CN118373890A - Regulation method of plant economic traits based on IbbHLH49 protein - Google Patents

Abstract

The invention provides a method for regulating and controlling economic traits of plants based on IbbHLH protein. IbbHLH49 protein is a protein of the following A1), A2) or A3): a1 Protein with the amino acid sequence shown as SEQ ID NO. 1 in the sequence table; a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence shown in SEQ ID NO. 1 in the sequence table, has more than 80% of identity with the protein shown in A1) and is related to the synthesis of plant starch; a3 Fusion proteins obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2). IbBHLH49 protein can regulate the root tuber size and yield of sweet potato, regulate the activity of key enzyme for starch synthesis and regulate the yield of starch, and has certain application space and market prospect in the agricultural field.

Description

Regulation method of plant economic traits based on IbbHLH49 protein

Technical Field

The present invention relates to the technical field of genetic engineering, and in particular to a method for regulating plant economic traits based on IbbHLH49 protein.

Background technique

Starch is an important product of plant photosynthesis. It is the most abundant and widely distributed carbohydrate in plants. It is not only the main energy source in human daily diet, but also the main source of feed. At the same time, as an important raw material for industrial production, it is widely used in winemaking, textiles, papermaking, and chemical industries. Starch mainly exists in the seeds, roots, tubers, and fruits of plants. The starch content of different crops varies.

Sweet potato (Ipomoea batatas) tubers are rich in starch, which has great utilization value. As a marginal land crop, the planting area of sweet potato will be further reduced if it does not compete with staple food. Therefore, it is an effective means to cultivate high-yield and high-starch sweet potato varieties using genetic engineering. However, the transcriptional regulatory mechanism of starch synthesis in sweet potato tubers is still unknown.

It is of great significance to explore the genes related to starch synthesis in sweet potato tubers, conduct in-depth research on the mechanism of starch synthesis, and regulate plant starch production in combination with plant genetic engineering.

Summary of the invention

The present invention provides a method for regulating plant economic traits based on IbbHLH49 protein, which is used to regulate the economic traits of plants, including at least one of root tuber size, root tuber yield, starch yield, and amylopectin ratio.

In a first aspect, the present invention provides a method for regulating plant economic traits, comprising: regulating the expression of a gene encoding an IbbHLH49 protein in a starting plant to obtain a transgenic plant, wherein the economic traits of the transgenic plant are different from those of the starting plant, and the economic traits include at least one of root size, root yield, starch yield, and amylopectin ratio;

The IbbHLH49 protein is the following protein A1), A2) or A3):

A1) a protein with an amino acid sequence as shown in SEQ ID NO: 1;

A2) a protein related to plant starch synthesis obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, which has more than 80% identity with the protein shown in A1);

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

In the above method, the IbbHLH49 protein is derived from sweet potato (Ipomoea batatas).

In the above method, the protein shown in SEQ ID NO: 1 consists of 521 residues, and the specific amino acid sequence is as follows:

MDKGGKDEAMAAKRGDDAMSFQSANVSSEWQMNGSNLANTPIGMIPNSNPMMVDAFCLNVWDQSASSASLGFCDANVHSNVTTSSPFGAGTSGFTTALRGGVDRGLGMAWHPANTMLKTGMLLPTAPAVVPPNLPQFPADSDFLQRAARFSCFSGGNLGDMMNPFESLSPYCRGITPTQRPQQVFVGNGLKPAPAGEISNGAADGSPLNNNSVIEYAVGSRNSAKEGG GAFGNEPNEPECSSRGGLDVSEGAGAESSASK KRKRSGQDAETDQNKGTPPPAEAATDQTDNQQKGDQNVTATPSKPGGKGGKQGSQASDNPKEDYIHIRARRGQATNSHSLAERVRREKISERMKFLQDLVPGCNKVTGKAVMLDEIINYVQSLQRQVEFLSMKLATVNPRLEFNIDSLLAKDILQSRAGSSSSLLSFPHDMTMPYPPVHHPPSTLIQAGLPSLGSSADAIRRTINPHLATA SGSFKEPTPQVPSMWDDELHNVVQMGFNPSAPLDSQDIGSLPPGHMKSEP

In the above method, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites 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 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 for calculation, the identity value (%) can be obtained.

In the above method, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In the above method, protein tag refers to a polypeptide or protein 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.

In the above method, the regulation may be up-regulation, enhancement or increase, or down-regulation, inhibition or reduction.

Furthermore, upregulating, enhancing or increasing the expression of the IbbHLH49 protein encoding gene specifically includes method M1):

M1) introducing the coding gene of the IbbHLH49 protein into the starting plant, upregulating or enhancing or improving the expression of the coding gene of the IbbHLH49 protein, and obtaining a transgenic plant; compared with the starting plant, the tuber size, tuber yield, starch yield and amylopectin ratio of the transgenic plant are all higher than those of the starting plant.

In the above method M1), the coding gene of the IbbHLH49 protein is one of B1) and B2);

B1) a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO: 2;

B2) A DNA molecule that has more than 80% identity with the DNA molecule shown in SEQ ID NO: 2 and encodes the IbbHLH49 protein.

