Protein related to rice grain traits, biological material thereof and application

Abstract

The present invention discloses a protein related to rice grain traits, its biological material and application, belonging to the field of biotechnology. The amino acid sequence of the protein is SEQ ID No.1. Experiments of the present invention show that overexpressing the coding gene of the aforementioned protein in wild-type Nipponbare significantly increases the grain length and width of crops, and further increases the biomass and yield of plants. It shows that the aforementioned protein provided by the present invention is of great significance for cultivating new high-yield rice materials with enlarged grains.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a protein related to rice grain traits, its biological material and application. Background Art

[0002] At present, the yields of main grains such as rice and wheat have approached their limits, and it has become very difficult to significantly increase grain yields through traditional breeding.

[0003] Rice grain shape is one of the most important factors determining yield. Grain weight is mainly determined by grain size (volume and filling degree). Grain shape size determines grain weight, which is determined by a specific three-dimensional structure: grain length, grain width, and grain thickness. Grain size is mainly determined by genetic factors and is strictly regulated by genetic factors, while filling is controlled by genetic and environmental factors. At present, multiple QTLs (quantitative trait loci) controlling rice grain size have been cloned. These QTLs can act on independent genetic pathways and mainly participate in the proteasome degradation, hormone, and G protein-mediated signal pathways together with other identified grain shape genes to regulate cell proliferation and cell elongation. The first cloned rice grain shape gene, GS3, is the main QTL controlling grain length and grain weight; a QTL affecting grain width, GS5, was detected using a double haploid population constructed from Zhenshan 97 (wide grain) and H94 (slender grain). This gene encodes a serine carboxypeptidase; qGL3 / qGL3.1 is a main QTL regulating grain shape, grain weight, and filling degree. This gene encodes a putative serine / threonine protein phosphatase. A series of genes such as GW2, GW5, GW8, GS2, and GL7 regulating grain size have been cloned successively.

[0004] With the cloning and research of more and more genes related to rice grain traits, it will provide rich gene resources for high-yield rice, improve the rice grain shape genes and their regulatory networks, and exert the best non-linear superposition effect of multiple gene polymerization on complex traits, thereby effectively achieving the directional improvement of high-yield and high-quality traits. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new gene related to grain traits. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a protein OsSTK38 related to rice grain traits, and the protein is any one of the following:

[0008] A1) A protein with an amino acid sequence of SEQ ID No. 1;

[0009] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1), having an identity of more than 80% with the protein shown therein and having the same function;

[0010] A3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid shown in A1) or A2).

[0011] The tag protein includes, but is not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0012] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein of the present invention by using known methods, such as directed evolution or site-directed mutagenesis. Those nucleotides that have been artificially modified and have an identity of 75% or more than 75% with the nucleotide sequence of the protein isolated from the present invention, as long as they encode the aforementioned protein and have the function of the aforementioned protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0013] The above-mentioned identity of 75% or more than 75% can be 80%, 85%, 90% or more than 95% identity.

[0014] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of the amino acid sequence, and then the identity value (%) can be obtained.

[0015] In this text, the identity of more than 80% may be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0016] The present invention also provides biological materials related to the aforementioned protein, which are any one of the following:

[0017] B1), a nucleic acid molecule encoding the aforementioned protein;

[0018] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0019] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0020] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0021] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0022] B6), a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

[0023] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0024] Among the above biological materials, the vector may be a plasmid, cosmid, phage or viral vector.

[0025] Among the above biological materials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0026] Among the above biological materials, the transgenic plant cell lines do not include propagation materials.

[0027] Among the above biological materials, the nucleic acid molecule described in B1) is a cDNA molecule or DNA molecule whose coding sequence is SEQ ID No.2.

[0028] Among the above-mentioned biomaterials, the expression cassette containing the nucleic acid molecule encoding the aforementioned protein (the gene expression cassette of the encoding gene of the aforementioned protein) refers to the DNA capable of expressing the encoding gene of the aforementioned protein in a host cell. This DNA may not only include a promoter for initiating the transcription of the encoding gene of the aforementioned protein, but also include a terminator for terminating the transcription of the encoding gene of the aforementioned protein. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters; tissue-, organ- and development-specific promoters, and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminator.

[0029] A recombinant vector containing the coding gene expression cassette of the aforementioned protein can be constructed using existing expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc., such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may further contain the 3′ untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3′ end of the mRNA precursor. For example, the non-translated regions transcribed at the 3′ end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos) and plant genes (such as the soybean storage protein gene) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (such as the GUS gene, the luciferase gene, etc.), a marker gene for antibiotics (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or a marker gene for anti-chemical reagents (such as an anti-herbicide gene), the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

[0030] In some specific embodiments of the present invention, the recombinant vector is obtained by replacing the SmaI and XbaI sites of the pCambia23A vector with the nucleotide shown in SEQ ID No.2.

