CN118726468A - Sweet potato root growth and development related protein IbbHLH112,112 and application thereof - Google Patents

Abstract

The application discloses a sweet potato root growth and development related protein IbbHLH and application thereof, belonging to the technical field of genetic engineering breeding. The application aims to solve the technical problems that: how to regulate the split skin and/or split character of sweet potato tuberous root. In order to solve the technical problems, the application provides IbbHLH protein or a substance for regulating and controlling the expression of a IbbHLH protein coding gene or the application of a substance for regulating and controlling the content of IbbHLH protein in regulating and controlling the split skin and/or split property of plant tuberous root, wherein IbbHLH protein is protein with an amino acid sequence of SEQ ID NO. 2. The application discloses IbbHLH protein and the function of the coding gene thereof in regulating and controlling the split skin and/or split character of sweet potato tuberous root and/or lignin content of tuber for the first time. Has important effects on improving and improving germplasm resources of crops such as sweet potatoes and the like.

Description

Sweet potato root growth and development related protein IbbHLH112,112 and application thereof

Technical Field

The application belongs to the technical field of genetic engineering breeding, and particularly relates to a sweet potato root growth and development related protein IbbHLH112,112 and application thereof.

Background

Split skin generally refers to a phenomenon in which the epidermis of a plant is split, exposing internal tissues and cells of an organ directly to the external environment. Tuberous roots are organs for storing nutrients of sweet potatoes, and the tuberous roots are rich in nutrient substances, but in recent years, the sweet potatoes often have split skin during the growing and developing process, so that the production of the sweet potatoes is severely restricted due to bad results such as reduced yield, reduced disease resistance and stress resistance, reduced quality and the like of the sweet potatoes. Therefore, research related to the growth and development of sweet potato roots and the splitting of sweet potato is carried out, and further, the cultivation of new varieties which are not easy to split is of great significance for improving the yield and quality of sweet potato. The digging of new genes for regulating and controlling the growth and development of sweet potato roots and splitting skin cracks and utilizing the new genes through a bioengineering means is an effective way for improving the quality of sweet potatoes.

Disclosure of Invention

The application aims to solve the technical problems that: how to regulate the split skin and/or split traits of plant tuberous root. Specifically, the technical problems to be solved by the application are as follows: how to regulate the split skin and/or split character of sweet potato tuberous root.

In order to solve the technical problems, the application provides the following technical scheme:

the application provides the use of IbbHLH protein or a substance regulating the expression of the IbbHLH protein-encoding gene or a substance regulating the content of the IbbHLH protein in any one of the following,

A1 Application in regulating and controlling split skin and/or split traits of plant tuberous root;

a2 For the preparation of a product for controlling the split skin and/or split traits of plant tubers;

a3 Application in regulating lignin content of plant tuberous root;

A4 For the preparation of a product for controlling the lignin content of plant tubers;

A3 For plant breeding or plant-assisted breeding);

A4 For the production of plant breeding or plant-assisted breeding products;

the IbbHLH112,112 protein may be any of the following:

a1 A protein with an amino acid sequence shown as SEQ ID NO. 2;

a2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residue of the amino acid sequence shown in a 1), has more than 80% of identity with the amino acid sequence shown in a 1) and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of a 1) or a 2).

In the present application, ibbHLH112,112 has the same meaning as IbbHLH112,112.

In the present application, the plant breeding indicator may include the split and/or split trait of the root tuber and/or lignin content of the root tuber.

In the present application, the object of plant breeding may include growing plants with improved split and/or split traits in tuberous roots. The improved split skin and/or split character of the root tuber may be specifically reduced number of split skin and/or split of the root tuber, reduced character or disappeared.

In the present application, plants with improved split and/or split traits of the tubers may be used for genetic breeding.

In the present application, the object of plant breeding may also include growing plants with increased split and/or split traits in tuberous roots. The increased splitting and/or cracking characteristics of the root tuber can be increased in number and increased in characteristics.

In the application, the plant with the increased split skin and/or split property of the root tuber can be used for researching the mechanism of the split skin and/or split of the root tuber.

In the present application, the modulation may be an increase or promotion or up-regulation.

In the present application, the modulation may also be reduction or inhibition or downregulation.

In the present application, the protein may be derived from sweet potato.

