CN118373890A - Method for regulating economic traits of plants based on IbbHLH 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

Method for regulating economic traits of plants based on IbbHLH protein

Technical Field

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

Background

Starch is an important product of photosynthesis of plants, is the most abundant and widely distributed carbohydrate in the plants, is not only a main energy source in human daily diet, but also a main feed source, and is widely applied to the fields of brewing, spinning, papermaking, chemical industry and the like as an important raw material for industrial production. Starch is mainly present in seeds, tubers and fruits of plants, and the starch content varies from crop to crop.

Sweet potato (Ipomoea batatas) tubers contain rich starch, and have great utility value. Sweet potato is used as a marginal land crop, and the planting area of the sweet potato tends to be further reduced on the premise of not competing with staple food. Therefore, the cultivation of sweet potato varieties with high yield and high starch by using genetic engineering is an effective means. However, the transcriptional regulatory mechanisms of starch synthesis in sweet potato tubers are currently unknown.

How to excavate the related genes of starch synthesis in sweet potato tubers, the starch synthesis mechanism is studied deeply, and the method has important significance for regulating and controlling the starch yield of plants by combining with plant genetic engineering.

Disclosure of Invention

The invention provides a method for regulating economic traits of plants based on IbbHLH protein, which is used for regulating the economic traits of the plants, including at least one of root tuber size, root tuber yield, starch yield and amylopectin proportion.

In a first aspect, the present invention provides a method for controlling 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).

In the above method, the IbbHLH 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 shown as follows:

MDKGGKDEAMAAKRGDDAMSFQSANVSSEWQMNGSNLANTPIGMIPNSNPMMVDAFCLNVWDQSASSASLGFCDANVHSNVTTSSPFGAGTSGFTTALRGGVDRGLGMAWHPANTMLKTGMLLPTAPAVVPPNLPQFPADSDFLQRAARFSCFSGGNLGDMMNPFESLSPYCRGITPTQRPQQVFVGNGLKPAPAGEISNGAADGSPLNNNSVIEYAVGSRNSAKEGGGAFGNEPNEPECSSRGGLDVSEGAGAESSASKKRKRSGQDAETDQNKGTPPPAEAATDQTDNQQKGDQNVTATPSKPGGKGGKQGSQASDNPKEDYIHIRARRGQATNSHSLAERVRREKISERMKFLQDLVPGCNKVTGKAVMLDEIINYVQSLQRQVEFLSMKLATVNPRLEFNIDSLLAKDILQSRAGSSSSLLSFPHDMTMPYPPVHHPPSTLIQAGLPSLGSSADAIRRTINPHLATASGSFKEPTPQVPSMWDDELHNVVQMGFNPSAPLDSQDIGSLPPGHMKSEP

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 BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the above method, the 80% or more identity may 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, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with the target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

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

Further, up-regulating or enhancing or increasing expression of IbbHLH protein-encoding genes specifically includes method M1):

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 a transgenic plant; compared with the original plant, the transgenic plant has the advantages that the root tuber size, the root tuber yield, the starch yield and the amylopectin ratio are higher than those of the original plant.

In the method M1), the coding gene of IbbHLH protein is one of B1) and 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.

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

ATGGATAAGGGTGGCAAGGATGAGGCCATGGCAGCAAAGAGAGGTGATGATGCTATGAGCTTTCAGTCAGCAAATGTGTCATCTGAGTGGCAAATGAATGGTTCCAATCTCGCGAATACGCCTATTGGAATGATTCCCAATAGCAATCCGATGATGGTGGATGCGTTTTGCCTGAATGTTTGGGACCAATCTGCAAGTTCAGCAAGCTTAGGCTTTTGTGATGCTAATGTTCATAGCAATGTTACCACTTCTAGCCCATTTGGAGCTGGAACAAGTGGGTTCACCACCGCCTTAAGAGGCGGTGTCGATAGAGGTCTTGGTATGGCGTGGCATCCAGCTAACACGATGTTGAAAACGGGGATGCTTCTGCCTACTGCTCCTGCAGTGGTTCCTCCGAACTTGCCTCAGTTCCCAGCTGATTCCGATTTTCTTCAAAGGGCAGCGAGGTTTTCGTGCTTCAGTGGAGGAAACTTGGGCGATATGATGAACCCCTTTGAGTCTCTGAGTCCTTATTGTAGAGGCATAACGCCAACTCAAAGGCCTCAACAGGTGTTTGTAGGTAATGGACTAAAACCTGCACCCGCGGGTGAAATCTCAAACGGGGCTGCTGATGGTAGCCCGCTAAATAACAATAGCGTGATTGAATATGCCGTTGGATCTAGAAATAGTGCAAAAGAAGGCGGAGGAGCCTTTGGAAATGAACCTAACGAGCCCGAATGTAGTAGCCGTGGTGGCCTTGATGTATCAGAGGGTGCAGGGGCAGAGTCTTCTGCCTCAAAGAAAAGGAAAAGAAGTGGGCAGGACGCTGAAACCGATCAAAACAAGGGAACTCCACCACCAGCTGAAGCAGCAACAGATCAGACTGATAACCAGCAGAAAGGAGATCAAAACGTGACCGCAACTCCCAGCAAGCCAGGTGGTAAAGGCGGTAAGCAGGGGTCCCAAGCTTCAGATAATCCTAAAGAAGATTACATCCACATTCGGGCTAGGAGAGGCCAGGCCACGAATAGCCACAGTCTTGCAGAAAGAGTTAGAAGGGAGAAAATCAGTGAAAGAATGAAGTTTCTTCAGGATCTCGTGCCCGGTTGTAACAAGGTCACTGGCAAAGCAGTAATGCTTGATGAAATCATTAATTATGTACAGTCACTCCAACGACAGGTTGAGTTCTTGTCGATGAAGCTTGCAACAGTAAACCCACGGCTCGAGTTCAACATTGATAGTCTCCTAGCAAAAGATATCCTCCAGTCCAGGGCTGGCTCTTCGTCTTCTCTGTTGTCTTTTCCACATGATATGACTATGCCTTATCCACCAGTACACCATCCACCATCAACGCTGATTCAAGCAGGTCTTCCTAGCCTGGGAAGTTCTGCAGATGCAATACGAAGAACCATCAACCCGCACTTGGCAACTGCGAGTGGGAGCTTCAAGGAGCCTACACCTCAGGTACCTAGTATGTGGGACGATGAGCTCCATAACGTTGTCCAAATGGGCTTCAATCCAAGCGCTCCCCTCGACAGCCAAGATATAGGTTCTCTACCACCAGGCCATATGAAATCTGAGCCCTGA

