CN112226455A

CN112226455A - Rice grain length and grain weight related protein, and coding gene and application thereof

Info

Publication number: CN112226455A
Application number: CN201910567153.7A
Authority: CN
Inventors: 程佑发; 周芳芳; 凌艳霞
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2021-01-15
Anticipated expiration: 2039-06-27
Also published as: CN112226455B

Abstract

The invention discloses a rice grain length and grain weight related protein, and a coding gene and application thereof. According to the invention, the OsMOB1A gene is introduced into wild rice by using the recombinant vector to obtain the transgenic rice, compared with the wild rice, the OsMOB1A gene is overexpressed, the grain length of the rice is obviously increased, and the grain weight is obviously increased. Therefore, the OsMOB1A gene is related to the grain type, and a foundation is laid for improving the cultivation of transgenic plants with changed grain types. The invention has important theoretical significance and practical significance for further clarifying the plant grain development molecular mechanism and cultivating high-quality and high-yield new crop varieties by means of genetic engineering.

Description

Rice grain length and grain weight related protein, and coding gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a rice grain length and grain weight related protein, and a coding gene and application thereof.

Background

Rice is one of the most important food crops in the world, and more than half of the world population takes rice as staple food. In recent years, with the increasing severity of problems such as reduced cultivated land area, energy shortage, climate change and increase of world population, the problem of food safety is getting more and more serious. Therefore, increasing the yield of rice and improving the quality of rice have become important research targets for plant breeding in China. Three major factors of rice yield: the number of single plant ears, the number of grains per ear and the grain weight. The grain type traits of rice include grain length, grain width, grain thickness and grain length-width ratio, and are positively correlated with grain weight, and are determinants of grain weight.

At present, nearly 60 genes are reported in research on genes and QTLs involved in regulation of rice grain type or grain weight. The genes closely related to the size variation of rice grains comprise grain width genes GW2, GS5, GW8, GW5/qSW5 and GW 7; grain length genes GL3.1, GS3, GS2, GLW7 and the like.

GW2 encodes a RING-type E3 ubiquitin ligase, is located in cytoplasm, and regulates the width of grains. GW2 is degraded by anchoring its substrate to the proteasome, thereby negatively regulating cell division. The loss-of-function mutation of the gene enables a substrate which is supposed to be degraded not to be specifically recognized, so that cell division of the glume is activated, the number of glume cells is increased, and the width of the glume is increased. In addition, the filling rate and the endosperm of the grains are improved, and finally, the grain width, the grain weight and the yield are improved. GS5 encodes a tryptophan carboxypeptidase, the high expression of the gene promotes cell division, increases the cell number, and the up-regulation of the expression of cell cycle related genes is a positive regulation factor of rice grain size. A recent study reported that GS5 has a competitive inhibitory effect on the interaction between OsBAK1-7 and OsMSBP1, indicating that GS5 may affect BR signals. GW8 encodes a transcription factor OsSPL16 containing SBP structural domain, and regulates rice grain width. The research shows that GW8 can regulate cell cycle, regulate the expression of cell division related gene and increase cell proliferation. The high expression of the gene can promote cell division and grain filling, thereby increasing the grain width and yield of rice. GW5/qSW5 encodes a nuclear localization protein that interacts with polyubiquitin. GW5 may regulate rice grain width and grain weight through ubiquitin proteasome pathway. The GW5 protein is located on a cytoplasmic membrane and can interact with rice GSK2 (homologous protein of Arabidopsis BIN2 kinase) and inhibit the activity of rice GSK2 kinase, so that the accumulation of non-phosphorylated proteins of downstream BZR1 and DLT transcription factors is increased, the downstream gene expression of brassinolide is regulated, and the change of the seed type is caused. GW7 encodes a TONNEAU recruiting protein, homologous to the C-terminal human centrosomal protein. Overexpression of the GW7 gene can produce elongated kernels, likely as a result of inhibition of transverse cell division due to promotion of longitudinal cell division of the kernel. GL3.1/qGL3 encodes a protein phosphatase with a Kelch repeat domain (OsPPKL1) by dephosphorylating the Cyclin-T1; 3 inhibiting cell division of glume, thereby negatively regulating the length of the kernel. GL3 contains two Kelch domains, and the rare allelic variation of aspartic acid at position 364 of the conserved AVLDT region in the second Kelch domain to glutamic acid results in a longer grain. Recent studies have shown that qGL3 is a homologous protein of the Arabidopsis BSU1 gene. BSU1 was positively regulating BR signaling in arabidopsis, while overexpression of qGL3 in rice produced a phenotype of BR loss-of-function mutations. qGL3 was able to interact with rice OsGSK3 kinase (a homologous protein of Arabidopsis BIN2 gene) and dephosphorylate OsGSK3 and stabilize its state, indicating that qGL3 inhibits BR signal by regulating phosphorylation and stability of OsGSK 3. GS3 encodes a transmembrane protein consisting of 232 amino acids, and controls the number of glume cells to negatively regulate the grain length. The protein comprises 4 domains: one organ size-regulating domain OSR, one transmembrane domain M, one cysteine-rich homologous region T of the tumor necrosis factor receptor family and one von Willebrand factor C VWFC. Where the OSR domain is a key domain for GS3 to perform its function, deletion of the OSR domain results in a long-grained phenotype. GS2 encodes a transcription activator OsGRF4 positioned in a nucleus, and the overexpression of the gene can promote cell division and expansion, thereby obviously improving the grain weight and the yield of rice. GL2/GS2 can interact with a negative regulatory factor GSK2 in a BR signal transduction pathway, the transcriptional activation activity of the GL2/GS2 is inhibited by GSK2, and the expression of the GL2/GS2 is regulated by OsmiR 396. GS2 can also interact with a transcription coactivator OsGIF1/2/3, and the overexpression of OsGIF1 increases the size and yield of grains. GLW7 encodes a transcription factor osppl 13 that is unique in plants. High expression of GLW7 increased kernel length and yield by promoting cell elongation, while the loss-of-function mutant produced short kernels. Furthermore, GLW7 can bind to the promoters of SRS5 and DEP1, promoting their expression, but their specific genetic interaction is still unknown.

