Drought resistance related protein IbbHLH118 and its encoding gene and application

Abstract

The present invention discloses a drought-resistant related protein IbbHLH118 and its encoding gene and application, belonging to the technical field of genetic engineering breeding. The present invention discloses the application of IbbHLH118 protein or a substance regulating the expression of the gene encoding the IbbHLH118 protein or a substance regulating the activity or content of the IbbHLH118 protein in regulating plant stress resistance; the amino acid sequence of the IbbHLH118 protein is shown in SEQ ID No.1. The present invention discovered the IbbHLH118 protein and its encoding gene and applied them to regulating the drought resistance of sweet potatoes, providing ideas for the study of the drought resistance mechanism of sweet potatoes and the preparation of sweet potatoes with improved drought resistance, which is of great significance for the development of drought-resistant sweet potatoes.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering breeding, and specifically relates to a drought resistance-related protein IbbHLH118 and a coding gene and application thereof. Background Art

[0002] Sweetpotato (Ipomoea batatas) is an economically important root and tuber crop that is widely used worldwide as an industrial and bioenergy source. This crop is mainly grown on marginal lands. Extreme or prolonged drought conditions can cause significant decreases in sweetpotato yield, prompting the need to improve the drought tolerance of this crop. Genetic engineering is an effective approach to improve drought tolerance in sweetpotato. However, the transcriptional regulatory mechanisms underlying the sweetpotato response to drought stress remain largely unknown.

[0003] bHLH transcription factors play an important role in the regulation of various plant growth and development processes and stress resistance networks. Studies have found that some bHLH transcription factors can regulate plant stress resistance. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to effectively improve the drought resistance of plants.

[0005] In order to solve the above technical problems, in a first aspect, the present invention provides an application, which is any one of the following:

[0006] A1), use of IbbHLH118 protein or a substance regulating the expression of a gene encoding the IbbHLH118 protein or a substance regulating the activity or content of the IbbHLH118 protein in regulating plant stress resistance;

[0007] A2), use of IbbHLH118 protein or a substance that regulates the expression of the gene encoding the IbbHLH118 protein or a substance that regulates the activity or content of the IbbHLH118 protein in the preparation of a product for regulating plant stress resistance;

[0008] A3), use of IbbHLH118 protein or a substance regulating the expression of a gene encoding the IbbHLH118 protein or a substance regulating the activity or content of the IbbHLH118 protein in plant breeding;

[0009] The IbbHLH118 protein is as follows P1) or P2) or P3):

[0010] P1), a protein having an amino acid sequence of SEQ ID No. 1;

[0011] P2), a protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in P1), which has more than 80% identity with the protein shown in P1) and has the function of regulating plant leaf and / or root development and / or plant yield;

[0012] P3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of P1) or P2).

[0013] Furthermore, in the above application, the IbbHLH118 protein is derived from sweet potato.

[0014] The purpose of plant breeding can be to cultivate plants with altered stress resistance, for example, to cultivate plants with enhanced drought resistance or reduced drought resistance.

[0015] Among them, SEQ ID NO.1 consists of 298 amino acid residues.

[0016] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0017] In the above proteins, a protein tag refers to a polypeptide or protein fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0018] Furthermore, in the above application, the substance that regulates the expression of the gene encoding the IbbHLH118 protein or the substance that regulates the activity or content of the IbbHLH118 protein is a biological material, and the biological material is any one of the following:

[0019] B1), an RNA molecule that inhibits or reduces the expression of the gene encoding the IbbHLH118 protein or an RNA molecule that inhibits or reduces the activity or content of the IbbHLH118 protein;

[0020] B2), a DNA molecule expressing the RNA molecule described in B1);

[0021] B3), an expression cassette containing the DNA molecule described in B2);

[0022] B4), a recombinant vector containing the DNA molecule described in B2), or a recombinant vector containing the expression cassette described in B3);

[0023] B5), a recombinant microorganism containing the DNA molecule described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

[0024] B6), a transgenic plant cell line containing the DNA molecule described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4);

[0025] B7), transgenic plant tissue containing the DNA molecule described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4);

[0026] B8), a transgenic plant organ containing the DNA molecule described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4);

[0027] B9), a nucleic acid molecule encoding the IbbHLH118 protein;

[0028] B10), expression cassettes, recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs containing the nucleic acid molecule described in B9).