in the above method M1), the 80% or more identity may 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 method M1), the expression of the IbbHLH protein coding gene is up-regulated or enhanced or improved by constructing a recombinant expression vector containing the IbbHLH protein coding gene and introducing the recombinant expression vector into a starting plant. Further, the recombinant expression vector containing IbbHLH protein encoding gene may be pCAMBIA1300-IbbHLH49-GFP. Specifically, pCAMBIA 1300-IbbHLH-GFP was obtained by inserting the gene encoding IbbHLH protein into pCAMBIA1300-GFP vector using restriction enzymes KpnI and BamHI. The recombinant expression vector may be transformed into a plant cell or tissue by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium-mediated transformation, etc., and the transformed plant cell or tissue is cultured into a plant.

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

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

In the above method M2), the gene knockout refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.

The gene silencing (GENE SILENCING) refers to the phenomenon that the gene is not expressed or is underexpressed under the condition that the original DNA is not damaged. Gene silencing is premised on the fact that the DNA sequence is not altered, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of a target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.

Furthermore, the invention plays an RNAi function by constructing the shRNA vector to reduce the expression level of IbbHLH protein coding genes, specifically, the sequence of one chain of the shRNA is the sequence obtained by transcription of the DNA fragment with the nucleotide sequence shown as the 1 st to 200 th positions of SEQ ID No. 2. Further, by constructing a recombinant expression vector containing a DNA molecule shown in the formula (I), the expression of the IbbHLH protein coding gene is down-regulated or inhibited or reduced after the shRNA is expressed, and the transgenic plant is obtained:

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.

And down-regulating or inhibiting or reducing the expression of IbbHLH protein coding gene to obtain transgenic plant with root tuber size, root tuber yield, starch yield and amylopectin ratio lower than those of original plant.

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

D1 Dicotyledonous plants;

D2 Tubular flower plants;

D3 A plant of the family Convolvulaceae;

D4 Sweet potato plant;

D5 Sweet potato.

It is understood that a primer pair for amplifying the full length of the IbbHLH protein-encoding gene or any fragment thereof is also included in the scope of the present invention.

In a second aspect, the invention provides the use of a IbbHLH protein as defined in any one of the preceding claims and/or a substance that modulates the expression of a gene encoding a 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.

In the above applications, the object of plant breeding may be to cultivate plants with altered economic traits, for example plants with increased or decreased starch content, plants with altered root size and/or root yield, plants with altered proportion of amylopectin. The plant breeding may be performed by crossing a plant containing IbbHLH protein or a gene IbbHLH encoding a protein with other plants.

In the above application, the substance regulating the IbbHLH protein content or activity is a biological material related to 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 Transcription of b 3) the gene encoding the 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.

In the above application, the nucleic acid molecule encoding IbbHLH protein B1) may be a nucleic acid molecule having a nucleotide sequence shown in SEQ ID NO. 2 as B1), or a DNA molecule encoding IbbHLH protein having more than 80% identity with a DNA molecule shown in SEQ ID NO. 2 as B2).

In the above application, the RNA molecule in b 3) that inhibits or reduces the expression of the IbbZIP protein-encoding gene is RNA transcribed from a DNA molecule of formula (I):

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.

In the above application, the nucleic acid molecule of b 4) is a DNA molecule of formula (I);

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.