The nucleotide sequence of the nucleic acid molecule shown in SEQ ID NO:2 is as follows:

ATGGATAAGGGTGGCAAGGATGAGGCCATGGCAGCAAAGAGAGGTGATGATGCTATGAGCTTTCAGTCAGCAAATGTGTCATCTGAGTGGCAAATGAATGGTTCCAATCTCGCGAATACGCCTATTGGAATGATTCCCAATAGCAATCCGATGATGGTGGATGCGTTTTGCCTGAATGTTTGGGACCAATCTGCAAGTTCAGCAAGCTTAGGCTTTTGTGATGCTAATGTTCATAGCAATGTTACCACTTCTAGCCCATT TGGAGCTGGAACAAGTGGGTTCACCACCGCCTTAAGAGGCGGTGTCGATAGAGGTCTTGGTATGGCGTGGCATCCAGCTAACACGATGTTGAAAACGGGGATGCTTCTGCCTACTGCTCCTGCAGTGGTTC CTCCGAACTTGCCTCAGTTCCCAGCTGATTCCGATTTTCTTCAAAGGGCAGCGAGGTTTTCGTGCTTCAGTGGAGGAAACTTGGGCGATATGATGAACCCCTTTGAGTCTCTGAGTCCTTATTGTAGAGGCATAACGCCAACTCAAAGGCCTCAACAGGTTTGTAGGTAATGGACTAAAACCTGCACCCGCGGGTGAAATCTCAAACGGGGCTGCTGATGGTAGCCCGCTAAATAACAATAGCGTGATTGAATATGCCGT TGGATCTAGAAATAGTGCAAAAGAAGGCGGAGGAGCCTTTGGAAATGAACCTAACGAGCCCGAATGTAGTAGCCGTGGTGGCCTTGATGTATCAGAGGGTGCAGGGGCAGAGTCTTCTGCCTCAAAGAAA AGGAAAAGAAGTGGGCAGGACGCTGAAACCGATCAAAACAAGGGAACTCCACCACCAGCTGAAGCAGCAACAGATCAGACTGATAACCAGCAGAAAGGAGATCAAAACGTGACCGCAACTCCCAGCAAGCCAGGTGGTAAAGGCGGTAAGCAGGGGTCCCAAGCTTCAGATAATCCTAAAGAAGATTACATCCACATTCGGGCTAGGAGAGGCCAGGCCACGAATAGCCACAGTCTTGCAGAAAGAGTTAGAAGGGAAAAAAA TCAGTGAAAGAATGAAGTTTCTTCAGGATCTCGTGCCCGGTTGTAACAAGGTCACTGGCAAAGCAGTAATGCTTGATGAAATCATTAATTATGTACAGTCACTCCAACGACAGGTTGAGTTCTTGTCGA TGAAGCTTGCAACAGTAAACCCACGGCTCGAGTTCAACATTGATAGTCTCCTAGCAAAAGATATCCTCCAGTCCAGGGCTGGCTCTTCGTCTTCTCTGTTGTCTTTTCCACATGATATGACTATGCCTTATCCACCAGTACACCATCCACCATCAACGCTGATTCAAGCAGGTCTTCCTAGCCTGGGAAGTTCTGCAGATGCAATACGAAGAACCATCAACCCGCACTTGGCAACTGCGAGTGGGAGCTTCAAGGAGCCTAC ACCTCAGGTACCTAGTATGTGGGACGATGAGCTCCATAACGTTGTCCAAATGGGCTTCAATCCAAGCGCTCCCCTCGACAGCCAAGATATAGGTTCTCTACCACCAGGCCATTGAAATCTGAGCCCTGA

In the above method M1), the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In the above method M1), a recombinant expression vector containing the coding gene of the IbbHLH49 protein is constructed and introduced into the starting plant to up-regulate, enhance or improve the expression of the IbbHLH49 protein coding gene. Further, the recombinant expression vector containing the coding gene of the IbbHLH49 protein can be pCAMBIA1300-IbbHLH49-GFP. Specifically, pCAMBIA1300-IbbHLH49-GFP is obtained by inserting the coding gene of the IbbHLH49 protein into the pCAMBIA1300-GFP vector using restriction endonucleases KpnI and BamHI. The recombinant expression vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant cells or tissues are cultivated into plants.

In the above method, down-regulating/inhibiting/reducing the expression of the IbbHLH49 protein encoding gene specifically includes method M2):

M2) down-regulating, inhibiting or reducing the expression of the IbbHLH49 protein encoding gene by gene knockout or gene silencing to obtain the transgenic plant.

In the above method M2), the gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

The gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

Furthermore, the present invention constructs an shRNA vector to exert RNAi function to reduce the expression level of the IbbHLH49 protein encoding gene. Specifically, the sequence of one strand of the shRNA is a sequence obtained by transcribing the DNA fragment of positions 1-200 of the nucleotide sequence such as SEQ ID No: 2. Further, by constructing a recombinant expression vector containing a DNA molecule represented by formula (I), the expression of the shRNA down-regulates or inhibits or reduces the expression of the IbbHLH49 protein encoding gene, and the transgenic plant is obtained:

SEQ reverse-X-SEQ forward (I);

The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.

The transgenic plants obtained by down-regulating, inhibiting or reducing the expression of the IbbHLH49 protein encoding gene have lower root size, root yield, starch yield and amylopectin ratio than the starting plants.

In the above method, the plant is any one of the following:

D1) Dicotyledons;

D2) Tubulariaceae;

D3) Convolvulaceae;

D4) Ipomoea batatas;

D5) Sweet potato.

It is understandable that primer pairs for amplifying the full length of the above-mentioned IbbHLH49 protein encoding gene or any fragment thereof also fall within the scope of protection of the present invention.

In a second aspect, the present invention provides the use of any of the above-mentioned IbbHLH49 proteins and/or substances for regulating the expression of genes encoding IbbHLH49 proteins in any of the following aspects:

D1) Application in regulating the size of plant root tubers;

D2) Application in regulating plant root yield;

D3) Application in regulating starch content of plants;

D4) Application in regulating the proportion of plant amylopectin;

D5) Application in regulating the activity of enzymes related to starch synthesis in plants;

D6) Application in plant breeding;

D7) Application in the preparation of products for regulating the starch content of plants.