In the present application, SEQ ID NO.2 consists of 415 amino acid residues.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

A3 The linkages may be linked by peptide bonds. Specifically, the connection of a 3) may be that the C-terminal end of the tag forms a peptide bond with the N-terminal end of a 1) or a 2) protein by dehydration condensation, or that the N-terminal end of the tag forms a peptide bond with the C-terminal end of a 1) or a 2) protein by dehydration condensation.

The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.

Further, in the application, the substance that regulates the expression of the IbbHLH protein-encoding gene or the substance that regulates the content of the IbbHLH protein is a biological material, and the biological material may be any one of the following:

b1 A nucleic acid molecule encoding said IbbHLH112,112 protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

B3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

B4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising B1) said nucleic acid molecule or a transgenic plant cell line comprising B2) said expression cassette or a transgenic plant cell line comprising B3) said recombinant vector;

B6 A transgenic plant tissue comprising B1) said nucleic acid molecule or a transgenic plant tissue comprising B2) said expression cassette or a transgenic plant tissue comprising B3) said recombinant vector;

B7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2) or a transgenic plant organ comprising the recombinant vector of B3).

Further, in said application, the nucleic acid molecule of B1) may be a DNA molecule as described in g 1) or g 2) below:

g1 A DNA molecule with the coding sequence of the coding strand SEQ ID NO. 1;

g2 A DNA molecule which has 80% or more identity with the DNA molecule of g 1) and encodes the same functional protein.

Further, in the application described, the expression cassette of B2) refers to a DNA capable of expressing the IbbHLH112,112 protein in a host cell, which may include not only a promoter for initiating transcription of the IbbHLH protein-encoding gene, but also a terminator and/or enhancer sequence for terminating transcription of the IbbHLH protein-encoding gene. Promoters useful in the present application 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, a wound-inducible promoter 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 promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, oleosin, and the promoters of soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) 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 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, the recombinant vector may contain a DNA molecule shown in SEQ ID NO.1 for encoding protein IbbHLH112,112.

The recombinant vector containing IbbHLH112,112 can be constructed using plant expression vectors. 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 pCAMBIA 1391-Xb.

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.

In a specific embodiment of the application, the recombinant vector is recombinant plasmid pCAMBIA1300-IbbHLH112,112.

The application clones IbbHLH gene and constructs pCAMBIA1300-IbbHLH expression vector, and uses in situ conversion method to transfer pCAMBIA1300-IbbHLH into sweet potato chestnut fragrance, further, in the application, the recombinant microorganism can be yeast, bacteria, algae and fungi.

Further, in the application, the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

Further, in the application, the transgenic plant organ may be the root, stem, leaf, flower, fruit and seed of a transgenic plant.

Further, in the described application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ may or may not include propagation material.

Further, in the application, the plant may be a dicot.

Further, in the application, the dicotyledonous plant may be a plant of the family Convolvulaceae.

Further, in the application, the Convolvulaceae plant may be a sweet potato plant.

Further, in the application, the sweet potato plant may be sweet potato (Ipomoea batatas).

The application also provides a method for regulating and controlling the split skin and/or split character of the sweet potato tuber, which comprises regulating and controlling the expression level of the coding gene of IbbHLH112,112 protein in the acceptor sweet potato and/or regulating and controlling the content of IbbHLH protein in the acceptor sweet potato to regulate and control the split skin and/or split character of the acceptor sweet potato tuber.

Further, the method may comprise increasing the expression level of the gene encoding the IbbHLH112,112 protein in the recipient sweetpotato and/or increasing the content of the IbbHLH112,112 protein in the recipient sweetpotato to increase or enhance the split and/or split traits of the root tuber of the recipient sweetpotato, and increasing the lignin content of the root tuber of the recipient sweetpotato.

Further, in the method, the improvement of the expression level of the coding gene of the IbbHLH protein in the acceptor sweet potato and/or the improvement of the content of the IbbHLH protein in the acceptor sweet potato can be realized by introducing the coding gene of the IbbHLH protein into the acceptor sweet potato.

Further, in the method, the IbbHLH protein-encoding gene may be the DNA molecule described in g 1) or g 2) as follows:

g1 A DNA molecule with the coding sequence of the coding strand SEQ ID NO. 1;

g2 A DNA molecule which has 80% or more identity with the DNA molecule of g 1) and encodes the same functional protein.