[0031] The present invention also provides any one of the following applications of the aforementioned protein or the aforementioned biological material:

[0032] D1) Regulating the yield of plants;

[0033] D2) Preparing a product for regulating the yield of plants;

[0034] D3) Cultivating plants with increased yield;

[0035] D4) Preparing a product for cultivating plants with increased yield;

[0036] D5) Improving plants with increased yield or preparing a product for plants with increased yield;

[0037] D6) Regulating the grain length of plants;

[0038] D7) Preparing a product for regulating the grain length of plants;

[0039] D8) Cultivating plants with increased grain length;

[0040] D9) Preparing a product for cultivating plants with increased grain length;

[0041] D10) Regulating the grain width of plants;

[0042] D11) Preparing a product for regulating the grain width of plants;

[0043] D12) Cultivating plants with increased grain width;

[0044] D13) Preparing a product for cultivating plants with increased grain width;

[0045] D14) Regulating the 1000-grain weight of plants;

[0046] D15) Preparing a product for regulating the 1000-grain weight of plants;

[0047] D16) Cultivating plants with increased 1000-grain weight;

[0048] D17) Preparing a product for cultivating plants with increased 1000-grain weight;

[0049] D18) Regulating the plant height of plants;

[0050] D19) Preparing a product for regulating the plant height of plants;

[0051] D20) Cultivating plants with increased plant height;

[0052] D21) Preparing a product for cultivating plants with increased plant height;

[0053] D22) Regulating the number of grains per panicle of plants;

[0054] D23) Preparing a product for regulating the number of grains per panicle of plants;

[0055] D24) Cultivating plants with an increased number of grains per panicle;

[0056] Product for preparing plants with an increased number of grains per ear.

[0057] In the above applications, the regulation can be up-regulation, enhancement or increase of the expression of the coding gene of the aforementioned protein, or the content or activity of the protein. The regulation can also be down-regulation, inhibition or decrease of the expression of the coding gene of the aforementioned protein, or the content or activity of the protein.

[0058] In the above applications, the substance for regulating the expression of the coding gene of the aforementioned protein or the substance for regulating the content or activity of the aforementioned protein is a substance that increases the expression of the coding gene of the aforementioned protein in cells.

[0059] The present invention also provides a method for regulating plant yield, which includes regulating plant yield by regulating the expression of the coding gene of the aforementioned protein or regulating the activity or content of the aforementioned protein.

[0060] The yield is reflected by the total weight of grains per plant.

[0061] The present invention also provides a method for regulating the length of plant grains, which includes regulating the length of plant grains by regulating the expression of the coding gene of the aforementioned protein or regulating the activity or content of the aforementioned protein.

[0062] Specifically, the present invention also provides a method for increasing the length of plant grains, which includes increasing the length of plant grains by increasing the expression of the coding gene of the aforementioned protein or increasing the activity or content of the aforementioned protein.

[0063] The present invention also provides a method for regulating the width of plant grains, which includes regulating the width of plant grains by regulating the expression of the coding gene of the aforementioned protein or regulating the activity or content of the aforementioned protein.

[0064] Specifically, the present invention also provides a method for increasing the width of plant grains, which includes increasing the width of plant grains by increasing the expression of the coding gene of the aforementioned protein or increasing the activity or content of the aforementioned protein.

[0065] The present invention also provides a method for regulating the thousand-grain weight of plant grains, which includes regulating the thousand-grain weight of plant grains by regulating the expression of the coding gene of the aforementioned protein or regulating the activity or content of the aforementioned protein.

[0066] Specifically, the present invention also provides a method for increasing the thousand-grain weight of plant grains, which includes increasing the thousand-grain weight of plant grains by increasing the expression of the coding gene of the aforementioned protein or increasing the activity or content of the aforementioned protein.

[0067] The present invention also provides a method for regulating plant plant height, which includes regulating plant plant height by regulating the expression of the coding gene of the aforementioned protein or regulating the activity or content of the aforementioned protein.

[0068] Specifically, the present invention also provides a method for increasing plant yield, which includes increasing plant yield by increasing the expression of the coding gene of the aforementioned protein or increasing the activity or content of the aforementioned protein.

[0069] Specifically, the present invention also provides a method for increasing plant plant height, which includes increasing plant plant height by increasing the expression of the coding gene of the aforementioned protein or increasing the activity or content of the aforementioned protein.

[0070] In the above method or application, the plant is any one of the following:

[0071] G1) Monocotyledonous plants;

[0072] G2) Gramineous plants;

[0073] G3) Oryza plants;

[0074] G4) Rice seed plants;

[0075] G5) Rice.

[0076] Specifically, in the above method or application, the plant is rice or its mutant. The mutant is a mutant in which the yield gene OsSTK38 is disrupted.

[0077] The advantages of the present invention are as follows: There is currently no research report on the correlation between OsSTK38 and grain size in rice. A mutant tiny1 with significantly reduced grain length and width was screened from the constructed rice T-DNA insertion mutant library. Compared with the wild type, the mutant tiny1 not only has reduced grain length and width, but also significantly reduced 1000-grain weight and per-plant yield. Genetic analysis showed that the mutant tiny1 is controlled by a pair of recessive single nuclear genes. The gene controlling this mutant trait was isolated using the map-based cloning method and named OsSTK38. At the same time, functional complementation research on this gene was carried out by the complementary method to restore the phenotype of the mutant. In addition, overexpressing the OsSTK38 gene in the wild-type Nipponbare significantly increased the grain length, width, and 1000-grain weight of the crop, thereby increasing the biomass and yield of the plant. It shows that the OsSTK38 protein provided by the present invention has important theoretical and practical significance for cultivating new high-yield rice materials with increased grain size. These functions play an important role in variety improvement, and the per-plant yield and plot yield of crops can be enhanced by genetic engineering or genetic engineering methods. While modifying the grain shape of the plant, this gene can also be used to regulate the source-sink flow pathway of the plant, improve the photosynthetic product transport efficiency of the plant, and select favorable traits required: plant types with excellent agronomic traits such as efficiency improvement, flow diversion, and capacity expansion. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 It is the phenotypic analysis of mutant tiny1 and the wild type. Figure 1 The small dots on each column in the figure represent the actually measured data values, and the same applies to the following figure.