In the above application, the purpose of plant breeding can be to cultivate plants with changed economic traits, for example, to cultivate plants with increased starch content or reduced starch content, to cultivate plants with changed root size and/or root yield, or to cultivate plants with changed amylopectin ratio. The application in plant breeding can specifically be to hybridize plants containing IbbHLH49 protein or protein encoding gene IbbHLH49 with other plants for plant breeding.

In the above application, the substance that regulates the content or activity of the IbbHLH49 protein is a biological material related to the IbbHLH49 protein;

The biological material is any one of the following b1) to b10):

b1) a nucleic acid molecule encoding the IbbHLH49 protein;

b2) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in b1);

b3) an RNA molecule that downregulates, inhibits or reduces the expression of the gene encoding the IbbZIP11 protein;

b4) transcribing the gene encoding the RNA molecule described in b3);

b5) an expression cassette containing the coding gene described in b4);

b6) a recombinant vector containing the coding gene described in b4) or a recombinant vector containing the expression cassette described in b5);

b7) a recombinant microorganism containing the coding gene described in b4), or a recombinant microorganism containing the expression cassette described in b5), or a recombinant microorganism containing the recombinant vector described in b6);

b8) a transgenic plant cell line containing the coding gene described in b4), or a transgenic plant cell line containing the expression cassette described in b5), or a transgenic plant cell line containing the recombinant vector described in b6);

b9) transgenic plant tissue containing the coding gene described in b4), or transgenic plant tissue containing the expression cassette described in b5), or transgenic plant tissue containing the recombinant vector described in b6);

b10) A transgenic plant organ containing the coding gene described in b4), or a transgenic plant organ containing the expression cassette described in b5), or a transgenic plant organ containing the recombinant vector described in b6).

In the above application, the nucleic acid molecule encoding the IbbHLH49 protein described in b1) can be B1) a nucleic acid molecule whose nucleotide sequence is as shown in SEQ ID NO: 2, or B2) a DNA molecule that has more than 80% identity with the DNA molecule shown in SEQ ID NO: 2 and encodes the IbbHLH49 protein.

In the above application, the RNA molecule that inhibits or reduces the expression of the gene encoding the IbbZIP11 protein in b3) is an RNA transcribed from a DNA molecule as shown in formula (I):

SEQ reverse-X-SEQ forward (I);

The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.

In the above application, the nucleic acid molecule described in b4) is a DNA molecule represented by formula (I);

SEQ reverse-X-SEQ forward (I);

The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.

In the above application, the expression cassette described in b2) or b5) refers to a DNA molecule capable of expressing the IbbHLH49 protein in a host cell or a DNA molecule capable of transcribing the RNA described in b3), and the DNA molecule may include not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, 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 No. 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; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 262:14 ... 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).

In the above application, the recombinant vector described in b2) or b6) includes the encoding gene expression cassette described in b1) or b3). Further, a recombinant vector containing the encoding gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-GFP, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using IbbHLH49 to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide, 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.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

Further, the recombinant vector described in b2) or b6) can be pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X. The construction process of the recombinant vector pCAMBIA1300-IbbHLH49-GFP is as follows: (1) the vector pCAMBIA1300-GFP is double-digested with restriction endonucleases KpnI and BamHI to obtain the vector backbone; (2) the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP is obtained by replacing the fragment between the restriction endonuclease KpnI and BamHI recognition sequences of pCAMBIA1300-GFP with the DNA molecule shown in SEQ ID NO:2 in the sequence list, and the recombinant vector pCAMBIA1300-IbbHLH49-GFP expresses the IbbHLH49 protein shown in SEQ ID NO:1 in the sequence list. The construction process of the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X is as follows: (1) the vector pCAMBIA1300-35SI-X is double-digested with restriction endonucleases BamHI and SalI to obtain a vector backbone; (2) the DNA molecule shown in the 1st to 200bp in SEQ ID NO:2 in the sequence list is used to replace the fragment between the restriction endonuclease BamHI and SalI recognition sequences of pCAMBIA1300-35SI-X; (3) the fragment between the restriction endonuclease KpnI and SacI recognition sequences of pCAMBIA1300-35SI-X is replaced with the reverse complementary sequence of the DNA molecule shown in the 1st to 200bp in SEQ ID NO:2 in the sequence list to obtain the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X, and the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X contains the DNA molecule shown in formula (I).

In the above application, the recombinant microorganism described in b2) or b7) can be yeast, bacteria, algae or fungi. The bacteria can be gram-positive bacteria or gram-negative bacteria. The gram-negative bacteria can be Agrobacterium tumefaciens. The Agrobacterium tumefaciens can specifically be Agrobacterium tumefaciens EHA105.

In the above application, the recombinant microorganism may specifically be EHA105/pCAMBIA1300-IbbHLH49-GFP or EHA105/pCAMBIA1300-IbbHLH49-35SI-X. EHA105/pCAMBIA1300-IbbHLH49-GFP is a recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP into Agrobacterium tumefaciens EHA105; EHA105/pCAMBIA1300-IbbHLH49-35SI-X is a recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X into Agrobacterium tumefaciens EHA105.

In the above applications, the transgenic plant tissue described in b2) or b9) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above application, the transgenic plant organ described in b2) or b10) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

In a third aspect, the present invention provides any of the above-mentioned IbbHLH49 protein-related biological materials.

