Plant glutelin transport and storage related protein OsVPS34 as well as coding gene and application thereof

Abstract

The invention relates to a plant glutelin sorting related protein OsVPS34 as well as a coding gene and application thereof. The protein provided by the invention is a protein as shown in (a) or (b): (a) a protein consisting of an amino acid sequence as shown in SEQ ID NO.3 in a sequence table; and (b) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence of SEQ ID NO.3, is related to plant glutelin sorting and is derived from SEQ ID NO.3. The protein provided by the invention affects the sorting of glutelin in plant endosperm, and the glutelin cannot be correctly transported into a type II protein body, so that the glutelin content in mature seeds is reduced, and a glutelin precursor is increased. The coding gene of the protein is introduced into a plant with abnormal glutelin sorting, and a transgenic plant with normal glutelin sorting can be cultivated. The protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a plant gluten sorting-related protein OsVPS34, its encoding gene, and its applications. Background Technology

[0002] Rice is one of the world's most important food crops, and ensuring high and stable rice yields is a crucial foundation for safeguarding global food security. The successful application of hybrid rice has largely solved the food problem caused by the world's rapidly increasing population. However, with the improvement of people's living standards, their requirements for rice quality have also increased. From simply having enough to eat to now wanting to eat well, cultivating high-quality, high-nutrition, and various functional rice varieties has become an urgent problem to solve. Storage protein, as the second largest nutrient after starch, has a content and composition that greatly affects rice quality. Glycerin, as the most important storage protein in rice, accounts for about 60%-80%. Therefore, understanding the molecular mechanisms of rice gluten synthesis, transport, processing, and accumulation is crucial for improving rice quality.

[0003] Glutenin in rice is mainly stored in irregular type II protein bodies (PBII). Glutenin contains essential amino acids and can be absorbed by the human body. Rice gluten is initially synthesized as a 57-kDa precursor on polyribosomes on the surface of the endoplasmic reticulum. Guided by a signal peptide, it enters the endoplasmic reticulum lumen during synthesis. Once inside the lumen, the signal peptide is cleaved, and with the help of molecular chaperones such as Bip and PDI, the gluten is formed into the correct conformation. Then, the gluten is transported to the Golgi apparatus in the form of COPII for further processing and modification. Dense vesicles are formed in the trans-Golgi network structure, and with the help of proteins such as Rab5a, vps9a, and GPA5, they enter PBII. In PBII, the gluten precursor is hydrolyzed into a 40-kDa acidic subunit and a 20-kDa basic subunit by the vacuole processing enzyme OsVPE1. If any of the above steps go wrong, gluten will accumulate in endosperm cells as a 57-kDa precursor, which can be observed as an increased 57-kDa precursor phenotype by SDS-PAGE. Summary of the Invention

[0004] This invention, through phenotypic analysis of the rice gluten sorting mutant vps34 and preliminary localization of the target gene, ultimately cloned the gluten sorting-related gene OsVPS34. Therefore, this invention provides a gluten sorting-related gene and its application.

[0005] The gluten sorting-related gene (OsVPS34) provided by this invention is derived from the genus *Oryza sativa* L. Ningjing 4, and is a protein as shown in (a) or (b) below:

[0006] (a) is a protein consisting of the amino acid sequence shown in SEQ ID NO.3;

[0007] (b) A protein derived from SEQ ID NO.3 with one or more amino acid residues substituted and / or deleted and / or added, and which is associated with gluten transport and storage.

[0008] In the sequence listing, SEQ ID NO.3 has a PI3K_C2 structure domain from the 51st to the 191st position from the N end, a PI3Ka structure domain from the 277th to the 456th position from the N end, and a PI3_PI4_kinase structure domain from the 560th to the 761st position from the N end.

[0009] To facilitate the purification of OsVPS34 in (a), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.3.

[0010] Table 1: Label Sequence

[0011] Label residues sequence Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK

[0012] The OsVPS34 mentioned in (b) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. The encoding gene of OsVPS34 mentioned in (b) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID NO.1, and, or by performing mistranslation mutations of one or more base pairs, and, or by attaching the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0013] In addition, the present invention also provides a gene (OsVPS34) encoding the above-mentioned storage protein sorting-related protein.

