Glucose metabolism-related protein IbpPGM and its biomaterials and applications

Abstract

The present invention discloses a sugar metabolism-related protein IbpPGM and its biological materials and applications. It belongs to the field of biotechnology. The problem to be solved by the present invention is how to regulate the starch and / or glucose and / or fructose content and / or sucrose content of plants. The present invention provides the application of IbpPGM protein or a substance that regulates the expression of the gene encoding the IbpPGM protein or a substance that regulates the activity or content of the IbpPGM protein in regulating the starch and / or glucose and / or fructose content and / or sucrose content of plants; the IbpPGM protein is a protein whose amino acid sequence is SEQ ID No. 2. The substance that promotes or increases or upregulates the expression of the IbpPGM gene can be used to increase or enhance the starch content and / or glucose content and / or fructose content and / or reduce or lower the sucrose content of plants or is used for plant breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a sugar metabolism-related protein IbpPGM and a biological material and application thereof. Background Art

[0002] Starch is the main storage form of carbohydrates in plants. During the day, leaves synthesize transitional starch in chloroplasts through photosynthesis, which is degraded into sucrose at night and transported to non-photosynthetic organs as an energy source. The synthetic substrate of transitional starch in chloroplasts is ADP-glucose (ADPG), which is mainly catalyzed by plastidial glucose phosphomimerase (pPGI), glucose phosphomutase (phosphoglucomutase, PGM) and ADP-glucose pyrophosphorylase (ADPase). ADPG then generates amylose and amylopectin under the catalysis of starch synthase and the like.

[0003] PGM is an enzyme that catalyzes the conversion of glucose-6-phosphate (G6P) and glucose-1-phosphate (G1P), and G1P is the precursor of ADPG. Plant PGM is divided into plastid type (pPGM) and cytosolic type (cPGM), and the two play a synergistic role in the metabolic distribution of photosynthetic carbon products. In plant cells, PGM maintains the balance between starch and sucrose by participating in three catalytic steps: 1. pPGM catalyzes the conversion of G6P in autotrophic organs (chloroplasts) or heterotrophic organs (amyloplasts) into G1P; 2. pPGM catalyzes the conversion of G1P formed by starch phosphorylase into G6P; 3. cPGM participates in sucrose synthesis by catalyzing the conversion of G6P in the cytoplasm into G1P. Potato, spinach, and pea all have one pPGM member and one cPGM member, while Arabidopsis, tobacco, and corn have one pPGM member and two cPGM members.

[0004] The starch content in the leaves of Arabidopsis and tobacco pPGM gene deletion mutants was almost undetectable, but the soluble sugar content was increased. Mutation of the site rug3 encoding the pPGM gene in pea resulted in a decrease in starch content in both leaves and seeds, proving that G6P is the source of hexose phosphate in pea amyloplasts. After inhibiting the expression of pPGM or cPGM genes in potato, the starch content of tubers decreased, and the photosynthetic rate of leaves decreased. Arabidopsis AtpPGM mutants also showed photosynthetic inhibition, but tobacco plants overexpressing Arabidopsis AtpPGM or AtcPGM genes did not show changes in photosynthetic rate. Although PGM plays an important role in the metabolic allocation of photosynthetic carbon products, how it affects the photosynthetic rate still needs further study. In addition, studies have found that the loss of PGM function can lead to gametophyte abortion, which may be due to insufficient supply of carbon energy substances in the reproductive organs. Therefore, regulating the starch or sucrose metabolic pathway in pollen is an effective means to induce male sterility, which is also widely used in breeding practice.

[0005] In recent years, more and more evidence has shown that there is a branch pathway for starch synthesis in plants, that is, ADPG, which is a substrate for starch synthesis, is directly produced in the cytoplasm by sucrose synthase and transported to the chloroplast. Inhibiting the expression of potato pPGM or cPGM genes separately will lead to a decrease in the starch content of its tubers. However, when the expression of these two genes is inhibited at the same time, there is no significant difference in the starch content of transgenic potato tubers compared with the wild type. This indicates that the precursors of starch synthesis (such as UDPG, ADPG, G1P) can be transported to heterotrophic organs to replace G6P. In cereal endosperm, G6P, the precursor of ADPG, can be transported from the cytoplasm to the amyloplast, and ADPG can also be directly synthesized by cytoplasmic APGase and transported by ADPG transporter. Zhi Zao Therefore, in addition to relying on PGM to catalyze the conversion of G6P and G1P in the plastid, autotrophic There are transport pathways across the plastid membrane in both organs and heterotrophic organs to provide precursors for starch synthesis into the plastid.

[0006] Sweet potato is an important root crop. Its tubers are rich in starch and can be eaten or used as a bioenergy raw material. The functions of key genes in the starch synthesis pathway in sweet potato have been reported. However, the function of PGM genes, which are important in the starch synthesis pathway, has not been clarified. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to regulate the sucrose content and / or glucose content and / or fructose content and / or starch content of plants and the expression levels of ADP-glucose pyrophosphorylase small subunit gene 1 (IbAGP-sTL1), glucose pyrophosphorylase small subunit gene 2 (IbAGP-sTL2), ADP-glucose pyrophosphorylase large subunit gene (IbAGP-TLI), granule-bound starch synthase (IbGBSSI), soluble starch synthase gene I (IbSSI), soluble starch synthase gene II (IbSSII), soluble starch synthase gene III (IbSSIII), soluble starch synthase gene IV (IbSSIV), starch branching enzyme gene I (IbSBEI), starch branching enzyme gene II (IbSBEII), isoamylase gene (IbIsa1) or pullulanase gene (IbPUL).

[0008] In order to solve the above problems, the present application provides a protein.

[0009] The protein is any one of the following:

[0010] A1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0011] A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein described in A1) and having more than 80% identity with the protein described in A1) and having the function of regulating the sucrose content and / or glucose content and / or fructose content and / or starch content in plants;

[0012] A3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of A1) or A2) to a protein tag.