The application also provides a method for obtaining the sweet potato with the purpose of changing the split skin and/or split character of the sweet potato root tuber, which can comprise the step of obtaining the sweet potato with the purpose of increasing the split skin and/or split character of the sweet potato root tuber and/or increasing the lignin content of the root tuber by increasing the expression level of the IbbHLH protein coding gene in the acceptor sweet potato and/or increasing the IbbHLH protein content in the acceptor sweet potato.

Further, in the method, the improvement of the expression level of the coding gene of IbbHLH112,112 protein in the acceptor sweet potato and/or the improvement of the content of IbbHLH protein in the acceptor sweet potato is realized by introducing the coding gene of IbbHLH protein into the acceptor sweet potato.

Further, the root tuber of the subject sweet potato has a greater number of split and/or split than the recipient sweet potato. The tuberous root of the target sweet potato has a lignin content higher than that of the recipient sweet potato.

The target sweet potato can be used for researching the mechanism of the root and peel splitting and/or cracking of the sweet potato.

The IbbHLH112,112 proteins described above and the biological materials described above are also the subject of the present application.

In the present application, identity refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the internet, such as the BLAST web page 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.

The 80% or more identity may be 80%, 85%, 90% or more than 95% identity.

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

The IbbHLH gene is cloned, and the constructed plant expression vector pCAMBIA1300-IbbHLH is introduced into the chestnut fragrance of the sweet potato variety to obtain the transgenic sweet potato plant of the overexpression IbbHLH. Compared with the control, the transgenic sweet potato tuberous root has obvious split skin, the lignin content is obviously improved, and IbbHLH112 over-expression is estimated to accelerate cell division in the tuber root so as to promote the split skin of the sweet potato. Therefore, the IbbHLH gene provided by the application plays an important role in regulating and controlling sweet potato split skin cracks, has important application value in the research of improving yield and plant quality, and has wide application space and market prospect in the agricultural field.

The beneficial technical effects obtained by the application are as follows:

The application discloses IbbHLH protein and the function of the coding gene thereof in regulating and controlling the split skin and/or split property of sweet potato tuberous root for the first time. The over-expression experiment shows that: ibbHLH112 protein and its coding gene can obviously regulate and control the split skin and/or split character of sweet potato root tuber and/or lignin content of root tuber. The sweet potato IbbHLH112,112 can be widely applied to the plant fields of sweet potato genetic breeding, germplasm resource improvement, transgene, genome editing breeding and the like, and plays an important role in improving and improving the germplasm resource of the sweet potato.

Drawings

FIG. 1 shows the PCR detection results of transgenic sweetpotato tubers. M is DNA molecular Marker, W is negative control water, P is positive control (pCAMBIA 1300-IbbHLH) and WT is genomic DNA of wild sweet potato variety chestnut tuber, OE-2, OE-3, OE-5 is IbbHLH chestnut tuber transgene.

FIG. 2 shows the relative expression levels of IbbHLH112 in transgenic sweetpotato plants and wild type sweetpotato plants. WT is cDNA of wild sweet potato and chestnut fragrant tuberous root, OE-2, OE-3, OE-5 is cDNA of IbbHLH chestnut fragrant transgenic sweet potato tuberous root.

FIG. 3 is a transgenic sweetpotato tuberous root phenotype observation; wherein WT is wild sweet potato chestnut tuberous root, OE-2, OE-3, OE-5 is transgenic sweet potato tuberous root (scale=5 cm).

FIG. 4 is lignin content of transgenic sweetpotato tubers; WT is chestnut-flavor wild sweet potato tuber, OE-2, OE-3 and OE-5 are transgenic sweet potato tuber.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The vector pCAMBIA1300-GFP is stored in the key laboratory of sweet potato biology and biotechnology in agricultural rural area of China university. The disclosure of this document "Zhen Wang, Xu Li, Xiao-ru Gao, Zhuo-ru Dai, Kui Peng, Li-cong Jia, Yin-kui Wu, Qing-chang Liu, Hong Zhai, Shao-pei Gao, Ning Zhao, Shao-zhen He, Huan Zhang, IbMYB73 targets abscisic acid-responsiveIbGER5to regulate root growth and stress tolerance in sweet potato,Plant Physiology, Volume 194, Issue 2, February 2024, Pages 787–804,https://doi.org/10.1093/plphys/kiad532" is that the public can apply for the biological material from the applicant, and the biological material obtained can only be used for the verification of the content of the present application and not for other uses.