[0014] The gene OsVPS34 can be a DNA molecule that is 1) or 2) or 3) or 4) below:

[0015] 1) The DNA molecule shown in SEQ ID NO.1;

[0016] 2) The DNA molecule shown in SEQ ID NO.2;

[0017] 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein;

[0018] 4) DNA molecules that have more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encode proteins related to gluten transport and storage.

[0019] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0020] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0021] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the Nos gene for lipase) and plant genes (such as the soybean storage protein gene).

[0022] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing promoter or constitutive promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter, can be added before the transcription initiation nucleotide. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0023] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0024] The recombinant vector is preferably a recombinant plasmid obtained by inserting the gene OsVPS34 between the restriction sites Xba1 and BamH1 of the pCAMBIA1305.1 vector, and named pCAMBIA1305.1-OsVPS34.

[0025] Expression cassettes, transgenic cell lines, and recombinant bacteria containing any of the genes (OsVPS34) described above are all within the scope of protection of this invention.

[0026] Primers that amplify the full length or any fragment of the gene (OsVPS34) are also within the scope of protection of this invention. Any primer capable of amplifying the full length or any fragment of the gene (OsVPS34) is within the scope of protection of this invention. In a specific example, the sequences are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0027] This invention also provides the application of the aforementioned protein, gene, recombinant vector, expression cassette, transgenic cell line, and recombinant bacteria in cultivating transgenic plants with normal gluten sorting. In some embodiments, the application specifically involves transferring the OsVPS34 gene into plants with abnormal OsVPS34 gene.

[0028] The present invention also provides a method for cultivating transgenic plants with normal gluten sorting. In some embodiments, the application specifically involves transferring the OsVPS34 gene into plants with abnormal OsVPS34 gene.

[0029] This invention provides the application of the described protein, gene, recombinant vector, expression cassette, transgenic cell line, and recombinant bacteria in cultivating transgenic plants with abnormal gluten sorting. In some embodiments, the application specifically involves silencing or knocking out the OsVPS34 gene to form transgenic plants with abnormal protein sorting.

[0030] The present invention also provides a method for cultivating transgenic plants with abnormal gluten sorting. In some embodiments, specifically, the OsVPS34 gene is silenced or knocked out to form transgenic plants with abnormal protein sorting.

[0031] This invention also provides knockout primers and knockout vectors for the aforementioned gene. Any primer capable of knocking out the gene (OsVPS34), preventing its normal expression, is within the scope of protection of this invention. In some embodiments, the knockout primers are shown in SEQ ID NO. 6 to SEQ ID NO. 21.

[0032] This invention provides a method for cultivating transgenic plants with normal gluten sorting. The method involves transferring the gene into a mutant with abnormal gluten sorting via transgenic means to obtain transgenic plants with normal gluten sorting. The plant with abnormal gluten sorting is one in which the gluten precursor in the endosperm is dramatically increased, accompanied by a decrease in the content of mature gluten. The transgenic plant with normal gluten sorting is one in which the gluten precursor can be normally processed into mature gluten. Specifically, the gene is introduced into a plant with increased gluten precursor and decreased mature gluten content via the recombinant expression vector; the mutant with abnormal gluten sorting can be vps34.

[0033] The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or recombinant bacteria, or the method can all be applied to rice breeding.

[0034] By using any vector capable of guiding the expression of exogenous genes in plants, the gene encoding the stated protein can be introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the stated gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The plant host being transformed can be either monocotyledonous or dicotyledonous, such as tobacco, birdsfoot, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0035] Advantages of this invention over existing technologies:

[0036] This invention marks the first discovery, localization, and cloning of a novel gene, OsVPS34, for a plant gluten transport-related protein. This protein affects vesicle transport, and inhibiting the expression of its encoding gene can lead to impaired gluten vesicle transport in plant seeds. Introducing the encoding gene into mutants with abnormal gluten sorting yields transgenic plants with normal gluten sorting. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0037] Figure 1 The seed phenotypes of wild-type Ningjing 4 and mutant vps34 are shown.

[0038] Figure 2 Scanning electron microscopy observation of the mature endosperm cross section of wild-type Ningjing 4 and mutant vps34.

[0039] Figure 3 SDS-PAGE gel images of mature seeds of wild-type Ningjing 4 and mutant vps34.