[0013] Sequence 2 is as follows (SEQ ID No. 2):

[0014] MASFCARIEPTSISISKQFKSARNAFPLSPPIRCLSFFNSPSSNFPLRKTVPSSSSPIFAASSSPSS

[0015] SSSSPPATVAESQALKIKSVPTKPIEGQKTGTSGLRKKVKVFMQDNYLANWIQALFNSLAPE

[0016] DYKDQLLVLGGDGRYFNREAAQLIIQIAAGNGVGQIMIGKDGIMSTPAVSAVIRKRKANGG

[0017] FIMSASHNPGGPDYDWGIKFNYSSGQPAPESITDKIYGNTLSISEIKMADIPDVDLSQLGVTR

[0018] YGNFSVEVVDPVGDYLELMQEVFDFSLIRDLLSRPNFRFVFDAMHAVTGAYAKPIFVDMLG

[0019] ASPESIVNGVPLEDFGHGHPDPNLTYAKDLVNVMFGENGPDFGAASDGDGDRNMILGRQF

[0020] FVTPSDSVAIIAANAKEAIPYFKSGPKGLARSMPTSGALDRVAEKLNLLFYEVPTGWKFFGN

[0021] LMDAGKLSVCGEESFGTGSDHIREKDGIWAVLAWLSIIAYRNKDKKPGEALVSVGDVVKQ

[0022] HWATYGRNFFSRYDYEECESEGANKMVAYLRELISTSKAGDKYGSYVLKFADDFSYVDPV

[0023] DGSVASKQGVRFVFTDGSRIIFRLSGTGSAGATVRVYIEQFESDASKHDVDAQIALKPLIELA

[0024] LSLSKLKEFTGREKPTVIT

[0025] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0026] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0027] In the above proteins, the above 80% identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0028] In the above protein, sequence 2 (SEQ ID No. 2) consists of 638 amino acid residues.

[0029] Among the above proteins, the protein is derived from sweet potato.

[0030] The protein may be named IbpPGM, and may be derived from sweet potato, specifically sweet potato tuber, or sweet potato high starch strain Xu 781, sweet potato high starch variety Zhenghong 22 or Zhenghong 23.

[0031] In order to solve the above problems, the present application also provides a biomaterial.

[0032] The biological material is any of the following:

[0033] B1), a nucleic acid molecule encoding the above protein;

[0034] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0035] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0037] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0038] B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0039] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0040] In the above, the nucleic acid molecule described in B1) is any one of the following:

[0041] C1) A DNA molecule whose nucleotide sequence of the coding strand is sequence 1;

[0042] C2) A nucleic acid molecule obtained by replacing and / or deleting and / or adding nucleotides of the nucleic acid molecule described in C1) and having more than 80% identity with the nucleic acid molecule described in C1) and having the function of regulating the starch content and / or glucose content and / or fructose content and / or sucrose content of plants

[0043] In the above, the nucleic acid molecule described in B1) is the DNA molecule shown in Sequence 1.

[0044] Sequence 1 is as follows (SEQ ID No. 1):

