CN118726468A - Protein IbbHLH112 related to sweet potato root growth and development and its application - Google Patents

Abstract

The present application discloses a protein IbbHLH112 related to the growth and development of sweet potato roots and its application, and belongs to the technical field of genetic engineering breeding. The technical problem to be solved by the present application is: how to regulate the cracking and/or fissure characteristics of sweet potato root tubers. In order to solve the above technical problems, the present application provides the application of IbbHLH112 protein or a substance that regulates the expression of the gene encoding the IbbHLH112 protein or a substance that regulates the content of the IbbHLH112 protein in regulating the cracking and/or fissure characteristics of plant root tubers, and the IbbHLH112 protein is a protein whose amino acid sequence is SEQ ID NO.2. The present application discloses for the first time the role of IbbHLH112 protein and its encoding gene in regulating the cracking and/or fissure characteristics of sweet potato root tubers and/or the lignin content of the root tubers. It plays an important role in improving and modifying the germplasm resources of crops such as sweet potatoes.

Description

Protein IbbHLH112 related to sweet potato root growth and development and its application

Technical Field

The present application belongs to the field of genetic engineering breeding technology, and specifically relates to a protein IbbHLH112 related to sweet potato root growth and development and its application.

Background Art

Cracked skin usually refers to a phenomenon in which cracks appear on the plant epidermis, exposing the internal tissues and cells of the organ directly to the external environment. The root tuber is the organ for storing nutrients in sweet potatoes, which is rich in nutrients. However, in recent years, cracked skin often occurs in the process of sweet potato growth and development, resulting in adverse consequences such as reduced sweet potato yield, decreased disease resistance and stress resistance, and decreased quality, which seriously restricts the production of sweet potatoes. Therefore, conducting research related to the growth and development of sweet potato roots and cracked skin of sweet potatoes and breeding new varieties that are not prone to cracking are of great significance to increase sweet potato yield and improve sweet potato quality. Discovering new genes that regulate the growth and development of sweet potato roots and cracked skin and utilizing them through bioengineering is an effective way to improve the quality of sweet potatoes.

Summary of the invention

The technical problem to be solved by the present application is: how to regulate the cracking and/or splitting characteristics of plant root tubers. Specifically, the technical problem to be solved by the present application is: how to regulate the cracking and/or splitting characteristics of sweet potato root tubers.

In order to solve the above technical problems, this application provides the following technical solutions:

The present application provides the use of IbbHLH112 protein or a substance for regulating the expression of a gene encoding the IbbHLH112 protein or a substance for regulating the content of the IbbHLH112 protein in any of the following items:

A1) Application in regulating the cracking and/or splitting characteristics of plant root tubers;

A2) Use in the preparation of products for regulating the cracking and/or splitting characteristics of plant root tubers;

A3) Application in regulating the lignin content of plant root tubers;

A4) Application in the preparation of products for regulating the lignin content of plant tubers;

A3) Application in plant breeding or plant assisted breeding;

A4) Use in the preparation of products for plant breeding or plant-assisted breeding;

The IbbHLH112 protein may be any of the following proteins:

a1), the amino acid sequence is the protein shown in SEQ ID NO.2;

a2), a protein having more than 80% identity with the amino acid sequence shown in a1) and having the same function as the amino acid sequence shown in a1) obtained by substitution and/or deletion and/or addition of amino acid residues;

a3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2).

In the present application, the IbbHLH112 protein has the same meaning as the protein IbbHLH112.

In the present application, the plant breeding index may include the crack bark and/or crack characteristics of the tuberous root and/or the lignin content of the tuberous root.

In the present application, the purpose of plant breeding may include cultivating plants with improved cracking and/or fissure traits of tubers. The improvement of cracking and/or fissure traits of tubers may specifically be a reduction in the number of cracking and/or fissures of the tubers, or a reduction or disappearance of the traits.

In the present application, the plants with improved bark cracking and/or cracking traits of the root tubers can be used for genetic breeding.

