CN112521470B - Plant starch synthesis related protein OsFLO18, and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and relates to a protein related to plant starch synthesis and its encoding gene and application. Specifically disclosed is a starch synthesis-related protein OsFLO18, which can affect the process of grain starch synthesis in rice endosperm. Transgenic plants with transparent and non-silty endosperm can be obtained by introducing the encoding gene of the protein into a farinaceous endosperm plant with abnormal starch synthesis. The protein and its encoding gene can be applied to plant genetic improvement.

Description

Plant starch synthesis related protein OsFLO18 and its encoding gene and application

technical field

The invention belongs to the field of genetic engineering, and relates to a protein related to plant starch synthesis and its encoding gene and application.

Background technique

Rice ( Oryza sativa L.) is one of the most important food crops in the world. Rice endosperm accumulates a large amount of starch during the maturation process, accounting for about 90% of the dry weight of seeds. These starches provide the main energy for seed germination and seedling growth and development; at the same time, these starches are also an important energy source for humans, livestock feed and industrial raw materials . Therefore, through the study of starch mutants, the key genes of starch synthesis can be discovered, which can not only elucidate the mechanism of rice starch synthesis and regulation in theory, but also provide genetic and material support for rice quality improvement.

Rice starch is mainly composed of amylose and amylopectin in chemical composition. Amylose is made up of α-1,4-glycosidic bonds (generally about 1000-5000 glucose monomers) and has few branches. Synthesized by granule-bound starch synthase (I GBSSI), which is encoded by the Waxy gene; amylopectin is highly branched, with approximately one α-1,6-glycoside for every 20-25 α-1,4-glycosidic bonds Bond branches, each independent branch can consist of 6-100 glucose monomers. Starch synthases (SSs), starch branching enzymes (BEs) and starch debranching enzymes (DBEs) are mainly involved in the synthesis of amylopectin. Mutations in the genes encoding these synthases in rice result in abnormal endosperm starch characteristics. In addition to synthases, there are other factors in rice that are indirectly involved in starch synthesis. More and more new farinoid genes have been cloned, indicating that the synthesis of starch is extremely sophisticated. These new farinogenic genes contain the PPR protein family.

PPR (Pentatricopeptide repeat) proteins are a large family of proteins widely distributed in various organisms. Most of them are located in chloroplasts and mitochondria in higher plants. There are more than 400 PPR proteins in rice, Arabidopsis and maize. PPR proteins have a sequence-specific recognition pattern for single-stranded RNA, and are closely related to RNA transcription, splicing, editing, and stability. Existing studies have shown that PPR plays an important role in the embryo and endosperm development of flowering plants. OsFLO18 belongs to a member of the PPR protein family, and its function in vivo has not been studied.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a starch synthesis related protein and its encoding gene and application.

The starch synthesis-related protein (OsFLO18) provided by the present invention is derived from Oryza sativa var. Ningjing No. 3, and is a protein of the following (a) or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ ID No. 1;

(b) The protein derived from SEQ ID No. 1, which is related to starch synthesis by substitution and/or deletion and/or addition of one or several amino acid residues from the amino acid sequence of SEQ ID No. 1.

SEQ ID No. 1 consists of 665 amino acid residues, from the amino terminus 188-1269 to the PPR family domain.

In order to make OsFLO18 in (a) easy to purify and study its subcellular location in rice cells, it can be linked at the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 as shown in Tables 1 and 2 shown label.

Table 1 Sequences of MBP tags

Table 2 Sequences of GFP tags

The OsFLO18 in the above (b) can be artificially synthesized, or its encoding gene can be synthesized first, and then biologically expressed. The gene encoding OsFLO18 in the above (b) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO. 2, and/or making one or several base pairs missense. It can be obtained by mutating, and/or ligating the coding sequences of the tags shown in Table 1 at its 5' and/or 3' ends.