[0079] Figure 2 It is the map-based cloning map of the OsSTK38 gene.

[0080] Figure 3 It is the detection of the expression level of the OsSTK38 gene.

[0081] Figure 4 It is the phenotypic analysis of the OsSTK38 back-complemented tiny1 mutant.

[0082] Figure 5 It is the phenotypic analysis of the OsSTK38 overexpression line. DETAILED DESCRIPTION OF THE INVENTION

[0083] The present invention will be further described in detail below in conjunction with the specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0084] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0085] In the following examples, quantitative tests were all set up with three repeated experiments, and the results were averaged.

[0086] In the following examples, the data was processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation, and the Student’s ttest was used for testing. P < 0.05 (*) indicates significant difference, and P < 0.01 (**) indicates extremely significant difference.

[0087] Example 1: Mapping and Cloning of the High-Yield Gene OsSTK38

[0088] I. Discovery and Phenotypic Analysis of the Mutant tiny1

[0089] The mutant tiny1 was derived from the T-DNA insertion mutant library constructed by the laboratory of the first inventor, and is described in the non-patent literature "Wan, S., Wu, J., Zhang, Z., Sun, X., Lv, Y., Gao, C., Ning, Y., Ma, J., Guo, Y., Zhang, Q., et al. (2009). Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Molecular Biology 69: 69 - 80. 10.1007 / s11103 - 008 - 9406 - 5". The public can obtain it from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes. The wild type is Nipponbare rice (Oryza sativa L. ssp. Japonica; hereinafter also referred to as wild type rice WT).

[0090] pCambia23A vector: described in "Xuean Cui, Zhiguo Zhang, Yanwei Wang, Jinxia Wu, Xiao Han, Xiaofeng Gu, Tiegang Lu. TWI1 regulates cell-to-cell movement of OSH15 to control leaf cell fate. New Phytol. 2019 Jan; 221(1):326-340.", its short name in this literature is "pCam23A", which can be obtained from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0091] Agrobacterium tumefaciens AGL1: purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., product number ZK296.

[0092] Construction and analysis of genetic population: The rice mutant tiny1 was continuously self-crossed and cultivated in experimental fields in Hebei and Hainan for multiple generations. The yield traits of the homozygous mutant were stably inherited and minimally affected by the environment. The aforementioned rice mutant tiny1 was used as the female parent and crossed with the indica rice variety dular as the male parent to obtain F1 generation plants, and then self-crossed to obtain the F2 population. For the F2 segregating population, after counting the phenotypes of each individual plant, the segregation ratio of wild-type phenotype plants to mutant phenotype plants was calculated, and the statistical data was processed and analyzed using Microsoft Office Excel 2010. The chi-square test was used to test the fitness of the segregation ratio of field experiment data to the Mendelian genetic theory segregation ratio using Minitab 16 Statistical Software.

[0093] Compared with wild-type rice, the OsSTK38 gene of the mutant tiny1 is mutated and other genes remain unchanged.

[0094] Both the mutant and wild-type materials were planted in the experimental field of the Institute of Biology, Chinese Academy of Agricultural Sciences. A series of agronomic traits were measured for the wild-type and mutant. It was found that compared with wild-type rice, the grain length and width of tiny1 were both reduced ( Figure 1 in A, B, E, F), the 1000-grain weight was decreased ( Figure 1 in G), the plant height was shorter ( Figure 1 in C and H), the panicle length was reduced ( Figure 1 in D and I), the number of grains per panicle was increased ( Figure 1 in J), the length and width of lemma epidermal cells were smaller ( Figure 1 in K-N), and the number of cells in the same length and width range of lemma epidermis was increased ( Figure 1 in O and P).

[0095] II. Mapping and Cloning of the High-Yield Gene OsSTK38

[0096] Since the grain size mutant tiny1 is a japonica rice genetic background material, it is crossed with the indica rice variety Dular (Oryza sativa L. ssp. Indica) to form a hybrid combination. In this way, more polymorphisms will be generated. After obtaining the F1 generation, the F2 generation segregation population is generated by self-crossing the F1 generation. All materials are planted in the experimental field of the Institute of Biology, Chinese Academy of Agricultural Sciences.