In the present application, the purpose of plant breeding may also include cultivating plants with aggravated cracking and/or fissures of tuberous roots. The aggravated cracking and/or fissures of tuberous roots may specifically be an increase in the number of cracking and/or fissures of the tuberous roots and an aggravation of the trait.

In the present application, the plant with aggravated bark cracking and/or fissures of the tuber roots can be used for studying the mechanism of bark cracking and/or fissures of the tuber roots.

In the present application, the regulation may be increasing, promoting or upregulating.

In the present application, the regulation may also be reduction, inhibition or down-regulation.

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 artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

a3) The connection can be through a peptide bond. Specifically, the connection described in a3) can be a peptide bond connection formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in a1) or a2), or a peptide bond connection formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in a1) or a2).

The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be a Flag 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.

Furthermore, in the application, the substance that regulates the expression of the gene encoding the IbbHLH112 protein or the substance that regulates the content of the IbbHLH112 protein is a biological material, and the biological material can be any one of the following:

B1), a nucleic acid molecule encoding the IbbHLH112 protein;

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

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

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

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

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

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

Furthermore, in the application, the nucleic acid molecule in B1) may be the DNA molecule described in g1) or g2) below:

g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID NO.1;

g2), a DNA molecule that has more than 80% identity with the DNA molecule described in g1) and encodes a protein with the same function.

Further, in the application, the expression cassette described in B2) refers to a DNA capable of expressing the IbbHLH112 protein in a host cell, which may include not only a promoter for initiating transcription of the IbbHLH112 protein encoding gene, but also a terminator or/and enhancer sequence for terminating transcription of the IbbHLH112 protein encoding gene. Promoters that can be used in this 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: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); 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 millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 10099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated 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 pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot et al. (1991) Mol. Gen. Genet, 262:141; (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 the DNA molecule encoding protein IbbHLH112 as shown in SEQ ID NO.1.

A plant expression vector can be used to construct a recombinant vector containing the IbbHLH112 . The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-GFP, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb.

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

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

The present application cloned the IbbHLH112 gene and constructed the pCAMBIA1300- IbbHLH112 expression vector, and used the in situ transformation method to transfer pCAMBIA1300 -IbbHLH112 into the sweet potato variety Lizixiang. Furthermore, in the application, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi.

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

Furthermore, in the application, the transgenic plant organ can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

Furthermore, in the application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ may or may not include propagation materials.

Furthermore, in the application, the plant may be a dicotyledonous plant.

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

Furthermore, in the application, the Convolvulaceae plant may be a plant of the genus Ipomoea batatas.

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

The present application also provides a method for regulating the skin cracking and/or cracking characteristics of sweet potato root tubers, which method comprises regulating the expression level of the gene encoding the IbbHLH112 protein in the recipient sweet potato and/or regulating the content of the above-mentioned IbbHLH112 protein in the recipient sweet potato to regulate the skin cracking and/or cracking characteristics of the recipient sweet potato root tubers.

Furthermore, the method may include increasing the expression level of the gene encoding the IbbHLH112 protein in the recipient sweet potato and/or increasing the content of the IbbHLH112 protein in the recipient sweet potato to increase or improve the skin cracking and/or cracking characteristics of the recipient sweet potato root tubers, and increase the lignin content of the recipient sweet potato root tubers.

Furthermore, in the method described above, the expression level of the gene encoding the IbbHLH112 protein in the recipient sweet potato and/or the content of the IbbHLH112 protein in the recipient sweet potato can be increased by introducing the gene encoding the IbbHLH112 protein into the recipient sweet potato.

Furthermore, in the method described above, the gene encoding the IbbHLH112 protein may be the DNA molecule described in g1) or g2) below:

g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID NO.1;

g2), a DNA molecule that has more than 80% identity with the DNA molecule described in g1) and encodes a protein with the same function.

The present application also provides a method for obtaining a sweet potato with altered skin cracking and/or cracking characteristics of sweet potato root tubers. The method may include increasing the expression level of the gene encoding the above-mentioned IbbHLH112 protein in the recipient sweet potato and/or increasing the content of the IbbHLH112 protein in the recipient sweet potato, thereby obtaining a sweet potato with aggravated skin cracking and/or cracking characteristics of sweet potato root tubers and/or increased lignin content in the root tubers.

