAGGAAGAACGAGGAGGAAGAGAGA

CGGGAGGAGACGCAGACCCGCCGCCGCAGGGGCCGCAAACCCAAGCGCCCCGCCGAG

GAGAGCGACGAGGATCCGGAGGAGAAAAAGGCGGAATCAGAGGAGGAGGAGGAGTCC

CGCGAGGAGGATAGCACGGAGGCGGTACCCGTCGGGGAACCTTTGAAGGTGACCGGCA

AGGGGAAGAAGCAGCGGAGGCATTACCTCTCCTTTGAGTACGAGGGCAACACCTTCGAG

CTTGAGGACCCGGTGCTGCTCACGCCCGAGCAGCAGAAAGAGAAGCCCTATGTCGCCAT

CATCAAGGATATAACAGAAAATGATGGGAGCTTATCGGTAACTGGTCAGTGGTTTTATAG

GCCAGAAGAAGCTGACAAAAAGGGTGGTGGGAATTGGACAGCAAGTGACACAAGAGA

GCTATTTTATAGTTTTCATATAGATGATGTGCCAGCGGAGTCAGTTATGCACAAATGTGTAG

TCCATTTCATTCCACTGAACAAGCAGATTCCCAGTCGAAAGGAACACCCTGGATTCATTG

TCCAAAAAGTTTACGACACAGTTGCGAAGAAATTGTGGAACTTAACAGATAAGGATTATG

AGGATAACAAGCAACACGAAATCGATCTCCTCGTGAAGAAAACAGTGGACCGTATTGGA

CAGCTTCCTGACCGTGAACCTATAGATGTACCTGCTGACAGTACTGATCAGTTCTCAAATA

AACGCGGTCTACGAAAGAGACCTCCGAACCCTTTAGACGTGTCGAGAGATACAACAGGC

AAACCCGAGCAATTCATAAAAGCAGAGACACCCGGGAGTGATAATCTAAAGCATTATGCC

ATCCTCGCCAAATATAAAGCTGTCACTAACGCCACTTATCGCGATAAGTGGCTTGATAAAC

TAGTAGACACTATTCCATTAACATCAAAAGAAGGTGCTGAGGCTTCTCATGCCGATGCTG

GCAGTGTGGCCAAAATTTCAAATAGCTCTTCATCTGCAAGGGATCCTAGTTCAAGTGATA

ATGAAAATTCATATCCACCAGATGTGGTTGTTTCAATAATGGCCTCCTTAGAAAGGTCTAC

ATATGACGCTCTTCATGCTGACTTCCAGAAGTATAATCAGAAAATGAGAAAATTAGAATTC

AACATCAAGAATAGCCCTGTACTGCGCAGGCGACTGATGAACAAGGAGCTTGACCCTCC

AGTTCTTTTAACCATGTCTCCAGATGAACTAAAGGCTGGGTTGACACCAGCAGAGAAAA

CGTCTGAACCAGAAGAAGCAAGGCGATTGCAGATGACTGATGCACGATGTGAAAGGTGC

ACAGAAAAGAGAGTAGGCATTTCTGATATTATTCATGCTGGTCATGGAGATCGATATCAGC

TTGAATGTATTTCCTGTGGTTACACATGGTTTTCGTCAAGAGATGCCATCTCATCGCTGAC

GGTCGATACTCCAAGTTCTGGTGGGAATGTAGGCACCGCTCCCTGGGCAACAGCAAAGT

TTGATGTCCTTCAGAAGCAGCTGGTGAGCCCTCGGGATCAGCCAGACAATAAGGCAAGT

GCTGATGCTCTCCAGAAGAACACGGTGGCGTCCATTCCTAAACTTGAGAGGCAGAAATC

ATTCATCAAACCGAAGCCAGAGGAGCCCTCTGCACCCACACTGGAGAAACAGAAGGCGTTCACCAAGCCAAAGCCGGAGGAACCCTCTGCACCTTCGGCTAGTCATTAG。

In a preferred embodiment, the wheat comprises diploid wheat, tetraploid wheat or hexaploid wheat; preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat; preferably, the commonly cultivated hexaploid wheat comprises Kenong wheat or other artificially bred wheat; preferably, regulating the growth cycle of wheat comprises shortening the vernalization time of the wheat; preferably, the ability to regulate effective tillering of wheat comprises increasing the number of effective tillers of wheat; preferably, the effective tillering number is the number of heading and firm tillers obtained after wheat tillering; preferably, regulating the yield of wheat comprises increasing the yield of wheat; preferably, increasing the yield of wheat comprises increasing the yield of wheat kernels.