[0040] Figure 4 Western blot results for several specific antibodies against mature seeds of wild-type Ningjing 4 and mutant VPS34.

[0041] Figure 5 Semi-thin section observation of wild-type Ningjing 4 and mutant vps34.

[0042] Figure 6 Transmission electron microscopy observation of wild-type Ningjing 4 and mutant VPS34.

[0043] Figure 7 This indicates the initial location of the mutated gene on chromosome 5.

[0044] Figure 8 The T1 grain phenotype of the T0 generation plants transformed with pCAMBIA1305.1-OsVPS34.

[0045] Figure 9 SDS-PAGE observation of T1 mature grains from T0 generation plants transformed with pCAMBIA1305.1-OsVPS34.

[0046] Figure 10 SDS-PAGE observation of T2 mature grains from T1 generation plants transgenic with pCRISPR-zero-OVPS34. Detailed Implementation

[0047] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0048] Example 1: Discovery of plant seed gluten sorting-related proteins and their encoding genes

[0049] I. Phenotypic Analysis of the Rice Glutenin Sorting Mutant vps34

[0050] A mutant line, vps34, with mealy grains was screened from tissue-cultured seedlings of the japonica rice variety Ningjing 4. Compared with the wild type, the main characteristics of vps34 are wrinkled, mealy, and opaque grains (see...). Figure 1 Scanning electron microscopy analysis confirmed that the starch granules in VPS34 were loosely arranged, which may be the main reason for the opacity of the endosperm powder (see...). Figure 2 SDS-PAGE analysis of VPS34 seed protein showed an increase in glutenin 57 kDa precursor, a decrease in the content of mature glutenin acidic and basic subunits, and a decrease in globulin content (see [link]). Figure 3 Western blot experiments further confirmed the above results (see...). Figure 4).

[0051] Transmission electron microscopy of the developing endosperm 12 days after flowering revealed that the type II protein bodies storing mature glutenin in VPS34 cells exhibited abnormal filling compared to wild-type cells. Furthermore, numerous aggregates of unknown origin, PMB, were also observed in the endosperm cells of VPS34 (see [link to article]). Figure 6 We also observed that dense vesicles could sprout normally from the Golgi apparatus. This was observed using a semi-thin section technique after staining with Coomassie brilliant blue (see...). Figure 5 The study found that type II gluten bodies in VPS34 exhibited a disordered state, and PMB structures were also observed. Therefore, the microscopic data indicated that gluten sorting was abnormal in the endosperm cells of the VPS34 mutant. Some gluten could not be correctly sorted into type II gluten bodies, but instead existed near the cell wall in the form of precursors, leading to an increase in gluten precursors and a decrease in the content of mature gluten.

[0052] II. Map-based cloning of mutant gene sites

[0053] 1. Location of the mutated gene

[0054] Because vps34(- / -) plants are homozygous lethal, the heterozygous mutant vps34(+ / -) was crossed with the widely compatible variety N22. In the F2 segregating population of vps34(+ / -) / N22, 12 recessive extreme individuals with the vps34(- / -) phenotype (mealous, wrinkled grains and increased glutenin precursors) were selected, and their seed tails were excised to extract DNA. Linkage analysis was performed on the 12 recessive extreme individuals using 565 pairs of SSR primers covering the entire rice genome, locating the glutenin sorting-related gene OsVPS34 on chromosome 5 between markers N5-7 and IN5-3. Figure 7 The molecular markers used for initial localization are shown in Table 1.

[0055] Table 1 Molecular markers used for initial localization

[0056]

[0057]

[0058] 2. Fine mapping of mutated genes

[0059] Based on the initial localization results, we sent seeds with powdery wrinkles and increased gluten precursors, as well as wild-type seeds, to the company for resequencing analysis. Within the initially localized region, we found only one indel, which was an insertion of a T base on the fourth exon of OsVPS34 (see Table 2), which caused premature termination of the gene translation and loss of target protein function.

[0060] Table 2 shows the resequencing results of Ningjing 4 (WT) and VPS34 for fine mapping.