Furthermore, in the method described above, increasing the expression level of the gene encoding the IbbHLH112 protein in the recipient sweet potato and/or increasing the content of the IbbHLH112 protein in the recipient sweet potato is achieved by introducing the gene encoding the IbbHLH112 protein into the recipient sweet potato.

Furthermore, the number of cracks and/or fissures of the tuber of the target sweet potato is greater than that of the recipient sweet potato. The lignin content of the tuber of the target sweet potato is higher than that of the recipient sweet potato.

The target sweet potato can be used for studying the mechanism of skin cracking and/or fissures of sweet potato tubers.

The above-mentioned IbbHLH112 protein and the above-mentioned biological material are also protected contents of this application.

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

The above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.

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

The present application cloned the IbbHLH112 gene, and introduced the constructed plant expression vector pCAMBIA1300- IbbHLH112 into the sweet potato variety Li Zixiang to obtain a transgenic sweet potato plant overexpressing IbbHLH112 . Compared with the control, the transgenic sweet potato root tubers showed obvious cracks and cracks, and the lignin content was significantly increased. It was speculated that the overexpression of IbbHLH112 would accelerate the cell division inside the root tubers and promote the cracks of the sweet potato skin. Therefore, the IbbHLH112 gene provided by the present application plays an important role in regulating the cracks of sweet potato skin, has important application value in increasing yield and improving plant quality research, and has broad application space and market prospects in the agricultural field.

The beneficial technical effects achieved by this application are as follows:

This application discloses for the first time the role of IbbHLH112 protein and its encoding gene in regulating the cracking and/or fissure traits of sweet potato root tubers. Overexpression experiments show that IbbHLH112 protein and its encoding gene can significantly regulate the cracking and/or fissure traits of sweet potato root tubers and/or the lignin content of the root tubers. Sweet potato IbbHLH112 can be widely used in plant fields such as sweet potato genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and improving sweet potato germplasm resources.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the PCR detection results of transgenic sweet potato tubers. M is a DNA molecule marker, W is a negative control water, P is a positive control (pCAMBIA1300- IbbHLH112 ), WT is the genomic DNA of the wild-type sweet potato variety Li Zi Xiang tubers, and OE-2, OE-3, and OE-5 are IbbHLH112 Li Zi Xiang transgenic sweet potato tubers.

Figure 2 shows the relative expression of IbbHLH112 in transgenic sweet potato plants and wild-type sweet potato plants. WT is the cDNA of wild-type sweet potato chestnut-scented root tubers, and OE-2, OE-3, and OE-5 are the cDNA of IbbHLH112 chestnut-scented transgenic sweet potato tubers.

Figure 3 shows the phenotype observation of transgenic sweet potato tubers; WT is the wild-type sweet potato tuber with chestnut fragrance, and OE-2, OE-3, and OE-5 are transgenic sweet potato tubers (scale = 5 cm).

Figure 4 shows the lignin content of transgenic sweet potato tubers; WT is the chestnut-scented wild-type sweet potato tuber, and OE-2, OE-3 and OE-5 are transgenic sweet potato tubers.

DETAILED DESCRIPTION

The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The vector pCAMBIA1300-GFP is deposited by the Key Laboratory of Sweet Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, China Agricultural University. It is disclosed in the 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-responsive IbGER5 toregulate 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". The public can apply for the biological material from the applicant. The obtained biological material can only be used for content verification of this application and cannot be used for other purposes.

Sweet potato chestnut fragrance is preserved by the Key Laboratory of Sweet Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, China Agricultural University. It is disclosed in the document "Zhang Huan, Yang Naike, Shang Lili, Gao Xiaoru, Liu Qingchang, Zhai Hong, Gao Shaopei, He Shaozhen. Cloning and functional analysis of sweet potato drought resistance-related gene IbNAC72[J]. Acta Agronomica Sinica, 2020, 46(11): 1649-1658." that the public can apply for the biological material from the applicant, and the obtained biological material can only be used for content verification of this application and cannot be used for other purposes.