Although many genes regulating seed grain type have been cloned, their genetic regulatory pathway network is not clear, and the correlation between them is still under further study. Therefore, cloning the rice grain type gene is of great significance for analyzing the genetic regulation mechanism of the rice grain type related gene.

Disclosure of Invention

The invention aims to provide a rice grain length and grain weight related protein, and a coding gene and application thereof.

In a first aspect, the invention provides an application of OsMOB1A protein in regulation and control of plant grain length and/or grain weight;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The OsMOB1A protein is derived from rice.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In a second aspect, the invention provides an application of the OsMOB1A protein-related biomaterial in regulation and control of plant grain length and/or grain weight; the OsMOB1A protein is a protein shown in any one of the above (A1) - (A4).

The related biological material is a nucleic acid molecule capable of expressing the OsMOB1A protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

The above application is embodied as follows:

the activity and/or expression of the OsMOB1A protein or the coding gene thereof in the plant are improved, and the length of plant grains is increased;

the activity and/or expression of the OsMOB1A protein or the coding gene thereof in the plant is improved, and the plant grain weight is increased.

In a third aspect, the method for protecting and cultivating the plant variety with the improved grain length and/or grain weight comprises the step of improving the expression and/or activity of OsMOB1A protein in a receptor plant; the OsMOB1A protein is a protein shown in any one of the above (A1) - (A4).

Further, the method for protecting and cultivating the transgenic plant comprises the following steps: introducing a nucleic acid molecule capable of expressing OsMOB1A protein into a receptor plant to obtain a transgenic plant; the transgenic plant has kernel length larger than that of a receptor plant and/or kernel size larger than that of the receptor plant; the OsMOB1A protein is a protein shown in any one of the above (A1) - (A4).

The "introducing into a recipient plant a nucleic acid molecule capable of expressing an OsMOB1A protein" is carried out by introducing into the recipient plant a recombinant expression vector containing a gene encoding the OsMOB1A protein.

The recombinant expression vector containing the coding gene of the OsMOB1A protein can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions.

When a recombinant plant expression vector is constructed by using the gene encoding the OsMOB1A protein, any one of an enhanced promoter or a constitutive promoter (e.g., cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin promoter from maize (Ubiquitin)) or a tissue-specific expression promoter (e.g., seed-specific expression promoter) may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters. In addition, when a plant expression vector is constructed using a gene encoding the OsMOB1A protein, an enhancer, including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence in order to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the invention, the recombinant expression vector can be specifically a recombinant expression vector obtained by replacing a fragment between BamHI and KpnI enzyme cutting sites of a pUN1301 vector with a DNA molecule shown in a sequence 2 in a sequence table.