[0029] Furthermore, in the above application, the RNA molecule in B1) is an RNA transcribed from a DNA molecule as shown in formula (I):

[0030] SEQ forward-X-SEQ reverse (I);

[0031] The SEQ forward sequence is the 27th to 298th position of the sequence 2 in the sequence list; the SEQ reverse sequence is the reverse complement of the SEQ forward sequence; the X is the spacer sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse.

[0032] Furthermore, in the above application, the sequence of one strand of the double-stranded RNA molecule may be a sequence obtained by transcribing the DNA fragment at positions 27-298 of sequence 2 in the sequence list;

[0033] Furthermore, in the above application,

[0034] B2) The DNA molecule is as shown in formula (I):

[0035] SEQ forward-X-SEQ reverse (I);

[0036] The SEQ forward sequence is the 27th to 298th position of the sequence 2 in the sequence list; the SEQ reverse sequence is the reverse complement of the SEQ forward sequence; the X is the spacer sequence between the SEQ forward sequence and the SEQ reverse sequence, and the X is not complementary to the SEQ forward sequence and the SEQ reverse sequence;

[0037] B9) The nucleic acid molecule is a DNA molecule as shown in any one of b1) to b3) below:

[0038] b1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;

[0039] b2) A DNA molecule that has more than 80% identity with the DNA molecule shown in b1) and encodes the IbbHLH118 protein.

[0040] Herein, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can then be obtained.

[0041] Herein, the 80% or greater 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.

[0042] Primer pairs for amplifying the full length or any fragment of the coding gene of the sweet potato root tuber and leaf development-related protein IbbHLH118 also fall within the protection scope of the present invention.

[0043] In the above, the expression cassette refers to a DNA molecule capable of expressing protein IbbHLH118 in a host cell, which may include not only a promoter for initiating transcription of the IbbHLH118 gene, but also a terminator for terminating transcription of the IbbHLH118 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; 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 of which can be induced 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 1 0099169.7)), 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, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, for example, Odell et al. (1985) Nature 55:3047-3053). 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).

[0044] In the above, the recombinant vector may contain the DNA molecule encoding protein IbbHLH118 as shown in SEQ ID NO.2.

[0045] A recombinant vector containing the IbbHLH118 encoding gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300-GFP, pCAMBIAsuper 1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using IbbHLH118 to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; In addition, when using the gene of the present invention to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation start region can come from a transcription start region or a structural gene.

[0046] 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.

[0047] In the present invention, the recombinant microorganism in B5) or B10) may specifically be yeast, bacteria, algae or fungi. The bacteria may be gram-positive bacteria or gram-negative bacteria. The gram-negative bacteria may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens may specifically be Agrobacterium tumefaciens GV3101.

[0048] In the present invention, the plant tissue described in B7) or B10) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0049] In the present invention, the transgenic plant organ described in B8) or B10) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0050] In the present invention, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ may or may not include propagation materials.

[0051] In the present invention, the inhibition, reduction or down-regulation of the expression of the gene encoding the IbbHLH118 protein can be achieved by gene knockout or gene silencing.

[0052] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0053] The gene silencing refers to the phenomenon of making the gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0054] In one embodiment of the present invention, the recombinant vector may specifically be a recombinant plasmid pCAMBIA 1300-IbbHLH118-GFP and pCAMBIA1300-SI-X-IbbHLH118. The structure of the recombinant plasmid pCAMBIA1300-IbbHLH118-GFP is to replace the small fragment between the restriction endonuclease Xba I and Pst I recognition sequences of the plasmid pCAMBIA 1300-GFP with the DNA molecule shown in SEQ ID NO.2, and keep the other nucleotide sequences of the plasmid pCAMBIA 1300-GFP unchanged, thereby obtaining the recombinant expression vector pCAMBIA1300-IbbHLH118-GFP. The recombinant expression vector pCAMBIA 1300-IbbHLH118-GFP expresses the IbbHLH118 protein having an amino acid sequence as shown in SEQ ID NO.1. The recombinant plasmid pCAMBIA1300-SI-X-IbbHLH118 contains a DNA molecule as shown in formula (I): SEQ forward-X-SEQ reverse (I); the sequence of the SEQ forward is the 27th to 298th position of sequence 2 in the sequence list; the sequence of the SEQ reverse is the reverse complement of the sequence of the SEQ forward; the X is the spacer sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse;

[0055] Furthermore, in the above application, the plant is any one of the following:

[0056] 1) Dicotyledonous plants;

[0057] 2) Tubular flowers;

[0058] 3) Convolvulaceae;

[0059] 4) Ipomoea batatas;

[0060] 5) Sweet potato (Ipomoea batatas).