In the above applications, the expression cassette of b 2) or b 5) refers to a DNA molecule capable of expressing IbbHLH protein in a host cell or a DNA molecule capable of transcribing the RNA of b 3), which may include not only a promoter for initiating transcription of the target gene but also a terminator for terminating transcription of the target gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful 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: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with a jasmonates); heat shock promoter (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. 5,057,422); seed-specific promoters such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, oleosin, and soybean beta conglycin) (Beachy et al (1985) EMBO J. 4:3047-3053) which may be used alone or in combination with other plant promoters all references cited herein are incorporated herein by reference 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 rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., odell et al (I985) Nature 313:810; rosenberg et al (1987) Gene,56:125; gueae et al (1991) mol. Genet. 262. Ud. 262, 141:141:141:141, and the like, and the Acid synthase (1997:141:141, F. Mu.g., 1991, F.1:141, F.g., F.1, F.35, F.Gene (1997), F.1, F.Gene, F.1, F.141, F.1, F.Gene (1982)).

In the above application, the recombinant vector of b 2) or b 6) includes the coding gene expression cassette of b 1) or b 3). Further, a plant expression vector may be used to construct a recombinant vector containing the coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1300、pCAMBIA1300-GFP、pCAMBIA1302、pCAMBIA2300、pCAMBIA2301、pCAMBIA1301、pBI121、pCAMBIA1391-Xa or pCAMBIA1391-Xb. When IbbHLH is used to construct recombinant vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants.

Further, the recombinant vector of b 2) or b 6) may be pCAMBIA 1300-IbbHLH-GFP or pCAMBIA 1300-IbbHLH-35 SI-X. The construction process of the recombinant vector pCAMBIA 1300-IbbHLH-GFP is as follows: (1) The vector pCAMBIA1300-GFP is digested with restriction enzymes KpnI and BamHI to obtain a vector skeleton; (2) The recombinant plasmid pCAMBIA 1300-IbbHLH-GFP obtained by replacing the fragment between the restriction enzyme KpnI and BamHI recognition sequences of pCAMBIA1300-GFP with the DNA molecule shown in SEQ ID NO. 2 in the sequence table, and the recombinant vector pCAMBIA 1300-IbbHLH-GFP expresses IbbHLH protein shown in SEQ ID NO. 1 in the sequence table. The construction process of the recombinant vector pCAMBIA 1300-IbbHLH-35 SI-X is as follows: (1) The vector pCAMBIA1300-35SI-X is cut by the restriction enzymes BamHI and SalI to obtain a vector skeleton; (2) Replacing a fragment between the recognition sequences of the restriction enzymes BamHI and SalI of pCAMBIA1300-35SI-X with a DNA molecule shown as 1-200bp in SEQ ID NO. 2 of the sequence Listing; (3) The recombinant vector pCAMBIA1300-IbbHLH49-35SI-X obtained by replacing the fragment between the restriction enzyme KpnI and the SacI recognition sequence of pCAMBIA1300-35SI-X with the reverse complementary sequence of the DNA molecule shown as 1-200bp in SEQ ID NO. 2 of the sequence list contains the DNA molecule shown as the formula (I).

In the above applications, the recombinant microorganism of b 2) or b 7) may be a yeast, a bacterium, an alga or a fungus. The bacteria may be gram positive or gram negative bacteria. The gram negative bacterium may be agrobacterium tumefaciens (Agrobacterium tumefaciens). The agrobacterium tumefaciens (Agrobacterium tumefaciens) can specifically be agrobacterium tumefaciens EHA105.

In the above application, the recombinant microorganism may specifically be EHA105/pCAMBIA 1300-IbbHLH-GFP, EHA105/pCAMBIA1300-IbbHLH49-35SI-X. EHA105/pCAMBIA 1300-IbbHLH-GFP is a recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA 1300-IbbHLH-GFP into Agrobacterium tumefaciens EHA 105; EHA105/pCAMBIA 1300-IbbHLH-35 SI-X is a recombinant Agrobacterium obtained by transforming Agrobacterium tumefaciens EHA105 with recombinant plasmid pCAMBIA 1300-IbbHLH-49-35 SI-X.

In the above applications, the transgenic plant tissue of b 2) or b 9) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above applications, the transgenic plant organs described under b 2) or b 10) can be the roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

In a third aspect, the invention provides a IbbHLH protein-related biological material as described in any one of the preceding claims.

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

The invention provides IbbHLH protein, the presently unknown function of the coding gene and the application thereof, and the content or the expression of the IbbHLH protein in sweet potato is regulated to obtain a sweet potato plant transformed with IbbHLH gene, and experiments show that compared with a wild sweet potato plant, the sweet potato plant over-expressed with IbbHLH gene has larger root tuber size and higher root tuber yield, the expression quantity of related enzyme of starch synthesis is increased, and the starch content and the amylopectin proportion are improved; the sweet potato plant with the disturbing expression IbbHLH gene has smaller root tuber size and lower root tuber yield, and the expression quantity of related enzyme related to starch synthesis is reduced, and the starch content and the amylopectin proportion are reduced; the IbbHLH protein and the coding gene play an important role in regulating economic traits of plants. The IbbHLH protein and the coding gene thereof provided by the invention have important application value in the economic traits of plants, and have certain application space and market prospect in the agricultural field.