Preferably, the above-mentioned common hexaploid wheat refers to wheat whose chromosome number is hexaploid (6 n), which is a heterohexaploid formed by two natural hybridization, and contains A, B and D subgenomic groups. I.e., each gene theoretically has a homologous gene in both the A, B and D subgenomic regions. Wherein the ancestor species of the A subgenomic group is Ula wheat, the ancestor species of the B subgenomic group is aegilops spelt, the ancestor species of the D subgenomic group is aegilops crudus, the number A, B and the number of the D subgenomic group are international universal numbers. The Kenong wheat is a winter wheat variety, and needs to be subjected to low temperature in winter (vernalization treatment) to bloom and fruit in spring and summer in the next year.

The common cultivated hexaploid wheat is actually wild hexaploid wheat, and the wild hexaploid wheat is subjected to long artificial breeding and cultivation, maintains excellent characters (plant height, tillering number, yield or stress resistance and the like), forms various cultivated varieties common in the current farmland, and is one of important grain crops in agricultural production. The wild hexaploid wheat is hexaploid wheat growing in a natural environment in the field, and the plant type is vertical and the spike grain is smaller and less artificial cultivation is performed because the growth of the hexaploid wheat is sparse and the height of the hexaploid wheat is lower.

All the above-mentioned wheat can be applied to the TaRVR1 protein or biological material provided by the application to carry out experiments in the application of regulating and controlling the wheat traits.

The TaRVR1 protein is an endogenous protein in the wheat, and the nucleic acid molecule is an endogenous nucleic acid in the wheat, so that the TaRVR1 protein or a corresponding biological material is applied to the wheat, the growth cycle, the effective tillering capacity and the yield of the wheat can be regulated, wheat plants with short vernalization time or even without vernalization, short growth cycle or large effective tillering quantity can be cultivated, and the wheat plants with high yield can be obtained by improving the effective tillering quantity of the wheat.

In a preferred embodiment, the above-described applications include any one or more of the following: down-regulating in wheat the expression level of the TaRVR1 protein in a 1), replacing the TaRVR1-A protein expressed in wheat with the sequence of SEQ ID NO:7 or SEQ ID NO:8 or a TaRVR1-A-1 protein or a TaRVR1-A-2 protein expressed in wheat by replacing the TaRVR1-B protein expressed in wheat with the sequence of SEQ ID NO:9 or replacing the TaRVR1-B-1 protein expressed in wheat with the sequence shown in SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, a TaRVR1-D-1 protein, a TaRVR1-D-2 protein or a TaRVR1-D-3 protein.

SEQ ID NO：7：

MGRRRRFTQQPTSDDDDDDDDEAVPQPLRTTKPTAPLSSGAKKQQRHRADEDDLELEE EEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDSQEKKAESEEEEESREEDS TEAVPVGQPFKVTGKGKKQRRHYFSFERTLCCSRPSSRKRSPMSPSSRI*。

SEQ ID NO：8：

MGRRRRFTQQPTSDDDDDDDDEAVPQPLRTTKPTAPLSSGAKKQQRHRADEDDLELEEEEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDSQEKKAESEEEEESREEDSTEAVPVGQPFKVTGKGKKQRRHYFSFEYEGNTFEQAAALLLLNKIFFVRFYRVFGCCKFCYGFV*。

SEQ ID NO：9：

MGRRRRFTQQPTSDDDDDDDKPVPQPLRTTKPTAPLFSGAKKQQRHRPDEDDLELEEE EEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEEPEEKKAESEEEEESREEDSTE AVPVGEPLKVTGKGKKQRRHYFSFERTRCCSRPSSRKRSPMSPSSRI*。

SEQ ID NO：10：

MGRRRRFTQQPTSDDDDDDDEAVPQPLRTTKPTAPLSPGAKKQQRHRADEDDLELEEEEEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDPEEKKAESEEEEESREEDSTEAVPVGEPLKVTGKGKKQRRHYLSFEYEGNTFEDPVLLTPEQQKEKPYVAIIKDITENDGSLSVTGQWFYRPEEADKKGGGNWTASDTRELFYSFHIDDVPAESVMHKCVVHFIPLNKQIPSRKEHPGFIVQKVYDTVAKKLWNLTDKDYEDNKQHEIDLLVKKTVDRIGQLPDREPIDVPADSTDQFSNKTRSTKETSEPFRRVERYNRQTRAIHKSRDTRE*。