In the above method, the introduction of the recombinant expression vector carrying the coding gene of the OsMOB1A protein into the recipient plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also transgenic progeny thereof.

The "coding gene of OsMOB1A protein" is a DNA molecule described in any one of the following items:

(B1) a DNA molecule shown in a sequence 2 of a sequence table;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the OsMOB1A protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the OsMOB1A protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a fourth aspect, the invention provides the use of an OsMOB1A protein or a biological material related thereto, or any of the methods described above, in plant breeding;

the related biological material is a nucleic acid molecule capable of expressing the OsMOB1A protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; the OsMOB1A protein is a protein shown in any one of the above (A1) - (A4).

The breeding aims to breed plants with long and/or large grain.

In the above aspects, the plant is (C1) or (C2) or (C3):

(C1) a dicot or monocot;

(C2) a gramineous plant;

(C3) a rice plant.

The rice can be rice with 11 # Zhonghua.

Experiments prove that the OsMOB1A gene is introduced into wild rice by using the recombinant vector to obtain transgenic rice, compared with the wild rice, the OsMOB1A gene is over-expressed, the grain length of the rice is obviously increased, and the grain weight is obviously increased. Therefore, the OsMOB1A gene is related to the grain type, and a foundation is laid for improving the cultivation of transgenic plants with changed grain types. The invention has important theoretical significance and practical significance for further clarifying the plant grain development molecular mechanism and cultivating high-quality and high-yield new crop varieties by means of genetic engineering.

Drawings

FIG. 1 shows the Real-timePCR expression identification of transgenic rice.

FIG. 2 is a phenotypic observation of OsMOB1A overexpression transgenic rice.

FIG. 3 shows the statistical data of grain length and thousand grain weight of OsMOB1A overexpression transgenic rice.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

pUN1301 vector: described in the literature "ZhangC, XuY, GuoS, ZhuJ, huangq, LiuH, WangL, LuoG, WangX, chongk. dynamic aerosol rassisterodependent oil dispersed porous genetic.2012, 8: e 1002686", publicly available from plant research institute of chinese academy of sciences.

Middle flower No. 11 rice: described in the literature, "Nichongchong", New cultivar of rice anther-Zhonghua No. 11, agricultural science and technology Commission, 1989(04):42 ", publicly available from the institute of plants of Chinese academy of sciences.

Example 1 obtaining of OsMOB1A protein and Gene encoding the same

Total RNA of the Zhonghua No. 11 rice seedling is extracted and is reversely transcribed into cDNA. Through a large number of sequence analysis, expression analysis and functional verification, a DNA coding sequence is found from cDNA, as shown in sequence 2 of the sequence table, and the protein coded by the DNA coding sequence is shown in sequence 1 of the sequence table.

The protein shown in the sequence 1 of the sequence table is named as OsMOB1A protein. The gene coding the OsMOB1A protein is named as OsMOB1A gene, and the open reading frame is shown as sequence 2 in the sequence table.

Example 2 construction of recombinant expression vector and obtaining of recombinant Agrobacterium

Construction of recombinant expression vector

The DNA molecule shown in the sequence 2 of the sequence table is replaced by the fragment between the BamHI and KpnI enzyme cutting sites of the pUN1301 vector, and the recombinant expression vector pUN1301-OsMOB1A is obtained (the sequencing is verified).

II, obtaining recombinant agrobacterium

1. The recombinant plasmid pUN1301-OsMOB1A was introduced into Agrobacterium EH105 to obtain recombinant Agrobacterium, which was designated recombinant Agrobacterium EH105/pUN1301-OsMOB 1A.

2. The pUN1301 vector is introduced into agrobacterium EH105 to obtain recombinant agrobacterium, and the recombinant agrobacterium is named as recombinant agrobacterium EH105/pUN 1301.

Example 3 functional verification of OsMOB1A protein in Rice

First, obtaining of OsMOB1A transgenic rice plant

The recombinant Agrobacterium EH105/pUN1301-OsMOB1A prepared in example 2 was transferred to the callus of the embryo of Zhonghua No. 11 rice (Oryzasatival. cvZhonghua11, hereinafter referred to as wild rice) through an Agrobacterium-mediated rice genetic transformation system, and a transgenic plant was obtained by induction, preculture, infection, co-culture, selection of hygromycin-resistant callus, differentiation, rooting, and seedling transplantation. 10 lines of T0 transgenic OsMOB1A rice plants are obtained by transformation. The method comprises the following specific steps:

1) induction and subculture of rice mature embryo embryonic callus: and selecting the wild type middle flower No. 11 rice seeds with plump seeds and glumes removed, and adding 70% ethanol for surface disinfection for 5 min. And then sterilizing with 20% sodium hypochlorite (NaClO) for 20min, washing with sterilized double distilled water solution for 4-5 times after sterilization, transferring to an induction culture medium NB2 after sterile filter paper is absorbed, cutting to make the section contact with the culture medium and make the embryo not be pressed into the culture medium, and lightly placing on the surface of the culture medium. After dark culture at 25 ℃ for 4-7 days, the primary callus is induced from the scutellum of the mature embryo, and the new bud is cut off by tweezers in a clean bench. Culturing at 25 deg.C in dark for 4-5 weeks. In order to obtain a better callus, the medium was changed two weeks later to ensure sufficient nutrient supply. Selecting light yellow compact embryogenic callus, transferring to subculture medium NB1, and dark culturing at 25 deg.C for 2-3 weeks; the pale yellow dense callus pieces were then picked and transferred to fresh NB1 subculture medium and cultured in the dark for 2-3 weeks at 25 ℃. After subculture for 2-4 times, selecting the yellow compact callus with good growth, transferring to a new NB1 culture medium, and continuously culturing in dark at 25 ℃ for 4-6 days to obtain the final product for agrobacterium infection transformation.

2) Activating agrobacterium strains: recombinant Agrobacterium EH105/pUN1301-OsMOB1A was streaked on YEP solid medium containing 50mg/L kanamycin, and cultured in an inverted state at 28 ℃ for 2-3 days. The recombinant agrobacterium tumefaciens EH105/pUN1301-OsMOB1A monoclonal colonies were picked and inoculated into 5mL YEP liquid culture medium containing 50mg/L kanamycin, shaking culture was carried out at 28 ℃ and 200rpm until late logarithmic growth, 0.5mL of bacterial liquid was sucked and transferred into 50mL YEP liquid culture medium containing 50mg/L kanamycin, and shaking culture was continued at 28 ℃ and 200rpm until OD600 was about 0.5. Transferring the bacterial liquid into a sterilized 50mL centrifuge tube, centrifuging at room temperature and 4,000rpm for 10min, discarding the supernatant, and resuspending the thallus by using an isometric liquid culture medium AAM-AS to prepare infection callus.

3) Co-culturing and transforming the callus and the agrobacterium: adding AAM-AS re-suspended bacteria solution into the pre-cultured callus blocks for 4-6 days, and performing dip dyeing for 20 min. The impregnated callus pieces were blotted dry with sterile absorbent paper, transferred to NB2C coculture medium with a layer of filter paper, and cultured in the dark at 25 ℃ for 2-4 days.

4) Screening and culturing callus tissues: the co-cultured callus was transferred to the selection medium NBS1 and cultured in the dark at 25 ℃ for 2 weeks. Most of the callus blocks become brown in the screening process, and after the transgenic callus blocks become brown, yellow-white callus, namely resistant callus, grows on the callus edges again. Resistant callus pieces were picked and transferred to selection medium NBS2, cultured at 25 ℃ in the dark and selection continued for 2 weeks.

5) Inducing differentiation and seedling formation of the callus: the yellow resistant callus was transferred to pre-differentiation medium RE1 and cultured in the dark for 1 week and then in the light for 2-3 weeks. Transferring the adventitious bud generated by differentiation to a differentiation medium RE2, and culturing for 2-3 weeks under illumination to differentiate into a plant. When the plant grows to 2-3cm, the plant can be transferred to 1/2MS culture medium containing rooting culture medium, and the plant can be cultured for about 2 weeks by illumination. When the seedlings grow to about 10cm, the sealing film is opened, and a small amount of distilled water is added. Hardening the seedlings under illumination for 5-7 days, and transplanting the tissue culture seedlings into soil for growth.

The media used in the above procedure are shown in Table 1.

TABLE 1 culture Medium formulation

Second, obtaining empty carrier rice plant

And (3) replacing the recombinant agrobacterium EH105/pUN1301 with the recombinant agrobacterium EH105/pUN1301-OsMOB1A, and operating according to the step one to obtain a transgenic empty vector rice plant.