Drawings

FIG. 1 is a PCR identification of transgenic sweetpotato plants; wherein A is the PCR identification result of the sweet potato positive plant with the IbbHLH gene over-expressed, and B is the PCR identification result of the sweet potato positive plant with the IbbHLH gene under interference expression; m is a DNA molecule Marker, W is negative control water, P is positive control plasmids (pCAMBIA 1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35 SI-X), WT is genomic DNA of a wild sweet potato plant, OE49-1 to OE49-13 are genomic DNA of a sweet potato positive plant over-expressed with IbbHLH genes, and Ri49-1 to Ri49-14 are genomic DNA of a sweet potato positive plant interfering with expression of IbbHLH genes;

FIG. 2 is an analysis of the expression level of IbbHLH gene in IbbHLH transgenic sweetpotato positive plants and wild type sweetpotato plants; wherein A is the relative expression quantity of IbbHLH gene in the over-expressed IbbHLH gene sweet potato positive plant, B is the relative expression quantity of IbbHLH gene in the interference expression IbbHLH gene sweet potato positive plant, WT is the cDNA of the wild sweet potato plant, OE49-1 to OE49-13 and Ri49-1 to Ri49-14 are the cDNA of the transgenic IbbHLH49 gene sweet potato positive plant;

FIG. 3 is a photograph of tuberous roots of a IbbHLH transgenic sweet potato plant and a wild type sweet potato plant; wherein, WT is a wild sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing IbbHLH gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with IbbHLH gene expression; the scale bar in the figure is 10cm;

FIG. 4 is a paraffin section of a IbbHLH transgenic sweetpotato plant and a wild-type sweetpotato plant tuberous root; wherein, WT is a wild sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing IbbHLH gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with IbbHLH gene expression; in the figure, intracellular particles are starch grains, and the proportion scale is 100 mu m;

FIG. 5 shows the results of determination of the yield, starch content and amylopectin ratio of the tuberous root of the IbbHLH transgenic sweet potato plant and wild sweet potato plant; wherein A is the result of measuring the yield of the transgenic IbbHLH gene sweet potato plant and the wild sweet potato plant tuberous root, B is the result of measuring the starch content of the transgenic IbbHLH gene sweet potato plant and the wild sweet potato plant, and C is the result of measuring the proportion of the amylopectin of the transgenic IbbHLH gene sweet potato plant and the wild sweet potato plant; WT is a wild sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing IbbHLH gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with IbbHLH gene expression;

FIG. 6 shows the results of AGPase and SBE activity assays in transgenic IbbHLH gene sweetpotato plants and wild sweetpotato plant tubers; wherein A is AGPase activity measurement result in IbbHLH transgenic sweet potato plant and wild sweet potato plant tuberous root, and B is SBE activity measurement result in IbbHLH transgenic sweet potato plant and wild sweet potato plant tuberous root; WT is a wild sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing IbbHLH gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with expression IbbHLH gene.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions thereof will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, which should not be construed as limiting the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In the description of the present invention, it is to be understood that the terminology used is for the purpose of description only and is not to be interpreted as indicating or implying relative importance.

In the following examples, sweet potato line 'H283' was maintained for the present laboratory. The public can obtain from the important laboratories of sweet potato biology and biotechnology in national agricultural university to repeat the experiment, and can not be used as other purposes.

The sweet potato variety "chestnut flavor" is preserved in the laboratory and is described in the following literature: "Xue Luyao. Research on the mechanism of function and molecular regulation of the genes of sweet potato IbbHLH, ibNF-YA1 and IbNF-YA10, doctor's academy of papers, china university of agriculture, 2022". The public can obtain from the important laboratories of sweet potato biology and biotechnology in national agricultural university to repeat the experiment, and can not be used as other purposes.

The cloning vector pMD19-T is a product of Bao bioengineering (Dalian) company with a catalog number of 6013.

The vector pCAMBIA1300-GFP is described in ：Luo HS,Meng DX,Liu HB,Xie MJ,Yin CF,Liu F,Dong ZB,Jin WW.Ectopic Expression of the Transcriptional Regulator silky3Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences.Plant Cell.2020,32:3750-3773; in the following literature, which is publicly available from the applicant for the purpose of repeating the present experiment and is not available for other uses.

PCAMBIA1300-35SI-X is purchased from the Wuhan transduction laboratory Co.Ltd, modified on the basis of pCAMBIA1300, 35S promoter+Intro+NOS terminator is added at MCS, and forward and reverse sequences are respectively inserted to form a hairpin structure.

