CN117305354A - Rice OsMYB-Hv1 gene and encoding protein and application thereof - Google Patents
Rice OsMYB-Hv1 gene and encoding protein and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention relates to the technical field of plant genetic engineering, in particular to cloning of a rice OsMYB-Hv1 gene and application thereof in increasing the grain length, grain weight and yield of rice, wherein the nucleotide sequence of the OsMYB-Hv1 gene is SEQ ID NO.1 in a sequence table; the nucleotide sequence of the protein coding region is SEQ ID NO.2 in the sequence table; the coded protein sequence is shown as SEQ ID NO.3 in the sequence table. The complete protein coding region sequence of the OsMYB-Hv1 gene is placed under the control of a constitutive promoter 35S to be over-expressed in rice, and the ear length, grain weight and yield of transgenic plants are obviously increased, so that the OsMYB-Hv1 gene has potential use value in genetic improvement of rice. The invention describes the role of the OsMYB-Hv1 gene in increasing spike length, grain weight and yield.
Description
[ field of technology ]
The invention belongs to the field of genetic engineering, and in particular relates to a rice OsMYB-Hv1 gene and a coding protein and application thereof
[ background Art ]
Rice (Oryza sativa L.) is one of the important food crops in our country. The rice spike type is always one of important contents of rice genetic and breeding researches, and is an important character affecting the rice yield. In recent years, with the completion of rice whole genome sequencing work and the development of rice molecular biology and functional genomics, a plurality of genes related to regulating spike development are positioned and cloned by utilizing a genetic population and mutant method. The cloned genes remain a minority relative to the large number of localized spike length QTLs. Ghd7 and Ghd7.1 encode CCT structural domain protein to affect spike number, plant height and heading period. spd6 (smallpanicle anddwarfness) codes for leucine carboxymethyltransferase, and near isogenic lines with spd6 have significantly shorter ears, reduced grain number, smaller grain and dwarfed plants. SP1 (short Panicle 1) encodes PTR2 (Peptide Transporter 2) domain and regulates rice spike length by regulating activity of spike meristem. DEP1 (Dense andErectPanicle 1), DEP2 and DEP3 control rice stand and spike density. LP (Largepanicle) the large ear is controlled to increase glume flowers and branches and resist lodging. OsCD1 encodes a cellulose-like synthase (cellulose synthase-like) D protein, and mutations in OsCD1 result in reduced ear length. OsARG encodes arginase (arginine hydrolysis enzyme), and in the nglf-1 mutant, osARG encoded protein has a shortened spike length due to premature termination, whereas overexpression of wild-type OsARG restores the phenotype. OsPIN5b encodes an auxin export vector-like (auxinefflux carrier-like) gene, and overexpression of OsPIN5b reduces spike length, whereas reduction of OsPIN5b increases spike length. OsGRF4 encodes a growth regulator, and high expression of OsGRF4 results in increased spike length, increased grain size and reduced grain size. Inhibiting the activities of miR398 and miR159 respectively increases and reduces the spike length.
Although some genes related to the development of rice ears are identified at present, the specific mode of action is still unknown, and the number of reported genes with the function of regulating the development of ears is not abundant enough. Therefore, the rice spike regulating gene is further excavated and is subjected to intensive research, so that the method has important significance for elucidating the genetic mechanism of rice spike development, and simultaneously provides a sufficient theoretical basis for rice high-yield breeding.
[ invention ]
The invention aims to provide a rice OsMYB-Hv1 gene capable of regulating and controlling rice ears, a coded protein and application thereof. The rice spike length, grain weight and yield are obviously increased through the protein coding region of the over-expressed OsMYB-Hv1 gene, and a novel method for carrying out genetic improvement on rice by utilizing the OsMYB-Hv1 gene in agricultural production is provided.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
the invention provides a rice OsMYB-Hv1 protein, which is any one of the following proteins 1) or 2):
1) Has an amino acid sequence shown as SEQ ID NO. 3;
2) A protein which has an amino acid sequence shown as SEQ ID NO.3 and is replaced, deleted or inserted by one or more amino acids, but has the same function as the OsMYB-Hv1 protein shown as SEQ ID NO. 3.
Further, the present invention also provides a gene encoding the aforementioned protein.