[0097] 1. Extraction of Rice Genomic DNA

[0098] It is extracted by the simple CTAB method. The specific steps are as follows:

[0099] 1) Take about 400 mg of fresh young rice leaves, quickly freeze them with liquid nitrogen and grind them into powder, and quickly transfer them to a 2.0 mL Eppendorf tube;

[0100] 2) Add 800 μL of CTAB extraction buffer (100 mM Tris-HCl PH8, 20 mM EDTA, 1.4 M NaCl, 2.0% CTAB, 1% PVP) preheated at 65 °C, incubate in a water bath at 65 °C for 30 min, and shake and mix every 15 min;

[0101] 3) Add an equal volume of chloroform-isoamyl alcohol mixture (20:1), shake and mix for 2 min, and let stand for 5 min;

[0102] 4) Centrifuge at 10000 rpm for 5 min, transfer the supernatant to another clean 2.0 mL centrifuge tube;

[0103] 5) Add an equal volume of isopropanol, gently invert and mix, and white flocculent precipitate appears after standing at room temperature for a while;

[0104] 6) Centrifuge at 10000 rpm for 5 min, discard the supernatant and collect the precipitate;

[0105] 7) Add 800 μL of 80% ethanol to wash the precipitate;

[0106] 8) Centrifuge at 13600 rpm for 10 min, then discard the supernatant;

[0107] 9) Dry the precipitate on the ultra-clean bench and then dissolve it in 50 μL of pure water.

[0108] 2. Gene Mapping and Cloning

[0109] Development of molecular markers and screening of linked markers: When performing fine mapping, according to the results of the initial mapping, InDel markers were continuously developed within the initial mapping interval. The specific steps were as follows: First, find the sequences of Nipponbare (japonica rice) and 9311 (indica rice) in the target region on the GREMENE website (http: / / ensembl.gramene.org / ). Then, through the BLAST function of NCBI, find ideal InDel and SNP markers, design a pair of primers using the biological software DNAMAN, and commission Beijing Tsingke Biotechnology Co., Ltd. to synthesize them. After the primers were synthesized, first detect the polymorphism of the DNA of the two parents, and select primers with better polymorphism for subsequent experiments.

[0110] Map-based cloning of the OsSTK38 gene: Using the F2 segregation population for mapping, the mutant traits could be observed when the plants were at the fruiting stage. Take the leaves of the mutant phenotype plants to extract genomic DNA for gene mapping. First, take 120 F2 mutant plants for preliminary mapping. Select molecular markers with better polymorphism approximately every 25 cM on the 12 linkage groups of rice, and use 24 mutant plants for linkage analysis. After finding the molecular markers linked to OsSTK38, use 168 mutant plants for further verification. After correct verification, use a large population for chromosome walking to further fine-map the target gene. The molecular markers used for mapping are shown in Table 1. A total of 300 F2 mutant plants were used to map OsSTK38 within a 40 kb region. There were a total of 5 complete Open Reading Frames (ORFs) predicted in this small region. Analyze the gene structures of the 5 ORFs and sequence them all. It was determined that there was a T-DNA insertion upstream of the promoter of the candidate gene LOC_Os01g09200, and this gene was used as the candidate gene for the next analysis ( Figure 2 ).

[0111] Table 1 shows the primer sequences for gene mapping

[0112]

[0113]

[0114] The nucleotide sequence of the candidate gene OsSTK38 is SEQ ID No.2, and the amino acid sequence of the protein encoded by this gene is SEQ ID No.1.

[0115] Example 2. Complementation test of the high-yield gene OsSTK38

[0116] I. Preparation of complementary transgenic plants

[0117] 1. Extraction of total RNA from rice

[0118] Total RNA of wild-type rice plants was extracted using the RNAprep pure plant kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. For each tissue sample used for detection, after sampling in the field, it was placed in tin foil, quickly frozen in liquid nitrogen, and then brought back to the Beijing laboratory and stored in an ultra-low temperature refrigerator. The tissue samples used to detect tissue expression levels included leaves, leaf sheaths, stems, flowers, and roots of wild-type plants at the mature stage, and the tissue samples at the seedling stage included leaves, leaf sheaths, and roots. The specific steps for RNA extraction are as follows:

[0119] (1) Grind 100 mg of the sample in a low-temperature liquid nitrogen environment, then add 500 μL of lysis buffer SL. β-Mercaptoethanol needs to be added before use, and immediately vortex vigorously to mix evenly;

[0120] (2) Centrifuge at 12,500 rpm for 5 min;

[0121] (3) Transfer the supernatant to a CS filter column, centrifuge at 12,500 rpm for 2 min, and transfer the supernatant to a new tube.

[0122] (4) Add 0.4 times the volume of anhydrous ethanol of the supernatant. Precipitation will occur after mixing. Transfer it to a CR3 adsorption column, centrifuge at 12,500 rpm for 30 sec, discard the waste liquid, and put the CR3 back into the original tube.

[0123] (5) Carefully add 400 μL of RW1 to the CR3, centrifuge at 13,000 rpm for 1 min, discard the lower liquid, and put the CR3 back into the tube.

[0124] (6) Preparation method of DNase I working solution: Take 10 μL of DNase I stock solution, add 70 μL of RDD solution, and gently flick to mix evenly.

[0125] (7) Add 80 μL of DNase I working solution to the center of the CR3 adsorption column, and incubate at room temperature for 15 min.

[0126] (8) Add 350 μL of RW1 to the CR3, centrifuge at 12,500 rpm for 30 sec, pour out the waste liquid, and put the CR3 back into the tube.