In a fourth aspect, the present invention provides a method for cultivating transgenic plants, comprising: regulating the expression of the IbbHLH49 protein encoding gene in a starting plant to obtain a transgenic plant, wherein at least one of the root tuber size, root tuber yield, starch yield, and amylopectin ratio of the transgenic plant is different from that of the starting plant.

The present invention provides an IbbHLH49 protein, a currently unknown function of a coding gene and its use. By regulating the content or expression of the IbbHLH49 protein in sweet potatoes, a sweet potato plant transfected with the IbbHLH49 gene is obtained. Experiments show that, compared with wild-type sweet potato plants, sweet potato plants overexpressing the IbbHLH49 gene have larger root size and higher root yield, and the expression of related enzymes related to starch synthesis increases, and the starch content and the proportion of amylopectin increase; while the sweet potato plants with interference expression of the IbbHLH49 gene have smaller root size and lower root yield, and the expression of related enzymes related to starch synthesis decreases, and the starch content and the proportion of amylopectin decrease; indicating that the IbbHLH49 protein and the coding gene play an important role in regulating plant economic traits. The IbbHLH49 protein and the coding gene provided by the present invention have important application value in plant economic traits, and have certain application space and market prospects in the agricultural field.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the PCR identification result of transgenic sweet potato plants; wherein A is the PCR identification result of sweet potato positive plants overexpressing the IbbHLH49 gene, and B is the PCR identification result of sweet potato positive plants interfering with the expression of the IbbHLH49 gene; M is a DNA molecule marker, W is a negative control water, P is a positive control plasmid (pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X), WT is the genomic DNA of wild-type sweet potato plants, OE49-1 to OE49-13 are the genomic DNA of sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-1 to Ri49-14 are the genomic DNA of sweet potato positive plants interfering with the expression of the IbbHLH49 gene;

Figure 2 is an expression analysis of the IbbHLH49 gene in sweet potato positive plants transgenic for IbbHLH49 and wild-type sweet potato plants; wherein A is the relative expression of the IbbHLH49 gene in sweet potato positive plants overexpressing the IbbHLH49 gene, B is the relative expression of the IbbHLH49 gene in sweet potato positive plants interfering with the expression of the IbbHLH49 gene, WT is the cDNA of the wild-type sweet potato plant, OE49-1 to OE49-13 and Ri49-1 to Ri49-14 are the cDNA of sweet potato positive plants transgenic for IbbHLH49;

Figure 3 is a photo of the root tubers of the IbbHLH49 transgenic sweet potato plants and the wild-type sweet potato plants; wherein WT is the wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene; the scale bar in the figure is 10 cm;

Figure 4 is a paraffin section of the root tubers of the IbbHLH49 transgenic sweet potato plants and the wild-type sweet potato plants; wherein WT is the wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with the expression of the IbbHLH49 gene; the intracellular granules in the figure are starch granules, and the scale bar is 100 μm;

Figure 5 shows the yield, starch content and amylopectin ratio of the tuberous roots of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants; wherein A is the yield of the tuberous roots of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants, B is the starch content of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants, and C is the amylopectin ratio of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants; WT is a wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene;

Figure 6 shows the results of AGPase and SBE activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants; wherein, A is the result of AGPase activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants, and B is the result of SBE activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants; WT is a wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

In the following examples, the sweet potato line 'H283' is stored in this laboratory. The public can obtain it from the Key Laboratory of Sweet Potato Biology and Biotechnology of China Agricultural University to repeat this experiment, but it cannot be used for other purposes.

The sweet potato variety “Lizixiang” is preserved in this laboratory and is recorded in the following document: “Xue Luyao. Function and molecular regulation mechanism of sweet potato IbbHLH118, IbNF-YA1 and IbNF-YA10 genes, PhD dissertation, China Agricultural University, 2022.” The public can obtain it from the Key Laboratory of Sweet Potato Biology and Biotechnology of China Agricultural University to repeat this experiment, but it cannot be used for other purposes.

The cloning vector pMD19-T is a product of Takara Biotechnology (Dalian) Co., Ltd., with the product catalog number being 6013.

The vector pCAMBIA1300-GFP is recorded in the following literature: Luo HS, Meng DX, Liu HB, Xie MJ, Yin CF, Liu F, Dong ZB, Jin WW. Ectopic Expression of the Transcriptional Regulatorsilky3Causes Pleiotropic Meristem and Sex Determination Defects in MaizeInflorescences. Plant Cell.2020,32:3750-3773; the public can obtain the above biological materials from the applicant to repeat this experiment, but they cannot be used for other purposes.

pCAMBIA1300-35SI-X was purchased from Wuhan Transduction Laboratory Co., Ltd. and was modified based on pCAMBIA1300. 35S promoter + Intro + NOS terminator were added to the MCS, and the forward and reverse sequences were inserted respectively to form a hairpin structure.

The plant total RNA extraction kit was the Transzol Plant Total RNA Extraction Kit (Catalog No.: ET111) from TransGen Biotech, Beijing.

HiFiScript gDNA Removal RT MasterMix Reverse Transcription Kit (Catalog Number: CW2020M) is a product of Kangwei Century Biotechnology Co., Ltd.

Seamless cloning enzyme comes from Hieff of Yisheng Biotechnology (Shanghai) Co., Ltd. Plus OneStep Cloning Kit (10911ES).

The following examples were processed using SPSS 26.0 statistical software, and the experimental results were expressed as mean ± standard deviation, and Student's t-test was used. * indicates significant difference (P < 0.05), and ** indicates extremely significant difference (P < 0.01). In the following examples, unless otherwise specified, three replicates were set for each treatment group.