The total plant RNA extraction kit is Transzol total plant RNA extraction kit (catalog number: ET 111) of full gold (TransGen Biotech, beijing).

HIFISCRIPT GDNA Removal RT MasterMix A reverse transcription kit (catalog number: CW 2020M) is a product of Kangji Biotechnology Co., ltd.

Seamless cloning of enzymes was from Hieff of the company Highway Biotechnology, inc., nextPlus One Step Cloning Kit kit (10911 ES).

The following examples used SPSS26.0 statistical software to process the data, and the experimental results were expressed as mean.+ -. Standard deviation, using Student's t-test, indicating significant differences (P < 0.05) and indicating very significant differences (P < 0.01). In the following examples, three replicates were set for the treatment groups unless otherwise specified.

Examples 1, ibbHLH Gene acquisition

1. Obtaining cDNA templates

H283 plants grown in vitro for 4 weeks were cryo-ground with liquid nitrogen, RNA extracted with total plant RNA extraction kit and reverse transcribed with HIFISCRIPT GDNA Removal RT MasterMix reverse transcription kit to give first strand cDNA.

2. Blastx analysis was performed on the IbbHLH gene by Sweetpotato Garden database (http:// sweetpotato-garden. Kazusa. Or. Jp/index. Html) to find out the complete gene ORF that is highly similar to the EST sequence.

3. Designing and artificially synthesizing primers O49-F and O49-R, carrying out PCR amplification by taking the cDNA obtained in the step 1 as a template to obtain a PCR amplification product of about 1566bp, and connecting the PCR amplification product with a cloning vector pMD19-T to obtain a recombinant vector and sequencing. The primer sequences were as follows:

O49-F：5′-ATGGATAAGGGTGGCAAGGAT-3′

O49-R：5′-TCAGGGCTCAGATTTCATATGG-3′

The result shows that the nucleotide sequence of the PCR amplified product is shown as SEQ ID NO. 2, the gene shown by the sequence is named IbbHLH gene, the coded protein is named IbbHLH protein or protein IbbHLH49, and the amino acid sequence of the protein is shown as SEQ ID NO.1.

Application of example 2, ibbHLH49 protein in improving starch content of plants

1. Construction of recombinant plasmid pCAMBIA 1300-IbbHLH-GFP

1. The double-stranded DNA molecule shown in positions 1 to 1563 from the 5' -end of SEQ ID NO. 2 is artificially synthesized. The double-stranded DNA molecule is used as a template, 1300-F-KpnI and 1300-R-BamHI are used as primers for PCR amplification, and the double-stranded DNA molecule fragment 1 containing restriction enzyme KpnI at the N end and restriction enzyme BamHI at the C end is obtained.

1300-F-KpnI:5'-ACGGGGGACGAGCTCGGTACCATGGATAAGGGTGGCAAGGAT-3' (underlined is the recognition sequence for restriction endonuclease KpnI);

1300-R-BamHI:5'-CATGTCGACTCTAGAGGATCCGGGCTCAGATTTCATATGG-3' (recognition sequence for the restriction enzyme BamHI is underlined).

2. The vector pCAMBIA1300-GFP was digested with restriction enzymes KpnI and BamHI to recover about 10444bp of vector backbone 2.

3. And (3) carrying out homologous recombination connection on the fragment 1 and the vector skeleton 2 by using a seamless cloning enzyme to obtain a recombinant plasmid pCAMBIA 1300-IbbHLH-GFP.

Based on the sequencing results, the recombinant plasmid pCAMBIA 1300-IbbHLH-GFP was structurally described as follows: the DNA molecule with the nucleotide sequence of SEQ ID No. 1 is used for replacing a small fragment between the recognition sequences of restriction enzymes KpnI and BamHI of plasmid pCAMBIA1300-GFP, other nucleotide sequences of pCAMBIA1300-GFP are kept unchanged, and a recombinant expression vector pCAMBIA 1300-IbbHLH-GFP is obtained, and the recombinant expression vector pCAMBIA 1300-IbbHLH-GFP expresses IbbHLH protein shown in SEQ ID No. 1.

2. Construction of recombinant plasmid pCAMBIA 1300-IbbHLH-35 SI-X

1. A double-stranded DNA molecule with the nucleotide sequence shown in SEQ ID NO. 2 is artificially synthesized. The double-stranded DNA molecule is used as a template, a sequence of 1-200bp of a gene is selected as an interference sequence, RNAi-F1-BamHI and RNAi-R1-SalI are used as primers for PCR amplification to obtain a sense strand, and RNAi-F2-SacI and RNAi-R2-KpnI are used as primers for PCR amplification to obtain an antisense strand.