Identification of OsMOB1A transgenic rice plant

Taking leaves of T0 generation transgenic OsMOB1A rice plants growing for 3-4 weeks, extracting genome DNA, taking the genome DNA as a template, adopting a primer pair consisting of a primer 1301-F and a primer OsMOB1A-R for PCR amplification, if a strip with the size of about 696bp is amplified by PCR, indicating that the plants are positive transgenic plants, and identifying 10 strains of T0 generation transgenic OsMOB1A rice plants in total. Transplanting the positive trans-OsMOB 1A rice to a greenhouse for cultivation, harvesting seeds of each plant to obtain T1 transgenic seeds, then propagating to obtain homozygous T2 transgenic seeds, and selecting T2 trans-OsMOB 1A rice with numbers of 1(OE1), 9(OE9) and 10(OE10) as materials in the subsequent experiments.

1301-F：5’-GCTACGAGTGGGACAACAAA-3’；

OsMOB1A-R:5’-GTATGGAATAATCGATTCGATG-3’。

Thirdly, mRNA expression quantity detection of OsMOB1A transgenic rice plant

And (3) the plant to be detected: rice wild type middle flower 11, number 1(OE1), 9(OE9), 10(OE10) T2 generation OsMOB1A rice plant and T2 generation empty carrier plant.

1. Extracting the total RNA of the seedling leaves in the trefoil stage of the plant to be detected, and carrying out reverse transcription to obtain cDNA.

2. And (3) carrying out real-time quantitative RT-PCR detection by taking the cDNA obtained in the step (1) as a template. The expression level of the OsMOB1A gene is detected by adopting a primer pair consisting of a primer OsMOB1A-qRT-F and a primer OsMOB1A-qRT-R, and the internal reference gene OsACTIN is detected by adopting a primer pair consisting of a primer OsACTIN-qRT-F and a primer OsACTIN-qRT-R.

OsMOB1A-qRT-F：5’-GATGCCACTCTTGGCAGTGG-3’；

OsMOB1A-qRT-R：5’-GTTGGCCTTCAATCCAGTCC-3’。

OsACTIN-qRT-F：5’-AGGCCAATCGTGAGAAGATGAC-3’；

OsACTIN-qRT-R：5’-AGGTCGAGACGAAGGATAGCAT-3’。

Each strain was tested in 3 biological replicates per material.

The results are shown in FIG. 1. The results show that the expression level of OsMOB1A gene in T2 transgenic OsMOB1A rice lines OE1, OE9 and OE10 is obviously up-regulated compared with wild rice (WT), which indicates that OsMOB1A gene is successfully expressed in large amount in transgenic rice. No significant difference exists between the T2 generation empty vector plants and the OsMOB1A gene expression level of wild rice.

Phenotype observation of rice plant with OsMOB1A transgenic gene

Planting the plant to be detected in a plant research institute test field of Chinese academy of sciences, and after heading and grouting of rice, performing character investigation and photographing on the grain type of the rice.

20 plants per strain were counted per material.

The results are shown in FIG. 2. The results show that kernel length of OsMOB1A rice transformed by T2 generation is remarkably increased compared with wild type rice. There was no significant difference between wild type rice (WT) and empty vector transgenic rice of the T2 generation.

Fifth, agronomic character statistics of OsMOB1A transgenic rice plant

And collecting mature seeds of the plants to be tested, harvesting and drying, and counting the average thousand seed weight of 20 plants and the average length of seeds.

The results are shown in FIG. 3 and Table 2. Fig. 3A shows the length of the kernel and fig. 3B shows the thousand kernel weight of 1000 full rice kernels.

TABLE 2 statistics of grain type-related traits in wild-type and transgenic plants

The results show that compared with wild rice, the grain length and thousand kernel weight of OsMOB1A transgenic rice of the T2 generation are obviously increased, and the over-expression of the OsMOB1A gene can improve the grain length and thousand kernel weight. There was no significant difference between wild type rice (WT) and empty vector transgenic rice of the T2 generation.

Sequence listing

<110> institute of plant of Chinese academy of sciences

<120> protein related to grain length and grain weight of rice grains, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 214