The gene encoding the above-mentioned starch synthesis-related protein ( OsFLO18 ) also belongs to the protection scope of the present invention. The gene OsFLO18 can be a DNA molecule of the following 1) or 2) or 3) or 4): 1) The DNA molecule shown in SEQ ID NO.2; 2) The DNA molecule shown in SEQ ID NO.3; 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) and encodes the protein under stringent conditions; 4) has more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encodes a regulatory DNA molecules of starch synthesis-related proteins. The stringent conditions can be hybridization and membrane washing in a solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS at 65°C. SEQ ID NO. 2 consists of 1998 nucleotides and is the CDS of OsFLO18. Recombinant expression vectors containing any of the above genes also belong to the protection scope of the present invention. Recombinant expression vectors containing the genes can be constructed using existing plant expression vectors.

The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall inducing (Ti) plasmid genes (such as Nos synthase Nos gene), plant genes (such as soybean storage). The untranslated regions transcribed at the 3' end of protein genes have similar functions.

When using the gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, maize Ubiquitin promoters, which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, These enhancer regions can be ATG initiation codons or contiguous region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can 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, the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce a color change or a luminescent compound. Gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or anti-chemical reagent marker gene (such as herbicide resistance gene), etc. Considering the safety of transgenic plants, the transformed plants can be directly screened under stress without adding any selectable marker gene.

The recombinant expression vector can be a recombinant plasmid obtained by recombination inserting the gene ( OsFLO18 ) between the multiple cloning sites Xba I and Bam HI of the pCAMBIA1305.1 vector. The recombinant plasmid can specifically be pCAMBIA1305.1 -OsFLO18 ; the pCAMBIA1305.1 -OsFLO18 is obtained by inserting the OsFLO18 genome coding sequence between the multiple cloning sites Xba I and Bam HI of pCAMBIA1305.1 by recombination technology (Clontech Company , Infusion Recombination Kit).

The pCAMBIA1305.1 containing OsFLO18 was named pCAMBIA1305.1 -OsFLO18 .

Expression cassettes, transgenic cell lines and recombinant bacteria containing any of the above genes ( OsFLO18 ) all belong to the protection scope of the present invention.

Another object of the present invention is to provide the protein, the application of at least one of the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria for improving starch synthesis in plant breeding, preferably The plant can be either a monocotyledonous plant or a dicotyledonous plant, such as: tobacco, L. japonicus, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, alfalfa and the like. Preferably the plant is rice. Correspondingly, the present invention also provides a method for cultivating transgenic plants with normal starch synthesis. The method for cultivating a transgenic plant with normal starch synthesis provided by the present invention is to introduce the gene into a plant with abnormal starch synthesis to obtain a transgenic plant with normal starch synthesis; the abnormal starch synthesis plant is an endosperm with a powdery phenotype. Plants; the transgenic plants with normal starch synthesis are transgenic plants with transparent and non-silty endosperm. Specifically, the gene is introduced into a plant with abnormal starch synthesis through the recombinant expression vector; the plant with abnormal starch synthesis can be flo18 .

The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria or the method can all be applied to rice breeding.

Transgenic cell lines and transgenic plants can be obtained by introducing the gene encoding the protein into plant cells using any vector that can guide the expression of exogenous genes in plants. The expression vector carrying the gene can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, Agrobacterium-mediated, etc. Plant tissue is grown into plants.

Beneficial effects: the starch synthesis-related protein of the present invention affects the process of grain starch synthesis in rice endosperm. Transgenic plants with transparent and non-silty endosperm can be obtained by introducing the encoding gene of the protein into a farinaceous endosperm plant with abnormal starch synthesis. The protein and its encoding gene can be applied to plant genetic improvement.

Description of drawings

Figure 1 shows the grain phenotypes of wild-type Ningjing 3 and mutant flo18 .

Figure 2 is the scanning electron microscope observation of the wild-type Ningjing 3 and the mutant flo18 .

Figure 3 is the observation of semi-thin sections of the endosperm of wild-type Ningjing 3 and mutant flo18 .

Figure 4 is a comparison of the grain type and thousand-grain weight of wild-type Ningjing 3 and mutant flo18 .

Figure 5 shows the comparison of physicochemical properties between wild-type Ningjing 3 and mutant flo18 .

Figure 6 shows the fine mapping of mutant genes on chromosome 7.