Example 1. Acquisition of IbbHLH49 gene

1. Obtaining cDNA template

H283 plants cultured in vitro for 4 weeks were frozen in liquid nitrogen and ground, RNA was extracted using a plant total RNA extraction kit, and the first-strand cDNA was reverse transcribed using a HiFiScript gDNA Removal RT MasterMix reverse transcription kit.

2. Perform Blastx analysis on the IbbHLH49 gene using the Sweetpotato Garden database (http://sweetpotato-garden.kazusa.or.jp/index.html) to find the complete gene ORF with high similarity to the EST sequence.

3. Design and synthesize primers O49-F and O49-R, use the cDNA obtained in step 1 as a template, perform PCR amplification, obtain a PCR amplification product of about 1566 bp, connect it with the cloning vector pMD19-T to obtain a recombinant vector and sequence it. The primer sequences are as follows:

O49-F：5′-ATGGATAAGGGTGGCAAGGAT-3′

O49-R：5′-TCAGGGCTCAGATTTCATATGG-3′

The results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO:2, the gene shown in the sequence was named IbbHLH49 gene, the protein encoded by it was named IbbHLH49 protein or protein IbbHLH49, and the amino acid sequence of the protein was as shown in SEQ ID NO:1.

Example 2: Application of IbbHLH49 protein in increasing starch content in plants

1. Construction of recombinant plasmid pCAMBIA1300-IbbHLH49-GFP

1. Artificially synthesize the double-stranded DNA molecule shown in positions 1 to 1563 from the 5′ end of SEQ ID NO: 2. Use the double-stranded DNA molecule as a template and 1300-F-KpnI and 1300-R-BamHI as primers to perform PCR amplification to obtain a double-stranded DNA molecule fragment 1 containing restriction endonuclease KpnI at the N-terminus and restriction endonuclease BamHI at the C-terminus.

1300-F-KpnI: 5′-ACGGGGGACGAGCTC GGTACC ATGGATAAGGGTGGCAAGGAT-3′ (the underline is the recognition sequence of restriction endonuclease KpnI);

1300-R-BamHI: 5′-CATGTCGACTCTAGA GGATCC GGGCTCAGATTTCATATGG-3′ (the underline is the recognition sequence of the restriction endonuclease BamHI).

2. Double-digest the vector pCAMBIA1300-GFP with restriction endonucleases KpnI and BamHI to recover the vector backbone 2 of about 10444 bp.

3. Fragment 1 and vector backbone 2 were homologously recombined using seamless cloning enzyme to obtain the recombinant plasmid pCAMBIA 1300-IbbHLH49-GFP.

According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP was described as follows: a small fragment between the restriction endonuclease KpnI and BamHI recognition sequences of the plasmid pCAMBIA1300-GFP was replaced with a DNA molecule whose nucleotide sequence was SEQ ID No: 1, while keeping the other nucleotide sequences of pCAMBIA1300-GFP unchanged, to obtain the recombinant expression vector pCAMBIA1300-IbbHLH49-GFP, and the recombinant expression vector pCAMBIA1300-IbbHLH49-GFP expressed the IbbHLH49 protein shown in SEQ ID NO: 1.

2. Construction of recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X

1. Artificially synthesize a double-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2. Using the double-stranded DNA molecule as a template, select a 1-200 bp sequence of the gene as an interference sequence, use RNAi-F1-BamHI and RNAi-R1-SalI as primers to perform PCR amplification to obtain the sense strand, and use RNAi-F2-SacI and RNAi-R2-KpnI as primers to perform PCR amplification to obtain the antisense strand.

RNAi-F1-BamHI: 5′-ACGGGGGACTCTAGT GGATCC ATGGATAAGGGTGGCAAGGATGAG-3′ (underlined is the recognition sequence of restriction endonuclease BamHI);

RNAi-R1-SalI: 5′-AATTACCCTCTACTA GTCGAC GAACTTGCAGATTGGTCCCAAACAT-3′ (underlined is the recognition sequence of restriction endonuclease SalI);

RNAi-F2-SacI: 5′-CGATCGGGGAAATTC GAGCTC ATGGATAAGGGTGGCAAGGATGAG-3′ (the underline is the recognition sequence of restriction endonuclease SacI);

RNAi-R2-KpnI: 5′-GTTAGGATTTCTAGA GGTACC GAACTTGCAGATTGGTCCCAAACAT-3′ (the underline is the recognition sequence of the restriction endonuclease KpnI).

2. Clone the above positive chain sequence into the multiple cloning site 1 (5'-BamHI-SalI-3') of the vector pCAMBIA1300-35SI-X in the 5'-3' direction;

3. After successful sequencing verification, the above successfully constructed clone was used as the target vector, and the antisense chain sequence was cloned again into the multiple cloning site 2 (5'-KpnI-SacI-3') in the 5'-3' direction. Sequencing verified that the second insertion was successful, and the pCAMBIA1300-IbbHLH49-35SI-X plasmid was constructed.

The sequencing results showed that the recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X contained the DNA molecule represented by formula (I):

SEQ reverse-X-SEQ forward (I);

The SEQ forward sequence is positions 1-200 of SEQ ID NO: 2; the SEQ reverse sequence is reverse complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to either the SEQ forward or the SEQ reverse.

3. Obtaining transgenic sweet potato plants

1. The recombinant plasmids pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X were transformed into Agrobacterium tumefaciens EHA105, respectively, to obtain recombinant Agrobacterium, and the recombinant Agrobacterium were named EHA105/pCAMBIA1300-IbbHLH49-GFP and EHA105/pCAMBIA1300-IbbHLH49-35SI-X, respectively.