[0061]

[0062] III. Obtaining the target gene OsVPS34

[0063] cDNA was extracted from the leaves of the japonica rice variety Ningjing 4. Using the cDNA as a template, PCR amplification was performed using primers primer1 and primer2. The amplified products were sequenced, and the sequencing results are shown in SEQ ID NO.2. The encoded protein is shown in SEQ ID NO.3, and the whole genome sequence is shown in SEQ ID NO.1.

[0064] Primer1:5'-ATGGCGAACGGCGGCGAGTTC-3';

[0065] Primer2:5'-GCGCCAGTATTGAGCCCATCT-3'.

[0066] The protein shown in SEQ ID NO.3 is named OsVPS34 protein. The gene encoding OsVPS34 protein is named OsVPS34 gene, and its open reading frame is shown in SEQ ID NO.2.

[0067] Example 2: Application of OsVPS34 protein and its encoding gene

[0068] I. Construction of Recombinant Expression Vectors

[0069] The OsVPS34 gene was obtained by PCR amplification using cDNA from Ningjing No. 4 leaves as a template. The PCR primer sequences are as follows:

[0070] Primer3:5'-cggagctagctctagaATGGCGAACGGCGGCG-3'(SEQ ID NO.4)

[0071] Primer4:5'-tgctcaccatggatccGCGCCAGTATTGAGCCC-3'(SEQ ID NO.5)

[0072] The primers were located at the 5' and 3' ends of the gene shown in SEQ ID NO.2. The amplification product contained only the CDS part of the gene (SEQ ID NO.2) and the vector adapter sequence. The PCR product was recovered and purified. The pCAMBIA1305.1 vector (reference: He Gao, Mingna Jin, et al., Days to heading 7, a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice. Proc Natl Acad Sci USA, 2014, 111(46):16337-16342) was inactivated by digestion with Xba I and BamH I. The digested vector and the recovered fragment were recombined to obtain the pCAMBIA1305.1-OsVPS34 complementary vector (which has been verified by sequencing).

[0073] II. Obtaining Transgenic Plants by Overexpression

[0074] 1. The pCAMBIA1305.1-OsVPS34 overexpression vector and the empty pCAMBIA1305.1 obtained in step one were introduced into Agrobacterium EHA105 strain (American Ingenium Company) to obtain the EH-pCAMBIA1305.1-OsVPS34 recombinant strain and the EH-pCAMBIA1305 empty vector control strain, respectively.

[0075] 2. The recombinant Agrobacterium EH-pCAMBIA1305.1-OsVPS34 obtained in step 1 and the empty vector control strain were used to transform rice gluten protein sorting aberrant heterozygous mutant (vps34(+ / -)), respectively. The specific method is as follows:

[0076] (1) Incubate EH-pCAMBIA1305-OsVPS34 (or control strain with empty vector) at 28℃ for 16 hours, collect the bacterial cells, and dilute them in N6 liquid medium (Sigma, C1416) to a concentration of OD600≈0.5 to obtain bacterial solution;

[0077] (2) Mix the rice callus (vps34(+ / -)) cultured for one month with the bacterial solution in step (1) for 30 minutes, shaking once every five minutes during the process. Then pour out the bacterial solution and use sterile filter paper to absorb the excess bacterial solution on the callus tissue. Place the callus tissue in (N6 solid co-culture medium, Sigma) and co-culture at 24°C for 3 days.

[0078] (3) Inoculate the callus cultured in step (2) onto solid screening N6 solid medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and culture for 25-30 days;

[0079] (4) Inoculate the healthy callus cultured in step (3) onto differentiation medium (Phyto Technology Laboratories, M524) and differentiate for 20-25 days;

[0080] (5) Transfer the transgenic seedlings obtained in step (4) into the rooting medium and culture for about 20-30 days to obtain T0 generation plants.

[0081] 3. Identify the T0 generation plants obtained in step 2, extract total DNA from the leaves of the plants to be tested, and perform PCR amplification using primers primer5 and primer6:

[0082] Primer5:5'-TTGGAGACAGGCATCTGGATAATC-3'

[0083] Primer6:5'-GTGCAGATGAACTTCAGGGTCAGC-3'

[0084] PCR reaction system: DNA (20 ng / μl) 1 μl, Primer 5 (10 pmol / μl) 2 μl, Primer 2 (10 pmol / μl) 2 μl, 2x Phanta Max Buffer 25 μl, dNTP Mix (10 mM) 1 μl, Phanta Max Super-Fidelity DNA Polymerase (1 U / μl) 1 μl, ddH2O 18 μl, total volume 50 μl. Amplification was performed on a PTC-200 (MJ Research Inc.) PCR instrument: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, 35 cycles; 72℃ for 5 min; 4℃ for 10 min.