RNAi-F1-BamHI:5'-ACGGGGGACTCTAGTGGATCCATGGATAAGGGTGGCAAGGATGAG-3' (recognition sequence for the restriction enzyme BamHI underlined);

RNAi-R1-SalI:5'-AATTACCCTCTACTAGTCGACGAACTTGCAGATTGGTCCCAAACAT-3' (recognition sequence underlined as restriction endonuclease SalI);

RNAi-F2-SacI:5'-CGATCGGGGAAATTCGAGCTCATGGATAAGGGTGGCAAGGATGAG-3' (recognition sequence underlined as restriction enzyme SacI);

RNAi-R2-KpnI:5'-GTTAGGATTTCTAGAGGTACCGAACTTGCAGATTGGTCCCAAACAT-3' (underlined is the recognition sequence for the restriction endonuclease KpnI).

2. Cloning the sense strand sequence into a vector pCAMBIA1300-35SI-X at a multiple cloning site 1 (5 '-BamHI-SalI-3') in the 5'-3' direction;

3. After successful sequencing verification, the constructed clone is used as a target vector, the antisense strand sequence is cloned into a multi-cloning site 2 (5 '-KpnI-SacI-3') in the 5'-3' direction again, and the second insertion is successfully verified by sequencing, so that the pCAMBIA 1300-IbbHLH-35 SI-X plasmid is constructed.

Sequencing results showed that: the recombinant plasmid pCAMBIA 1300-IbbHLH-35 SI-X contains a DNA molecule shown in the formula (I):

SEQ reverse-X-SEQ forward (I);

the sequence of the SEQ forward direction is 1-200 th bit of SEQ ID NO. 2; 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.

3. Obtaining transgenic sweet potato plants

1. The recombinant plasmids pCAMBIA 1300-IbbHLH-GFP and pCAMBIA 1300-IbbHLH-35 SI-X are respectively transformed into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium, and the recombinant agrobacterium is respectively named EHA105/pCAMBIA 1300-IbbHLH-GFP and EHA105/pCAMBIA1300-IbbHLH49-35SI-X.

2. Removing stem tip meristem with length of about 0.5mm, placing on MS solid culture medium containing 2.0mg L ^-1, 2,4-D, and culturing in dark at 27+ -1deg.C for 4-6 weeks to obtain embryogenic callus. Then transferring the embryogenic callus to MS liquid culture medium containing 2.0mg L ^-1 -2, 4-D for shaking culture and propagation for 3-4 weeks to obtain embryogenic cell suspension culture system with diameter of 0.7-1.3 mm.

3. Culturing agrobacterium: the agrobacterium liquid is activated on a resistance plate, single colony is selected and inoculated into 20mL of LB liquid culture medium added with corresponding antibiotics, and the culture is carried out at 28 ℃ under 200rpm shaking overnight until the OD ₆₀₀ value is in the range of 0.5-0.7. Centrifugation at 5000rpm and removal of supernatant, washing the cells twice with an equal volume of LB medium, washing the cells once with an equal volume of MS liquid medium containing 2.0mg L ^-1, 4-D, and resuspending the cells with an equal volume of MS liquid medium containing 2.0mg L ^-1, 4-D.

4. Infection and co-cultivation: suspending chestnut embryogenic cell masses in the prepared agrobacterium tumefaciens bacterial liquid, shaking for a moment to sufficiently disperse the embryogenic cell masses so that the embryogenic cell masses are in full contact with the bacterial liquid, standing for 5min, sucking out the bacterial liquid by using a suction pipe, transferring the infected embryogenic cell masses to an MS solid culture medium containing 30mg of L ^-1 AS and 2.0mg of L ^-1 2,4-D for co-culture, paving 1 layer of common filter paper on the solid culture medium, and performing dark culture for 3 days at the temperature of 27+/-1 ℃.

5. Selective culture and regeneration of quasi-transgenic plants: the embryogenic cell mass after 3 days of co-culture was gently scraped with a razor blade, washed 3 times with MS liquid medium containing 500mg L ^-1 of cephalosporin and 2.0mg L ^-1, 4-D, transferred into MS liquid medium containing 100mg L ^-1 of cephalosporin and 2.0mg L ^-1, 4-D, The culture was delayed for 1 week. Then sucking the liquid culture medium as much as possible, placing the liquid culture medium on a solid MS culture medium which is paved with 1-2 layers of filter paper and contains 5mg L ^-1 of hygromycin, 100mg L ^-1 of cephalosporin and 2.0mg L ^-1 2,4-D for selective culture, the culture conditions were 27.+ -. 1 ℃ and dark culture. After 2 weeks, calli well grown were transferred to solid MS medium with 1 layer of filter paper laid on and containing 11mg L ^-1 hygromycin, 100mg L ^-1 cephalosporin and 2.0mg L ^-1 2,4-D for selective culture. Followed by 1 time every 2 weeks. Will contain 11mg of L ^-1 hygromycin, Resistant calli formed after 8 weeks of selective culture on solid MS medium of 100mg L ^-1 of cephalosporin and 2.0mg L ^-1 2,4-D were transferred to MS solid medium containing 100mg L ^-1 of cephalosporin and 1mg L ^-1 of ABA, culturing at 27+ -1deg.C under light irradiation of 13 hr and 3000lux daily to induce formation of somatic embryo. Inducing for 2-4 weeks, transferring the mature somatic embryo to MS solid culture medium, culturing at 27+/-1 deg.c under the condition of 13 hr and 3000lux light for 4-8 weeks, and regenerating the complete quasi-transgenic plant. Cutting off regenerated plantlets, subculturing on MS solid medium at 27+ -1deg.C under illumination of 3000lux per day for 13h, and subculturing 1 time every 6 weeks