The sweet potato chestnut flavor is preserved in the key laboratory of sweet potato biology and biotechnology in rural areas of China agricultural university. The cloning and functional analysis of the sweet potato drought-resistance related gene IbNAC [ J ]. Crop theory, 2020, 46 (11): 1649-1658. "is disclosed in the documents" Zhang Huan, yang Naike, shang Lili, gao Xiaoru, liu Qingchang, gong, gao Shaopei, he Shaozhen, which are available to the public from the applicant for application, and which are usable only for the content verification of the present application and not for other uses.

The quantitative tests in the examples below were repeated three times, and the results averaged, unless otherwise specified.

The following examples used SPSS statistical software to process the data, and the experimental results were expressed as mean ± standard deviation, with significant differences as indicated by t-test (P < 0.01).

Examples 1, ibbHLH acquisition of the 112 Gene

1. Obtaining cDNA templates

Extracting total RNA of 18 tubers of the sweet potato variety by using a plant total RNA extraction kit, and reversely transcribing the total RNA into first-strand cDNA by using a PRIMESCRIPTTM RT REAGENT KIT WITH GDNA ERASER kit.

2. Designing and artificially synthesizing primers IbbHLH-112-F and IbbHLH-112-R, performing PCR amplification by taking the cDNA obtained in the step 1 as a template to obtain a PCR amplification product of about 1248 and bp, and connecting the PCR amplification product with a cloning vector pMD19-T to obtain a recombinant vector and sequencing. The PCR amplification primer sequences were as follows:

IbbHLH112-F: 5’-ATGGCGGATGATTTTATGACAGG-3’

IbbHLH112-R: 5’-CTACCTGAAGGATGCCCCG-3’

the result shows that the nucleotide sequence of the PCR amplified product is shown in positions 1 to 1248 from the 5' end of SEQ ID NO.1, the gene shown in the sequence is named IbbHLH112 gene, the encoded protein is named IbbHLH protein or protein IbbHLH112, and the amino acid sequence is shown in SEQ ID NO. 2.

Examples 2, ibbHLH, obtaining of transgenic sweetpotato tubers

1. Construction of recombinant plasmid pCAMBIA1300-IbbHLH112

1. The double-stranded DNA molecule shown in positions 1 to 1248 from the 5' -end of SEQ ID NO.1 is artificially synthesized. The double-stranded DNA molecule is used as a template, and p-IbbHLH-112-F (KpnI) and p-IbbHLH-112-R (XbaI) are used as primers for PCR amplification, so that the double-stranded DNA molecule containing restriction enzyme KpnI at the N end and restriction enzyme XbaI at the C end is obtained.

P-IbbHLH112-F (KpnI): 5'-ACGGGGGACGAGCTCGGTACCATGGCGGATGATTTTATGACAGG-3' (underlined is the recognition sequence for restriction endonuclease KpnI);

p-IbbHLH112-R (XbaI): 5'-AAGATCTTCGTCGACTCTAGACTACCTGAAGGATGCCCCG-3' (recognition sequence for the restriction endonuclease XbaI underlined).

2. Vector pCAMBIA1300-GFP was digested with restriction enzymes KpnI and XbaI to recover vector backbone 1 of about 10442 bp.

3. A double-stranded DNA molecule having a restriction enzyme KpnI at the N-terminus and a restriction enzyme XbaI at the C-terminus was digested with both the restriction enzymes KpnI and XbaI, whereby fragment 2 containing about 1290 bp was recovered.

4. And connecting the fragment 2 with the vector framework 1 to obtain the recombinant plasmid pCAMBIA1300-IbbHLH112.

Based on the sequencing results, the recombinant plasmid pCAMBIA1300-IbbHLH112 was structurally described as follows: the small fragment between the recognition sequences of restriction enzymes KpnI and XbaI of the recombinant plasmid pCAMBIA1300-GFP is replaced by the DNA molecule shown in positions 1 to 1248 from the 5' end of SEQ ID NO.1, and the recombinant plasmid pCAMBIA1300-IbbHLH can express IbbHLH protein with the amino acid sequence of SEQ ID NO. 2.

2. Obtaining of transgenic sweet potato plants over-expressed IbbHLH112,112

1. Selecting sweet potato variety chestnut fragrant stem segments which grow vigorously, fresh and healthy in the field, wherein each stem segment is about 20-25 cm a long: 3-5 holes are pricked at the stem node by a needle head for standby.