SEQ ID NO：11：

MGRRRRFTQQPTSDDDDDDDEAVPQPLRTTKPTAPLSPGAKKQQRHRADEDDLELEEE EEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDPEEKKAESEEEEESREEDSTE AVPVGEPLKVTGKGKKQRRHYLSFERTRCCSRPSSRKRSPMSPSSRI*。

SEQ ID NO：12：

MGRRRRFTQQPTSDDDDDDDEAVPQPLRTTKPTAPLSPGAKKQQRHRADEDDLELEEE EEDEKDLAELRKNEEEERREETQTRRRRGRKPKRPAEESDEDPEEKKAESEEEEESREEDSTE AVPVGEPLKVTGKGKKQRRRTRCCSRPSSRKRSPMSPSSRI*。

In a preferred embodiment, the TaRVR1 protein in a 3) has at least 85% identity and the same function as the TaRVR1 protein in a 1); preferably, at least 95% identity, more preferably 99%, still more preferably 99.9%; preferably, the TaRVR1 protein in a 3) is derived from wheat.

In a preferred embodiment, the nucleic acid molecule of c 2) has at least 85% identity and the same function as the coding gene of c 1); preferably, there is at least 95%, more preferably 99%, even more preferably 99.9% identity.

In a second exemplary embodiment of the present application, there is provided a method of breeding wheat comprising reducing the expression level of a TaRVR1 protein in a wheat of interest, reducing the activity of a TaRVR1 protein, or reducing the expression level of a nucleic acid molecule encoding a TaRVR1 protein in a wheat of interest, to obtain a wheat with improved traits; wherein the TaRVR1 protein or the nucleic acid molecule encoding the TaRVR1 protein is a TaRVR1 protein in any of the above applications, and the nucleic acid molecule encoding the TaRVR1 protein is a nucleic acid molecule in any of the above applications; trait improvement includes, but is not limited to, shortening of growth cycle, increased effective tillering capacity, or increased yield.

The TaRVR1 protein can influence the growth cycle, the effective tillering capacity and the yield of wheat, and the low expression level of the TaRVR1 protein in the wheat or the reduced activity of the protein can shorten the vernalization time of the wheat or eliminate vernalization, and has strong effective tillering capacity and improved yield. Therefore, by means of the prior art, the growth cycle of the wheat can be shortened and the effective tillering capacity of the wheat can be enhanced by reducing the transcription and expression quantity of nucleic acid molecules in target wheat and/or reducing the TaRVR1 protein content in the wheat, so that the wheat with short vernalization time, increased effective tillering quantity and improved yield can be obtained. By adjusting any one or more of the plurality of nucleic acid molecules, the expression quantity of the corresponding TaRVR1 protein can be changed, so that the regulation and control on the growth cycle, the effective tillering capacity and the yield of the wheat are realized, and the wheat with short vernalization time or strong effective tillering capacity and high yield without vernalization is cultivated and obtained.

In a preferred embodiment, the method of reducing the expression level of a TaRVR1 protein or nucleic acid molecule in a wheat of interest comprises gene editing or RNAi (RNA interference); preferably, the gene editing comprises cleavage of the nucleic acid molecule, and/or insertion, deletion or substitution of bases on the nucleic acid molecule; preferably, the short growth cycle wheat has a shorter growth cycle than the target wheat; preferably, the effective tillering quantity of the wheat with strong effective tillering capacity is more than that of the target wheat; preferably, the short growth cycle includes a short vernalization time or no vernalization is required; preferably, the effective tillers are tillers capable of heading and setting; preferably, the yield increase comprises a yield increase of the kernel. Preferably, the gene editing includes, but is not limited to, CRISPRi (CRISPR interference).

RNAi (RNA interference) refers to a phenomenon of gene silencing induced by small double-stranded RNAs (siRNAs, about 21-23 bp) in an organism by melting the siRNAs into sense and antisense strands by the action of RNA helicases, and combining the antisense strands with some enzymes in the body (including endonucleases, exoenzymes, helicases, etc.) to form RNA-induced silencing complexes (RNA-induced silencing complex, RISC). RISC specifically binds to the homologous region of mRNA expressed by the target gene and cleaves mRNA at the binding site, and the cleaved mRNA is then degraded to inhibit gene expression. In preferred embodiments of the present application, RNAi includes, but is not limited to, the use of antisense RNA in combination with mRNA to inhibit transcription and translation, thereby reducing the expression of the TaRVR1 protein or nucleic acid molecule in the wheat of interest.

The CRISPR technology can be utilized to enhance or reduce the expression level of genes so as to influence the expression level of proteins; typically, the insertion or deletion of a base in a gene directly modulates the activity of a protein (including enhancement, attenuation, or even inactivity of the protein). In the application, a CRISPR method is adopted to carry out gene editing on the TaRVR1 gene of the Kenong wheat 9204, so that the base deletion of the exon region of the gene is caused, the translation of the TaRVR1 protein is stopped in advance, and the inactive or low-activity protein is produced.