6. Identification of transgenic plants: a method combining PCR detection and qRT-PCR detection was used.

1) The PCR detection method comprises the following steps:

DNA of wild sweet potato and the to-be-transferred IbbHLH gene sweet potato strain is extracted and PCR identification is carried out. The pCAMBIA 1300-IbbHLH-GFP and pCAMBIA 1300-IbbHLH-35 SI-X recombinant plasmids were used as positive controls, water and wild type WT as negative controls, primers were as follows:

OE49-F：5'-AGGAAGTTCATTTCATTTGGAGA-3'

OE49-R：5'-TCAGGGCTCAGATTTCATATGG-3'

Ri49-F：5'-CTTCGCAAGACCCTTCCTCT-3'

Ri49-R：5'-TCGGGGAAATTCGAGCTC-3'

the amplified PCR products are electrophoretically separated in 1% (w/v) agarose gel, the PCR positive plants should have a specific electrophoresis band of 1612bp and 623bp, and the line numbers of the PCR positive plants are recorded.

As shown in FIG. 1, the results showed that only positive control and quasi-transgenic sweetpotato plants OE49-1 to OE49-13 appeared in the electrophoresis band around 1612bp, ri49-1 to Ri49-14 appeared in the electrophoresis band around 623bp, and wild-type sweetpotato and negative control did not appear in the electrophoresis band, and the sweet potato transgenic positive plants OE49-1 to OE49-13 and Ri49-1 to Ri49-14 were preliminarily determined.

2)qRT-PCR

Extracting RNA of positive sweet potato plants, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR by taking a wild type as a control.

The Ibactin gene is an internal reference:

Ibactin-F：5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

Ibactin-R：5′-GGAAAATTAGAAGCACTTCCTGTGAAC-3′

IbbHLH49 the primer sequence is:

qRT49-F：5′-ATCCACATTCGGGCTAGGAG-3′

qRT49-R：5′-CGGGTTTACTGTTGCAAGCT-3′

The results are shown in fig. 2, and the results show that the expression quantity of IbbHLH genes is obviously up-regulated in the over-expressed transgenic sweet potato plants and obviously down-regulated in the interference-expressed transgenic sweet potato plants. 2 over-expression transgenic sweet potato plants (OE 49-9 and OE 49-12) and 2 interference expression transgenic sweet potato plants (Ri 49-7 and Ri 49-14) are selected for propagation to obtain IbbHLH T1 generation transgenic sweet potato lines OE49-9, OE49-12, ri49-7 and Ri49-14, and subsequent experiments are carried out.

4. Identification of starch content of transgenic sweet potato plants through over-expression IbbHLH49

WT is a wild chestnut aroma, transgenic sweetpotato is IbbHLH T ₁ generation transgenic sweetpotato strain OE49-9, OE49-12, ri49-7 and Ri49-14 plant.

1. Observation of IbbHLH49 transgenic sweet potato tuberous root

And taking sweet potato tuberous roots of IbbHLH T1 generation transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 planted in the isolated field for 4 months, cleaning and observing.

As a result, as shown in FIG. 3, the sweet potato tubers of the over-expressed transgenic plants are significantly larger than those of the wild type plants, while the sweet potato tubers of the interference-expressed transgenic plants are significantly smaller than those of the wild type plants.

2. Paraffin section observation of IbbHLH49 transgenic sweetpotato tubers

And taking sweet potato tubers of IbbHLH T1 generation transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 planted in the isolated field for 4 months, cleaning, and taking the middle part of the tubers to prepare paraffin sections. The method comprises the following steps:

1) The material is soaked in FAA fixing solution for more than 48 hours.

2) The immobilized material was dehydrated with alcohol having a gradient concentration of 50%, 70%, 80%, 95% and 100% in this order, and each concentration was treated for 30 minutes.

3) Xylene with 100% alcohol 1:1 post-mixing 2 samples, 30 minutes each, to decolorize plant cells to become transparent.

4) The treated material was placed in paraffin for 30 minutes and treated 2 times, each time with an oven at 40 ℃ open overnight.

5) Pouring the melted paraffin into an embedding device, rapidly moving the material into the embedding device by using hot tweezers, placing the material in a position, and placing the observation surface downwards.

6) The embedder was then moved into the water, slowly immersed in water, and after the paraffin had completely coagulated, removed from the water and the name and date of the embedded block were noted.

7) After the embedded material is trimmed, stuck and trimmed, the wax block is fixed on the wood block by a little paraffin, then the wood block is fixed on a slicing machine, a slicing knife fixer or a material fixer is adjusted to enable the slicing knife to be close to the wax block, the slicing thickness is adjusted to be 2mm, slicing is carried out, and the slicing is moved into a tray paved with black paper by a writing brush.