Specifically, it has any one of the following nucleotide sequences 1) to 3):
1) A nucleotide sequence shown as SEQ ID NO.1;
2) A nucleotide sequence as shown in SEQ ID NO.1 with one or more nucleotide substitutions, deletions or insertions;
3) Under stringent conditions with a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in 1) or 2) and encoding the same functional protein.
Further, the present invention also provides a vector containing the gene, and a host cell containing the gene or the vector. The vector comprises a plant expression vector pCaMU or a derivative vector thereof and the like; the host cells include Agrobacterium cells, E.coli cells, and the like.
The vectors and host cells are understood to be those used by the person skilled in the art in the transgenic process and are not capable of developing into a plant individual. However, with the development of technology, the choice of the vector and the host cell may be changed, or the application field of the vector and the host cell may be related to the application field of the non-transgenic purpose, but the vector and the host cell are all within the protection scope of the invention as long as the vector contains the gene of the invention or the vector of the invention.
In addition, the invention provides application of the gene in regulating and controlling the rice spike type. The application comprises the application of the OsMYB-Hv1 protein or the coding gene in improving the grain length, grain weight and yield of rice.
Experiments show that the ear length of the OsMYB-Hv1 mutant is increased by 16.9% compared with that of a wild type.
In view of the above, the invention also provides application of the OsMYB-Hv1 gene in preparing transgenic plants.
The preparation of the transgenic plant is a conventional technical means in the field, the invention is not limited in any way, and the technical scheme for carrying out rice transgenic breeding by utilizing the gene is within the protection scope of the invention.
Therefore, the invention provides application of the OsMYB-Hv1 protein of rice or the encoding gene thereof or the biological material containing the encoding gene in regulating and controlling the spike length, grain weight and yield of rice.
The invention provides application of rice OsMYB-Hv1 protein or a coding gene thereof or a biological material containing the coding gene in increasing rice spike length, grain weight and yield.
The invention provides application of rice OsMYB-Hv1 protein or a coding gene thereof or a biological material containing the coding gene in improving rice spike-type germplasm resources.
The invention provides application of rice OsMYB-Hv1 protein or a coding gene thereof or a biological material containing the coding gene in preparation of long-spike, long-grain, high-grain-weight or high-yield transgenic rice.
Experiments further show that the OsMYB-Hv1 gene can be transformed into rice variety G1025, so that the ear length, grain weight and single plant yield can be increased.
The rice OsMYB-Hv1 gene mutant gene provided by the invention is 636 th base from CDS initiation codon of rice OsMYB-Hv1 (LOC_Os09G 36730) gene, and is mutated from G to C, namely the sequence of the rice OsMYB-Hv1 gene mutant gene is shown as SEQ ID NO. 2.
The invention provides application of the mutant gene of the OsMYB-Hv1 gene of rice or a biological material containing the gene in transgenic rice for increasing the ear length, grain weight and yield.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers the OsMYB-Hv1 gene and the encoding protein thereof which have influence on the ear length, grain weight and yield of rice for the first time, and verifies that the gene has the function of regulating the ear length, grain weight and yield through experiments. Mutation of the OsMYB-Hv1 gene results in significant increase of spike length, grain weight and yield. The technical scheme provided by the invention provides a new direction for rice breeding and transgenic rice preparation, and the transgenic rice transformed by the gene is constructed, so that the rice yield can be improved.
[ description of the drawings ]
FIG. 1 shows the phenotype of wild type IR24 and OsMYB-Hv1 mutant K1561, wherein A is a plant phenotype, and B is an ear length phenotype.
FIG. 2 shows the OsMYB-Hv1 gene localization, structure and transgenic phenotype. Wherein, (a), (b) is gene targeting, (c), (d), (e), (f) is transformation mutant CDS sequence (LOC_Os 09G 36730-K1561), wild-type CDS sequence LOC_Os09G36730-G1025 and acceptor wild-type G1025 plant, ear, grain phenotype. (G, (h), (i), (j) are statistical comparisons of transformation mutant CDS sequences (LOC_Os 09G 36730-K1561), wild-type CDS sequences LOC_Os09G36730-G1025 and receptor wild-type G1025 spike length, thousand kernel weight, individual plant weight and kernel length, respectively.
FIG. 3 nucleotide (a) and amino acid (b) sequence differences of mutant K1561 and wild-type G1025 candidate gene LOC_Os09G36730 (OsMYB-Hv 1).