Figure 7 shows the phenotype of T1 grains transfected with pCAMBIA1305.1 -OsFLO18 .

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

Example 1. Discovery of plant starch synthesis-related proteins and their encoding genes

1. Phenotypic analysis and genetic analysis of rice starch synthesis mutant flo18

The seed powder opaque mutant flo18 was screened from the MNU mutant library of rice variety Ningjing 3.

Figure 1 The left picture is the scanning image of the whole and cross-section of the mature seed of Ningjing 3, showing the phenotype of completely transparent endosperm, and the right picture is the scanning image of the whole and cross-section of the mature seed of flo18 , showing the phenotype of the whole endosperm powder .

Figure 2 is the scanning electron microscope analysis of Ningjing 3 and flo18 . The SEM of the mature seeds of Ningjing 3 showed that the starch granules were closely arranged and uniform in size, while the starch granules in flo18 were loosely arranged and the granules were mostly round. Thus scattering occurs when light passes through, resulting in an opaque phenotype in the appearance of flo18 grains.

The morphology of Ningjing 3 and flo18 composite starch granules was observed by I ₂ -KI staining after semi-thin sectioning (Fig. 3). In the inner layer of endosperm cells of wild-type Ningjing 3, multiple independent starch granules were produced inside each amyloplast. Further observation of the mutant flo18 found that there were many small and scattered single starch granules in the cytoplasm of the inner layer of the endosperm, and the starch granules were not closely arranged, indicating that the starch in the mutant was abnormally developed and relatively lag compared to wild type. (image 3).

The grain thickness of the mature seeds of the flo18 mutant was significantly lower than that of the wild type (Fig. 4). The seed weight of mature flo18 mutant was significantly lower than that of Ningjing 3 (Fig. 4).

The seeds of the flo18 mutant had a higher content of fat, while the starch and protein contents were significantly lower than those of the wild type (Fig. 5). Correspondingly, the amylose content was significantly reduced (Fig. 5).

The location of the mutant gene

1. Preliminary localization of mutant genes

The mutant flo18 was crossed with Nipponbare, and 10 silty seeds were randomly selected from the F2 segregated population of flo18 /N22. After germination, DNA was extracted from the leaves of each plant. First, polymorphism analysis was performed between Ningjing 3 and N22 with SSR and Indel primers covering the whole genome of rice, and then a pair of primers that were polymorphic between the two parents was selected every 10 cM. A total of 12 DNA samples from the two parental DNAs together with the population DNA were analyzed using the selected primers that covered 12 chromosomes and had polymorphisms. Finally, the key starch synthesis gene OsFLO18 was located on chromosome 7 SSR markers I7-12 and I7 Between -13.

The methods for the above SSR and Indel marker analysis are as follows:

(1) Extract the total DNA of the selected individual plant as a template, and the specific method is as follows:

①Take about 0.2 grams of young rice leaves, put them in an Eppendorf tube, place a steel ball in the tube, freeze the Eppendorf tube with the sample in liquid nitrogen for 5 minutes, and place it on a 2000 GENO/GRINDER instrument to crush the sample for 1 minute .

②Add 660μl of extract (containing 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4MNaCl, 0.2g/ml CTAB solution), vortex vigorously on a vortexer to mix well, and ice bath for 30min.

③ Add 40 μl of 20% SDS, incubate at 65°C for 10 minutes, and mix by gently inverting every two minutes.

④Add 100μl 5M NaCl and mix gently.

⑤ Add 100 μl of 10×CTAB, incubate at 65°C for 10 minutes, and mix by gently inverting it intermittently.

⑥ Add 900 μl of chloroform, mix well, and centrifuge at 12,000 rpm for 3 min.

⑦ Transfer the supernatant to a 1.5mL Eppendorf tube, add 600μl isopropanol, mix well, and centrifuge at 12000rpm for 5min.

⑧ Discard the supernatant, rinse the precipitate once with 70% (volume percent) ethanol, and dry at room temperature.

⑨Add 100 μl of 1×TE (121 g of Tris dissolved in 1 liter of water, and adjust the pH value to 8.0 with hydrochloric acid) to dissolve the DNA.

