CN112194713B - Protein FSE5 related to rice endosperm starch granule development and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a rice endosperm starch granule development related protein FSE5, and a coding gene and application thereof, belonging to the field of genetic engineering. The amino acid sequence of the rice endosperm starch granule development related protein FSE5 is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2; provides the application of the protein FSE5, or the coding gene, the expression cassette, the transgenic cell line or the recombinant bacteria thereof in cultivating rice for improving the development of starch granules. The coding gene of the protein FSE5 is introduced into a plant with abnormal starch granule development, so that a transgenic plant with normal starch granules can be cultivated. The invention has important theoretical significance and practical significance for further clarifying the molecular mechanism of the development of the starch granules of the endosperm of the gramineous plants and genetically improving the grain quality through the technology and means of genetic engineering.

Description

Protein FSE5 related to rice endosperm starch granule development and encoding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a protein related to the development of rice endosperm starch granules, and a coding gene and application thereof.

Background

Rice is one of three important food crops in China, and more than half of population takes rice as staple food. The rise of the green revolution and the large-area popularization of the hybrid rice greatly improve the yield of the rice and basically meet the grain requirements of people. With the improvement of living standard, people need to eat enough rice and good rice, and higher requirements are provided for rice quality. Starch constitutes about 90% of the rice dry weight, in the form of starch granules in the endosperm cells, the development of which determines the yield, taste quality and end use of rice. To date, the study of the enzymatic reaction processes for starch synthesis is basically clear, but one cannot synthesize starch in vitro. Therefore, the intensive research on rice starch synthesis and starch granule formation can help us to improve rice quality and yield by means of genetic engineering.

The rice endosperm contains compound starch granules, i.e. one amyloplast contains a plurality of sub-granules. The starch is processed in the powder-making body by enzyme catalysis to form compound starch granules with the diameter of 10-20 mu m. At present, the rice starch is mainly used for making daily staple food, rice wine and cakes for people. The size of the starch granules affects the application, and the small starch granules have application advantages over the large starch granules in the aspects of solubility, fluidity, swelling potential and gelatinization, and can be used as additives in the fields of food industry, cosmetics, bioengineering and the like. The preparation of small starch granules mainly comprises two methods of mechanical crushing and direct extraction from plant raw materials, and the industrial application of rice starch is greatly increased if small starch granules can be directly obtained from rice endosperm.

The rice floury endosperm mutant is a mutant with floury opaque phenotype in endosperm caused by abnormal starch synthesis and accumulation, and is an ideal genetic material for researching endosperm development and regulation and control network. At least 30 mutations have been cloned in rice to date which result in the endosperm-floury phenotype. Most of these genes are synthetases involved directly in starch synthesis, while others are involved indirectly in starch synthesis via other pathways. PPR (Pentatricopeptide repeat) protein widely exists in plants, subcellular location is usually in chloroplast or mitochondria, and participates in the regulation and control process after transcription, such as RNA transcription, shearing, editing, stability and the like. Research has shown that PPR plays an important role in the development of plant seeds and endosperm. FSE5 belongs to a member of the PPR protein family, but its function in rice has not been studied.

Disclosure of Invention

The invention aims to provide a protein related to the development of rice endosperm starch granules, and a coding gene and application thereof. The coding gene of the protein FSE5 is introduced into a plant with abnormal starch granule development, so that a transgenic plant with normal starch granules can be cultured. The invention has important theoretical significance and practical significance for further clarifying the molecular mechanism of development of the starch granules of the gramineous endosperm and carrying out genetic improvement on the grain quality through the technology and means of genetic engineering.

The present invention provides a protein (FSE 5) associated with the development of starch granules from rice of the genus Oryza: (Oryza sativa

var. Kitaake), is a protein of the following (a) or (b):

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

(b) And (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence in the sequence 1, is related to the sorting of gluten and is derived from the sequence 1.

Sequence 1 in the sequence table is composed of 395 amino acid residues, and the 82 nd to 330 th positions from the amino terminal are PPR structural domains.

The protein of (b) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The gene encoding FSE5 in (b) above may be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 2 of the sequence Listing, and/or by performing missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to its 5 'end and/or 3' end.

A gene encoding the above-mentioned starch granule-associated protein: (FSE5) Also belong to the protection scope of the present invention.

The geneFSE5Can be the following DNA molecules 1) or 2) or 3) or 4)A son:

1) A DNA molecule shown as a sequence 2 in a sequence table;

2) DNA molecule shown in sequence 3 in the sequence table;

3) A DNA molecule which hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes said protein;

4) DNA molecule which has more than 90% of homology with the DNA sequence defined by 1) or 2) or 3) and codes the protein related to the development of starch granules.

Sequence 2 in the sequence table is composed of 1188 nucleotides.

The stringent conditions can be in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, in 65% SDS ^o Hybridization and washing of membranes at C.

The recombinant expression vector containing any one of the genes also belongs to the protection scope of the invention.

The recombinant expression vector containing the gene can be constructed by using the existing plant 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 polyadenylation signal can direct polyadenylation to the 3 'end of the mRNA precursor, and similar functions can be found in untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopaline synthase Nos), plant genes (e.g., soybean storage protein genes).

When the gene is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter and maize Ubiquitin promoter (Ubiquitin), and the enhanced promoter or constitutive promoter can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, 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 to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. 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. From the safety of transgenic plants, the transformed plants can be screened directly in stress without adding any selective marker gene.