In a preferred embodiment, the method comprises: introducing genetic material for expressing nuclease into cells or tissues of target wheat, and culturing the introduced cells or tissues into complete plants to obtain wheat with short growth cycle and/or strong effective tillering capability; preferably, the nuclease is capable of specifically cleaving a functional segment of the nucleic acid molecule; genetic material includes circular plasmids of DNA, or linear fragments of DNA, or RNA transcribed in vitro. Preferably, the genetic material is a recombinant vector capable of transcribing the guide RNA and expressing the Cas protein; the guide RNA is RNA with palindromic structure formed by combining crRNA and tracrRNA through partial base pairing; crRNA includes RNA fragments capable of complementary binding to functional segments; preferably, the method of introducing genetic material into cells or tissues of the wheat of interest includes a gene gun method, an agrobacterium infection method, a PEG-induced protoplast method, an electrode method, a silicon carbide fiber-mediated method, or a vacuum infiltration method; preferably, the cells include any cell that can be used as an infusion recipient and can be regenerated into a whole plant by tissue culture; the tissue is any tissue which can be used as an introduction receptor and can be regenerated into a complete plant through tissue culture; preferably, the cells comprise protoplast cells or suspension cells; tissues include callus, immature embryo, mature embryo, leaf, stem tip, young ear or hypocotyl.

Preferably, the functional segment comprises a segment on the TaRVR1 gene sequence capable of causing a decrease in the activity or inactivation of the expressed protein.

Gene editing is performed on the target wheat nucleic acid molecules, and the gene editing tools or systems in the prior art can be flexibly selected, including but not limited to CRISPR/Cas9, CRISPR/Cas12 or RED recombination systems.

In a preferred embodiment, the wheat of interest comprises diploid wheat, tetraploid wheat or hexaploid wheat; preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat; preferably, the commonly cultivated hexaploid wheat includes a kenaf wheat (including, but not limited to, kenaf 9204 wheat, kenaf 1002 wheat, or kenaf 8162 wheat) or other artificially bred wheat.

In a third exemplary embodiment of the present application, there is provided a TaRVR1 protein or biological material in the above-described method of nutritional or wheat breeding.

The advantageous effects of the present application will be explained in further detail below in connection with specific examples.

Example 1 Kenong 9204 wheat was not subjected to vernalization test

The winter wheat variety is characterized in that winter wheat can bloom and fruit in the spring and summer in the next year only through low temperature in winter (vernalization treatment), and if the winter wheat variety does not undergo low temperature in winter, the winter wheat variety can have a certain influence on blooming and fruit setting.

Planting the Kenong 9204 wheat which is not subjected to vernalization treatment in an artificial environment with constant temperature of 24 ℃ in 16 hours of illumination/8 hours of darkness, and culturing until the wheat flowers and fruits.

Planting the Kenong 9204 wheat in an artificial environment with 16 hours of illumination/8 hours of darkness and constant temperature at 24 ℃, placing the wheat in the artificial environment with 8 hours of illumination/16 hours of darkness and constant temperature at 4 ℃ for 6 weeks after germination and growth of the wheat, transferring the wheat to the artificial environment with 16 hours of illumination/8 hours of darkness and constant temperature at 24 ℃ for culturing the wheat until flowering and fruiting of the wheat.

The culture forms and statistical results of the Kenong 9204 wheat without vernalization treatment and the 6-week vernalization treatment at different culture periods are shown in FIG. 1. In fig. 1, a is a morphological diagram of a plant 9204 which is cultivated to bloom and fruit after being not vernalized and vernalized for 6 weeks in the plant 9204; FIG. 1B is a statistical plot of days before flowering of non-vernalized Kenong 9204 wheat and vernalized 6-week Kenong 9204 wheat; figure 1C is a morphology of ears and kernels after harvest of non-vernalized crop 9204 wheat and 6 week vernalized crop 9204 wheat; in FIG. 1, D is a statistical plot of the tillering numbers of non-vernalized Kenong 9204 wheat and 6 week vernalized Kenong 9204 wheat cultivated to flowering and fruiting.

As can be seen from fig. 1, the non-vernalized coronet 9204 wheat has a late flowering time, and the number of tillers is greater than that of the vernalized coronet 9204 wheat, but the number of ineffective tillers is also increased; the wheat ears on the effective tillers are imperfect in development and are in a curved shape, the grains on the wheat ears are fewer, and the grains are not plump as those of wheat knots after six weeks of vernalization.