2. Peel off the stem tip meristem of Lizixiang about 0.5 mm long, place it on MS solid medium containing 2.0 mg L ^-1 2,4-D, and induce culture in the dark at 27±1℃ for 4 to 6 weeks to obtain embryonic callus. Then transfer the embryonic callus to MS liquid medium containing 2.0 mg L ^-1 2,4-D and culture for 3 to 4 weeks with shaking to obtain embryonic cell suspension culture lines with a diameter of 0.7 to 1.3 mm that can be used for transformation.

3. Cultivation of Agrobacterium: Activate Agrobacterium culture on a resistant plate, pick a single colony and inoculate it into 20 mL of LB liquid medium to which the corresponding antibiotic has been added, and culture it overnight at 28°C with shaking at 200 rpm until the OD ₆₀₀ value is within the range of 0.5 to 0.7. Centrifuge at 5000 rpm and remove the supernatant, wash the cells twice with an equal volume of LB medium, wash the cells once with an equal volume of MS liquid medium containing 2.0 mg ^{L -1} 2,4-D, and resuspend the cells with an equal volume of MS liquid medium containing 2.0 mg L ^-1 2,4-D.

4. Infection and co-cultivation: Suspend the chestnut-fragrant embryonic cell mass in the prepared Agrobacterium bacterial solution, shake it for a while to fully disperse it so that the embryonic cell mass can fully contact the bacterial solution, let it stand for 5 minutes, then use a pipette to suck out the bacterial solution, and move the infected embryonic cell mass to MS solid culture medium containing 30 mg L ^-1 AS and 2.0 mg L ^-1 2,4-D for co-cultivation. Cover the solid culture medium with a layer of ordinary filter paper and culture it in the dark at 27±1℃ for 3 days.

5. Selection culture and regeneration of transgenic plants: After 3 days of co-culture, the embryonic cell mass was gently scraped off with a blade, washed 3 times with MS liquid medium containing 500 mg L ^-1 cephalosporin and 2.0 mg L ^-1 2,4-D, and then transferred to MS liquid medium containing 100 mg L ^-1 cephalosporin and 2.0 mg L ^-1 2,4-D, and cultured for 1 week. Then the liquid medium was dried as much as possible and placed on a solid MS medium covered with 1 to 2 layers of filter paper and containing 5 mg L ^-1 hygromycin, 100 mg L ^-1 cephalosporin and 2.0 mg L ^-1 2,4-D for selection culture. The culture conditions were 27±1℃ and dark culture. After 2 weeks, the callus with good growth status was transferred to a solid MS medium covered with 1 layer of filter paper and containing 11 mg L ^-1 hygromycin, 100 mg L ^-1 cephalosporin and 2.0 mg L ^-1 2,4-D for selection culture. Subculture once every 2 weeks thereafter. The resistant callus formed after 8 weeks of selective culture on solid MS medium containing 11 mg L ^-1 hygromycin, 100 mg L ^-1 cephalosporin and 2.0 mg L ^-1 2,4-D was transferred to MS solid medium containing 100 mg L ^-1 cephalosporin and 1 mg L ^-1 ABA. The culture conditions were 27±1℃, 13h per day, and 3000lux of light to induce the formation of somatic embryos. After 2 to 4 weeks of induction, the mature somatic embryos were transferred to MS solid medium. The culture conditions were 27±1℃, 13h per day, and 3000lux of light. After 4 to 8 weeks of culture, complete transgenic plants were regenerated. The regenerated plantlets were cut and subcultured on MS solid medium. The culture conditions were 27±1℃, 13h per day, and 3000lux of light. Subculture once every 6 weeks.

6. Identification of transgenic plants: using a combination of PCR and qRT-PCR detection.

1) The PCR detection method is as follows:

DNA of wild-type sweet potato and the sweet potato lines to be transformed with IbbHLH49 gene were extracted for PCR identification. pCAMBIA1300-IbbHLH66-GFP and pCAMBIA1300-IbbHLH49-35SI-X recombinant plasmids were used as positive controls, water and wild-type WT were used as negative controls, and the primers were as follows:

OE49-F: 5'-AGGAAGTTTCATTTCATTTGGAGA-3'

OE49-R：5'-TCAGGGCTCAGATTTCATATGG-3'

Ri49-F: 5'-CTTCGCAAGACCCTTCCTCT-3'

Ri49-R: 5'-TCGGGGAAATTCGAGCTC-3'

The amplified PCR products were separated by electrophoresis in 1% (w/v) agarose gel. The PCR-positive plants should have a specific electrophoresis band of 1612 bp and 623 bp. The strain number of the PCR-positive plants was recorded.

The results are shown in Figure 1, which show that only the positive control and the pseudo-transgenic sweet potato plants OE49-1 to OE49-13 showed electrophoresis bands near 1612 bp, and Ri49-1 to Ri49-14 showed electrophoresis bands near 623 bp, while the wild-type sweet potato and the negative control did not show any bands. It was preliminarily determined that the present invention obtained the sweet potato transgenic positive plants OE49-1 to OE49-13 and Ri49-1 to Ri49-14.

2) qRT-PCR

RNA was extracted from positive sweet potato plants, reverse transcribed to obtain cDNA, and qRT-PCR was performed, with the wild type as the control.