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

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

Example 1. Acquisition of IbbHLH112 gene

1. Obtaining cDNA template

Total RNA from the tubers of sweet potato variety Xushu 18 was extracted using a plant total RNA extraction kit, and the total RNA was reverse transcribed into the first-strand cDNA using a PrimeScriptTM RT reagent Kit with gDNA Eraser kit.

2. Design and synthesize primers IbbHLH112-F and IbbHLH112-R, use the cDNA obtained in step 1 as a template, perform PCR amplification, obtain a PCR amplification product of about 1248 bp, connect it with the cloning vector pMD19-T to obtain a recombinant vector and sequence it. The PCR amplification primer sequences are as follows:

IbbHLH112-F: 5’-ATGGCGGATGATTTTATGACAGG-3’

IbbHLH112-R: 5’-CTACCTGAAGGATGCCCG-3’

The results showed that the nucleotide sequence of the PCR amplification product was shown in SEQ ID NO.1 from position 1 to 1248 from the 5' end. The gene shown in the sequence was named IbbHLH112 gene, the protein encoded by it was named IbbHLH112 protein or protein IbbHLH112, and the amino acid sequence was shown in SEQ ID NO.2.

Example 2: Obtaining IbbHLH112 transgenic sweet potato root tubers

1. Construction of recombinant plasmid pCAMBIA1300- IbbHLH112

1. Artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO.1 from position 1 to 1248 from the 5' end. Use the double-stranded DNA molecule as a template and p- IbbHLH112 -F ( Kpn I) and p- IbbHLH112 -R ( Xba I) as primers for PCR amplification to obtain a double-stranded DNA molecule containing restriction endonuclease Kpn I at the N-terminus and restriction endonuclease Xba I at the C-terminus.

p- IbbHLH112 -F ( Kpn I): 5′-ACGGGGGACGAGCTC GGTACC ATGGCGGATGATTTTATGACAGG-3′ (underlined is the recognition sequence of restriction endonuclease Kpn I);

p- IbbHLH112 -R ( Xba I): 5′-AAGATCTTCGTCGAC TCTAGA CTACCTGAAGGATGCCCCG-3′ (the recognition sequence of restriction endonuclease Xba I is underlined).

2. Double-digest the vector pCAMBIA1300-GFP with restriction endonucleases Kpn I and Xba I to recover the vector backbone 1 of about 10442 bp.

3. The double-stranded DNA molecule containing restriction endonuclease Kpn I at the N-terminus and restriction endonuclease Xba I at the C-terminus was double-digested with restriction endonucleases Kpn I and Xba I to recover fragment 2 containing about 1290 bp.

4. Connect fragment 2 to vector backbone 1 to obtain recombinant plasmid pCAMBIA1300- IbbHLH112 .

According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300- IbbHLH112 was described as follows: the small fragment between the restriction endonuclease Kpn I and Xba I recognition sequences of the recombinant plasmid pCAMBIA1300-GFP was replaced with the DNA molecule shown in positions 1 to 1248 from the 5' end of SEQ ID NO.1, and the recombinant plasmid pCAMBIA1300- IbbHLH112 can express the IbbHLH112 protein with an amino acid sequence of SEQ ID NO.2.

2. Obtaining transgenic sweet potato plants overexpressing IbbHLH112

1. Select fresh and healthy chestnut-scented stem segments of the sweet potato variety that are growing vigorously in the field. Each stem segment is about 20-25 cm long. Use a needle to poke 3-5 holes at the stem nodes and set aside.

2. The recombinant plasmid pCAMBIA1300- IbbHLH112 was transformed into Agrobacterium rhizogenes K599 and named K599/pCAMBIA1300 -IbbHLH112 .

3. Before infection, add the monoclonal clone of K599/pCAMBIA1300- IbbHLH112 into 40 mL of LB liquid culture medium containing 100 mg/L Kan antibiotic, culture at 28°C, shaker speed 200 rpm, in the dark for 12-18 hours, and the OD600 should be around 0.8.