⑩ Take 2 μl of DNA to measure the quality of DNA by electrophoresis, and measure the concentration with DU800 spectrophotometer (Bechman Instrument Inc. U.S.A).

(2) Dilute the DNA extracted above to about 20ng/μl, and use it as a template for PCR amplification;

PCR reaction system (10μl): DNA (20ng/ul) 1ul, upstream primer (2pmol/ul) 1ul, downstream primer (2pmol/ul) 1ul, 10xBuffer (MgCl2 free) 1ul, dNTP (10mM) 0.2ul, MgCl2 (25mM) ) 0.6ul, rTaq(5u/ul) 0.1ul, ddH2O 5.1ul, a total of 10ul.

PCR reaction program: denaturation at 94.0 °C for 5 min; denaturation at 94.0 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, a total of 35 cycles; extension at 72 °C for 5 min; storage at 10 °C. PCR reactions were performed in a MJ Research PTC-225 thermal cycler.

(3) Detection of PCR products labeled with SSR and Indel

Amplification products were analyzed by 8% native polyacrylamide gel electrophoresis. The molecular weight of the amplified product was compared with the 50bp DNA Ladder as the control, and the color was developed by silver staining.

2. Fine mapping of mutant genes

According to the results of preliminary mapping, SSR and Indel markers were developed in the region where the mutation site was located at a certain interval, so as to screen more markers in the relevant section of the chromosome to further locate the mutant site. F2 seeds identified as mutant phenotypes were picked from the F2 segregating population obtained from the flo18 /N22 cross combination for fine mapping of the mutation sites. Using the molecular markers on the public map and the SSR and Indel molecular markers developed by ourselves based on the rice genome sequence data, the mutation sites were finely mapped, and the mutation sites were preliminarily determined according to the positioning results. The specific methods are as follows:

(1) SSR mark development

The SSR markers of the public map were integrated with the rice genome sequence, and the BAC/PAC clone sequence near the mutation site was downloaded. Use SSR Hunter (Li Qiang et al., Genetics, 2005, 27(5): 808-810) or SSRIT software to search for potential SSR sequences in clones (number of repetitions ≥ 6); these SSRs and their adjacent 400-500 bp sequences are listed in NCBI The BLAST program is used to compare with the corresponding indica sequences online. If the SSR repeats between the two are different, it is preliminarily inferred that the PCR products of the SSR primers are polymorphic between indica and japonica. Then use Primer Premier 5.0 software to design SSR primers, And synthesized by Shanghai Yingjun Biotechnology Co., Ltd. The self-designed SSR paired primers were mixed in equal proportions, and their polymorphisms between Ningjing 3 and N22 were detected, and those with polymorphisms were used as molecular markers for fine mapping of the OsFLO18 gene. Molecular markers used for fine mapping are shown in Table 3.

Table 3 Molecular markers for fine mapping

primer preprimer post primer physical distance bp I7-12 CGTTCGTGTTTTTCGCTGAT GATCGGAGGCTTTTGTTTGA 28469598 S7-21 GAGAGACTAAAGTTTAGTCA TTCGGGAAAGGACAAGAG 29084405 S7-30 GATGTGAGCAAACGAACACAA AAGATGGTGATCGATACTCT 29201884 S7-27 GTATCAAAGTGAACAGGCCCTT ACAACTCTGATGCCTCGT 29259713 I7-13 TGGAGGCTTTCTCGCTTTC GAGGAAGTCGAGGATGAGGA 29661741

According to the molecular data and phenotypic data of endosperm farinaceous individuals in the F2 population, and according to the "recessive extreme individual gene mapping" method reported by Zhang et al., the OsFLO18 gene was finally finely mapped between S7-27 and S7-30. The physical distance is about 57kb (Figure 6). Genome sequencing of the candidate segment revealed that, in flo18 , a single-base substitution from A to T occurred in the gene LOC_Os07g48850, resulting in premature termination of protein translation.