[0085] PCR products were detected by 1% agarose gel electrophoresis. The pCAMBIA1305.1-OsVPS34 plasmid was used as a positive control, and pCAMBIA1305.1 was used as a negative control. Plants that could amplify the target size band were transgenic positive plants.

[0086] III. Phenotypic Identification

[0087] The T0 generation transgenic plants pCAMBIA1305.1-OsVPS34, vps34, and wild-type Ningjing 4 were planted in a transgenic field at the Jiangning Tuqiao Breeding Base of Nanjing Agricultural University. Results showed that the seeds of the T2 transgenic line contained transparent grains with a background of vps34 (- / -) (see...). Figure 8 SDS-PAGE analysis showed that transparent seeds (COM1 / COM2 / COM3) behaved similarly to wild-type seeds (see [link]). Figure 9 This verifies that the opaque starchy texture and increased gluten precursors before transgenication are controlled by the OsVPS34 gene, meaning that the OsVPS34 gene is a gluten sorting-related gene. Transforming pCAMBIA1305.1-OsVPS34 into the rice vps34(+ / -) mutant can increase the mature gluten content to normal levels.

[0088] Example 3: Construction of OsVPS34 weak mutant

[0089] I. Construction of multi-target knockout vectors for promoters

[0090] We designed eight knockout target sites targeting the first 2000 bp of the ATG in the OsVPS34 gene. The PCR primers are as follows:

[0091] Primer7:5'-taGGTCTCTGGCAacaaagcaccagtgg-3'(SEQ ID NO.6)

[0092] Primer8:5'-cgGGTCTCtctggactgggcctgcaccagccggg-3'(SEQ ID NO.7)

[0093] Primer9:5'-taGGTCTCtccagacctgcgcgttttagagctagaa-3'(SEQ ID NO.8)

[0094] Primer10:5'-cgGGTCTCgtcgctagagacgtgcaccagccggg-3'(SEQ ID NO.9)

[0095] Primer11:5'-taGGTCTCagcgacgccagctgttttagagctagaa-3'(SEQ ID NO.10)

[0096] Primer12:5'-cgGGTCTCcaacgatggaaggtgcaccagccggg-3'(SEQ ID NO.11)

[0097] Primer13:5’-taGGTCTCtcgttgcggaggtgttttagagctagaa-3’(SEQ ID NO.12)

[0098] Primer14:5’-cgGGTCTCgcgacatcgaccatgcaccagccggg-3’(SEQ ID NO.13)

[0099] Primer15:5’-taGGTCTCtgtcgccttgctggttttagagctagaa-3’(SEQ ID NO.14)

[0100] Primer16:5’-cgGGTCTCtgattatataatctgcaccagccggg-3’(SEQ ID NO.15)

[0101] Primer17:5’-taGGTCTCtaatcacgagtgtgttttagagctagaa-3’(SEQ ID NO.16)

[0102] Primer18:5’-cgGGTCTCtagtattttggtgtgcaccagccggg-3’(SEQ ID NO.17)

[0103] Primer19:5’-taGGTCTCatactaaaatgtcgttttagagctagaa-3’(SEQ ID NO.18)

[0104] Primer20:5’-cgGGTCTCcaagttgttgcattgcaccagccggg-3’(SEQ ID NO.19)

[0105] Primer21:5’-taGGTCTCaacttgtaaggtagttttagagctagaa-3’(SEQ ID NO.20)

[0106] Primer22:5’-taGGTCTCAAAACtcatttagtgcgagagtatgtgcaccagccggg-3’(SEQ IDNO.21)

[0107] Using pUC57-gRNA-tRNA plasmid as a template, PCR reactions were performed. The obtained PCR products were then subjected to a Golden Gate reaction with the pCRISPR-zero vector (Zhou, Shirong, et al. Fine-tuning rice heading date through multiplexediting of the regulatory regions of key genes by CRISPR-Cas9. Plant Biotechnology Journal 22.3(2024):751-758.) to obtain the vector pCRISPR-zero-OsVPS34.