[0045] ATGGCGTCGTTTTGTGCGAGGATCGAACCGACCTCCATTTCCATCTCGAAGCAGTTCAAA

[0046] TCCGCCAGGAATGCCTTTCCTCTCTTCCCACCAATCCGATGCCTCTCCTTCTTCAATTCCC

[0047] CTTCCTCTAATTTCCCTCTCAGGAAAACTGTGCCTTCTTCTTCGTCTCCCATCTTCGCCGC

[0048] TTCTTCATCTCCTTCCTCCTCCTCCTCTTCCTCCCGCTACCGTCGCCGAATCTCAAGCA

[0049] CTCAAGATTAAATCGGTTCCGACAAAGCCAATCGAAGGACAGAAGACGGGAACTAGTG

[0050] GGCTCCGTAAGAAGGTTAAAGTTTTTATGCAAGATAATTACCTTGCGAATTGGATTCAGG

[0051] CATTGTTTAATTCGTTGGCGCCTGAGGATTATAAGGACCAGTTGTTGGTTCTCGGAGGTG

[0052] ATGGCCGATATTTTAATCGCGAAGCTGCACAGTTAATCATTCAAATTGCTGCTGGCAATG

[0053] GGGTTGGTCAAATTATGATTGGCAAGGATGGAATAATGTCTACTCCAGCTGTGTCTGCTG

[0054] TGATACGAAAGAGAAAGGCTAATGGTGGCTTTATAATGAGTGCAAGCCATAATCCTGGTG

[0055] GTCCAGACTATGATTGGGGCATCAAGTTCAATTACAGCAGTGGTCAACCAGCACCAGAA

[0056] TCTATTACTGACAAAATATACGGGAACACGCTTTCTATTTCTGAAATTAAGATGGCTGACA

[0057] TTCCTGATGTTGATCTCTCTCAACTTGGAGTTACTAGATATGGGAATTTTAGTGTTGAAGT

[0058] GGTTGACCCAGTAGGTGACTATTTGGAGCTAATGCAGGAAGTGTTTGATTTTTCACTTAT

[0059] CAGAGATCTTCTTTCCAGACCAAATTTCAGGTTTGTGTTTGATGCCATGCATGCTGTCAC

[0060] TGGTGCTTATGCAAAGCCTATTTTTGTTGACATGCTAGGAGCTAGCCCGGAATCTATTGTT

[0061] AATGGCGTGCCTCTTGAAGATTTTGGACATGGTCATCCAGACCCCTAATCTTACATATGCG

[0062] AAAGATTTGGTCAATGTAATGTTTGGCGAGAATGGACCCTGATTTTGGTGCTGCAAGTGAT

[0063] GGGGATGGTGACAGAAATATGATTCTAGGTAGGCAATTTTTTGTTACTCCGTCAGATTCT

[0064] GTAGCAATTATTGCTGCCAATGCAAGAGGCCATTCCATACTTCAAGGTGGTCCCAA

[0065] GGGATTGGCTCGCTCTATGCCCACTAGTGGTGCTTTGGACCGTGTTGCGGAAAAGCTAA

[0066] ATCTTCTGTTTTTACGAGGTTCCTACTGGATGGAAATTCTTTGGGAATCTAATGGATGCAG

[0067] GAAAGTTGTCAGTTTGTGGGGAAGAAAGTTTTGGGACAGGTTCTGACCACATTCGTGA

[0068] GAAAGATGGTATATGGGCTGTATTAGCTTGGCTTTCAATAATTGCATATAGGAACAAGGA

[0069] CAAGAAACCAGGGGAGGCATTGGTTTCTGTTGGTGATGTTGTCAAGCAGCATTGGGCAA

[0070] CTTATTGGAGGAATTTCTTTTCTAGATATGACTATGAGGAATGTGAATCTGAAGGAGCCA

[0071] ATAAGATGGTTGCATATCTTAGAGAACTAATCTCTACCAGTAAGGCTGGTGATAAGTATG

[0072] GAAGTTATGTCCTCAATTTGCCGATGACTTCTCCTATGTTGATCCAGTAGATGGAAGTG

[0073] TTGCATCCAAACAGGGGTCCGATTTGTGTTCACTGATGGATCAAGGATCATCTTTAGAT

[0074] TATCGGGTACTGGTTCTGCTGGTGCAACAGTAAGAGTGTATATTGAACAGTTTGAGTCTG

[0075] ATGCCTCTAAGCATGATGTGGATGCCCAAATTGCATTGAAACCATTGATAGAACTCGCTC

[0076] In the nucleic acid molecule of TGTCTTTATCAAAGCTAAAGGAATTTACCGGAAGAGAGAAGCCAACTGTCATAACATAAB1), those of ordinary skill in the art can easily adopt known methods, such as directed evolution or point mutation, to mutate the nucleotide sequence encoding the protein IbpPGM of the present invention. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of the protein IbpPGM isolated from the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein IbpPGM and have the function of the protein IbpPGM.

[0077] The above 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0078] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0079] In the above biological material, the nucleic acid molecule in B1) may be a gene encoding the protein. Specifically, the nucleic acid molecule in B1) may be a DNA molecule whose coding sequence of the coding chain is shown in Sequence 1.

[0080] Herein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be vector pET-28a and / or pMDC83 and / or pBI121;

[0081] In the above biological materials, the expression cassette described in B2) refers to a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription, but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example: Odell et al. (1985) Nature, 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671;

[0082] Sanfacon et al., Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0083] In the above B3), a plant expression vector can be used to construct a recombinant expression vector containing the gene expression cassette. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using IbpPGM to construct a recombinant expression vector, at its transcription start Any nucleotide can be preceded by An enhancing, constitutive, tissue-specific, or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation start region can come from a transcription start region or a structural gene.

[0084] As a specific example, the prokaryotic expression vector used in the present application may be pET-28a. The binary vector may be pMDC83 or pBI121.

[0085] In order to solve the above problems, the present application provides the following uses:

[0086] Any of the following uses of the material:

[0087] U1, the material regulates the plant starch content and / or glucose content and / or fructose content and / or sucrose content

[0088] U2, the material is used in the preparation of products that regulate the plant starch content and / or glucose content and / or fructose content and / or sucrose content;

[0089] U3. Use of the material in plant breeding;

[0090] U4, the material regulates the expression of at least one of the following genes in plants: ADP-glucose pyrophosphorylase small subunit gene 1, glucose pyrophosphorylase small subunit gene 2, ADP-glucose pyrophosphorylase large subunit gene, granule-bound starch synthase, soluble starch synthase gene I, soluble starch synthase gene II, soluble starch synthase gene III, soluble starch synthase gene IV, starch branching enzyme gene I, starch branching enzyme gene II, isoamylase gene and pullulanase gene;

[0091] The material is any of the following:

[0092] C1) the above proteins;

[0093] C2) the above biological materials;

[0094] C3) a substance that regulates the expression of the gene encoding the protein;

[0095] C4) a substance that regulates the activity and / or content of the protein.

[0096] In this article, ADP-glucose pyrophosphorylase small subunit gene 1 is IbAGP-sTL1 gene, glucose pyrophosphorylase small subunit gene 2 is IbAGP-sTL2 gene, ADP-glucose pyrophosphorylase large subunit gene is IbAGP-TLI gene, granule-bound starch synthase is IbGBSSI gene, soluble starch synthase gene I is IbSSI gene, soluble starch synthase gene II is IbSSII gene, soluble starch synthase gene III is IbSSIII gene, soluble starch synthase gene IV is IbSSIV gene, starch branching enzyme gene I is IbSBEI gene, starch branching enzyme gene II is IbSBEII gene, isoamylase gene is IbIsa1 gene and pullulanase gene is IbPUL gene. The CDS sequence of IbAGP-sTL1 gene is GenBank: Z79635 (Feb-4-2011); the CDS sequence of IbAGP-sTL2 gene is GenBank: Z79636 (Feb-4-2011); the CDS sequence of IbAGP-TLI gene is GenBank: AJ252316 (Jul-26-2016); the CDS sequence of IbGBSSI gene is GenBank: Because the CDS sequence is GenBank: AB071604 (Aug-9-2006); the CDS sequence of IbSSII gene is GenBank: AF 068834 (Mar-10-2010); the CDS sequence of IbSBEI gene is GenBank: AB194725 (Aug-22-2006); the CDS sequence of IbSBEII gene is GenBank: AB071286 (Aug-9-2006); the CDS sequence of IbIsa1 gene is Gen Bank: DQ074643 (Jan-27-2009)).

[0097] The above gene can be derived from sweet potato.

[0098] Herein, the regulation of plant starch content and / or glucose content and / or fructose content and / or sucrose content may be down-regulating, inhibiting or reducing plant sucrose content, and / or up-regulating, enhancing or increasing plant starch content and / or glucose content and / or fructose content.