The recombinant expression vector can be a recombinant plasmid obtained by recombining and inserting the gene (FSE 5) between the EcoRI multi-cloning sites of the pCAMBIA1305-35S vector. The recombinant plasmid can be pCAMBIA1305-FSE5; the pCAMBIA1305-FSE5 is obtained by inserting the sequence of the coding region of the FSE5, the upstream 2730 bp promoter region and the downstream 742 bp fragment into the EcoRI multi-cloning site of pCAMBIA1305-35S through a recombination technology (Nanjing Nozaki company, a non-ligase dependent single-fragment rapid cloning kit).

Comprising any one of the genes described above (FSE5) The expression cassette, the transgenic cell line and the recombinant strain belong to the protection scope of the invention.

Amplifying the gene (a)FSE5) Primer pairs of either full length or any fragment are also within the scope of the invention. Therefore, the invention also provides a primer pair for amplifying the coding gene with the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the primer pair is shown in SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequences of the primer pair for amplifying the gene with the sequence shown in SEQ ID NO.3 are shown in SEQ ID NO.6 and SEQ ID NO. 7.

The method for cultivating the transgenic plant with normal starch granule development provided by the invention is to introduce the gene into the plant with abnormal starch granule development to obtain the starch granule developmentA normal transgenic plant; the abnormal starch granule development plant is a plant with small starch granules; the transgenic plant with normal starch granule development is a transgenic plant with transparent appearance restored by endosperm and composite starch changed by starch granules. Specifically, the gene is introduced into an amyloplast dysplastic plant through the recombinant expression vector; the starch granule dysplastic plant can befse5。

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

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the gene for coding the protein into plant cells, so that a transgenic cell line and a transgenic plant can be obtained. The expression vector carrying the gene can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues can be cultured into plants. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: tobacco, lotus roots, arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, lawn grass, alfalfa and the like.

The invention has the following beneficial effects:

the protein related to the development of the starch granules influences the formation process of the starch granules in the endosperm of rice. The coding gene of the protein is introduced into a plant with reduced starch granules, so that a transgenic plant with normal starch granule development can be obtained. The protein and the coding gene thereof can be applied to the genetic improvement of grains.

Drawings

FIG. 1 shows wild type Kitaake and mutantsfse5The brown rice is observed by a scanning electron microscope.

FIG. 2 shows wild type Kitaake and mutantsfse5Morphological observation of endosperm starch granules in development period, wherein A is half-thin slice observation of wild type milk filled 9-day endosperm; b is half thin slice observation of mutant milk for 9 days endosperm; c is wild-type 15-day endospermObserving the semi-thin slice; d is half thin slice observation of endosperm at 15 days after filling of the mutant; a1 is the enlargement of the position of the box in the diagram A; b1 is the enlargement of the position of the frame of the diagram B; c1 is an enlargement of the position of the box in FIG. C; d1 magnification of the box position of FIG. D.

FIG. 3 is a schematic diagram of the fine localization of FSE5, wherein A is the fine localization of the mutant gene; b is a structural schematic diagram of a mutant gene, and C is a sequencing peak diagram of a mutant site; d is the verification of the enzyme cutting mark on the mutation site.

FIG. 4 shows the result of PCR positive detection of transgenic plants.

FIG. 5 shows positive T plant transformed with pCAMBIA1305-FSE5 ₁ Representative of brown rice phenotype.

FIG. 6 shows the mutant and T transformed with pCAMBIA1305-FSE5 ₁ Scanning electron microscope for brown rice cross section.

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.

Example 1 discovery of plant starch granule development-related protein and Gene encoding the same

1. Rice floury endosperm mutantsfse5Phenotypic analysis of

Screening in a japonica rice variety Kitaake mutant library in an earlier laboratory to obtain a powdery endosperm mutant family with stable inheritance, which is named asfse5(Floury shrunken endosperm5)。

Wild Type (WT) brown rice seeds are transparent, and mutantfse5The seeds exhibited a floury opaque phenotype. When the starch granules of the cross sections of the seeds of the two types are observed by using a scanning electron microscope, the starch granules in the wild type are densely arranged and are polygonal, while the starch granules in the mutant are loosely arranged and have larger gaps (figure 1). We subsequently observed starch granules in endosperm after 9 and 15 days of development of wild type and mutant by half-thin section-light microscopy. The results show that the content of the active ingredients,wild-type endosperm cells contained complex starch granules with multiple subparticles per starch body (FIGS. 2A, C, A1 and C1), whereas mutant cells exhibited a large number of small single starch granules in the endosperm (FIGS. 2B, D, B1 and D1).

In conclusion, in the mutantfse5During development, mature seeds exhibit a powdery opaque phenotype due to the formation of small single starch grains in the endosperm, which results in larger spaces between starch grains.

2. Targeting of genes

1. Preliminary location of target gene

The mutant is subjected tofse5Hybridizing with wide-affinity indica rice variety Dular (purchased from germplasm resources pool of institute of crop science, academy of Chinese agrology), andfse5f of/Dular ₂ Randomly selecting 20 brown rice presenting a flour phenotype from the separated population, and germinating the seeds under the aseptic condition. And extracting DNA from seedlings growing for one week, measuring the concentration of the DNA, and uniformly mixing the DNA in equal amount to construct a mixed genome pool. Using Indel primers covering the whole genome of ricefse5And Dular's polymorphism screening, from which a pair of primers with polymorphisms between the two parents is selected every 10cM or so. Two parentsfse5And Dular's DNA and pool-mixed DNA are counted into three templates, selected polymorphic primers covering 12 chromosomes of rice are used for linkage analysis, and finally, the DNA of Dular and pool-mixed DNA are used for linkage analysisFSE5Located between the 5 th chromosomal markers ZL5-7 and ZL 5-3.

The method of Indel marker analysis described above is as follows:

(1) The DNA of the selected individual plant is extracted by a CTAB method, and the operation is as follows:

taking about 0.1 g of young and tender rice leaves, placing the young and tender rice leaves in a 2 ml Eppendorf tube, covering the lid tightly with a steel ball, placing the tube in liquid nitrogen for 5min, and placing the tube in an up-and-down vibration grinder (Shanghai Jingxin) to grind a sample for 1min.