Example 2 wheat TaRVR1 Gene cloning

Based on the arabidopsis AtRVR1 amino acid sequence, searching for homologous genes in Chinese spring wheat, taking the nucleotide sequence of the TaRVR1 gene in Chinese spring wheat as a reference, designing a specific primer, and cloning TaRVR1 (TraesKN 3A01HG21050, traesKN3B01HG21090 and TraesKN3D01HG 18870) genes of wheat of Kenong 9204 (KN 9204).

The specific operation is as follows:

1. primer design:

the primer sequences are shown in Table 1

TABLE 1

2. PCR amplification

The PCR amplification system is shown in Table 2.

TABLE 2

3. The PCR instrument runs the following series of reactions: 98 ℃ for 1min; (98 ℃,30s;60 ℃,30s;72 ℃,2 min) x32 cycles; 72 ℃ for 5min;4 ℃, and the preservation temperature.

4. The PCR amplification product was recovered using a Takara (cat# 9762) PCR gel recovery kit.

5. The PCR amplification product was ligated to the linearized vector using T4 ligase under the ligation conditions according to the instructions.

6. The ligation product was transformed into DH 5. Alpha. Competent cells, and single colonies were picked for sequencing after incubation.

7. The comparison of the TaRVR1 amino acid sequence of wheat with BdRVR1 of Brevibacterium bifidum and AtRVR1 amino acid sequence of Arabidopsis thaliana shows that the sequences have higher similarity, as shown in figure 2, A in figure 2 is a schematic diagram of three TaRVR1 genes (TaRVR 1A, taRVR B and TaRVR 1D) and the gene structures of AtAIPP3 gene and BdRVR1 gene. In FIG. 2B is a schematic representation of the amino acid sequence alignment of three TaRVR1 proteins (TaRVR 1A, taRVR B and TaRVR 1D) with AtAIPP3 and BdRVR1 proteins. In FIG. 2, C is a schematic diagram showing the prediction of the BAH protein structures of AtAIPP3 and TaRVR1, and "H3K27me3peptide" represents a peptide fragment representing 3 methylation modification of lysine 27 on histone H3.

Example 3 construction of wheat TaRVR1 Gene editing vector wheat was transformed.

1. Two specific sgrnas were designed for the TaRVR1 coding region using the http:// CRISPR.

TABLE 3 Table 3

sgRNA	Sequence(s)
		sgRNA1(SEQ ID NO:15)	AACACCTTCGAGCTTGTAAGCGG
sgRNA2(SEQ ID NO:16)	GATCAGTTCAAATAAACGCGCGG

In the present application, the design of two sgrnas is primarily to ensure complete inactivation of the TaRVR1 protein. The corresponding sites for binding of sgRNA1 and sgRNA2 to the gene are both exon segments, as shown in fig. 2B.

2. The two specific sgrnas described above were ligated into a CRISPR/Cas9 vector, which were transcribed driven by the TaU and TaU6 promoters, respectively.

3. The ligation product was transformed into DH 5. Alpha. Competent cells, and single colonies were picked for sequencing after incubation.

4. The plasmid with correct sequencing result is transformed into agrobacterium.

(1) 100. Mu.L of Agrobacterium (EHA 105) +2. Mu.L of plasmid, ice-bath for 5min, liquid nitrogen flash-frozen for 5min, water-bath at 37℃for 5min, ice-bath for 5min. 500. Mu.L of antibiotic-free LB was added, and the mixture was shaken at 28℃for 2 hours at 200rpm, and directly smeared on plates of the corresponding bacterial antibiotic +rifampicin, and incubated at 28℃for two days.

(2) And (3) selecting monoclonal shaking bacteria, and preserving glycerinum bacteria after PCR identification, so that a later transformation experiment can be performed.

5. Streaking the positive agrobacterium on an activation culture medium, picking a monoclonal, shaking slightly and shaking greatly, centrifuging, and suspending by using a suspension culture medium; about 14 days after stripping and flowering Adding the scutellum of the young embryo of the right wheat into the agrobacterium suspension, and standing for 5min; pouring the young embryo in the centrifuge tube onto a co-culture medium, sucking out superfluous agrobacterium tumefaciens bacterial liquid on the surface by using a pipettor, and performing dark co-culture for 2 days at the temperature of 23 ℃; after co-cultivation, the above materials are transferred into a recovery medium and cultivated in the dark at 28 ℃; transferring the strain to a differentiation medium with corresponding resistance after 5 days, and continuously culturing the strain in the dark at 25 ℃; after 2 weeks the material was cut in half and placed in differentiation medium of the corresponding resistance, with continuous light at 25℃ (100. Mu. Mol/m ² S) culturing for 3 weeks; transferring the differentiated plantlets onto rooting medium, and continuously irradiating at 25deg.C (100 μmol/m) ² /s) culturing for 2 weeks until rooting; transferring the young seedling into a small basin for growth, and transplanting the young seedling into a greenhouse after a certain growth stage.