[0099] Herein, the regulation of the expression of the plant IbAGP-sTL1 gene and / or IbAGP-sTL2 gene and / or IbAGP-TLI gene and / or IbGBSSI gene and / or IbSSI gene and / or IbSSII gene and / or IbSSIII gene and / or IbSSIV gene and / or IbSBEI gene and / or IbSBEII gene and / or IbIsa1 gene and / or IbPUL gene may be upregulation or enhancement or increase of the expression of the IbAGP-sTL1 gene and / or IbAGP-sTL2 gene and / or IbAGP-TLI gene and / or IbGBSSI gene and / or IbSSI gene and / or IbSSII gene and / or IbSSIII gene and / or IbSSIV gene and / or IbSBEI gene and / or IbSBEII gene and / or IbIsa1 gene and / or IbPUL gene.

[0100] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, and the protein encoded by the gene is any of the following:

[0101] A1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0102] A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein described in A1) and having more than 80% identity with the protein described in A1) and having the function of regulating plant starch content, glucose content, fructose content and sucrose content;

[0103] A3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of A1) or A2) to a protein tag.

[0104] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0105] In the above-mentioned use, the plant breeding includes cultivating or breeding plants with higher starch content and / or glucose content and / or fructose content and / or lower sucrose content.

[0106] In the above, the plant having high starch content and / or glucose content and / or fructose content and / or lower sucrose content has a higher starch content and / or glucose content and / or fructose content than the starting plant, and a lower sucrose content than the starting plant.

[0107] The starting plant can be a recipient plant bred by genetic engineering or bred by sexual reproduction. of donor plants.

[0108] The plant may be a plant root.

[0109] In order to solve the above problems, the present application also discloses a method for regulating the sugar content of plants.

[0110] The method for regulating the sugar content of plants is A and / or B, wherein A is to downregulate, inhibit or reduce the sucrose content of plants, and B is to upregulate, enhance or increase the starch content and / or glucose content and / or fructose content of plants; the method comprises upregulating, enhancing or increasing the expression of the coding gene of the above-mentioned protein in the target plant or the activity and / or content of the above-mentioned protein to regulate the sugar content of plants.

[0111] In order to solve the above problems, the present application also discloses a method for regulating the sugar content of plants.

[0112] The method for cultivating plants with altered sugar content comprises up-regulating or enhancing or increasing the expression of the coding gene of the above-mentioned protein or the activity and / or content of the above-mentioned protein in the target plant to obtain plants with altered sugar content. Compared with the target plant, the plants with altered sugar content have at least one of the following characteristics: reduced sucrose content, increased starch content, increased glucose content and increased fructose content.

[0113] In the above, the up-regulation or enhancement or increase in the expression level of the gene encoding the protein of the target plant can be achieved by over-expressing the above protein encoding gene through transgenic technology.

[0114] In the above, the gene vector for overexpressing the above protein encoding gene may be a plant expression vector.

[0115] The plant expression vector may be the vector described in B3). As a specific embodiment, the plant expression vector of the present application may be a pCAMBIA3301 vector.

[0116] In the above method, the protein encoding gene is any one of the following:

[0117] C1) A DNA molecule whose nucleotide sequence of the coding strand is sequence 1;

[0118] C2) A nucleic acid molecule obtained by replacing and / or deleting and / or adding nucleotides of the nucleic acid molecule described in C1) that has more than 80% identity with the nucleic acid molecule shown in C1) and has the function of regulating plant starch content and / or glucose content and / or fructose content and / or sucrose content.

[0119] In the above, the protein encoding gene may be a DNA molecule of sequence 1.

[0120] In order to solve the above problems, the present application also discloses a method for up-regulating, enhancing or improving gene expression in sweet potato.

[0121] The method for up-regulating, enhancing or improving gene expression in sweet potato comprises up-regulating, enhancing or improving the expression of the coding gene of the above-mentioned protein in the target plant or the activity and / or content of the above-mentioned protein, so as to obtain a sweet potato in which the expression of at least one of the following genes is up-regulated, enhanced or improved: the gene is at least one of the following genes: ADP-glucose pyrophosphorylase small subunit gene 1, glucose pyrophosphorylase small subunit gene 2, ADP-glucose pyrophosphorylase large subunit gene, granule-bound starch synthase, soluble starch synthase gene I, soluble starch synthase gene II, soluble starch synthase gene III, soluble starch synthase gene IV, starch branching enzyme gene I, starch branching enzyme gene II, isoamylase gene and pullulanase gene.

[0122] In the above-mentioned use and method, the plant is any one of the following:

[0123] C1) dicots or monocots; C2) Tubulales, C3) Convolvulaceae, C4) Ipomoea batatas, C5) Sweet potato.

[0124] The sweet potato can be the sweet potato high starch line Xu 781, the sweet potato high starch variety Zhenghong 22 or Zhenghong 23.

[0125] Beneficial effects:

[0126] This study cloned the plastid PGM gene IbpPGM from sweet potato and performed expression analysis and preliminary functional identification, providing a new candidate gene for increasing the starch content of sweet potato root tubers by genetic engineering means. The present application obtains transgenic sweet potatoes with overexpression of the IbpPGM gene by bioengineering technology. Experiments have shown that the starch content and / or glucose content and / or fructose content of the transgenic plants overexpressing the IbpPGM gene are higher than the wild type and / or the sucrose content is lower than the wild type. The present invention cloned and identified a sugar metabolism-related protein IbpPGM, which significantly increased the starch content and / or glucose content and / or fructose content of sweet potatoes and / or significantly reduced the sucrose content after overexpression. It provides new strategies and new ideas for breeding sweet potatoes with high starch content and / or high glucose content and / or high fructose content and / or low sucrose content. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 Schematic diagram of the T-DNA insertion segment of the recombinant overexpression vector pC3301-121-IbpPGM.

[0128] Figure 2 Multiple sequence comparison of PGM proteins.