Add 500. Mu.l of extract (containing 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4M NaCl, 0.2g/ml CTAB solution), vortex vigorously on a vortex mixer and mix, water bath at 65 ℃ for 30 min, while inverting the sample up and down every 10 min.

Add 500. Mu.l chloroform/isoamyl alcohol (volume ratio 24.

Mu.l of the supernatant (not to wash the precipitate from the layer separation) was carefully pipetted off into a new 1.5 mL Eppendorf tube, 600. Mu.l of pure ethanol was added, mixed well and placed in a freezer at minus 20 ℃ for 30 min.

12000 Centrifuging at rpm for 10min, removing supernatant, adding 70% ethanol to wash precipitate twice, and blow-drying at room temperature.

The DNA was dissolved by adding 100. Mu.l of ultrapure water.

Mu.l of the solubilized DNA was taken and the concentration of the DNA was measured by One Drop spectrophotometer.

Finally, 20 powdery phenotype individual DNA strains are mixed evenly into a 2 mL Eppendorf tube to construct a mixed genome pool, and the final concentration of each sample in the mixed sample is 100 ng/. Mu.l.

(2) Are respectively provided withfse5Dulaer and Mixed genome pool as a templateThe plates were individually subjected to PCR amplification of the complete genome polymorphism primers.

PCR reaction (10. Mu.l): DNA (20 ng/ul) 1 ul, pre-primer (2 pmol/ul) 1 ul, post-primer (2 pmol/ul) 1 ul,10xBuffer (MgCl) ₂ free) 1ul，dNTP(10 mM) 0.2 ul， MgCl ₂ 0.6 ul (25 mM), 0.1 ul of rTaq enzyme (5 u/ul), ddH ₂ O5.1 ul, total 10 ul.

PCR reaction procedure: denaturing at 98.0 deg.c for 5 min; denaturation at 98.0 deg.C for 30 s, annealing at 58 deg.C for 40 s, and extension at 72 deg.C for 40 s, and circulating for 33 times; extending for 10min at 72 ℃; storing at 4 deg.C. The PCR reaction was amplified in a Langzhi A100 thermal cycler.

(3) Indel-labeled PCR product detection

The amplification products were analyzed by 8% native polyacrylamide gel electrophoresis. The molecular weight of the amplified product is compared by taking 50 bp DNA Ladder as a control, and silver staining is performed for color development.

2. Fine localization of mutant genes

According to the result of primary positioning, indel markers are developed by self at certain intervals in the region where the mutation sites are located, so that more markers can be screened in the primary positioning section for fine positioning. Fromfse5/F obtained by Dular hybridization combination ₂ Picking F confirmed as mutant phenotype in segregating populations ₂ Seeds as a finely localized population. The primer for fine positioning is designed into Indel markers by comparing sequence differences between the genomic sequences of indica rice and japonica rice of subspecies of rice published in the NCBI database of the United states, and the specific method is as follows:

(1) Indel marker development

And integrating the rice public genetic map with a rice genome sequence, and downloading a BAC/PAC cloning sequence near a mutation site. Comparing the sequence segment by segment with corresponding indica rice sequence (9311) at NCBI through BLAST program on line, if the sequences of the two have difference of 4 and more than 4 bases (which can be base insertion or base deletion), preliminarily deducing that the PCR product of the Indel primer has polymorphism between indica rice and japonica rice; then, an Indel Primer is designed by using Primer Premier 5.0 software and synthesized by Beijing Bomaide biotechnology, inc. Will be designed by oneselfMixing Indel pair primers in equal proportion, and detecting the primer inFSE5And Dular, and a primer showing polymorphism used as a primerFSE5Fine positioning molecular markers of genes. The finely positioned primers are shown in Table 1.

TABLE 1 molecular markers for Fine localization

Marking	Pre-primer	Rear primer	Cloning of the cell
				I5-3	5’ GCTCCCCTCAACTTTTCCTC 3’	5’ TCGGTTGCCTGAATACCTTT 3’	OSJNOa0023G10
Z5-1	5’ TTTTCTTCCTCCTCCAGAGCC 3’	5’ GCGTCCTCCTGTCGTCTCAT 3’	OSJNBb0067H15
				Z5-8	5’ ACCATCTGCTAGTGTGGCTT 3’	5’ GCTGGTTGTTTGGTGTTAGC 3’	OSJNBa0017O06
Z5-14	5’ GCATACTTTGTCGCTCCACA 3’	5’ TCCTCATCATCAGTGCTGCT 3'	OSJNBb0016G07
				Z5-18	5’ TGCCTTCCTCCATAGCTCTG 3’	5’ CCAGGAAACCGAGCAAAGAG 3’	OJ1430B02

PCR reaction for marker analysis: DNA (20 ng/ul) 2ul, pre-primer (10 pmol/ul) 2ul, post-primer (10 pmol/ul) 2ul,10xBuffer (MgCl) ₂ free) 2 ul，dNTP(10mM) 0.4 ul， MgCl ₂ (25mM) 1.2ul，rTaq(5 u/ul) 0.4 ul，ddH ₂ O10 ul, total volume 20ul.

Indel primer amplification reactions were performed on a langyi a100 thermal cycler PCR instrument: 5min at 98 ℃; 30 s at 98 ℃,58 ℃ (primer different, adjusted) for 40 s, 1min at 72 ℃,33 cycles; 10min at 72 ℃.