[0127] (9) Add 500 μL of RW to the CR3, centrifuge at 12,500 rpm for 30 sec, pour out the waste liquid, and put the CR3 back into the tube.

[0128] (10) Repeat step (9).

[0129] (11) Centrifuge at 12,500 rpm for 3 min without sample, put the CR3 into a clean RNase-Free centrifuge tube, add 70 μL of RNase-Free ddH2O to the center of the membrane, incubate at room temperature for 5 min, and centrifuge at 13,000 rpm at room temperature for 2 min to obtain the RNA extraction product.

[0130] Note: The volume of the elution buffer should be greater than 30 μL. Too small a volume will affect the recovery efficiency. The RNA sample is stored in an ultra-low temperature freezer at -80 °C. To increase the RNA yield, the obtained RNA solution can be added with CR3, incubated at room temperature for 1 - 2 min, and centrifuged at 13,000 rpm for 2 min to obtain an RNA solution with higher yield and concentration.

[0131] 2. RNA reverse transcription

[0132] The first-strand cDNAs are synthesized using a reverse transcription kit IIIFirst Strand SynthesisKit (Invitrogen, USA). Since the GC content of rice genes is relatively high, the high-GC reverse transcription method recommended by the kit is adopted, and Oligo(dT)20 (50 μM) is selected for synthesis. The specific method is as follows:

[0133] (1) Preparation of RNA-primermixture: 2 μg total RNA: n μL (the loading volume n is determined according to the RNA concentration), Oligo(dT)20 (50 μM): 1 μL, 10 mM dNTP mix: 2.5 μL, supplemented with RNase-Free ddH2O to 25 μL;

[0134] (2) Incubate the RNA sample in a PCR instrument at 65 °C for 3 min and immediately transfer it to 55 °C;

[0135] (3) Preparation of cDNA-Synthesis Mix (25 μL): RNase-Free ddH2O 3 μL, 10×RTbuffer 5 μL, 25 mM MgCl2 10 μL, 0.1 M DTT 5 μL, RNase OUTRecombinant RNaseI nhibitor 1 μL, SuperScriptIIIRT 1 μL. Mix the above and set aside;

[0136] (4) Gently and thoroughly mix the RNA-primer mixture with the cDNA-Synthesis Mix, react at 55 °C on a PCR instrument for 1 h, and terminate at 85 °C for 3 min;

[0137] (5) Add RNaseH: 1 - 2 μL, incubate at a constant temperature of 37 °C in a PCR instrument for 30 min, and store at -20 °C for later use.

[0138] 3. Construction of complementary vector and transformation of Agrobacterium

[0139] Design primers NDRcdsF and NDRcdsR using the primer design software DNAMAN:

[0140] NDRcdsF: 5'-cccgggATGGAGAGCGAGATGGCGGAC-3';

[0141] NDRcdsR: 5'-tctagaTCATTGAATCATCGGATCACCAG-3'.

[0142] Using the cDNA obtained in the above 2 as a template, PCR amplification was performed with primers NDRcdsF and NDRcdsR (using high-fidelity KOD enzyme in the amplification system) to obtain a PCR product.

[0143] After sequencing, this PCR product has the nucleotides shown in SEQ ID No. 2 and is the gene OsSTK38.

[0144] The obtained PCR product was digested with SmaI and XbaI, and the digested product was ligated to the pCambia23A vector digested with the same enzymes to obtain the recombinant vector pCambia23A-OsSTK38. After sequencing, this recombinant vector pCambia23A-OsSTK38 is a vector obtained by replacing the SmaI and XbaI sites of the pCambia23A vector with the nucleotides shown in SEQ ID No. 2.

[0145] Transformation of Agrobacterium tumefaciens AGL1 by electroporation: Pipette 1 μl of the complementary vector plasmid into 20 μl of Agrobacterium competent cells and mix gently. At the same time, take out the electroporation cuvette from absolute ethanol and dry it; pre-cool the dried electroporation cuvette in a -20°C refrigerator for 5 min, add the mixed competent cells to the electroporation cuvette, and perform electroporation using an electroporator; add 500 μl of antibiotic-free YEP to the electroporation cuvette, transfer it to a 2 ml EP tube, culture at 28°C for 3 - 4 h, then plate (50 mg / l kanamycin, 50 mg / l rifampicin), and pick monoclonal colonies for PCR identification.

[0146] The above recombinant vector pCambia23A-OsSTK38 was introduced into Agrobacterium tumefaciens AGL1 by electroporation to obtain the recombinant bacterium AGL1 / pCambia23A-OsSTK38. After digestion verification, positive recombinant bacteria were obtained.

[0147] 4. Agrobacterium-mediated genetic transformation of the mutant tiny1

[0148] S1. Induction of rice callus

[0149] Shell the mutant tiny1 seeds, then treat them with 75% ethanol for 1 min and surface-sterilize them with 20% sodium hypochlorite for 15 min in sequence; wash the seeds 5 times with sterile water; place the seeds on the callus induction medium and culture them in the dark for 4 weeks at a culture temperature of 28 ± 1°C.