[0129] Figure 3 To use GSDS to analyze the phylogenetic relationship and gene structure of IbpPGM and PGM proteins of other species.

[0130] Figure 4 This is the expression pattern analysis of the IbpPGM gene.

[0131] Figure 5 This is the prokaryotic expression of the IbpPGM gene in Escherichia coli.

[0132] Figure 6 The leaf cell localization of IbpPGM protein in Nicotiana benthamiana leaves. The scale bar is 50 μm.

[0133] Figure 7 It is the expression level of IbpPGM gene in transgenic sweet potato lines and control lines.

[0134] Figure 8 is the expression level of starch metabolism related genes in transgenic sweet potato lines and control lines.

[0135] Fig. 9 The process of sweet potato transgenic overexpressing IbpPGM gene.

[0136] Fig.10 The differences in leaf phenotype and plant growth potential between transgenic sweet potato lines and control lines in test tube seedlings. DETAILED DESCRIPTION

[0137] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0138] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0139] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0140] The following examples used Excel and SPSS17.0 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. Student's t test was used, and P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0141] Plant materials and reagents

[0142] Sweet potato high starch variety Xu 781: preserved by the Potato Research Laboratory, Institute of Food Crops, Henan Academy of Agricultural Sciences.

[0143] Sweet potato low-starch variety Li Zixiang: preserved by the Potato Research Laboratory, Institute of Grain Crops, Henan Academy of Agricultural Sciences.

[0144] Nicotiana benthamiana: preserved by the Potato Research Laboratory, Institute of Food Crops, Henan Academy of Agricultural Sciences.

[0145] The high starch sweet potato line Xu 781, the low starch sweet potato varieties Lizixiang and Nicotiana benthamiana Plant (Nicotiana benthamiana) was recorded in the following documents before the application date: Wang YN, Li Y, Zhang H, Zhai H, Liu QC, He SZ. A soluble starch synthase I gene, IbSSI, alters the content, composition, granule size and structure of starch in transgenic sweet potato. Scientific Reports, 2017, 7, 2315. The public can obtain the biological material from the applicant, and the biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes. Prokaryotic expression vector pET-28a: Yisheng Biotechnology (Shanghai) Co., Ltd. (11905ES03).

[0146] Subcellular localization vector pMDC83: Wuhan Puint Bioengineering Co., Ltd. (ZT5375).

[0147] Plant expression vector pCAMBIA3301: Changsha Abiwei Biotechnology Co., Ltd. (HG-VZC0333).

[0148] Plant expression vector pBI121: Shanghai Maokang Biotechnology Co., Ltd. (MF3721).

[0149] Agrobacterium competent EHA105: Shanghai Weidi Biotechnology Co., Ltd. (AC1012).

[0150] Gene cloning and fluorescence quantitative PCR related reagents were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. and Tiangen Biochemical Technology (Beijing) Co., Ltd., Escherichia coli competent cells Trans5α and Transetta (DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and primer synthesis and first-generation sequencing were completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0151] Example 1. Cloning and sequence analysis of sweet potato IbpPGM gene

[0152] Total RNA of sweet potato line Xu 781 was extracted and reverse transcribed into cDNA. Degenerate primers DF / DR (as shown in Table 1) were designed using the pPGM gene sequence cloned in plants. After amplification of the EST sequence, the full-length cDNA sequence of the IbpPGM gene was cloned using the RACE method and sequenced. Afterwards, primers GF / GR were designed to amplify the full-length sequence of the IbpPGM genome and sequenced. The open reading frame (ORF), protein molecular weight, signal peptide and genome structure of the IbpPGM gene were analyzed online using ORFfinder, ExPASy, TargetP-2.0 and GSDS (Gene Structure DisplayServer). At the same time, multiple sequence alignment of PGM proteins was performed using DNAMAN software.

[0153] The results showed that the full-length cDNA sequence of the IbpPGM gene cloned from the sweet potato high-starch line Xu 781 using the RACE method was 2182bp, and the ORF was 1917bp. The full-length IbpPGM genome is 5583bp long, containing 22 exons and 21 introns. The gene encodes a protein with a length of 638 amino acids and a molecular weight of 69.3kDa. TargertP-2.0 combined with ChloroP 1.1 online prediction showed that there was a chloroplast transit peptide containing 73 amino acid residues at the N-terminus of the IbpPGM protein. After removing the transit peptide, the mature IbpPGM protein was 61.7kDa. Multiple sequence comparisons of PGM proteins showed that ( Figure 2 ), IbpPGM protein shares the same conserved domain with Arabidopsis, potato and other plant PGMs and eukaryotic yeast PGMs. At the same time, pPGM has a chloroplast transit peptide compared to cPGM. GSDS online analysis showed that IbpPGM is closely related to tomato pPGM protein (XP_004234144), and pPGM and cPGM form two distinct branches. The pPGM gene contains 22 exons, while the cPGM contains 18 exons ( Figure 3 ).

[0154] Example 2. Expression analysis of IbpPGM gene

[0155] In order to detect the expression level of IbpPGM gene in various parts of sweet potato, Xu 781 plants grown in the field for 100 days were taken, and total RNA (Tiangen Biochemical Technology Co., Ltd., DP432) was extracted from tuberous roots, fibrous roots, stems, leaves and petioles. After reverse transcription into cDNA (Takara, RR047A), primers (qPGM-F / R) were designed and qRT-PCR was performed using the ABI7500 system (Takara, RR420A). The internal reference gene was IbActin (AY905538, Actin-F / R).

[0156] To detect the response of IbpPGM gene to exogenous sucrose treatment, leaves (with petioles) of Xu 781 grown for 1 m in the field were immersed in sterile water for 1 day for starvation treatment, and then the leaves were immersed in sterile water (CK) or 175 mM sucrose solution prepared with sterile water for dark culture, and samples were taken at 0h, 2h, 4h, 6h, 12h, 24h and 48h for liquid nitrogen freezing and preservation, and leaves were repeated 3 times for each treatment and each time point. Then RNA extraction, reverse transcription and qRT-PCR were performed on the samples.