According to F ₂ Molecular data and phenotype data of the endosperm germplasm single plants in the population are finally mapped according to a 'recessive extreme individual gene mapping' method reported by Zhang and the likeFSE5The gene was finely located between markers Z5-14 and Z5-18 of PAC clone OJ1430B02 at a physical distance of about 85 kb (FIG. 3A). The candidate segment contains 10 predicted Open Reading Frames (ORFs). Sequencing the genome sequence of 10 ORFs, and finding out the ORF6 gene in the mutantOs05g0207200There was a deletion of one base G (FIG. 3B, C), and a CAPS restriction tag was designed at this site to confirm the presence of the deleted site again (FIG. 3D). Deletion of a single base G causes premature termination of the protein translated by the mutant.

(3) Obtaining of mutant Gene

Designing primers according to the positioned sites, wherein the sequences are as follows:

primer1：

5'-ATGGAGTCCGTCCTCGCCCG-3'（SEQ ID NO.4）

primer2：

5'-TCAAGCCAAGCTCAAATT-3'（SEQ ID NO.5）

the target gene was obtained by PCR amplification using primer1 and primer2 as primers and cDNA of Kitaake endosperm as a template.

The amplification reaction was performed on a langyi a100 thermal cycler PCR instrument: 5min at 98 ℃; 30 s at 98 ℃, 30 s at 58 ℃,2 min at 72 ℃ and 33 cycles; 7 min at 72 ℃. And (3) carrying out agarose gel electrophoresis on the PCR product, recovering and purifying, cloning the PCR product to an intermediate vector pEASY-Blant (Beijing hologold), transforming Escherichia coli DH5 alpha competent cells (CD 201 of Beijing hologold), selecting positive clones, and sequencing.

The sequencing result shows that the fragment obtained by PCR reaction has CDS nucleotide sequence shown in SEQ ID No.2 of the sequence table, and codes protein (from ATG to TGA) consisting of 395 amino acid residues (see SEQ ID No.1 of the sequence table). The protein shown in SEQ ID NO.1 was named FSE5 (as described in the Fine localization)Os05g0207200Gene) of the protein represented by SEQ ID NO.1, and naming the gene encoding the protein represented by SEQ ID NO.1FSE5。

Example 2 acquisition and Positive identification of transgenic plants

1. Recombinant expression vector construction

Taking DNA of japonica rice Kitaake (purchased from germplasm resource library of institute of crop science of Chinese academy of agricultural sciences) as a template, and performing PCR amplification to obtain the DNAFSE5Genes, PCR primer sequences are as follows:

primer3 (sequence shown underlined for EcoRI cleavage site):

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

primer4 (sequence shown underlined is EcoRI cleavage site):

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

the primers are positioned at the upstream 2730 bp and the downstream 742 bp of the gene shown in SEQ ID NO.2, the amplification product comprises a promoter part of the gene, the PCR product is recovered and purified, and the PCR product is cloned into a vector pCAMBIA1305-35S by adopting a ligase independent single-fragment rapid cloning kit (Nanjing Novezam company) to construct pCAMBIA1305-FSE5.

Recombination reaction (20.0 μ L): PCR product 1. Mu.L (50-100 ng), pCAMBIA1305-35S vector 2. Mu.L (30-50 ng) after EcoR1 single enzyme digestion, 5 × CE II buffer plus 4.0. Mu.L, exnase II enzyme 2.0. Mu.L, ddH ₂ O added 11.0. Mu.L. After the reaction system is finished, lightly blowing and mixing up and down by using a pipette gun, and reacting for 30 min at 37 ℃. Immediately after the reaction, the mixture was taken out and placed in an ice water bath for 5min, and 10. Mu.L of the reaction system was used to transform E.coli DH 5. Alpha. Competent cells (CD 201, kyoto Kogyo gold Co., ltd.) by a heat shock method. All the transformed cells were spread evenly on LB solid medium containing 50mg/L kanamycin. Culturing at 37 deg.C overnight, picking out clone positive clone, and sequencing. The sequencing result shows that the recombinant expression vector of the gene containing the nucleotide sequence shown in SEQ ID NO.3 is obtained and will containFSE5pCAMBIA1305 of (1) is named pCAMBIA1305-FSE5,FSE5the gene was inserted between the multiple cloning sites EcoRI.

2. Obtaining of recombinant Agrobacterium

The pCAMBIA1305-FSE5 is transformed into an agrobacterium EHA105 strain (purchased from Jun corporation, america) by a heat shock method to obtain a recombinant strain, and plasmids are extracted for PCR and enzyme digestion identification. The recombinant strain identified correctly by PCR and enzyme digestion was named EH-pCAMBIA1305-FSE5.

3. Obtaining transgenic plants

EH-pCAMBIA1305-FSE5 transformation mutantfse5The specific method comprises the following steps:

(1) Activating and culturing the recombinant strain EH-pCAMBIA1305-FSE5 on a shaker at 28 deg.C for 16 h, centrifuging at 4000 g for 5min to collect thallus, and diluting into N6 liquid culture medium (Sigma, C1416) containing 100. Mu. Mol/L acetosyringone to OD concentration ₆₀₀ ≈0.5；

(2) Will be cultured for one monthfse5Mixing the embryogenic callus of the mature rice embryo with the bacterial liquid obtained in the step (1) for infection for 30 min, sucking the bacterial liquid by filter paper, transferring the bacterial liquid into a co-culture medium (N6 solid co-culture medium, sigma company), and co-culturing for 3d at 24 ℃;

(3) Inoculating the callus obtained in the step (2) on an N6 solid screening culture medium containing 100 mg/L kanamycin resistance for primary screening (20 d);

(4) Selecting healthy callus, transferring the healthy callus to an N6 solid screening culture medium containing 100 mg/L kanamycin resistance for secondary screening, and subculturing every 20 d;

(5) Selecting healthy calluses, transferring the calluses to an N6 solid screening culture medium containing 50mg/L kanamycin resistance for third screening, and subculturing every 20 d;

(6) Selecting the resistant callus to transfer to a differentiation medium for differentiation;

obtaining T differentiated into seedlings ₀ And (5) generating positive plants.