Genotyping and seed collection were performed on the transgenic positive plants (T1), the sequences of the mutation site identification primers are shown in Table 4, and the PCR amplification system is shown in Table 5. Mutation site identification sgRNA-1 primer F

TABLE 4 Table 4

TABLE 5

The PCR instrument runs the following series of reactions: 94 ℃ for 1min; (94 ℃,30s;60 ℃,30s;72 ℃,30 s) x32 cycles; 72 ℃ for 1min;4 ℃, and the preservation temperature.

And (5) sending the PCR product to sample and sequencing.

And planting a T2 transgenic plant, and carrying out genotype identification to obtain a corresponding homozygous mutant.

A schematic diagram of gene editing by CRISPR/Cas9 technology of three TaRVR1 genes (TaRVR 1A, taRVR B and TaRVR 1D) is shown in fig. 3, a in fig. 3 is a schematic diagram of the structure of the TaRVR1 gene and the setting of a target point by CRISPR/Cas9 technology, TSS represents a transcription initiation site, and sgRNA represents guide RNA in CRISPR/Cas 9; FIG. 3B is a schematic diagram of the gene structure of sgRNA1 and sgRNA2 binding to three TaRVR1 genes (TaRVR 1A, taRVR B and TaRVR 1D), BAH represents BAH protein domain, TFS2M represents Transcription elongation factor S-II, and transcription elongation factor S-II; in FIG. 3, C is the schematic representation of the amino acid sequences of wild-type control acceptor wheat (i.e., target wheat, labeled WT) and three genetically edited TaRVR1 homozygous mutant wheat (rv1#2-45, r1#16-23 and r1#25-44) (SEQ ID NO: 1-3 is the amino acid sequence of TaRVR1A, taRVR B and TaRVR1D of target wheat; SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:10 are the amino acid sequences of TaRVR1-A-1, taRVR1-B-1 and TaRVR1-D-1 in r1#2-45; SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 are the amino acid sequences of TaRVR1-A-1, taRVR1-B-1 and TaRVR1-D-2 in r1#16-23; SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:12 are the amino acid sequences of TaRVR 1-A-1-2 and TaRVR 1-D-44, respectively).

Wherein, the number of each mutant is exemplified by #2-45, and 2 represents the number of the wheat after gene editing. Sowing the harvested wheat seeds after planting the No. 2 edited wheat seeds, and then carrying out phenotype and genotype identification to find that the No. 45 wheat obtained by the wheat seeds subjected to gene editing is a homozygotic mutant.

Example 4 phenotypic characterization of wheat TaRVR1 Gene editing mutants

The TaRVR1 homozygous mutant wheat and the receptor control wheat (namely target wheat, kenong 9204) are planted under the same environmental condition, the growth condition of the TaRVR1 homozygous mutant wheat is observed, and the TaRVR1 homozygous mutant wheat and the receptor control wheat are sampled for gene expression detection. The phenotype identification schematic diagram of the wheat TaRVR1 gene editing mutant is shown in fig. 4, wherein A in fig. 4 is a schematic diagram of the growth morphology of wild type receptor control group wheat (namely, target wheat, CK (KN 9204) is marked in parentheses of the wheat variety) and three TaRVR1 homozygous mutant wheat (rv1#2, rv1#16 and rv1#25) in the same period; FIG. 4B is a schematic diagram of the morphology of the wild type acceptor control group wheat (i.e., wheat of interest, labeled CK) during the maturation period; in FIG. 4, a statistical plot of days before flowering of the target wheat (designated KN) and three TaRVR1 homozygous mutant wheat (rv1#2, rv1#16 and rv1#25) is shown.

The result shows that the TaRVR1 gene knockout of wheat can significantly advance the flowering time of the wheat and significantly improve the effective tillering number. The tiller number and individual yield statistics of the recipient control wheat and the three TaRVR1 homozygous mutants are shown in Table 6.