[0157] qRT-PCR analysis showed that the IbpPGM gene was expressed in all major tissues of Xu 781 plants ( Figure 4 -A), with the highest expression in tuberous roots, followed by leaves, fibrous roots, stems and petioles. After the leaves (with petioles) were treated with 175mM sucrose solution in the dark, the IbpPGM gene was strongly induced to express, and the expression level began to increase significantly after 12h of treatment, reaching the highest level at 48h, which was about 28 times that of 0h ( Figure 4 -B).

[0158] Example 3. Prokaryotic expression of IbpPGM gene

[0159] In order to verify whether the IbpPGM gene can encode mature protein, it was expressed in Escherichia coli. Primers pET-F / R and pET-ΔF / R were designed to amplify the ORF sequence of the IbpPGM gene and the ORF sequence without the signal peptide (ΔIbpPGM), respectively. After sequencing verification, they were connected to the prokaryotic expression vector pET-28a. The recombinant vectors pET-28a-IbpPGM, pET-28a-ΔIbpPGM and pET-28a empty were transformed into Escherichia coli competent cells Transetta (DE3), and the positive transformation strains were inoculated into 350mL liquid LB medium at a ratio of 1:100 (v / v) and cultured at 28℃ with shaking until OD600=0.8. After adding IPTG (1mM), it was placed on a shaker to induce expression for 12h (28℃, 110rpm). Then, protein SDS-PAGE electrophoresis was performed using the Junyi Oriental JY-SCZ2+ vertical electrophoresis tank (primer sequences are shown in Table 1).

[0160] Vector construction: pET-28a vector was used to insert the IbpPGM gene ORF fragment (B fragment, the sequence is shown in SEQ ID NO: 1) or the IbpPGM gene ORF fragment after removing the signal peptide (ΔB fragment, the sequence is shown in SEQ ID NO: 3)

[0161] The nucleotide sequence is sequence B or ΔB to replace the fragment between the restriction endonuclease NcoI and XhoI recognition sites of the pET-28a vector (Novagen), while keeping the other nucleotide sequences of the pET-28a vector unchanged, to obtain pET-28a-B and pET-28a-ΔB recombinant vectors.

[0162] The results showed that the two vectors were induced to express in E. coli ( Figure 5 The IbpPGM gene with a complete coding frame did not induce the expression of the corresponding protein (69.3 kDa) in E. coli, while the ΔIbpPGM gene without the chloroplast transit peptide sequence induced the expression of a protein of the corresponding size (61.7 kDa), indicating that the chloroplast transit peptide may have a certain inhibitory effect on the expression of the IbpPGM gene in E. coli.

[0163] Example 4. Subcellular localization of IbpPGM gene

[0164] The total RNA of the sweet potato line Xu 781 was reversely transcribed into cDNA as a template, and the ORF of the IbpPGM gene (as shown in sequence 1) was amplified with primers 83-F / 83-R (as shown in Table 1), and the amplified IbpPGM gene ORF fragment (as shown in sequence 1) replaced the fragment between the restriction endonuclease Pac I and Asc I recognition sites of the pMDC83 vector, and the other nucleotide sequences of the pMDC83 vector were kept unchanged to obtain the recombinant vector pMDC83-IbpPGM. The recombinant vector pMDC83-IbpPGM and the empty vector control were transformed into the Agrobacterium EHA105 strain, and the positive transformed strains were screened and injected into the lower epidermis of Nicotiana benthamiana leaves, and then placed at 28°C for 36h (16h light-8h dark), and after the culture was completed, a laser confocal microscope (Nikon Inc., Melville, NY, USA) was used for fluorescence observation.

[0165] The expression vector pMDC83-IbpPGM was constructed and injected into the lower epidermis of Nicotiana benthamiana for transient expression. Laser confocal microscopy revealed that ( Figure 6 ), the green fluorescence of the target gene IbpPGM and GFP fusion protein was scattered in dots and overlapped with the red fluorescence of chloroplasts, indicating that the IbpPGM protein was localized in chloroplasts.

[0166] Example 5. Construction of overexpression vector and acquisition of overexpression transgenic sweet potato plants

[0167] Total RNA of sweet potato line Xu 781 was reverse transcribed into cDNA as a template, and OPGM-F (nucleotide sequence is 5'-cgggatccATGGCGTCGTTTTGTGC-3') and OPGM-R (nucleotide sequence is 5'-cgagctcTTATGTTATGACAGTTGGCTTCTCTCT-3') were used as primers for PCR amplification of the CDS of the IbpPGM gene with BamH I and Sac I recognition sites at both ends (the CDS sequence is shown in Sequence 1). The PCR product of the CDS sequence of the IbpPGM gene was inserted into pBI121 to obtain the recombinant vector pBI121-IbpPGM. pBI121-IbpPGM is obtained by replacing the fragment (small fragment) between the restriction endonuclease BamHI and Sac I recognition sites of the pBI121 vector with the CDS sequence of the IbpPGM gene whose nucleotide sequence of the coding chain is sequence 2 (as shown in sequence 1), while keeping the other nucleotide sequences of pBI121 unchanged. pBI121-IbpPGM was digested with HindIII and EcoR I, and a small fragment was collected, which is referred to as 35S-IbpPGM-NOS (partial map as shown in Figure 1). 35S-IbpPGM-NOS was inserted into pCAMBIA3301 to obtain the IbpPGM gene recombinant vector pC3301-121-IbpPGM. pC3301-121-IbpPGM is a recombinant vector pC3301-121-IbpPGM obtained by replacing the fragment (small fragment) between the restriction endonuclease HindIII and EcoR I recognition sites of the pCAMBIA3301 vector with 35S-IbpPGM-NOS, while keeping the other nucleotide sequences of the pCAMBIA3301 vector unchanged.