(3) Acquisition of mutant genes

Primers are designed according to the located sites, and the sequences are as follows:

primer1: 5'-ATGTCTTGGCCACACGACC-3' (SEQ ID NO. 4)

primer2: 5'-TTAGTTGTTCTTCAACTTAA-3' (SEQ ID NO.5)

Using primer1 and primer2 as primers and the cDNA of Ningjing 3 as template, PCR amplification was performed to obtain the target gene, and the amplified product was the target fragment of 1998 bp.

The amplification reaction was performed on a PTC-200 (MJ Research Inc.) PCR machine: 94°C for 3min; 94°C for 30sec, 60°C for 45sec, 72°C for 2min, 35 cycles; 72°C for 5min. The PCR product was recovered and purified, cloned into the vector pEASY (Beijing Quanshijin Company), transformed into E. coli DH5α competent cells (CB101, Beijing Tiangen Company), and sequenced after selecting positive clones. The sequence determination results show that the fragment obtained by PCR reaction has the nucleotide sequence shown in SEQ ID NO. 1). The protein shown in SEQ ID NO.1 was named OsFLO18 (that is, the OsFLO18 gene described in the gene mapping), and the gene encoding the protein shown in SEQ ID NO.1 was named OsFLO18 .

Example 2. Acquisition and identification of transgenic plants

1. Construction of recombinant expression vector

Using the cDNA of Ningjing No. 3 as a template, PCR amplification was performed to obtain the coding sequence of the OsFLO18 gene. The PCR primer sequences were as follows:

primer3:

5' CGGAGCTAGCTCTAGAATGTTCTTGGCCACACGACC 3' (SEQ ID NO. 6)

primer4:

5' GTTGTTCTTCAACTTAAGGATCCATGGTGAGCA 3' (SEQ ID NO. 7)

The above primers are respectively located at the upstream of ATG to the downstream of TGA of the gene shown in sequence 2, and the amplification product contains all the coding region of the gene, and the PCR product is recovered and purified. The PCR product was cloned into the vector pCAMBIA1305.1 using the Infusion recombination kit (Clontech) to construct pCAMBIA1305.1 -OsFLO18 ; recombination reaction system (10.0 μL): PCR product 5.4 μL (50-100 ng), pCAMBIA1305.1 vector 1.6 μL (30-50ng), 5×Infusion buffer 2.0 μL, Infusion enzyme mix 1 μL. After a brief centrifugation, the mixed system was water bathed at 37°C for more than 0.5 h, and 2.5 μL of the reaction system was taken to transform E. coli DH5α competent cells (Beijing Tiangen Company; CB101) by heat shock method. All transformed cells were evenly spread on LB solid medium containing 50 mg/L kanamycin.

After culturing at 37°C for 16 h, clone-positive clones were picked and sequenced. The sequencing results showed that a recombinant expression vector containing the gene shown in SEQ ID NO.3 was obtained, and the pCAMBIA1305.1 containing OsFLO18 was named pCAMBIA1305.1 -OsFLO18 , and the OsFLO18 gene was inserted between the multiple cloning sites Xba I and Bam HI .

Second, the acquisition of recombinant Agrobacterium

The pCAMBIA1305.1 -OsFLO18 was transformed into Agrobacterium EHA105 strain (purchased from American Handsome Company) by electric shock method to obtain the recombinant strain, and the plasmid was extracted for PCR and enzyme digestion identification. The correct recombinant strain identified by PCR and enzyme digestion was named pCAMBIA1305.1 –OsFLO18 .

The acquisition of transgenic plants

The specific method for transforming pCAMBIA1305.1 –OsFLO18 into mutant flo18 is:

(1) Cultivate pCAMBIA1305.1 –OsFLO18 at 28°C for 16 hours, collect the cells, and dilute them into N6 liquid medium (Sigma, C1416) containing 100 μmol/L acetosyringone to a concentration of OD600≈0.5 to obtain bacteria liquid;

(2) The flo18 rice mature embryogenic callus cultured for one month is mixed with the bacterial liquid of step (1) and infected for 30 minutes, and the filter paper absorbs the bacterial liquid and transfers it to a co-cultivation medium (N6 solid co-cultivation medium). , Sigma Company), co-cultured at 24°C for 3 days;

(3) inoculate the callus of step (2) on the N6 solid screening medium containing 100 mg/L paromomycin (Phyto Technology Laboratories) for the first screening (16 days);

(4) pick the healthy callus and transfer it to the N6 solid screening medium containing 100mg/L paromomycin for the second screening, and subculture once every 15 days;

(5) pick healthy callus and transfer it to the N6 solid screening medium containing 50mg/L paromomycin for the third screening, and subculture once every 15 days;

(6) Pick resistant callus and transfer it to differentiation medium for differentiation; obtain T0 generation positive plants differentiated into seedlings.