4. Identification of transgenic plants

1. PCR molecular characterization

The T obtained in the third step ₁ Genomic DNA was extracted from the plant generation, and amplified using Primer5 and Primer6 as Primer pairs (Primer 5: TACGGCGAGTTCTGTAGGTC (SEQ ID NO. 8) and Primer6: GGATTGCACGCAGGTTCTC (SEQ ID NO. 9)) using the genomic DNA as a template. And (3) PCR reaction system: 2ul of DNA (50 ng/ul), 2ul of Primer5 (10 pmol/ul), 2ul of Primer6 (10 pmol/ul), 10xBuffer (MgCl) ₂ free) 2ul，dNTP(10mM) 0.4ul，MgCl ₂ (25mM) 1.2ul，rTaq(5u/ul) 0.4ul，ddH ₂ O10 ul, total volume 20ul. The amplification reaction was performed on a langyi a100 thermal cycler PCR instrument: 5min at 98 ℃; 30 s at 98 ℃, 45 s at 58 ℃, 1.5 min at 72 ℃ and 33 cycles; and 8 min at 72 ℃.

The PCR product was detected by 1% agarose electrophoresis, and lanes 1 and 2 in FIG. 4 represent wild-type and mutant nontransgenic plantsfse5Amplification products (Primer 5 on the genome and Primer6 on the vector, so that non-transgenic plants are not amplified); lanes 3-5 are transgenic positive lines, as positive controls.

2. Phenotypic identification of transgenic brown rice

T transformed into pCAMBIA1305-FSE5 ₁ Generational plants, wild type Kitaake andfse5is planted in a transgenic test field of Chinese academy of agricultural sciences. After the seeds are mature, harvesting the seeds by single plant, and then removing the glumes of the seeds to obtain the brown rice.The observation result shows that transparent seeds appear in the brown rice with pCAMBIA1305-FSE5 positive plants (FIG. 5, L1, L2 and L3 are three different transgenic lines), and the cross section scanning electron microscope result of the brown rice also shows that the starch forms of the complementary lines L1 and L2 are recovered to be normal (FIG. 6). Thereby verifying that the characters of grain opacity and endosperm starch granule reduction before transgenosis are caused byFSE5Under gene control, i.e.FSE5The gene is related to the development of starch granules.

<110> institute of crop science of Chinese academy of agricultural sciences

<120> protein FSE5 related to rice endosperm starch granule development and encoding gene and application thereof

<160> 9

<210> 1

<211> 395

<212> PROTEIN

<213> Oryza sativa Rice (Oryza sativa L. Japonica. Cv. Kitaake)

<223> protein FSE5 amino acid sequence related to starch granule development

<400> 1

Met Glu Ser Val Leu Ala Arg Leu Pro Ser Ser Leu Arg Pro Arg Glu Pro Leu Leu Cys

Arg Val Ile Ser Ala Tyr Gly Arg Ala Arg Leu Pro Ala Ala Ala Arg Arg Ala Phe Ala

His Pro Ala Phe Pro Ala Pro Arg Thr Ala Arg Ala Leu Asn Thr Leu Leu His Ala Leu

Leu Ala Cys Arg Ala Pro Leu Pro Glu Leu Leu Ser Glu Cys Arg Gly Ser Gly Ile His

Pro Asp Ala Cys Thr Tyr Asn Ile Leu Met Arg Ala Ala Val Ala Asp Ser Gly Ser Val

Asp Asn Ala Cys Leu Leu Phe Asp Glu Met Leu Gln Arg Gly Ile Ala Pro Thr Val Val

Thr Phe Gly Thr Leu Val Thr Ala Phe Cys Glu Ala Gly Arg Leu Glu Glu Ala Phe Lys

Val Lys Glu Val Met Ser Leu Gln Tyr Asn Ile Arg Pro Asn Ala His Val Tyr Ala Ser

Leu Met Lys Ala Leu Cys Glu Lys Gly Lys Val Asp Asp Ala His Arg Leu Lys Glu Glu

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

Leu Phe Arg Leu Gly Lys Lys Gly Glu Val Val Ser Leu Leu Glu Glu Met Lys Glu Lys

Gly Ile Lys Val Gly Arg Glu Val His Asn Ser Met Ile Ala Gly Phe Cys Glu Asp Glu

Gly Asp Leu Asp Ala Ala Phe Ala Ala Leu Asp Asp Met Gln Lys Gly Gly Cys Lys Pro

Asp Ser Val Ser Tyr Asn Thr Leu Val Gly Gly Leu Cys Lys Met Gly Arg Trp Arg Asp

Ala Ser Glu Leu Val Glu Asp Met Pro Arg Arg Gly Cys Arg Pro Asp Val Val Thr Tyr

Arg Arg Leu Phe Asp Gly Ile Cys Asp Ala Gly Gly Phe Ser Glu Ala Arg Arg Val Phe

Asn Glu Met Val Phe Lys Gly Phe Ala Pro Ser Lys Asp Gly Val Arg Lys Phe Val Ala

Trp Ile Glu Arg Glu Gly Asp Ala Ala Ser Leu Glu Ser Val Leu Cys Gln Leu Ala Ser

Val Asn Ala Leu Glu Ser Ser Glu Trp Glu Lys Ala Met Ser Gly Val Leu His Asp Pro

Ala Glu Gln Lys Ile Val Lys Leu Leu Asp Asn Leu Ser Leu Ala

<210> 2

<211> 1188

<212> DNA

<213> Oryza sativa L. Japonica. Cv. Kitaake)