FIG. 4 sequence differences between nucleotide (a) and amino acid (b) of mutant K1561 and wild-type G1025 candidate gene LOC_Os09G 36750.
FIG. 5 is a schematic diagram of the structure of the vector pCaMU according to the present invention.
FIG. 6 shows the analysis of the expression pattern of the OsMYB-Hv1 gene in various tissues of rice. R, leaf; s, stems; l, leaves; YP, young ear (3-5 cm); MP, mature spike (10-15 cm).
[ detailed description ] of the invention
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: obtaining and phenotyping the OsMYB-Hv1 mutant.
The IR24 is taken as an acceptor, the small grain wild rice is taken as a donor, and a part of imported material K1561 with the spike length remarkably higher than that of parents is obtained by distant hybridization and backcrossing and combined with embryo rescue methods. K1561 spike length is 30.3cm, IR24 spike length is 25.3cm, and small wild rice spike length is 15.7cm. (Guo Si construction of an introduction line of Oryza sativa L.and identification of traits [ doctor's thesis ]. Wuhan, university of agriculture, china, 2009).
Example 2: obtaining of OsMYB-Hv1 Gene of Rice
A new, major spike length QTLPL9 (Zhu Z, li X, weiY, guo S, shaA. Identification ofa novel QTL forpanicle length from wild rice (Oryza minuta) by specific locus amplified fragment sequencing and high density genetic mapping. Front Plant Sci.2018, 9:1492) was located on chromosome 9 using the recombinant inbred population constructed by crossing K1561 with a short spike restorer G1025, and a near isogenic line backcross population and encrypted markers were further constructed by backcrossing to locate PL9 within the 283.7kb region between RM24739 and RM24751 (FIG. 2 a). According to the gene annotation information provided by the rice genome annotation website (http:// rice. Plant biology. Msu. Edu /), the interval has 46 non-transposon genes, and 36 genes encode proteins with annotated functions. Gene expression of these 36 genes was analyzed by RT-PCR in different tissues (root, stem, leaf, young ear, mature ear) of G1025 and K1561, and 17 genes were found to be expressed in at least one tissue of G1025 or K1561, with 12 genes being expressed simultaneously in G1025 and K1561 (Table 1). Comparing the differences in G1025 and K1561 of CDS sequences of these 12 genes, it was found that LOC_Os09G36730 had a SNP (SEQ ID NO. 2) and LOC_Os09G 3679 had an Indel (FIG. 2b, FIG. 3a, FIG. 4 a). SNP in LOC_Os09g36730 is located 793 bases from the start codon in the genome (SEQ ID NO. 1), indel in LOC_Os09g36750 is located 930bp from the start codon in the genome (FIG. 2 b). The markers dCAP730 and Indel750 were developed from this SNP and Indel, respectively, and verified in the parental G1025, K1561 and 201 RIL populations, which found more than 90% identity between genotype and phenotype. Thus, these two genes were determined as candidate genes. The SNP G/C mutation in LOC_Os09G36730 in G1025/K1561 resulted in mutation of the amino acid arginine/serine, whereas the Indel of 621bp in LOC_Os09G36750 resulted in a change of 28 amino acids at the N-terminus (FIG. 3b, FIG. 4 b).
TABLE 1 tissue-specific expression and CDS sequence variation statistics of PL9 candidate genes
Example 3: transformation of wild rice by overexpression of candidate genes
For the purpose of functional complementation experiments, an overexpression vector was constructed from the Ubiquitin promoter plus the gene CDS. After amplifying the wild-type CDS sequence (LOC_Os 09G 36730-G1025), the mutant CDS sequence (LOC_Os 09G 36730-K1561), the LOC_Os09G 367550 wild-type CDS sequence (LOC_Os 09G 367550-G1025) and the mutant CDS sequence (LOC_Os 09G 367550-K1561) in LOC_Os09G36730, respectively, kpnI site was introduced through the 5' end and BstElI site was introduced into the pCaMU vector (FIG. 4).