The identification of transgenic plants

1. Hygromycin resistance identification

The invention utilizes 1‰ concentration hygromycin solution to identify transgenic plants. Specific method: Put fresh leaves of transgenic plants in a petri dish, soak them in a freshly prepared 1‰ hygromycin solution, and incubate them in the dark for 48 hours in a 28°C incubator. Compared with the control, leaf necrosis indicates no resistance , no necrosis showed resistance, and the hygromycin-resistant family was named pCAMBIA1305.1 –OsFLO18 .

2. Phenotypic identification

PCAMBIA1305.1 -OsFLO18 -positive plants from T0 generation, Ningjing No. 3 and flo18 were respectively planted in the Pailou experimental base of Nanjing Agricultural University. The phenotype of the T ₁ generation seeds was identified, and it was found that the T ₁ generation showed a segregation phenotype, and the phenotype of the transparent seeds was the same as that of Ningjing 3 (Fig. 7), indicating that the phenotype of the flo18 mutant was indeed controlled by the OsFLO18 gene , that is, the OsFLO18 gene is a starch synthesis-related gene.

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

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ATGTTCTTGGCCACACGACCATCTGGGTCGTCGTCTCTCATTACCAGGCTCTGCGTGCTCCACACCGTTTCCTGGATCGTTTCATCGAGGCAGGTAGCGAGGTTCAGCACCGGCGTCGACAATGCCAATCCAGGGGCTCACTGCCGGTTGTCTGAGCTTTTCCGGCCAGTTCGCACAGAGACCAGTTGCGTTATTATTGGGCGAGCGCTGGAGTGTGGGAGGTGGTCAGAGTCGGTTGAGCTGGAGCTGGAGGGCCTCCATGTCGAGCTGGATCCTTTCGTCGTCAACAAGGTGCTCCGTGGCTTGTTGGACTCGGGGATGGCAGTTCGGTTTTACTGGTGGGCAGAGTCACGCCCTGGATTTTATCATAACAATTTTGCCATAGCGTACATCATAAGCTTGCTGTTTGTAGATGACAATTTCGCCCTGCTTTCGGAGTTCCTGGGAAGAGTAAGGAGCCAGGGGGTAGCATTTCATCGCTCTCTCTATCGGGTACTTTTGGCTGGCTATGCCCGAGCTGGCAAGTTTGATTCCGTCATAGAAACATTTGATGAGATGGTCACATCAGGTTGTCGTGAGTTCGGTGTCGATTACAACAGGTTCATAGGTGTTATGATTAAGAACTGTTGCTTTGATTTGGTCGAGAAGTATTACAACATGGCACTTGCAAAAGGATTTTGTTTAACTCCATTCACTTACTCAAGGTGGATTACTGCATTGTGTCAATCAAACAGGATTGAGCTTGTAGAGGAGCTTCTGACTGACATGGATAAATTTGGGTGCTTCCCAGATTTTTGGGCATGTAACATATACGTTCACTATTTGTGTGGTCACAATAGGTTATATGATGCTTTGCAAATGGTAGAGAAGATGACCATGAAAGGAACCGGCCCAGATGTCGTCACCTACACAACAGTTGTTAGCTGTTTATGCGATCACAGGAGGTTCTCAGAAGCAGTTGGGCTCTGGGAAGAAATGGTGAGGAGGGGACTTAAACCTG ATGTTGTAGCTTGTGGTGCATTGATATTTGGTTTGTGCAAGAATCAAAAGGTTGATGAGGCTTTTGAATTAGCATCAAGGATGCTAACTCTGGATATCCAACTTAATGTTTCGATTTATAATGCCCTAATAAGTGGTTTCTGGAGAGCTGGCAGCATAGAGAAAGCCTACAAAACTGTGTCCTTCATGCAGAGAAATGGTTGTGAACCAGATGTTGTGACGTACAATATCCTTCTAAACCATTACTGTAGTATAGGTATGACAGATAAAGCTGAAAATTTGATCAGAAAGATGGAAATGAGTGGGGTGAATCCTGACAGGTACAGCTATAATATACTGTTAAAAGGGCTATGCAAAGCTCATCAACTGGACAAAGCATTTGCTTTCGTTTCTGATCATATGGAAGTTGGTGGGTTCTGCGATATTGTATCCTGCAACATACTCATCGATGCATTTTGCAGGGCAAAAAAGGTAAATTCTGCACTGAACCTATTCAAGGAAATGGGTTACAAGGGAATTCAAGCTGATGCTGTGACTTATGGGATTCTGATTAATGGTCTTTTTGGCATAGGATACTCAAATCTAGCCGAAGAGCTTTTTGACCAGATGCTAAACACCAAAATTGTTCCTAATGTAAATGTGTACAATATTATGCTGCATAATTTATGTAAGGTTGGTCATTTCAAACATGCACAGAAAATATTTTGGCAGATGACTCAGAAAGAAGTATCACCAGATACAGTTACTTTTAACACACTAATATACTGGCTTGGAAAAAGCTCAAGAGCCGTTGAGGCCCTTGACCTTTTTAAGGAAATGAGGACAAAAGGGGTAGAACCAGATAACTTGACATTTAGGTATATAATCAGTGGTCTTCTGGATGAAGGGAAAGCTACGTTGGCTTATGAGATATGGGAGTATATGATGGAGAATGGTATCATTCTCGATAGAGATGTTTCTGAAAGGCTGATAAGTGTGCTTAAGTTGAAGAACAACTAA