2. The recombinant plasmid pCAMBIA1300-IbbHLH112 was transferred into Agrobacterium rhizogenes K599 and designated K599/pCAMBIA 1300-IbbHLH.

3. Before infection, the monoclonal K599/pCAMBIA1300-IbbHLH112,112 is added into 40, 40 mL LB liquid medium containing 100 mg/L Kan antibiotics, at 28deg.C, shaking rotation speed 200 rpm, dark culture 12-18h, and OD600 around 0.8.

4. Pouring the bacterial liquid with the OD600 of 0.8 into a 50mL centrifuge tube, centrifuging the bacterial liquid with the OD600 of 5000 rpm for 8min, collecting bacterial bodies and discarding the supernatant; then adding a proper amount of sterile water to resuspend the thalli, and repeating for 2-3 times to ensure that the OD600 of the bacterial liquid is about 0.8.

5. Soaking the stem segment treated in the step 1 in the bacterial liquid in the step 4, and standing at room temperature for about 8 h.

6. The infected sweet potato stems were planted in isolated fields, at least 10 plants per transformation event. Tubers were harvested after 3 months of growth in the field, observed and identified.

Setting a wild control group, planting the chestnut fragrant stem segments of the wild sweet potato variety into an isolated field, and planting at least 10 plants. After 3 months of growth in the field, the wild sweet potato tuberous root can be harvested for observation and identification.

7. Identification of transgenic plants: method using PCR detection and qRT-PCR detection in combination

1) The PCR detection method comprises the following steps:

DNA of wild sweet potato and the tuber of the sweet potato with IbbHLH gene to be transferred is extracted and PCR identification is carried out. The pCAMBIA1300-IbbHLH112 plasmid was used as positive control, water and wild type WT as negative control, primers were as follows:

35S-F：5’-TCCTTCGCAAGACCCTTCCTC-3’

IbbHLH112-R：5’-CTACCTGAAGGATGCCCCGAATGTTGGTGTCCAGAAA-3’

The amplified PCR products were electrophoretically identified in a 0.8% agarose gel, and the results are shown in FIG. 1, wherein only positive control and tuberous roots of the transgenic IbbHLH gene sweet potatoes OE-2, OE-3 and OE-5 show the target bands, and the wild sweet potato tuberous roots do not show the bands, so that the results indicate that OE-2, OE-3 and OE-5 are transgenic sweet potato plants.

2）qRT-PCR

And (3) extracting RNA of the transgenic sweet potato tubers, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR analysis.

The primer sequences used were:

qRT-IbbHLH112-F：5’-ACTGATGGGTTTGAATTAGAAAAATCT-3’；

qRT-IbbHLH112-R：5’-GATTTGCTTGAACGAAGAATCTGA-3’。

As a result, as shown in FIG. 2, the relative expression level of IbbHLH112 gene was significantly increased in the transgenic sweetpotato tubers.

The OE-2, OE-3 and OE-5 transgenic sweetpotato were propagated asexually for subsequent phenotypic observation and identification, respectively.

3. Phenotype observation of transgenic sweetpotato tuberous root with over-expression IbbHLH112

And respectively planting 5 strains of each strain of OE-2, OE-3, OE-5 and wild chestnut fragrance in an isolated field according to a conventional planting mode of sweet potatoes, and observing the size, shape, split skin and the like of the sweet potatoes after harvesting, photographing and counting. If a plurality of obvious cracks, cracks and depressions appear in the same potato block, the potato block is regarded as the split skin of the potato block. The experiment was repeated three times.

As shown in FIG. 3, the wild chestnut fragrant potato block has no split skin, while IbbHLH112 over-expressed chestnut fragrant transgenic potato blocks OE-2, OE-3 and OE-5 all have split skin split to different degrees. The result shows that the over-expression of IbbHLH112,112 promotes the split skin and crack of the tuber root of sweet potato, and affects the development of the root.

IV, over-expression IbbHLH112,112 determination of lignin content of transgenic sweet potato tuberous root

The lignin content of the transgenic sweetpotato OE-2, OE-3 and OE-5 tubers harvested in step three was determined using a lignin content determination kit (Suzhou Ming Bioproduct, cat# MGS-2-G) and by reference to the product specifications.