[0150] Formulation of the induction medium: Based on the N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, casein hydrolysate of 0.8 g / L, proline of 0.3 g / L, sucrose of 30 g / L, and phytagel of 3 g / L.

[0151] Formulation of the N6 medium: The solutes are potassium nitrate 2830 g / L, ammonium sulfate 463 g / L, calcium chloride (CaCl2·2H2O) 166 g / L, magnesium sulfate (MgSO4·7H2O) 185 g / L, potassium dihydrogen phosphate 400 g / L, ferrous sulfate (FeSO4·7H2O) 27.8 g / L, manganese sulfate (MnSO4·H2O) 4.4 g / L, zinc sulfate (ZnSO4·7H2O) 1.6 g / L, boric acid 0.8 g / L, potassium iodide 1.6 g / L, vitamin B1 (thiamine hydrochloride) 1.0 g / L, vitamin B6 (pyridoxine hydrochloride) 0.5 g / L, nicotinic acid 0.5 g / L, glycine 2.0 g / L, and the solvent is deionized water.

[0152] S2. Subculture of callus

[0153] Select bright yellow, firm, and relatively dry embryogenic callus of the mutant tiny1, place it on the subculture medium (the nutrient components are the same as those of the induction medium), and culture it in the dark for 3 weeks at a culture temperature of 28 ± 1°C.

[0154] S3. Preparation of Agrobacterium culture solution

[0155] Culture Agrobacterium AGL1 / pCambia23A-OsSTK38 in the LA medium (10 g / L Tryptone, 5 g / L Yeast extract, 10 g / L NaCl, 15 g / L agar powder, the rest is deionized water, pH 7.0) containing 50 mg / L kanamycin for 2 d at a temperature of 28 ± 1°C; transfer Agrobacterium AGL1 / pCambia23A-OsSTK38 to the suspension medium and culture it on a shaker at 28°C and 200 rpm for 2 h to obtain the Agrobacterium suspension.

[0156] Suspension medium: Based on the N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, casein hydrolysate of 0.8 g / L, proline of 0.3 g / L, sucrose of 30 g / L, glucose of 10 g / L, and acetosyringone of 100 μM.

[0157] S4. Agrobacterium infection

[0158] Transfer the callus into a sterilized Erlenmeyer flask; adjust the OD600nm value of the Agrobacterium suspension to about 0.3; soak the callus in the Agrobacterium suspension for 30 min; transfer the callus to sterilized filter paper to dry it; then place it on the co-culture medium and culture for 3 d at a temperature of 22 °C.

[0159] Co-culture medium: Based on the N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, 0.3 g / L of proline, 30 g / L of sucrose, 10 g / L of glucose, 100 μM of acetosyringone, and 8 g / L of agar powder.

[0160] S5. Screening and culture of callus

[0161] The co-cultured callus is washed 10 times with sterilized water; then the callus is soaked in sterilized water containing 200 mg / L of ticarcillin for 30 min; transfer the callus to sterilized filter paper to dry it; then transfer the callus to the selection medium and select and culture it twice, with each culture lasting for 2 weeks.

[0162] Selection medium: Based on the N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, 0.3 g / L of proline, 50 mg / L of G418, 200 mg / L of ticarcillin, 30 g / L of sucrose, and 8 g / L of agar powder.

[0163] S5. Differentiation culture of callus

[0164] Transfer the resistant callus to the differentiation medium and culture it under light (2000 Lux) at a temperature of 28 ± 1 °C.

[0165] Differentiation medium formula: Based on the MS medium, add 2 mg / L of KT, 0.2 mg / L of NAA, 2 mg / L of 6-BA, 0.2 mg / L of IAA, 0.8 g / L of hydrolyzed casein, 0.3 g / L of proline, 30 g / L of sucrose, and 3 g / L of phytagel, with the balance being sterile water.

[0166] MS medium formula: The solutes are 440 mg / L of CaCl2·2H2O, 170 mg / L of KH2PO4, 370 mg / L of MgSO4·7H2O, 1650 mg / L of NH4NO3, 1900 mg / L of KNO3, 0.83 mg / L of KI, 0.025 mg / L of CoCl2·6H2O, 6.2 mg / L of H3BO4, 0.25 mg / L of Na2MoO4·7H2O, 22.3 mg / L of MnSO4·4H2O, 0.025 mg / L of CuSO4·5H2O, 8.6 mg / L of ZnSO4·7H2O, 27.8 mg / L of FeSO4·7H2O, 37.3 mg / L of Na2EDTA, 0.1 mg / L of thiamine hydrochloride, 0.5 mg / L of pyridoxine hydrochloride, 0.5 mg / L of nicotinic acid, 100 mg / L of inositol, 2.0 mg / L of glycine, 30000 mg / L of sucrose, 7000 mg / L of agar powder, and the balance is deionized water.

[0167] S6. Rooting culture

[0168] Cut off the roots generated during differentiation, and then transfer them to the rooting medium, and culture them under 2000 Lux light for 3 weeks (temperature 28 ± 1 °C).

[0169] Rooting medium formula: Based on 1 / 2MS medium, add sucrose with a final concentration of 20 g / L and 3 g / L of phytagel.

[0170] 1 / 2MS medium: The final concentration of the solutes in MS is halved.