[0168] 35S-1bpPGM-NOS sequence (SEQ ID NO 8):

[0169]

[0170] pC3301-121-IbpPGM was transformed into Agrobacterium tumefaciens EHA105 strain and the positive transformed strain pC3301-121-IbpPGM / EHA105 was screened. The embryonic suspension cell culture system of the sweet potato low starch variety Lizixiang was constructed according to the method of the following document: Liu QC, Zhai H, Wang Y, Zhang DP. Efficient plant regeneration from embryogenic suspension cultures of sweet potato. In Vitro Cell Dev Biol-Plant, 2001, 37: 564-567.

[0171] Sweet potato genetic transformation was performed by infecting embryonic suspension cells of the sweet potato variety Lizixiang with the pC3301-121-IbpPGM / EHA105 strain according to the method of the following document: Wang YN, Li Yan, Zhang H, Zhai H, Liu QC, He S Z. Aplastidic ATP / ADP transporter gene, IbAATP, increases starch and amylose contents and alters starch structure in transgenic sweetpotato. J Integr Agri, 2016, 15(9): 1968-1982, and obtaining regenerated plants, which are called pseudo-transgenic plants. The proposed transgenic plants were subjected to GUS staining and PCR identification, and then the positive transgenic plants identified were subjected to qRT-PCR using primers qPGM-F / R (primer sequences are shown in Table 1), the internal reference gene was IbActin (primer sequences are shown in Table 1), and the kit used was PerfectStart Green qPCRSuperMix (AQ601-02), and the reaction conditions were: 94℃30s; 94℃5s, 60℃30s (42cycles); Dissociation stage. Instrument: Bio-Rad CFX96. The three strains with the highest expression of IbpPGM were selected for subsequent phenotypic identification (primer sequences are shown in Table 1).

[0172] The results showed that the recombinant expression vector pC3301-121-IbpPGM was constructed and the low-starch sweet potato variety Lizixiang was genetically transformed by Agrobacterium infection of embryonic suspension cells. A total of 97 pseudo-transgenic lines were obtained, of which 10 were positive transgenic lines. The expression level of the IbpPGM gene in these 10 lines was detected by qRT-PCR ( Figure 7 ), the results showed that the expression level of the IbpPGM gene in the overexpression strains was 1.3 to 15.3 times that of the wild-type control, among which the three strains with the highest expression levels were OX17 (6.2 times), OX53 (10.3 times) and OX85 (15.3 times). The starch content and other indicators of these three strains were determined.

[0173] Example 6. Determination of starch and soluble sugar content in transgenic sweet potato root tubers

[0174] The starch content of the tubers of the overexpressed transgenic lines (OX17, OX53 and OX85) and the wild-type control (WT) was determined according to the method of Smith and Zeeman, and each line was repeated 3 times. The basic principle of this method is to hydrolyze starch into glucose, and then use hexokinase and glucose-6-phosphate dehydrogenase to catalyze glucose to produce NADPH, and calculate the starch content in the sample by measuring the difference in NADPH absorbance. At the same time, the content of sucrose, glucose and fructose in the tubers was determined by liquid chromatography.

[0175] The method for determining the starch content of tubers is as follows:

[0176] 1. Determination steps

[0177] (1) Weigh 0.2-0.5 g of the above fresh root tubers and quickly freeze them with liquid nitrogen.

[0178] (2) Transfer the tubers to a centrifuge tube containing 5 mL of 80% ethanol (v / v) and place in a boiling water bath for 3 min. Centrifuge at ≥3000 g for 5-10 min at room temperature (20-25°C) and discard the supernatant. Repeat the ethanol extraction process twice, discard the supernatant, and ensure that the ethanol in the tube has evaporated completely.

[0179] (3) Transfer the root tuber to a small mortar and grind it into a homogenate with a small amount of water. Transfer the homogenate to a graduated centrifuge tube and make up to 5 mL with water.

[0180] (4) Transfer 0.5 mL of the above homogenate to four 1.5 mL centrifuge tubes and heat at 100°C for 10 min to gelatinize the starch granules. (5) Cool to room temperature and add 0.5 mL of 200 mM sodium acetate (pH 5.5) to the four centrifuge tubes. Then, add 6 U of α-amyloglucosidase and 5 U of α-amylase to two of the centrifuge tubes (this is the sample tube). Add an equal volume of ddH2O to the other two centrifuge tubes. 2 O (this is the control tube). Place the 4 centrifuge tubes at 37°C for 4 hours (the incubated samples can be stored at -20°C for a few days or at -80°C for several months before the next measurement).

[0181] (6) Centrifuge at room temperature for 5 min (≥10,000 g).

[0182] (7) Determine the glucose content in the supernatant. Pipette 0.01 mL of the solution from the sample tube and the control tube into a 1.5 mL centrifuge tube, add enzyme buffer (containing 100 mM HEPES (pH 7.5), 0.5 mM ATP, 1 mM NAD, 4 mM MgCl 2 ) to 1 mL, then add 1.5 U of hexokinase and 1.5 U of glucose-6-phosphate dehydrogenase, and react at 30°C for 10 min.

[0183] (8) Transfer the reaction solution in the two tubes to a 1 cm wide cuvette and measure the absorbance at 340 nm. Mark the absorbance value of the sample tube as OD S , the absorbance value of the control tube is marked as OD C (When measuring the OD values ​​of the sample tube and the control tube, the blank solution without hexokinase and glucose-6-phosphate dehydrogenase was used to adjust the value to zero.) Each sample was repeated three times.