<223> CDS sequence of FSE5 protein involved in development of starch granule

<400> 2

ATGGAGTCCG TCCTCGCCCG GCTCCCCTCC TCGCTCCGCC CGCGCGAGCC CCTCCTGTGC 60

CGCGTCATCT CCGCGTACGG CCGCGCCCGC CTCCCCGCCG CCGCCCGCCG CGCCTTCGCG 120

CACCCGGCGT TCCCGGCGCC GCGCACCGCC CGCGCCCTCA ACACGCTCCT CCACGCCCTC 180

CTCGCCTGCC GCGCGCCCCT CCCGGAGCTC CTCTCCGAGT GCCGCGGCTC CGGCATCCAC 240

CCCGACGCGT GCACCTACAA CATCCTAATG CGCGCCGCGG TGGCCGACTC TGGCTCCGTC 300

GACAACGCCT GCCTCCTGTT CGACGAAATG CTGCAGCGGG GGATCGCCCC GACTGTCGTC 360

ACGTTTGGCA CCCTCGTCAC TGCTTTCTGC GAGGCAGGAC GTCTAGAGGA GGCATTCAAG 420

GTTAAGGAGG TGATGTCTTT GCAGTACAAC ATTAGGCCGA ACGCTCATGT GTATGCGAGC 480

CTGATGAAGG CGCTCTGCGA GAAGGGGAAG GTGGATGATG CGCACAGACT CAAGGAGGAG 540

ATGGTGAGCA ATTCAGAACC TTTGGTGGAT TCAGGAGCTT ATGCGACGCT GGCAAGGGCA 600

TTGTTCCGGT TAGGGAAGAA AGGAGAGGTT GTTAGTTTGC TGGAAGAGAT GAAGGAAAAG 660

GGAATCAAGG TTGGTAGAGA AGTTCATAAT TCGATGATTG CAGGGTTTTG TGAGGATGAA 720

GGGGATCTGG ATGCTGCATT TGCAGCGCTC GATGACATGC AGAAGGGTGG GTGCAAGCCG 780

GACTCGGTGA GCTATAATAC ACTGGTTGGT GGTCTATGCA AGATGGGGCG GTGGCGGGAT 840

GCAAGTGAGT TGGTTGAGGA TATGCCACGA AGGGGATGCC GTCCTGATGT AGTCACCTAC 900

AGGAGGCTGT TTGATGGGAT TTGTGATGCT GGGGGGTTCA GCGAGGCGAG GAGGGTTTTC 960

AATGAGATGG TATTCAAGGG CTTTGCACCA AGCAAGGATG GTGTGAGGAA ATTTGTTGCA 1020

TGGATTGAGA GGGAAGGAGA TGCGGCGTCA CTGGAGTCAG TGTTGTGCCA ATTGGCTAGC 1080

GTTAACGCCT TGGAATCAAG TGAGTGGGAG AAGGCAATGA GTGGTGTGCT CCATGATCCT 1140

GCAGAGCAGA AGATTGTGAA GTTGCTGGAT AATTTGAGCT TGGCTTGA 1188

<210> 3

<211> 4658

<212> DNA

<213> Oryza sativa L. Japonica. Cv. Kitaake)