Ibactin gene as internal reference:

Ibactin-F: 5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

Ibactin-R: 5′-GGAAAATTAGAAGCACTTCCTGTGAAC-3′

The IbbHLH49 primer sequence is:

qRT49-F: 5′-ATCCACATTCGGGCTAGGAG-3′

qRT49-R: 5′-CGGGTTTACTGTTGCAAGCT-3′

The results are shown in Figure 2. The results showed that the expression level of the IbbHLH49 gene was significantly upregulated in the overexpression transgenic sweet potato plants and significantly downregulated in the interference expression transgenic sweet potato plants. Two overexpression transgenic sweet potato plants (OE49-9 and OE49-12) and two interference expression transgenic sweet potato plants (Ri49-7 and Ri49-14) were selected for propagation to obtain the IbbHLH49 T1 generation transgenic sweet potato lines OE49-9, OE49-12, Ri49-7 and Ri49-14 for subsequent experiments.

4. Identification of starch content in transgenic sweet potato plants overexpressing IbbHLH49

WT is the wild type Li Zixiang, and the transgenic sweet potato is the plants of the IbbHLH49 T ₁ generation transgenic sweet potato lines OE49-9, OE49-12, Ri49-7 and Ri49-14.

1. Observation of IbbHLH49 transgenic sweet potato root tubers

Sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in an isolated field for 4 months were taken, cleaned and observed.

The results are shown in Figure 3. The sweet potato tubers of the overexpressing transgenic plants were significantly larger than those of the wild-type plants, while the sweet potato tubers of the interference-expressing transgenic plants were significantly smaller than those of the wild-type plants.

2. Paraffin section observation of IbbHLH49 transgenic sweet potato root tubers

The sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months were taken, cleaned, and the middle part of the tubers was taken to make paraffin sections. The method is as follows:

1) Immerse the material in FAA fixative for more than 48 hours.

2) Dehydrate the fixed material using gradient concentrations of alcohol: 50%, 70%, 80%, 95%, and 100%, with each concentration treated for 30 minutes.

3) Treat the sample twice with a mixture of xylene and 100% alcohol in a ratio of 1:1, each time for 30 minutes, to decolorize the plant cells and make them transparent.

4) Place the treated material in paraffin for 30 minutes, and perform two treatments, each time in an oven at 40°C uncovered overnight.

5) Pour the melted paraffin into the embedding container, use hot tweezers to quickly move the material into the embedding container, and position it with the observation side facing downward.

6) Then move the embedding device to the water surface and slowly immerse it in the water. After the paraffin is completely solidified, take it out of the water and mark the name and date of the embedded block.

7) After trimming, gluing and finishing the embedded material, fix the wax block on the wood block with a little paraffin wax, then fix the wood block on the microtome, and adjust the slicer holder or material holder to bring the slicer close to the wax block, adjust the slice thickness to 2 mm, slice, and move the slices with a brush to a plate covered with black paper.

8) Place the cut wax strips on the drying table to flatten the slices. Then take a small drop of glycerin and apply it on the slide. Use the outside of your little finger to apply it into a thin layer. The area applied should be larger than the area occupied by the wax strips. The thinner and more evenly applied, the better. Dry at 37°C.

9) Place the slide in a dyeing jar containing xylene to remove the paraffin (10 to 20 minutes), and repeat the operation twice.

10) Finally, dyeing is performed. The completely dewaxed material is dyed through the following procedure: 1/2 xylene + 1/2 pure alcohol → 100% alcohol → 95% alcohol → 85% alcohol → 70% alcohol → 50% alcohol → 1% safranin dye (more than 4 hours) → 50% alcohol → 70% alcohol → 85% alcohol → 95% alcohol → 0.5% Fast Green (1 minute) → 95% alcohol → 100% alcohol → 100% alcohol (the reaction time for steps without time indication is 3 minutes).

11) Place in xylene for 3 minutes until transparent.

12) Add neutral gum to seal the prepared specimen.

After the slices were prepared, they were observed and photographed using an upright and inverted microscope (Revolve; Echo).

The results are shown in Figure 4. The number of starch granules in the root cells of sweet potato overexpressing transgenic plants is significantly greater than that of wild-type plants, while the number of starch granules in the root cells of transgenic interference expression plants is significantly less than that of wild-type plants.

2. Yield determination of IbbHLH49 transgenic sweet potato root tubers

The sweet potato root yield of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants grown in an isolated field for 4 months was measured.

The results are shown in Figure 5A. The root yield per plant of sweet potato in the overexpression transgenic plants increased by 9.4-17.2% compared with the wild-type plants, while the root yield per plant of sweet potato in the interference expression transgenic plants decreased by 11.0-13.3% compared with the wild-type plants.

3. Determination of total starch content in IbbHLH49 transgenic sweet potato roots

The starch content in the tubers of sweet potato plants was measured using a starch content test kit (Suzhou Keming Biotechnology Co., Ltd., DF-2-Y). The tubers of sweet potato plants were selected from the tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.

The results are shown in FIG. 5B . The starch content of sweet potatoes in the overexpression transgenic plants increased by 5.1 to 16.7% compared with the wild-type plants, while the starch content of sweet potatoes in the interference expression transgenic plants decreased by 4.0 to 8.4% compared with the wild-type plants.

4. Determination of the proportion of amylopectin in IbbHLH49 transgenic sweet potato roots

The sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months were taken, cleaned, dried, ground and crushed, dissolved in deionized water and the mixture was passed through a 100-mesh sieve. The filtered suspension was allowed to settle and the supernatant was removed. The obtained precipitate was placed in an oven at 40°C and dried to constant weight to obtain sweet potato tuber starch. The amylopectin content test kit (ZHDF-2-Y) was used to determine the amylopectin ratio of the obtained sweet potato tuber starch.

The results are shown in Figure 5C. The amylopectin ratio of the overexpression transgenic plants increased by 2.3-3.6% compared with the wild-type plants, while the amylopectin ratio of the interference expression transgenic plants decreased by 2.9-6.5% compared with the wild-type plants.