The primers used are 730-F and 5'-CGCGGTGGAGCTCGGATGGGGAGGTCACCGTGC-3' respectively;
730-R:5’-ATTCGAGCTGGTCACTCATTTCATTTCCAAGCTTCTGAAG-3’；
750-F:5’-CGCGGTGGAGCTCGGATGATCCGGTTCATCCTGCTG-3’；
750-R:5’-ATTCGAGCTGGTCACTTATTCCAGCTTCTCAAGCTCACCC-3’；
the constructed complementary vector is transferred into agrobacterium EHA105 and infects the wild G1025 seed induced callus. 13, 17, 14, 11 independent transformants were obtained in T0 generation transgenic plants of LOC_Os09G36730-G1025, LOC_Os09G36730-K1561, LOC_Os09G 3679-G1025, LOC_Os09G 3679-K1561, respectively, wherein the ear length of the majority of the transgenic lines increased only when LOC_Os09G36730 sequences (OE-K1561) were overexpressed in the mutants (Table 2, FIGS. 2 c-e), thus ultimately determining LOC_Os09G36730 as a PL9 candidate gene. 3 lines with the longest spike length were selected from the over-expressed wild-type LOC_Os09G36730 CDS (OE-G1025) and mutant LOC_Os09G36730 CDS (OE-K1561) T0 transgenic lines, respectively, and were planted to the T3-substitution test spike length and other yield traits, and the OE-K1561 spike length, grain length, thousand grain weight and yield were found to be significantly increased (FIGS. 2 d-i), thus proving that the G/C mutation in LOC_Os09G36730 was able to increase the yield (SEQ ID NO. 2) by increasing the spike length, grain length and thousand grain weight.
LOC_Os09g36730 encodes the MYB-Hv1 protein (SEQ ID NO. 3) and is thus designated OsMYB-Hv1.
TABLE 2 transgenic lines and wild-type ear Length statistics
Example 4: rice OsMYB-Hv1 gene expression pattern
In order to determine the tissue expression mode of the OsMYB-Hv1 gene, a semi-quantitative RT-PCR method is adopted to detect the expression level of the gene in each tissue of rice, including roots, stems, leaves and young spikes (3-5 cm) and mature spikes (10-15 cm). The results show that the OsMYB-Hv1 gene is mainly expressed in stems, young ears and mature ears, the expression in K1561 young ears is stronger than that of G1025, and the difference in other tissues is not great (figure 6), which shows that the expression intensity of the OsMYB-Hv1 gene in different organs and parts is consistent with the functional effect of the gene.
The primers used for semi-quantitative RT-PCR were as follows:
OsMYB-Hv1-F：5’-ATGGGGAGGTCACCGTGCTGCGAG-3’；
OsMYB-Hv1-F:5’-TCATTTCATTTCCAAGCTTCTGAAGTCG-3’；
Actin-F：5’-AGAGGAAGTCAGCGGCTAAG-3’；
Actin-R:5’-CGCATACCATTGACAGTGGT-3’。
the foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and changes can be made by those skilled in the art without departing from the inventive concept herein.
Claims (4)
1. Application of rice OsMYB-Hv1 protein or encoding gene thereof or biological material containing encoding gene thereof in regulation and control of rice spike length, grain weight and yield; the amino acid sequence of the rice OsMYB-Hv1 protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene is shown as SEQ ID NO.1; the biological material is an expression cassette, a vector, a host bacterium or a host cell incapable of propagating into a plant individual.
2. Application of rice OsMYB-Hv1 protein or encoding gene thereof or biological material containing encoding gene thereof in increasing ear length, grain weight and yield; the amino acid sequence of the rice OsMYB-Hv1 protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene is shown as SEQ ID NO.1; the biological material is an expression cassette, a vector, a host bacterium or a host cell incapable of propagating into a plant individual.
3. Application of rice OsMYB-Hv1 protein or encoding genes thereof or biological materials containing encoding genes thereof in improving rice spike-type germplasm resources; the amino acid sequence of the rice OsMYB-Hv1 protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene is shown as SEQ ID NO.1; the biological material is an expression cassette, a vector, a host bacterium or a host cell incapable of propagating into a plant individual.
4. Application of rice OsMYB-Hv1 protein or encoding gene thereof or biological material containing encoding gene thereof in preparing transgenic rice with long spike, long grain, high thousand grain weight and high yield; the amino acid sequence of the rice OsMYB-Hv1 protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene is shown as SEQ ID NO.1; the biological material is an expression cassette, a vector, a host bacterium or a host cell incapable of propagating into a plant individual.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102936605A (en) * | 2007-10-29 | 2013-02-20 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
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