[0108] II. Obtaining Plants by Tapping Multiple Times

[0109] 1. The pCRISPR-zero-OsVPS34 multiple knockout vector obtained in step one was introduced into Agrobacterium EHA105 strain (American Junction Company) to obtain the pCRISPR-zero-OsVPS34 strain.

[0110] 2. The recombinant Agrobacterium pCRISPR-zero-OsVPS34 obtained in step 1 was used to transform rice Ningjing 7. The specific method was the same as the method described above.

[0111] 3. Identify the T0 generation plants obtained in step 2, extract total DNA from the leaves of the plants to be tested, and perform PCR amplification using primers primer 23 and primer 24:

[0112] Primer23:5'-gatactggttactacatgtg-3'

[0113] Primer24:5'-gatactggttactacatgtg-3'

[0114] PCR reaction system: DNA (20 ng / μl) 1 μl, Primer 5 (10 pmol / μl) 2 μl, Primer 2 (10 pmol / μl) 2 μl, 2x Phanta Max Buffer 25 μl, dNTP Mix (10 mM) 1 μl, Phanta Max Super-Fidelity DNA Polymerase (1 U / μl) 1 μl, ddH2O 18 μl, total volume 50 μl. Amplification was performed on a PTC-200 (MJ Research Inc.) PCR instrument: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 2 min, 35 cycles; 72℃ for 5 min; 4℃ for 10 min.

[0115] PCR products were detected by 1% agarose gel electrophoresis, with Ningjing 7 leaf DNA used as a positive control; plants with bands significantly smaller than the positive control were transgenic knockout positive plants.

[0116] III. Phenotypic Identification

[0117] T1 generation transgenic plants pCRISPR-zero-OsVPS34 and wild-type Ningjing 7 were planted in transgenic fields at the Jiangning Tuqiao Breeding Base of Nanjing Agricultural University. SDS-PAGE analysis of the harvested seeds showed that different transgenic families exhibited varying degrees of increased glutenin precursor phenotype (see...). Figure 10 The presence of abnormal gluten sorting indicates that this gene is closely related to gluten sorting.

Claims

1. The gluten-sorting related protein OsVPS34 is a protein that is either (a) or (b) below: (a) is a protein consisting of the amino acid sequence shown in SEQ ID NO.3; (b) A protein derived from SEQ ID NO.3 with one or more amino acid residues substituted and / or deleted and / or added, and which is associated with gluten transport and storage.

2. The gene encoding the protein OsVPS34 as described in claim 1.

3. The gene according to claim 2, characterized in that, DNA molecules that are 1), 2), 3), or 4) as follows: 1) The DNA molecule shown in SEQ ID NO.1; 2) The DNA molecule shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein; 4) DNA molecules that have more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encode proteins related to gluten transport and storage.

4. A recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria comprising the gene of claim 2 or 3.

5. Primers for amplifying the gene according to claim 2 or 3; preferably, the sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. Primers for knocking out the gene described in claim 2 or 3, preferably, the knockout primers are shown in SEQ ID NO. 6 to SEQ ID NO.

21.

7. The application of the protein of claim 1, the gene of claim 2 or 3, the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria of claim 4, and the primers of claim 5 or 6 in the cultivation of transgenic rice with normal gluten sorting; preferably, the application specifically involves transferring the gene OsVPS34 of claim 2 or 3 into rice with abnormal gene OsVPS34.

8. A method for cultivating transgenic rice with normal gluten sorting, comprising transferring the gene OsVPS34 as described in claim 2 or 3 into rice with abnormal gene OsVPS34.

9. The application of the protein of claim 1, the gene of claim 2 or 3, the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria of claim 4, and the primers of claim 5 or 6 in the cultivation of transgenic rice with abnormal gluten sorting; preferably, specifically silencing or knocking out the gene of claim 2 or 3 to form transgenic rice with abnormal protein sorting.

10. A method for cultivating transgenic plants with abnormal gluten sorting, comprising silencing or knocking out the gene described in claim 2 or 3 to form transgenic rice with abnormal gluten sorting.

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