<223> Gene sequence of protein FSE5 related to starch granule development

<400> 3

CTAGGATGCC ATGTCAACGA AATTAACCAG GCATCTATAC TACTGTGGAA CCTAAATTAC 60

ACGGTTTGGT ATAGTTTAGA GGTAAAGATT TCTGATATTG AGGATAAGGG ATATCAAACA 120

AACTCGGTGT AAAGTTGAGG GACAGCATGT GAACTTATTC CATTGAGAAA TAGCGAGACT 180

CGATCCGGCA CTTTAACCTT TTCATTTTTG AGGTTTTTGT CCCTCTCTTA GCACCACGTG 240

GCGCAGTTTC GTAGGCCCAA CAGAGGGCCA ATCAGTCGGC GCCGGCTTTA GATAAATAGA 300

TAGATTTGCA TTACATTATA ATGTCATGTG GGCTAGTTTC CTTTGTACTA AGCCATAGCT 360

TTATTACCTC ATGCCATTGA TAACTTGGGA ATCAATATAA TATGACTAGA TGTTGGTTTA 420

GAAAATGCTT AGTTATGCTT AGATCAACTA GCATACTATA CTATCTCTGT CCTAGAAAGA 480

CCACAATTTT GCTCCAATGT TTGACCGTCC ATCTTATTTG AAAAAAATTA TGATTAGTAT 540

TTTATTGCTA TTAGATGGTA AAATATGAAT AATACTTTAT GTGTGACTAC TTTTTTTATT 600

TTTGCATAAA GTTTTGAAAT AAAACGGACG GTCAAATGTT GAACACGGAA ATCCATAACT 660

GCACTAAAAT TGGGACGGAG ATATACCGTG GTGATGACAT TAGACATGTA CCATTGTGTT 720

TGTAATTCAA CTAAAACATG ACTTAATGAT TGCGCTGTGA GTACATCGTT TTGCTAGACG 780

TGCCCATGGC AGTTGATGAC TGGTTCACGT GAAACCTCTC GAAGATATAC CGTGCTTAAG 840

CAAGCATGGG CAACTACTGG ACTTGTAGTA TGACTCGACC CTTTTCTAGG CACCGAGGCA 900

AGGGTGCGCG TGATAGAGTT GGGTTGTCCC CGATTTAAGG GTTGATTGGA TGTCAGAGTC 960

ACCACGGCAC ACGAGGAGGG GCTGTCCACC CCGATTTAAG GGTGGGTAAA ACCTCGGTGT 1020

GGTGTGCATG GTTATGGGAG GGTTATGCGA AGGGGCCTGT TACGATTTTT CCCTTTACAG 1080

TACCATGGTG GTACTTTGGG GCATGGCAAC ATGCGTGGAA TCATGTCTCG TGAGTATAAT 1140

TGTACACCTC CGCCTAGAGT AGAACTATTT GAATAGCCGT GCGTATGGTT ATGGGCGATC 1200

AACCAACTTC GTCATGATTA GTCTCACCTT AATAAGTTTA GGTGGATGGT TGGGCTTGTC 1260

ATAACGTGGT GTAGCGTTGG ACAGCGATGA TTAATATTGC TTAATTTTGA TTTAACATTT 1320

TTACTGCTTT CAACTGCTGC TTTACCCTAA AAAAAAACTA GCCTCCTTAG AAATGCCTGC 1380

ATCATACCTT CTCTTATTAT GACTTGCTGA GTATAGTGAG TACCACTCAA TCTTTATTTT 1440

TCCCCAACCT CAAAGCTGGA GAATTCCTCA GACGAAGGCA AGTCGGAGGA GTAAGCTTCA 1500

TCCGTACCGT AAAGGATGCG TATGGAGTGG AGTCATGTGC TGCTAGCATC AGTTACCTTA 1560

GTTTAGCTAG CTTTTACCTT TTCTCGTGGC ATTTGTAAGA CTTTCTATAT GTTGTGTAAG 1620

ACGTGCGGAT GTTTGTCAGC TTTATCATTT GTGTACCCTA GTCGGTCCTG GACAGAGGTT 1680

TAATGTACAT TCTGCTTGGA ATTCTGGTTC ATGAATTTCT GAGCGTGACA CCGATGACAT 1740

CACTAAAAGA AACTAGTACT CCCTTCGTCC CAAAATAAGT ATAGTTTTGT ACTATTCATG 1800

TCCAATGTTT GACTGTTCGT CTTATTTGAA AACTTTTTAT GATTAATATT TTATTGTTAT 1860

TAGATGATAA AACATGAATA GTACTTTATG TGTATGTGTG ACGTGACTAT TTTCTTTTAA 1920

TTTTTTCATA AAATTTTCAA ATAAGATGGG TGGTAAAATG TTGGACACGG ATACCCACAG 1980

CTGCACTTAT TTTGGGACGG ATGGAGTAGG ATTTTGGGCT ATTCCAATTG TTTTTAGTTT 2040

CCTTGTTGAG TATTGCCTTC GTACGAACGC ACATCCTCTG CAGTACTGCT AATGCAGTAG 2100

CAGCGGCTGA CATGTGGGCC TGCTACTGCA TTAGCAGTAC TGCAGATAAT CTCCACTGAC 2160

CTTCGTACAT ATTACAATAC ACTTCGAATT TTTTAAGTAA AACTTTTTTA AGTTTAACCA 2220

AATTTATAAA AAAACATATC AATACTTTTA CATAAAAAAC AGTATAAAAA TATTTAATAT 2280

TTTATTTAAT GACACTATTC CCTTGCTATG AAAGTTGTTT TTCTTTTTAC AAACTTAATC 2340

AAACATATGG AAGTTTGAAA AAACAAATCG AACCGTATAA ACGGAGGGAG TGGTAGGCCT 2400

TGGGCTGCGT TGGGCTACTT ACCGCCAGGA AGCCCAATAG AGGTGTACGG CTTCAAAGCT 2460

TGCGCACGCC ACGTCGCCGG CCTCGAGTCG ACGACGCCGT AGCCCACCGA CATGAGCGGC 2520

GCCACCGCCA AGTTCTCTTC CTCCTACCAC CTCGCCGCCG CGCTCCGCCG CGAGCCCGAC 2580

CCGGCGGCCG CGCTCCGCCT CTTCCTCAGC CCCACCCCGA CCGCCGCCGC CGGGCCATCC 2640

TCCTCCCCCG CGCCGCCGTT CCGCTACTCC CTCCGCTGCT ACGACATCAT CGTCTGCAAG 2700

CTCGCCGCCG CGCGCCTCTT CCCGGCCATG GAGTCCGTCC TCGCCCGGCT CCCCTCCTCG 2760

CTCCGCCCGC GCGAGCCCCT CCTGTGCCGC GTCATCTCCG CGTACGGCCG CGCCCGCCTC 2820

CCCGCCGCCG CCCGCCGCGC CTTCGCGCAC CCGGCGTTCC CGGCGCCGCG CACCGCCCGC 2880

GCCCTCAACA CGCTCCTCCA CGCCCTCCTC GCCTGCCGCG CGCCCCTCCC GGAGCTCCTC 2940

TCCGAGTGCC GCGGCTCCGG CATCCACCCC GACGCGTGCA CCTACAACAT CCTAATGCGC 3000

GCCGCGGTGG CCGACTCTGG CTCCGTCGAC AACGCCTGCC TCCTGTTCGA CGAAATGCTG 3060

CAGCGGGGGA TCGCCCCGAC TGTCGTCACG TTTGGCACCC TCGTCACTGC TTTCTGCGAG 3120

GCAGGACGTC TAGAGGAGGC ATTCAAGGTT AAGGAGGTGA TGTCTTTGCA GTACAACATT 3180

AGGCCGAACG CTCATGTGTA TGCGAGCCTG ATGAAGGCGC TCTGCGAGAA GGGGAAGGTG 3240

GATGATGCGC ACAGACTCAA GGAGGAGATG GTGAGCAATT CAGAACCTTT GGTGGATTCA 3300

GGAGCTTATG CGACGCTGGC AAGGGCATTG TTCCGGTTAG GGAAGAAAGG AGAGGTTGTT 3360

AGTTTGCTGG AAGAGATGAA GGAAAAGGGA ATCAAGGTTG GTAGAGAAGT TCATAATTCG 3420

ATGATTGCAG GGTTTTGTGA GGATGAAGGG GATCTGGATG CTGCATTTGC AGCGCTCGAT 3480

GACATGCAGA AGGGTGGGTG CAAGCCGGAC TCGGTGAGCT ATAATACACT GGTTGGTGGT 3540