[0171] S7. Transplanting of tissue-cultured seedlings

[0172] Wash off the residual medium on the roots, transfer the seedlings with good roots to the greenhouse, and keep the soil moist in the first few days. The obtained regenerated seedlings are T0 generation transgenic plants (i.e., T0 generation OsSTK38 gene complementary rice). After self-crossing of T0 generation OsSTK38 gene complementary rice, T1 generation OsSTK38 gene complementary rice is obtained and used for subsequent phenotypic observation and analysis.

[0173] 5. Molecular identification of transgenic plants

[0174] Extract the RNA of young leaves of wild-type rice (i.e., Figure 3 WT in Figure 3 ), mutant tiny1 (i.e., Figure 3 tiny1 in

[0175] QRT1: 5'-AGCGCAAGGAGAGGCGCTTT-3';

[0176] QRT2: 5'-GCCGCATGTACTCTGTTTCC-3'.

[0177] Quantitative PCR was performed on an iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad), and the fluorescence quantitative reagent used was SYBR Green Mix; each sample was independently repeated 3 times.

[0178] The reaction system was 25 μL: 2×Mix 12.5 μl, Primer1 1 μl, Primer2 1 μl, cDNA 3 μl, ddH2O 7.5 μl.

[0179] The reaction program: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 30 s, 80°C for 10 s to collect fluorescence, 72°C for 5 min, rising from 55°C to 95°C at 0.5°C / s to make a melting curve, and steps 2 - 5 were run for 40 cycles.

[0180] The internal reference primers used were actin-F and actin-R:

[0181] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0182] actin-R: 5'-AATGAGTAACCACGCTCCGC-3'.

[0183] The results are as Figure 3 shown. It can be seen that the expression level of the OsSTK38 gene in the OsSTK38 gene complementary transgenic rice ( Figure 3 gTINY1; tiny#1 and gTINY1; tiny#2 in Figure 3 WT in

[0184] II. Phenotypic study of complementary transgenic plants

[0185] The OsSTK38 gene complementary transgenic rice, wild-type rice (WT), and mutant tiny1 were planted in the experimental field (field) of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences to observe the phenotypes.

[0186] The results are as Figure 4As shown, WT is the wild-type Japanese blue variety, tiny1 is the mutant with the high-yield gene OsSTK38 disrupted, gTINY1; tiny1#1 and gTINY1; tiny1#2 are two lines of transgenic OsSTK38 gene complementary rice. It can be seen that compared with wild-type rice, the grain shape and panicle shape of the tiny1 mutant are smaller, the plant is shorter, and the 1000-grain weight is reduced. However, the phenotypes of the transgenic OsSTK38 gene complementary rice obtained from the complementation experiment (i.e., Figure 4 gTINY1; tiny#1 and gTINY1; tiny#2 in

[0187] ) are all restored to the wild-type level.

[0188] Example 3. Application of Overexpressing the OsSTK38 Gene in Increasing Yield

[0189] I. Preparation of Overexpressing Plants of the OsSTK38 Gene in Wild-Type Rice

[0190] 1. Preparation of Recombinant Bacteria

[0191] The recombinant vector pCambia23A-OsSTK38 prepared in Example 2 was introduced into Agrobacterium tumefaciens AGL1 to obtain the recombinant bacterium AGL1 / pCambia23A-OsSTK38. After digestion verification, positive recombinant bacteria were obtained.

[0192] 2. Agrobacterium-Mediated Genetic Transformation of Wild-Type Rice Nipponbare

[0193] The rice variety "Nipponbare" is preserved by the Joint Laboratory for Photosynthesis Enhancement and C4 Rice Creation of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The public can obtain it from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes. The optimal concentration of G418 for screening resistant callus is 50 mg / L.

[0194] The recombinant bacterium AGL1 / pCambia23A-OsSTK38 was transferred into wild-type rice Nipponbare by the same rice genetic transformation method as in Step 4 of Example 2 to obtain the T0 generation of transgenic OsSTK38 gene overexpressing rice lines. The specific operation is as follows: Replace the mutant tiny1 seeds with the seeds of the rice variety "Nipponbare", and the rest of the operations are the same as those in "4. Agrobacterium-Mediated Genetic Transformation of the Mutant tiny1" in Example 2.

[0195] 3. Molecular Identification of Transgenic Plants Overexpressing the OsSTK38 Gene

[0196] Extract the RNA from the young leaves of T0 generation transgenic OsSTK38 overexpressing rice, reverse transcribe to obtain cDNA as a template, and perform quantitative PCR amplification using QRT1 primer and QRT2 primer.

[0197] QRT1: 5'-AGCGCAAGGAGAGGCGCTTT-3';

[0198] QRT2: 5'-GCCGCATGTACTCTGTTTCC-3'.

[0199] Quantitative PCR was performed on the iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad), and the fluorescence quantitative reagent used was SYBR Green Mix; each sample was independently repeated 3 times.

[0200] The reaction system was 25 μL: 2×Mix 12.5 μl, Primer1 1 μl, Primer2 1 μl, cDNA 3 μl, ddH2O 7.5 μl.