5. Determination of AGPase Activity in IbbHLH49 Transgenic Sweet Potato Roots

The activity of AGPase in the tuberous roots of sweet potato plants was measured using an AGPase activity assay kit (Suzhou Keming Biotechnology Co., Ltd., AGP-2A-Y). The tuberous roots of sweet potato plants were selected from the tuberous roots of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.

The results are shown in Figure 6A. The activity of AGPase in the sweet potato tubers of the overexpressing transgenic plants was significantly higher than that of the wild-type plants, while the activity of AGPase in the sweet potato tubers of the interference-expressing transgenic plants was significantly lower than that of the wild-type plants.

6. Determination of SBE activity in IbbHLH49 transgenic sweet potato roots

The activity of SBE in the tuberous roots of sweet potato plants was measured using an SBE activity assay kit (Suzhou Keming Biotechnology Co., Ltd., SBE-2-Y). The tuberous roots of sweet potato plants were selected from the sweet potato tuberous roots of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.

The results are shown in Figure 6B. The activity of SBE in the sweet potato tubers of overexpressing transgenic plants was significantly higher than that of wild-type plants, while the activity of SBE in the sweet potato tubers of interference-expressing transgenic plants was significantly lower than that of wild-type plants.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A method for regulating economic traits of plants, comprising: regulating and controlling expression of IbbHLH protein coding genes in a starting plant to obtain a transgenic plant, wherein economic characters of the transgenic plant are different from those of the starting plant, and the economic characters comprise at least one of root tuber size, root tuber yield, starch yield and amylopectin proportion;

The IbbHLH protein is a protein of the following A1), A2) or A3):

a1 Protein with the amino acid sequence shown as SEQ ID NO. 1;

A2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence shown in SEQ ID NO.1, has more than 80% of identity with the protein shown in A1) and is related to the synthesis of plant starch;

a3 Fusion proteins obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2).

2. The method of claim 1, wherein the modulation is up-regulation or enhancement or increase, or down-regulation or inhibition or decrease.

3. The method of claim 2, wherein the modulation method comprises M1) or M2):

M1) introducing the IbbHLH protein coding gene into a starting plant, and up-regulating or enhancing or improving the IbbHLH protein coding gene expression to obtain the transgenic plant;

M2) down-regulating or inhibiting or reducing the IbbHLH protein-encoding gene expression by gene knockout or gene silencing to obtain the transgenic plant.

4. A method according to claim 3, wherein in M1) the coding gene of IbbHLH protein is one of B1), B2);

b1 A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 2;

b2 A DNA molecule which has more than 80% of the identity with the DNA molecule shown in SEQ ID NO. 2 and codes IbbHLH protein.

5. The method of claim 3, wherein M2) the transgenic plant is obtained by introducing into the starting plant a DNA molecule of formula (I) to down-regulate or inhibit or reduce the expression of the IbbHLH protein-encoding gene;

SEQ reverse-X-SEQ forward (I);

The sequence of the SEQ forward direction is the 1 st to 200 th positions of the sequence shown in SEQ ID NO. 2 in the sequence table; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; and X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary with both the SEQ forward direction and the SEQ reverse direction.

6. The method of any one of claims 1-5, wherein the plant is any one of:

C1 Dicotyledonous plants;

c2 Tubular flower plants;

c3 A plant of the family Convolvulaceae;

c4 Sweet potato plant;

C5 Sweet potato.

7. Use of IbbHLH protein according to any one of claims 1 to 6 and/or a substance that modulates expression of a gene encoding IbbHLH protein in any one of the following aspects:

d1 The application of regulating the root tuber size of the plant;

d2 Regulating and controlling the plant root tuber yield;

D3 Regulating and controlling the starch content of plants;

D4 Regulating and controlling the proportion of plant amylopectin;

D5 Regulating and controlling the activity of starch synthesis related enzymes in plants;

D6 Use in plant breeding;

D7 The application of the starch in preparing products for regulating and controlling the starch content of plants.

8. The use according to claim 7, wherein the substance regulating the expression of the gene encoding IbbHLH protein is a biological material associated with the IbbHLH protein;

The biological material is any one of the following b 1) to b 10):

b1 A nucleic acid molecule encoding said IbbHLH protein;

b2 A) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of b 1);

b3 An RNA molecule that down-regulates or inhibits or reduces expression of a gene encoding the IbbZIP protein;

b4 B 3) expressing a gene encoding said RNA molecule;

b5 An expression cassette containing the coding gene of b 4);

b6 A recombinant vector comprising the coding gene of b 4) or a recombinant vector comprising the expression cassette of b 5);

b7 A recombinant microorganism comprising b 4) the coding gene, b 5) the expression cassette, b 6) the recombinant vector;

b8 A transgenic plant cell line containing b 4) the coding gene, or a transgenic plant cell line containing b 5) the expression cassette, or a transgenic plant cell line containing b 6) the recombinant vector;

b9 A transgenic plant tissue containing b 4) the coding gene, or b 5) the expression cassette, or b 6) the recombinant vector;

b10 A transgenic plant organ containing b 4) the coding gene, or a transgenic plant organ containing b 5) the expression cassette, or a transgenic plant organ containing b 6) the recombinant vector.

9. The IbbHLH protein-related biological material of claim 9.

10. A method of growing a transgenic plant comprising: regulating expression of IbbHLH protein coding gene of claim 1 in the starting plant to obtain transgenic plant, wherein at least one of root tuber size, root tuber yield, starch yield and amylopectin ratio of the transgenic plant is different from that of the starting plant.