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<223> OsFLO18 gene

<400> 3

ATGTTCTTGGCCACACGACCATCTGGGTCGTCGTCTCTCATTACCAGGCTCTGCGTGCTCCACACCGTTTCCTGGATCGTTTCATCGAGGCAGGTAGCGAGGTTCAGCACCGGCGTCGACAATGCCAATCCAGGGGCTCACTGCCGGTTGTCTGAGCTTTTCCGGCCAGTTCGCACAGAGACCAGTTGCGTTATTATTGGGCGAGCGCTGGAGTGTGGGAGGTGGTCAGAGTCGGTTGAGCTGGAGCTGGAGGGCCTCCATGTCGAGCTGGATCCTTTCGTCGTCAACAAGGTGCTCCGTGGCTTGTTGGACTCGGGGATGGCAGTTCGGTTTTACTGGTGGGCAGAGTCACGCCCTGGATTTTATCATAACAATTTTGCCATAGCGTACATCATAAGCTTGCTGTTTGTAGATGACAATTTCGCCCTGCTTTCGGAGTTCCTGGGAAGAGTAAGGAGCCAGGGGGTAGCATTTCATCGCTCTCTCTATCGGGTACTTTTGGCTGGCTATGCCCGAGCTGGCAAGTTTGATTCCGTCATAGAAACATTTGATGAGATGGTCACATCAGGTTGTCGTGAGTTCGGTGTCGATTACAACAGGTTCATAGGTGTTATGATTAAGAACTGTTGCTTTGATTTGGTCGAGAAGTATTACAACATGGCACTTGCAAAAGGATTTTGTTTAACTCCATTCACTTACTCAAGGTGGATTACTGCATTGTGTCAATCAAACAGGATTGAGCTTGTAGAGGAGCTTCTGACTGACATGGATAAATTTGGGTGCTTCCCAGATTTTTGGGCATGTAACATATACGTTCACTATTTGTGTGGTCACAATAGGTTATATGATGCTTTGCAAATGGTAGAGAAGATGACCATGAAAGGAACCGGCCCAGATGTCGTCACCTACACAACAGTTGTTAGCTGTTTATGCGATCACAGGAGGTTCTCAGAAGCAGTTGGGCTCTGGGAAGAAATGGTGAGGAGGGGACTTAAACCTG ATGTTGTAGCTTGTGGTGCATTGATATTTGGTTTGTGCAAGAATCAAAAGGTTGATGAGGCTTTTGAATTAGCATCAAGGATGCTAACTCTGGATATCCAACTTAATGTTTCGATTTATAATGCCCTAATAAGTGGTTTCTGGAGAGCTGGCAGCATAGAGAAAGCCTACAAAACTGTGTCCTTCATGCAGAGAAATGGTTGTGAACCAGATGTTGTGACGTACAATATCCTTCTAAACCATTACTGTAGTATAGGTATGACAGATAAAGCTGAAAATTTGATCAGAAAGATGGAAATGAGTGGGGTGAATCCTGACAGGTACAGCTATAATATACTGTTAAAAGGGCTATGCAAAGCTCATCAACTGGACAAAGCATTTGCTTTCGTTTCTGATCATATGGAAGTTGGTGGGTTCTGCGATATTGTATCCTGCAACATACTCATCGATGCATTTTGCAGGGCAAAAAAGGTAAATTCTGCACTGAACCTATTCAAGGAAATGGGTTACAAGGGAATTCAAGCTGATGCTGTGACTTATGGGATTCTGATTAATGGTCTTTTTGGCATAGGATACTCAAATCTAGCCGAAGAGCTTTTTGACCAGATGCTAAACACCAAAATTGTTCCTAATGTAAATGTGTACAATATTATGCTGCATAATTTATGTAAGGTTGGTCATTTCAAACATGCACAGAAAATATTTTGGCAGATGACTCAGAAAGAAGTATCACCAGATACAGTTACTTTTAACACACTAATATACTGGCTTGGAAAAAGCTCAAGAGCCGTTGAGGCCCTTGACCTTTTTAAGGAAATGAGGACAAAAGGGGTAGAACCAGATAACTTGACATTTAGGTATATAATCAGTGGTCTTCTGGATGAAGGGAAAGCTACGTTGGCTTATGAGATATGGGAGTATATGATGGAGAATGGTATCATTCTCGATAGAGATGTTTCTGAAAGGCTGATAAGTGTGCTTAAGTTGAAGAACAACTAA