CTATGCAAGA TGGGGCGGTG GCGGGATGCA AGTGAGTTGG TTGAGGATAT GCCACGAAGG 3600

GGATGCCGTC CTGATGTAGT CACCTACAGG AGGCTGTTTG ATGGGATTTG TGATGCTGGG 3660

GGGTTCAGCG AGGCGAGGAG GGTTTTCAAT GAGATGGTAT TCAAGGGCTT TGCACCAAGC 3720

AAGGATGGTG TGAGGAAATT TGTTGCATGG ATTGAGAGGG AAGGAGATGC GGCGTCACTG 3780

GAGTCAGTGT TGTGCCAATT GGCTAGCGTT AACGCCTTGG AATCAAGTGA GTGGGAGAAG 3840

GCAATGAGTG GTGTGCTCCA TGATCCTGCA GAGCAGAAGA TTGTGAAGTT GCTGGATAAT 3900

TTGAGCTTGG CTTGATTATA TATCCCTATG GATTGTTGTC ACTGTACTGG TTGTTTTTCT 3960

GATCTGCCAC AGTAGTTTAC TGATTTAAAT TTGATGACTC TTGAGTAATT ATCATGGGAA 4020

GGAAAGAGAT ACAACATCAT CAATACGAGG AGTTTGTCAT TAAAAATACA TGCCAATCTA 4080

ACATCAATGG TGCATTCTTG AAGCAAATGA AGCAAGGAGT CATAGGGATG ATTGCAAGTG 4140

ATGATACAGA CCAAGTCTTA TTTTTCATAA CGCTGCCGTC TTCAGCAATG ATTGCAAACA 4200

GAGTTTGAGC ATGCAAATAT GCTCTGTCCT TGGCAAAGAT TTGGATCAAC CGCCTAATCA 4260

TCGTTGAGTA AGGCACTGCA AATCCAGTTG AGCTTCTATT TCTCTACGAT GTGGCATGGA 4320

GTCCATCCAA ATTTTTTACA CCGAAGAAAG ATGTCTGAAA ACTTGCAGCG GAGATATCAA 4380

AATTAAGAAA AAAAAAACTC GGTAGAGTAG GACATGAAAT TGCCTGGAAT GCAAAATTAT 4440

CAGGACTTAA TGCTGTTATG TTGTATTTTC AGTCCAAGTT CAGGGTTGTA TATAGCAATA 4500

TGAGCTCCGG GATTGTACCC CCTATACCCG TGATATCTAA TACTCCTAAA GATCTCTCTT 4560

TTCCCATAAT GAGGTAACTA GCCGAGATCA GCGAAAAGAA GAACAACACG AGTGCAACAG 4620

ATCTGGTGAC ATTTTTGTCA GTCGCTAACA TCGGGAAA 4658

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<223> Primer1

<400> 4

atggagtccg tcctcgcccg 20

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence

<223> Primer2

<400> 5

tcaagccaag ctcaaatt 18

<210> 6

<211> 37

<212> DNA

<213> Artificial sequence

<223> Primer3

<400> 6

ccatgattac gaattcctag gatgccatgt caacgaa 37

<210> 7

<211> 36

<212> DNA

<213> Artificial sequence

<223> Primer4

<400> 7

taccgagctc gaattctttc ccgatgttag cgactg 36

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<223> Primer5

<400> 8

tacggcgagt tctgttaggt c 21

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<223> Primer6

<400> 9

ggattgcacg caggttctc 19

Claims (7)

1. An application of a rice endosperm starch granule development related protein FSE5, or an encoding gene, a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium thereof in cultivating rice for improving starch granule development is characterized in that the amino acid sequence of the rice endosperm starch granule development related protein FSE5 is shown as SEQ ID No. 1; and the improved rice has the function loss of rice endosperm starch granule development related protein FSE5.

2. A method for cultivating transgenic rice with normal starch granule development is characterized in that a gene of rice endosperm starch granule development related protein FSE5 shown by SEQ ID NO.1 is introduced into rice with abnormal starch granule development to obtain transgenic rice with normal starch granule development; the rice with abnormal starch granule development is rice with single small starch granules in endosperm, and has the function loss of rice endosperm starch granule development related protein FSE5; the transgenic rice with normal starch granule development is a compound transgenic rice with large starch granule form changed by restoring transparent endosperm.

3. The method according to claim 2, wherein the gene encoding rice endosperm starch granule development-associated protein FSE5 represented by SEQ ID No.1 is introduced into rice having abnormal starch granule development by a recombinant expression vector containing the gene.

4. The method according to claim 2 or 3, wherein the nucleotide sequence of the gene of the rice endosperm starch granule development related protein FSE5 is shown as SEQ ID No. 2.

5. The method of claim 3, wherein the recombinant expression vector is a binary Agrobacterium vector or a vector useful for microprojectile bombardment of plants.

6. The method of claim 5, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between the multiple cloning sites EcoRI of the pCAMBIA1305-35S vector.

7. The method of claim 6, wherein said recombinant expression vector further comprises a 3' untranslated region of said gene; an enhanced promoter or a constitutive promoter in front of its transcription initiation nucleotide.

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