The experiment was repeated three times and the results averaged.

As shown in FIG. 4, the lignin content of OE-2, OE-3 and OE-5 tubers was higher than that of chestnut-flavor wild-type tubers, indicating that IbbHLH112 overexpression can significantly increase the lignin content of transgenic sweetpotato tubers.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1. Use of an IbbHLH112 protein or a substance that modulates the expression of a gene encoding said IbbHLH protein or a substance that modulates the protein content of said IbbHLH protein in any one of,

   A1 Application in regulating and controlling split skin and/or split traits of plant tuberous root;

   a2 For the preparation of a product for controlling the split skin and/or split traits of plant tubers;

   a3 Application in regulating lignin content of plant tuberous root;

   A4 For the preparation of a product for controlling the lignin content of plant tubers;

   A3 For plant breeding or plant-assisted breeding);

   A4 For the production of plant breeding or plant-assisted breeding products;

   The IbbHLH112,112 protein is any one of the following proteins:

   a1 A protein with an amino acid sequence shown as SEQ ID NO. 2;

   a2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residue of the amino acid sequence shown in a 1), has more than 80% of identity with the amino acid sequence shown in a 1) and has the same function;

   a3 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of a 1) or a 2).
2. 2. The use according to claim 1, wherein the substance regulating the expression of the IbbHLH protein-encoding gene or the substance regulating the protein content of IbbHLH is a biological material, said biological material being any of the following:

   B1 A nucleic acid molecule encoding the IbbHLH112,112 protein of claim 1;

   b2 An expression cassette comprising the nucleic acid molecule of B1);

   B3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

   B4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

   b5 A transgenic plant cell line comprising B1) said nucleic acid molecule or a transgenic plant cell line comprising B2) said expression cassette or a transgenic plant cell line comprising B3) said recombinant vector;

   B6 A transgenic plant tissue comprising B1) said nucleic acid molecule or a transgenic plant tissue comprising B2) said expression cassette or a transgenic plant tissue comprising B3) said recombinant vector;

   B7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2) or a transgenic plant organ comprising the recombinant vector of B3).
3. 3. The use according to claim 2, wherein the nucleic acid molecule of B1) is a DNA molecule according to g 1) or g 2) as follows:

   g1 A DNA molecule with the coding sequence of the coding strand SEQ ID NO. 1;

   g2 A DNA molecule which has 80% or more identity with the DNA molecule of g 1) and encodes the same functional protein.
4. 4. The use according to any one of claims 1 to 3, wherein the plant is a dicotyledonous plant.
5. 5. A method for controlling the split and/or split trait of a sweet potato tuber, comprising controlling the expression level of a gene encoding the IbbHLH112,112 protein of claim 1 in a recipient sweet potato and/or controlling the content of the IbbHLH112,112 protein in a recipient sweet potato, to control the split and/or split trait of a recipient sweet potato tuber.
6. 6. The method of claim 5, comprising increasing the expression of the IbbHLH112,112 protein encoding gene in the recipient sweetpotato and/or increasing the IbbHLH protein content in the recipient sweetpotato to increase or enhance the split and/or split traits of the root tuber of the recipient sweetpotato, and increasing the lignin content of the root tuber of the recipient sweetpotato.
7. 7. The method of claim 6, wherein increasing the expression level of the gene encoding IbbHLH112,112 protein in the recipient sweetpotato and/or increasing the content of the IbbHLH protein in the recipient sweetpotato is by introducing the gene encoding IbbHLH protein into the recipient sweetpotato.
8. 8. A method for obtaining sweet potato with altered split and/or split traits in sweet potato tubers, comprising obtaining sweet potato with increased split and/or split traits in sweet potato tubers and/or increased lignin content in sweet potato tubers by increasing the expression level of the gene encoding IbbHLH112,112 protein in the recipient sweet potato and/or increasing the content of IbbHLH protein in the recipient sweet potato.
9. 9. The method of claim 8, wherein the increasing the expression level of the gene encoding IbbHLH112,112 protein in the recipient sweetpotato and/or the increasing the content of the IbbHLH112,112 protein in the recipient sweetpotato is achieved by introducing the gene encoding IbbHLH112,112 protein into the recipient sweetpotato.
10. 10. IbbHLH112 protein as claimed in claim 1 and biomaterial as claimed in claim 2 or 3.