<210> 4

<211> 20

<212> DNA

<213> Artificial sequences

<220>

<223> primer1

<400> 4

ATGTCTTGGCCACACGACC 20

<210> 5

<211>20

<212> DNA

<213> Artificial sequences

<220>

<223> primer2

<400> 5

TTAGTTGTTCTTCAACTTAA 20

<210> 6

<211>36

<212> DNA

<213> Artificial sequences

<220>

<223> primer3

<400> 6

CGGAGCTAGCTCTAGAATGTTCTTGGCCACACGACC 36

<210> 7

<211>33

<212> DNA

<213> Artificial sequences

<220>

<223> primer4

<400> 7

GTTGTTCTTCAACTTAAGGATCCATGGTGAGCA 33

Claims (5)

1. a regulation and control starch synthesis-related protein OsFLO18, its encoding gene, or at least one in the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria containing said encoding gene for improving the plant breeding of starch synthesis application, the plant is rice; The protein OsFLO18 related to regulating starch synthesis is a protein composed of the amino acid sequence shown in SEQ ID NO.1. 2. The application according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. 3. a method for cultivating the normal transgenic plant of starch synthesis is to import the gene of regulating and controlling starch synthesis-related protein OsFLO18 in the abnormal starch synthesis plant, to obtain the normal transgenic plant of starch synthesis; the normal transgenic plant of described starch synthesis is endosperm performance. A transparent non-silty transgenic plant; the abnormal starch synthesis plant is a plant whose endosperm is a powdery phenotype, and the plant is rice; The protein OsFLO18 related to regulating starch synthesis is a protein composed of the amino acid sequence shown in SEQ ID NO.1. 4 . The method according to claim 3 , wherein the gene is introduced into a plant with abnormal starch synthesis through a recombinant expression vector. 5 . 5. The method according to claim 4, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between the multiple cloning sites Xba I and Bam HI of the pCUBi1390 vector.

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