[0184] 2. Calculation of starch content in root tubers

[0185] The reaction principle for determining glucose content is as follows:

[0186]

[0187] According to Lambert-Beer law: A = ε × C × L, where A is absorbance (OD), ε is extinction coefficient (Lmol -1 cm -1 ), C-sample concentration (mol L -1 ), L-light path (cm). The extinction coefficient of NADPH is 6.22×10 3 Lmol -1 cm -1 . It can be concluded that: ΔA=OD S -OD C =ε×ΔC×L, and then ΔC=ΔA / (ε×L), ΔA is the change in absorbance within 10 minutes, and ΔC is the change in NADPH concentration within 10 minutes, and the following calculation formula is obtained:

[0188] ΔC=ΔA / (6.22×10 3 Lmol -1 cm -1 ×1cm)=ΔA / (6.22×10 3 Lmol -1)=ΔA / 6.22(μmol mL -1 )

[0189] Since the reaction volume is 1 mL, the change in the molecular weight of NADPH within 10 min is ΔA / 6.22 (μmol), which is the glucose content in the 1 mL reaction system. The glucose in the 1 mL reaction system is actually contained in 0.01 mL in step (7). Therefore, the glucose contained in 1 mL in step (5) is:

[0190]

[0191] Then the glucose contained in 5 mL of solution in step (3) is:

[0192]

[0193] Convert starch content to μmol glucose g -1 Fresh weight is:

[0194]

[0195] Convert to μg starch g -1 For fresh weight, multiply the above formula by 162 to get:

[0196]

[0197] Excel and SPSS17.0 statistical software were used to process the data. The experimental results were expressed as mean ± standard deviation. Student's t test was used. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated an extremely significant difference.

[0198] The results showed that the starch content of the root tubers of the transgenic lines and the control was measured (Table 2), and the overexpression of the IbpPGM gene significantly increased the starch content of the sweet potato root tubers, among which the starch content of OX85 increased by 12% compared with the control. At the same time, the sucrose content in the root tubers of the overexpression lines decreased significantly, and the glucose and fructose contents increased significantly.

[0199] Example 7. qRT-PCR detection of starch synthesis related genes

[0200] To investigate the effect of overexpression of IbpPGM on starch synthesis-related genes in transgenic lines, total RNA from root tubers of wild type (WT) and transgenic lines (OX17, OX53 and OX85) was extracted and reverse transcribed into cDNA. The wild type was used as the control, and qRT-PCR analysis was performed on the following 12 genes (primer sequences are shown in Table 1): IbAGP-sTL1 gene (CDS sequence is GenBank: Z79635 (Feb-4-2011)), IbAGP-sTL2 gene (CDS sequence is GenBank: Z79636 (Feb-4-2011)), IbAGP-TLI gene (CDS sequence is GenBank: AJ252316 (Jul-26-2016)), IbGBSSI gene (granule-bound starch synthase, CDS sequence is GenBank: AB071604 (Aug-9-2006)), IbSSI gene (soluble starch synthase, the CDS sequence is shown in SEQ ID NO 4), IbSSII gene (CDS sequence is GenBank: AF068834 (Mar-10-2010)), IbSSIII gene (CDS sequence is shown in SEQ ID NO 5), IbSSIV gene (CDS sequence is shown in SEQ ID NO 6), IbSBEI gene (starch branching enzyme, CDS sequence is GenBank: AB194725 (Aug-22-2006)), IbSBEII gene (CDS sequence is GenBank: AB071286 (Aug-9-2006)), IbIsa1 gene (isoamylase, CDS sequence is GenBank: DQ074643 (Jan-27-2009)) and IbPUL gene (pullulanase, CDS sequence is shown in SEQ ID NO 7).

[0201] The internal reference gene was IbActin (primer sequences are shown in Table 1), the kit used was PerfectStart Green qPCR SuperMix (AQ601-02), the reaction conditions were: 94°C 30s; 94°C 5s, 60°C 30s (42 cycles); Dissociation stage. Instrument: 7500 Real-Time PCR System.

[0202] qRT-PCR was used to analyze the expression levels of starch synthesis-related genes in the overexpression lines ( Figure 8), compared with the wild type, the above-mentioned genes were up-regulated to varying degrees in the over-expression strains (OX17, OX53 and OX85). Among them, IbAGP-sTL1, IbAGP-sTL2, and IbAGP-TLI are located downstream of IbpPGM and are responsible for the synthesis of the starch precursor ADPG, IbGBSSI is mainly responsible for the synthesis of amylose, and the remaining IbSSI and other genes are responsible for the synthesis of amylopectin. This indicates that the over-expression of the IbpPGM gene promotes the accumulation of starch synthesis precursors, thereby causing the up-regulated expression of downstream related genes.

[0203] Table 1 Primers used in this study

[0204]

[0205]

[0206] Table 2 Starch content and soluble sugar content in the root tubers of transgenic lines and control lines

[0207]

[0208] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Any of the following uses of biological materials: U1. Use of the biological material in down-regulating the sucrose content of plants, and / or up-regulating the starch content and / or glucose content and / or fructose content of plants; U2. Use of the biological material in preparing products for lowering the sucrose content of plants, and / or raising the starch content and / or glucose content and / or fructose content of plants; The biological material is any of the following: B1), nucleic acid molecules encoding proteins; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); The protein is any one of the following: A1), the amino acid sequence is the protein shown in SEQ ID NO.2; A2), connecting the N-terminus and / or C-terminus of the protein described in A1) to a protein tag to obtain a fusion protein; The plant is sweet potato tuber.

2. A method for regulating plant sugar content, characterized in that: The method comprises up-regulating the expression of the gene encoding the protein of claim 1 in the target plant to down-regulate the sucrose content of the plant, and / or up-regulating the starch content and / or glucose content and / or fructose content of the plant, wherein the plant is a sweet potato tuber.

3. A method for cultivating a plant with altered sugar content, characterized in that: The method comprises up-regulating the expression of the gene encoding the protein of claim 1 in the target plant to obtain a plant with altered sugar content. Compared with the target plant, the plant with altered sugar content has at least one of the following characteristics: reduced sucrose content, increased starch content, increased glucose content and increased fructose content. The plant is a sweet potato tuber.

4. The method according to claim 2 or 3, characterized in that The protein coding gene is a DNA molecule whose coding chain nucleotide sequence is SEQ ID NO.1.

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