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 1

Met Ser Leu Phe Gly Leu Gly Arg Asn Gln Lys Thr Phe Arg Pro Lys

1 5 10 15

Lys Ser Ala Pro Ser Gly Ser Lys Gly Ala Gln Leu Arg Lys His Ile

20 25 30

Asp Ala Thr Leu Gly Ser Gly Asn Leu Arg Glu Ala Val Arg Leu Pro

35 40 45

Pro Gly Glu Asp Ile Asn Glu Trp Leu Ala Val Asn Thr Val Asp Phe

50 55 60

Phe Asn Gln Val Asn Leu Leu Tyr Gly Thr Leu Ala Glu Phe Cys Thr

65 70 75 80

Pro Glu Ser Cys Pro Thr Met Thr Ala Gly Pro Lys Tyr Glu Tyr Arg

85 90 95

Trp Ala Asp Gly Val Gln Ile Lys Lys Pro Ile Glu Val Ser Ala Pro

100 105 110

Lys Tyr Val Glu Tyr Leu Met Asp Trp Ile Glu Gly Gln Leu Asp Asp

115 120 125

Glu Ser Ile Phe Pro Gln Lys Leu Gly Thr Pro Phe Pro Pro Asn Phe

130 135 140

Lys Glu Val Val Lys Thr Ile Phe Lys Arg Leu Phe Arg Val Tyr Ala

145 150 155 160

His Ile Tyr His Ser His Phe Gln Lys Ile Val Ser Leu Lys Glu Glu

165 170 175

Ala His Leu Asn Thr Cys Phe Lys His Phe Ile Leu Phe Thr Thr Glu

180 185 190

Phe Gly Leu Ile Asp Lys Lys Glu Leu Ala Pro Leu Gln Glu Leu Ile

195 200 205

Glu Ser Ile Ile Pro Tyr

210

<210> 2

<211> 645

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 2

atgagtctct tcgggctcgg ccggaatcag aagacgttcc gtcccaagaa gagtgctcct 60

tcaggaagca agggtgcaca gcttcggaag cacatagatg ccactcttgg cagtggaaac 120

cttagggaag ctgtaaggtt gccgcctgga gaagatatca atgaatggct ggctgtgaat 180

actgtggatt tctttaacca agttaacctg ctgtacggca cactcgctga gttctgcaca 240

cctgagagct gcccaacaat gactgctggc ccaaagtatg agtacagatg ggctgatggt 300

gtacagataa agaagccgat agaagtatca gcaccaaaat atgtggagta cctaatggac 360

tggattgaag gccaacttga tgatgaatct atatttcccc aaaagcttgg cacaccattc 420

ccgccaaact tcaaggaggt tgtaaagaca attttcaagc gcttgtttcg tgtttatgcc 480

cacatatatc actcccattt tcagaagatt gtcagcctca aggaggaggc ccatcttaac 540

acgtgcttca agcacttcat cctgtttaca actgaatttg gcctgattga caagaaggag 600

ctggctccac tccaggagct catcgaatcg attattccat actga 645

Claims

The application of the OsMOB1A protein in regulating and controlling the length and/or weight of plant seeds;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
The application of the OsMOB1A protein-related biological material in regulating and controlling the length and/or weight of plant grains;

the related biological material is a nucleic acid molecule capable of expressing the OsMOB1A protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
3. Use according to claim 1 or 2, characterized in that:

the specific embodiment of the application is as follows:

the activity and/or expression of the OsMOB1A protein or the coding gene thereof in the plant are improved, and the length of plant grains is increased;

the activity and/or expression of the OsMOB1A protein or the coding gene thereof in the plant is improved, and the plant grain weight is increased.
4. A method for breeding a plant variety with improved kernel length and/or improved kernel weight, which comprises the step of improving the expression and/or activity of OsMOB1A protein in a receptor plant;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
5. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing OsMOB1A protein into a receptor plant to obtain a transgenic plant; the transgenic plant has kernel length larger than that of a receptor plant and/or kernel size larger than that of the receptor plant;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
6. The method of claim 5, wherein: the "introducing into a recipient plant a nucleic acid molecule capable of expressing an OsMOB1A protein" is carried out by introducing into the recipient plant a recombinant expression vector containing a gene encoding the OsMOB1A protein.
7. The method of claim 6, wherein: the "coding gene of OsMOB1A protein" is a DNA molecule described in any one of the following items:

(B1) a DNA molecule shown in a sequence 2 of a sequence table;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the OsMOB1A protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the OsMOB1A protein.
Use of an OsMOB1A protein or a biological material related thereto, or a method according to any one of claims 4 to 7, in plant breeding;

the related biological material is a nucleic acid molecule capable of expressing the OsMOB1A protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the OsMOB1A protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
9. The use of claim 8, wherein: the breeding aims to breed plants with long and/or large grain.
10. Use or method according to any of claims 1 to 9, wherein: the plant is (C1) or (C2) or (C3):

(C1) a dicot or monocot;

(C2) a gramineous plant;

(C3) a rice plant.