4. Pour the bacterial solution with an OD600 of 0.8 into a 50 mL centrifuge tube, centrifuge at 5000 rpm for 8 min, collect the bacteria and discard the supernatant; then add an appropriate amount of sterile water to resuspend the bacteria, repeat 2-3 times to make the OD600 of the bacterial solution around 0.8.

5. Soak the stem segments treated in step 1 in the bacterial solution in step 4 and let them stand at room temperature for about 8 hours.

6. Plant the infected sweet potato stem segments in an isolated field, with at least 10 plants planted for each transformation event. Harvest the tubers after growing in the field for 3 months for observation and identification.

A wild-type control group was set up, and the wild-type sweet potato variety chestnut-scented stem segments were planted in an isolated field, and at least 10 plants were planted. After growing in the field for 3 months, the wild-type sweet potato tubers could be harvested for observation and identification.

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

1) The PCR detection method is as follows:

DNA was extracted from wild-type sweet potatoes and sweet potatoes to be transfected with IbbHLH112 gene, and PCR identification was performed. pCAMBIA1300- IbbHLH112 plasmid was used as a positive control, water and wild-type WT were used as negative controls, and the primers were as follows:

35S-F: 5’-TCCTTCGCAAGACCCTTCCTC-3’

IbbHLH112- R: 5'-CTACCTGAAGGATGCCCCGAATGTTGGGTCCAGAAA-3'

The amplified PCR products were identified by electrophoresis in 0.8% agarose gel. The results are shown in Figure 1. Only the positive control and the tuberous roots of sweet potatoes OE-2, OE-3 and OE-5 transgenic with the IbbHLH112 gene showed the target band, while no band appeared in the tuberous roots of wild-type sweet potatoes. The results showed that OE-2, OE-3 and OE-5 were transgenic sweet potato plants.

2) qRT-PCR

RNA was extracted from transgenic sweet potato roots, reverse transcribed to obtain cDNA, and then subjected to qRT-PCR analysis.

The primer sequences used are:

qRT- IbbHLH112 -F: 5'-ACTGATGGGTTTGAATTAGAAAATCT-3';

qRT- IbbHLH112 -R: 5'-GATTTGCTTGAACGAAGAATCTGA-3'.

The results are shown in FIG2 , and the relative expression level of the IbbHLH112 gene was significantly increased in transgenic sweet potato tubers.

OE-2, OE-3 and OE-5 transgenic sweet potatoes were asexually propagated for subsequent phenotypic observation and identification.

3. Observation of root phenotype of transgenic sweet potato overexpressing IbbHLH112

Five strains of each of OE-2, OE-3, OE-5 and wild-type Lizixiang were planted in isolated fields according to the conventional sweet potato planting method. After harvest, the tuber size, morphology, cracks and cracks were observed, photographed and counted. If multiple obvious cracks, cracks or depressions appeared in the same tuber, it was considered to have cracked skin. The experiment was repeated three times.

The results are shown in Figure 3. The wild-type chestnut-scented sweet potato tubers had no cracks, while the IbbHLH112- overexpressing chestnut-scented transgenic tubers OE-2, OE-3, and OE-5 all had cracks to varying degrees. The results showed that overexpression of IbbHLH112 promoted cracks in the sweet potato root tubers and affected the development of the roots.

4. Determination of lignin content in root tubers of transgenic sweet potato overexpressing IbbHLH112

The lignin content of the root tubers of WT (wild-type chestnut fragrance) and IbbHLH112 overexpressing transgenic sweet potato OE-2, OE-3 and OE-5 harvested in step three was determined using a lignin content determination kit (Suzhou Keming Biological Products, catalog number MGS-2-G) and referring to the product instructions.

The experiment was repeated three times and the results were averaged.

The measurement results are shown in Figure 4. The lignin content of the tubers of OE-2, OE-3 and OE-5 is higher than that of the wild-type tubers with chestnut fragrance, indicating that the overexpression of IbbHLH112 can significantly increase the lignin content of the transgenic sweet potato tubers.

The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departing from the disclosed scope in the present application and changes made with conventional techniques known in the art.

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.