8) Placing the cut wax strips on a drying table, spreading and ironing the cut slices, then taking a small drop of glycerin to coat on a glass slide, coating the glass slide into a thin layer by using the outer side of the little finger, wherein the coating area is more than the area occupied by the wax strips, and drying at 37 ℃ more uniformly and thinner.

9) The slide was placed in a dye vat containing xylene to remove paraffin (10-20 minutes) and the procedure was repeated 2 times.

10 Finally, the dewaxed material is dyed by the following procedure: 1/2 xylene+1/2 pure alcohol → 100% alcohol → 95% alcohol → 85% alcohol → 70% alcohol → 50% alcohol → 1% safranin staining (more than 4 hours) → 50% alcohol → 70% alcohol → 85% alcohol → 95% alcohol → 0.5% solid green (1 minute) → 95% alcohol → 100% alcohol (the step reaction time for the unnoticed time is 3 minutes).

11 Placed in xylene for 3 minutes to clear.

12 Dripping neutral gum to seal the prepared specimen.

After the section was made, it was observed and photographed using a positive and negative integrated microscope (Revolve; echo).

As a result, as shown in FIG. 4, the number of starch grains in the tuberous root cells of the transgenic plants is significantly higher than that of the wild type plants, while the number of starch grains in the tuberous root cells of the transgenic plants with interference expression is significantly lower than that of the wild type plants.

2. Yield determination of IbbHLH49 transgenic sweetpotato tubers

Yield measurements were performed on the sweet potato tubers of IbbHLH T1 generation transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 and wild type plants grown in isolated fields for 4 months.

As shown in FIG. 5A, the yield of the sweet potato individual root tuber of the over-expression transgenic plant is increased by 9.4-17.2% compared with that of the wild type plant, and the yield of the sweet potato individual root tuber of the interference expression transgenic plant is reduced by 11.0-13.3% compared with that of the wild type plant.

3. Determination of total starch content of IbbHLH49 transgenic sweet potato tubers

Starch content in the tuberous root of the sweet potato plant was measured using a starch content test box (DF-2-Y, sumac Ming biotechnology limited). Tuberous roots of sweet potato plants were selected from the tuberous roots of IbbHLH T1 transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 and wild plants, which were planted in the isolated field for 4 months.

As a result, as shown in FIG. 5B, the sweet potato starch content of the over-expressed transgenic plant is increased by 5.1-16.7% compared with that of the wild type plant, while the sweet potato starch content of the interference expression transgenic plant is reduced by 4.0-8.4% compared with that of the wild type plant.

4. Amylopectin ratio determination of IbbHLH49 transgenic sweet potato tubers

And taking sweet potato tuberous roots of IbbHLH T1 generation transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 planted in the isolated field for 4 months, cleaning, drying, grinding and crushing, dissolving by using deionized water, and sieving the mixture through a 100-mesh sieve. And standing the suspension obtained by filtering, precipitating, removing the supernatant, and drying the obtained precipitate in an oven at 40 ℃ to constant weight to obtain the sweet potato tuberous root starch. The amylopectin ratio of the obtained sweet potato tuberous root starch was measured using an amylopectin content test box (ZHDF-2-Y).

As a result, as shown in FIG. 5C, the proportion of sweet potato amylopectin in the over-expressed transgenic plant was increased by 2.3 to 3.6% as compared with the wild-type plant, while the proportion of sweet potato amylopectin in the transgenic plant was reduced by 2.9 to 6.5% as compared with the wild-type plant.

5. Determination of AGPase Activity in IbbHLH transgenic sweetpotato tubers

AGPase activity in the tuberous root of the sweet potato plants was measured using an AGPase activity assay kit (AGP-2A-Y, ming Biotechnology Co., suzhou). Tuberous roots of sweet potato plants were selected from the tuberous roots of IbbHLH T1 transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 and wild plants, which were planted in the isolated field for 4 months.

As a result, as shown in FIG. 6A, the activity of AGPase in the sweet potato tubers of the over-expressed 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-expressed transgenic plants was significantly lower than that of the wild type plants.

6. Determination of SBE Activity in IbbHLH transgenic sweetpotato tubers

SBE activity in tuberous roots of sweet potato plants was measured using an SBE activity assay kit (SBE-2-Y, suzhou Kogyo Ming Biotechnology Co., ltd.). Tuberous roots of sweet potato plants were selected from the tuberous roots of IbbHLH T1 transgenic plants OE49-9, OE49-12, ri49-7 and Ri49-14 and wild plants, which were planted in the isolated field for 4 months.

The results are shown in FIG. 6B, where SBE activity in sweetpotato tubers of the over-expressed transgenic plants is significantly higher than that of wild-type plants, and SBE activity in sweetpotato tubers of the interference-expressed transgenic plants is significantly lower than that of wild-type plants.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart 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.