TABLE 6

Wheat variety	Average effective tillering (plant)	Yield of single plant (gram)
			Kenong 9204	18.25±2.05	9.29±1.31
rvr1-#2-45	24.25±1.42	14.56±1.08
			rvr1-#16-23	23.25±1.93	14.52±1.58
rvr1-#25-44	22.83±1.59	14.00±0.95

As can be seen from Table 6, the average effective tillering number of the three TaRVR1 homozygous mutant wheat is significantly increased compared with that of the acceptor control wheat at the maturation stage, and the yield of the individual plant is also significantly improved.

The expression level of TaVRN1 gene of the recipient control group wheat and three TaRVR1 homozygous mutant wheat at different growth periods is detected as shown in FIG. 5. The result shows that the flowering time of the TaVRN1 homozygous mutant wheat is advanced, because the expression of the TaVRN1 gene of the mutant wheat is activated in advance in the seed germination stage, and the expression level of the TaVRN1 gene in the mutant wheat is still obviously higher than that of the control wheat after the low-temperature treatment. TaVRN1 is a positive regulatory factor critical to wheat flowering, so that TaRVR1 of wheat is likely to regulate and control the wheat flowering time by negatively regulating and controlling the gene expression of TaVRN1, and the gene and homologous genes are shown to have important application values in wheat breeding and trait improvement.

After gene editing is carried out on the wheat of the Kenong 9204, the winter characteristic of the wheat of the Kenong 9204 is changed into the spring wheat characteristic, namely, the mutant is not required to be subjected to low temperature in winter and can be directly cultivated in spring, so that the growth period is greatly advanced, and meanwhile, compared with the wheat of the Kenong 9204 subjected to low temperature in winter (shown as a wheat form of the Kenong 9204 in the 6 week vernalization of the diagram in fig. 1), the effective tillering number of the wheat of the mutant obtained after the gene editing is greatly improved.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the coding region of the wheat TaRVR1 gene is edited to mutate into a termination codon sequence, so that the translation of the TaRVR1 protein is terminated in advance, a functional TaRVR1 protein cannot be formed, the vernalization time of the wheat is shortened, or vernalization treatment is not needed, the flowering time of the wheat is advanced, the number of effective tillers is increased, the yield is improved, and new wheat plants with short growth period, strong effective tillering capability and high yield are obtained.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

The application of TaRVR1 protein or biological material in regulating and controlling wheat character is characterized in that,

the TaRVR1 protein includes any one of the following a 1) -a 4):

a1 SEQ ID NO:1, the TaRVR1-A protein shown in SEQ ID NO:2 or the TaRVR1-B protein shown in SEQ ID NO:3, any one or more of the TaRVR1-D proteins shown in figure 3; or (b)

a2 A) a TaRVR1 protein having the same function by substituting and/or deleting and/or adding one or more amino acid residues to the TaRVR1 protein in the a 1); or (b)

a3 A TaRVR1 protein having at least 75% identity and the same function as the TaRVR1 protein in a 1); or (b)

a4 A TaRVR1 protein having the same function obtained by linking protein tags at the N-terminus or/and C-terminus of the TaRVR1 protein in the a 1), the a 2) or the a 3);

the biomaterial comprises any one of the following b 1) -b 7):

b1 A nucleic acid molecule encoding the TaRVR1 protein of any of a 1) -a 4);

b2 An expression cassette comprising said nucleic acid molecule;

b3 A recombinant vector comprising said nucleic acid molecule or said expression cassette;

b4 A recombinant microorganism comprising said nucleic acid molecule, said expression cassette or said recombinant vector;

b5 A transgenic wheat cell line comprising said nucleic acid molecule or said expression cassette;

b6 A transgenic wheat tissue comprising said nucleic acid molecule or said expression cassette;

b7 A transgenic wheat organ comprising said nucleic acid molecule or said expression cassette;

the regulating the wheat trait comprises one or more of regulating a growth cycle of the wheat, regulating an effective tillering capacity of the wheat, or regulating a yield of the wheat.
2. The use according to claim 1, wherein the nucleic acid molecule of b 1) comprises:

c1 A gene encoding said TaRVR1-A protein, said TaRVR1-B protein, or said TaRVR1-D protein;

the coding gene of the TaRVR1-A protein comprises SEQ ID NO: 4;

the coding gene of the TaRVR1-B protein comprises SEQ ID NO:5, a nucleic acid shown in FIG. 5;

the coding gene of the TaRVR1-D protein comprises SEQ ID NO: 6; or (b)

c2 A nucleic acid molecule having at least 75% identity to the encoding gene in c 1) and encoding the TaRVR1 protein of any of the a 1) -a 4); or (b)

c3 A nucleic acid molecule which hybridizes under stringent conditions to the coding gene of c 1) and which codes for the TaRVR1 protein of any of a 1) to a 4).
3. The use according to claim 1, wherein said wheat comprises diploid wheat, tetraploid wheat or hexaploid wheat;

preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat;

preferably, the commonly cultivated hexaploid wheat comprises a kenaf wheat;

preferably, the regulating the growth cycle of wheat comprises shortening the vernalization time of the wheat;

preferably, the ability to regulate effective tillering of wheat comprises increasing the number of effective tillers of wheat;

preferably, the number of effective tillers is the number of tillers containing a heading ear obtained after wheat tillers;

preferably, said modulating the yield of wheat comprises increasing the yield of wheat;

preferably, the increasing the yield of wheat comprises increasing the yield of wheat kernel.
4. The use according to claim 1, wherein the TaRVR1 protein of a 3) has at least 85% identity and the same function as the TaRVR1 protein of a 1);

preferably, at least 95% identity, more preferably 99%, still more preferably 99.9%;

preferably, said TaRVR1 protein in said a 3) is derived from wheat.
5. The use according to any one of claims 1-4, characterized in that the use comprises any one or more of the following:

down-regulating the expression level of the TaRVR1 protein in a 1) in the wheat,

replacing said TaRVR1-a protein expressed in said wheat with SEQ ID NO:7 or SEQ ID NO:8 or TaRVR1-A-1 or TaRVR1-A-2 protein,

replacing said TaRVR1-B protein expressed in said wheat with SEQ ID NO:9, or TaRVR1-B-1 protein shown in the specification

Replacing said TaRVR1-D protein expressed in said wheat with SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, taRVR1-D-1, taRVR1-D-2 or TaRVR1-D-3 protein.
6. A method for breeding wheat, comprising reducing the expression level of a TaRVR1 protein in a wheat of interest, reducing the activity of the TaRVR1 protein, or reducing the expression level of a nucleic acid molecule encoding the TaRVR1 protein in the wheat of interest, to obtain a wheat with improved traits;

wherein the TaRVR1 protein is the TaRVR1 protein in any one of claims 1 to 5, and the nucleic acid molecule encoding the TaRVR1 protein is the nucleic acid molecule in any one of claims 1 to 5;

The trait improvement includes one or more of a shortened growth cycle, an improved effective tillering capacity, or an improved yield.
7. The method according to claim 6, wherein the expression amount of the TaRVR1 protein, the activity of the TaRVR1 protein or the expression amount of the nucleic acid molecule in the target wheat is reduced by a method comprising gene editing or RNAi;

preferably, the gene editing comprises cleaving the nucleic acid molecule, and/or inserting, deleting or replacing bases on the nucleic acid molecule;

preferably, the reduced growth cycle comprises reduced vernalization time;

preferably, the increase in effective tillering capacity comprises an increase in tiller number for heading;

preferably, the yield increase comprises a yield increase of kernels.
8. The method according to claim 6, characterized in that the method comprises:

introducing genetic material expressing nuclease into cells or tissues of the target wheat, and culturing the introduced cells or tissues into complete plants to obtain the wheat with improved properties;

preferably, the nuclease is capable of specifically cleaving a functional segment of the nucleic acid molecule; the genetic material comprises a circular plasmid of DNA, or a linear fragment of DNA, or RNA transcribed in vitro;

Preferably, the genetic material is a recombinant vector capable of transcribing the guide RNA and expressing the Cas protein;

the guide RNA is RNA with a palindromic structure formed by combining crRNA and tracrRNA through partial base pairing;

the crRNA comprises an RNA fragment capable of complementary binding to a functional segment;

preferably, the method of introducing the genetic material into the wheat cell or tissue of interest comprises a gene gun method, an agrobacterium infection method, a PEG-induced protoplast method, an electrode method, a silicon carbide fiber-mediated method, or a vacuum infiltration method;

preferably, the cells include any cell that can serve as an infusion recipient and can be regenerated into a whole plant by tissue culture; the tissue is any tissue which can be used as an introduction receptor and can be regenerated into a complete plant through tissue culture;

preferably, the cells comprise protoplast cells or suspension cells; the tissue comprises callus, immature embryo, mature embryo, leaf blade, stem tip, young ear or hypocotyl.
9. The method according to any one of claims 6 to 8, wherein the wheat of interest comprises diploid wheat, tetraploid wheat or hexaploid wheat;

preferably, the hexaploid wheat comprises commonly cultivated hexaploid wheat;

Preferably, the commonly cultivated hexaploid wheat comprises a kenaf wheat.
10. The TaRVR1 protein or the biological material in the use of any one of claims 1 to 5, or the method of wheat breeding of any one of claims 6 to 9.