[0201] The reaction program: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 30 s, collect fluorescence at 80°C for 10 s, 72°C for 5 min, raise the temperature from 55°C to 95°C at 0.5°C / s to make a melting curve, and the 2nd - 5th steps were run for 40 cycles.

[0202] The internal reference primers used were actin-F and actin-R:

[0203] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0204] actin-R: 5'-AATGAGTAACCACGCTCCGC-3'.

[0205] The lines identified as OsSTK38 overexpressing plants were named OsSTK38-OE#2 and OsSTK38-OE#6.

[0206] The results are as Figure 5 shown in C, OsSTK38-OE#2 and OsSTK38-OE#6 are two random lines of T0 generation transgenic OsSTK38 overexpressing rice. It can be seen that the expression level of the OsSTK38 gene in T0 generation transgenic OsSTK38 overexpressing rice is higher than that in wild-type rice, indicating successful overexpression. After self-crossing of T0 generation transgenic OsSTK38 overexpressing rice, T1 generation transgenic OsSTK38 overexpressing rice was obtained and used for subsequent phenotypic observation and analysis.

[0207] II. Phenotypic study of OsSTK38 overexpressing plants

[0208] The transgenic OsSTK38 overexpressing rice and wild-type rice were planted in the experimental field (field) of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences to observe the phenotypes. The wild-type Nipponbare rice was used as the control (i.e., Figure 5 WT in the figure).

[0209] Fifteen rice plants were counted for each plant line. The results are as Figure 5 shown. WT is the wild-type Nipponbare variety, and OsSTK38-OE#2 and OsSTK38-OE#6 are two random plant lines of transgenic OsSTK38 overexpressing rice. It can be seen that compared with the wild-type rice, the expression level of OsSTK38 in the transgenic OsSTK38 overexpressing rice was significantly increased ( Figure 5 C in the figure), the plant height became taller ( Figure 5 A in the figure), the grain length and width became larger ( Figure 5 B, D, and E in the figure), the 1000-grain weight increased ( Figure 5 F in the figure), and the excellent traits of the length ( Figure 5 G and H in the figure) and width ( Figure 5 G and I in the figure) of the lemma epidermal cells became larger, indicating that overexpressing the OsSTK38 gene in wild-type rice can increase traits such as grain size, significantly improve plant yield, and it is a novel high-yield gene not reported yet.

[0210] SEQ ID No.1 (protein sequence)

[0211] MESEMADAAPAPAVAAAATAEPLAAVAEEGEEGGEAAVGSTLTMERVAAAKKFIENHYRSQMKNIQERKERRFRLERQLESSQVPREQQINLLKDLERKETEYMRLKRHKICVDDFELLTIIGRGAFGEVRLCREKTSSNIYAMKKLKKSDMVVRGQVEHVRAERNLLAEVASHCIVKLYYSFQDSEYLYLIMEYLPGGDIMTLLMREDTLTEHVARFYIAETILAIESIHKHNYIHRDIKPDNLLLDKNGHMKLSDFGLCKPIDCSKLSTLNEDEPMGDDNLRESMDIDSSFSETTNGRRWRSQHEQLQHWQMNRRKLAFSTVGTPDYIAPEVLLKKGYGMECDWWSLGAIMYEMLVGYPPFYSDDPITTCRKIVHWRNHLKFPEDSKVSPEARDLICRLLCDVDHRIGSAGADQIKAHPWFRGVAWEKLYEMEAAFKPQVNDELDTQNFMKFEEMDNAPTRTGSGPSRKMMLNSKDLSFVGYTYKNFDAVKGLKHSDQQRNQSLIRPSIGSIFGPADMDPSREPNGRDKHMHTVSSGDPMIQSEQ ID No.2 (CDS sequence)

[0212]

[0213] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Any of the following uses of a protein or a biological material related to the protein: D1) regulates the grain length of plants; D2) preparing a product for regulating seed length of a plant; D3) breeding plants with increased kernel length; D4) preparing a product for breeding plants with increased kernel length; D5) regulates the grain width of the plant; D6) preparing a product for regulating the grain width of a plant; D7) cultivating plants with increased kernel width; D8) preparing a product for breeding plants with increased kernel width; D9) regulating the thousand-grain weight of the plant; D10) preparing a product for regulating thousand-grain weight of a plant; D11) cultivating plants with increased thousand-grain weight; D12) preparing a product for breeding plants with increased thousand-grain weight; D13) regulating plant height; D14) preparing a product for regulating plant height; D15) cultivating plants with increased plant height; D16) preparing a product for growing plants with increased plant height; The protein is any of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in A1); The protein-related biological material is any one of the following: B1), a nucleic acid molecule encoding the protein described in A1) or A2); B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2); the plant is rice.

2. The use according to claim 1, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.

2.

3. The use according to claim 1 or 2, characterized in that: The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein or the content or activity of the protein.

4. A method for regulating plant seed length, regulating plant seed width, regulating plant seed thousand-grain weight, or regulating plant height, characterized in that: The method comprises regulating the plant seed length or the plant seed width or the plant seed thousand-grain weight or the plant height by regulating the expression of the protein encoding gene or regulating the activity or content of the protein; The protein is any of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in A1); The plant is rice.

5. The method according to claim 4, characterized in that The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein or the content or activity of the protein.

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