[0061] In order to solve the above technical problems, in a second aspect, the present invention provides the protein in the above application or the biomaterial in the above application.

[0062] To solve the above technical problems, in a third aspect, the present invention provides a method for regulating plant drought resistance, the method comprising regulating the plant drought resistance by regulating the expression of the IbbHLH118 protein encoding gene or regulating the activity or content of the IbbHLH118 protein.

[0063] Furthermore, the above method includes M1) and / or M2):

[0064] M1), comprising introducing the DNA molecule represented by formula (I) described in the above application into a recipient plant, inhibiting or reducing or down-regulating the expression of the IbbHLH118 protein encoding gene or the activity or content of the IbbHLH118 protein, to obtain a target plant with higher drought resistance than the recipient plant;

[0065] M2), introducing the nucleic acid molecule described in B9) in the above application into the recipient plant to promote or increase the expression of the IbbHLH118 protein encoding gene or the activity or content of the IbbHLH118 protein, thereby obtaining a target plant with lower drought resistance than the recipient plant.

[0066] To solve the above technical problems, in a fourth aspect, the present invention provides a method for preparing drought-resistant plants, the method comprising introducing the DNA molecule represented by formula (I) in the above application into a recipient plant, inhibiting or reducing or down-regulating the expression of the IbbHLH118 protein encoding gene or the activity or content of the IbbHLH118 protein, and obtaining a target plant with higher drought resistance than the recipient plant.

[0067] In the above method, the plant is any one of the following:

[0068] 1) Dicotyledonous plants;

[0069] 2) Tubular flowers;

[0070] 3) Convolvulaceae;

[0071] 4) Ipomoea batatas;

[0072] 5) Sweet potato (Ipomoea batatas).

[0073] In the above method, the high drought resistance is mainly reflected under drought stress conditions. Compared with the wild type, the biomass of the RNA interference plants under drought stress conditions increases; the proline content, superoxide dismutase (SOD) activity, and stress-related hormone content (abscisic acid content) of the plants increase, and the malondialdehyde content and hydrogen peroxide content decrease.

[0074] In the above, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi; for example, the bacteria may be Agrobacterium GV3101 strain.

[0075] In the above, the transgenic plant organ can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0076] In the above, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0077] In the above method, the introduction of the DNA molecule represented by formula (I) in the above application into the recipient plant is achieved by introducing a recombinant plasmid containing the DNA molecule represented by formula (I) into the recipient plant. In one embodiment of the present invention, the recombinant plasmid containing the DNA molecule represented by formula (I) is pCAMBIA1300-SI-X-IbbHLH118.

[0078] In the above method, the method for expressing or overexpressing protein IbbHLH118 is to introduce the gene encoding protein IbbHLH118 into the target plant.

[0079] In the above method, the gene encoding protein IbbHLH118 can be introduced into the target plant through a plant expression vector carrying the IbbHLH118 gene of the present invention. The plant expression vector carrying the gene IbbHLH118 of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant cells or tissues are cultivated into plants.

[0080] In the above method, the nucleotide sequence of the gene encoding the protein IbbHLH118 is the DNA molecule shown in SEQ ID NO.2.

[0081] In a specific embodiment of the present invention, the plant expression vector carrying the gene IbbHLH118 of the present invention can be pCAMBIA 1300-IbbHLH118-GFP. The reduction in drought resistance is mainly reflected in that under drought stress conditions, compared with the wild type, the biomass of plants overexpressing the IbbHLH118 protein is reduced, the proline content, SOD activity, POD enzyme activity and stress-related hormone ABA content of the plants are reduced, and the reactive oxygen content is increased; the improvement in drought resistance is mainly reflected in that under drought stress conditions, compared with the wild type, the biomass of plants interfering with IbbHLH118 by RNAi is increased, the proline content, SOD activity, POD enzyme activity and stress-related hormone ABA content of the plants are increased, and the reactive oxygen content is reduced.

[0082] The present invention discovered the IbbHLH118 protein and its encoding gene, and introduced the encoding gene of the IbbHLH118 protein into sweet potato to obtain transgenic sweet potato plants that overexpress and RNAi silence IbbHLH118. The transgenic plants were subjected to drought stress treatment. Compared with the control, the drought resistance of the overexpressed / RNAi silenced transgenic strains was weakened / enhanced, which was specifically reflected in the reduction / increase of proline content, POD activity, SOD activity, stress-related hormone ABA content, and the increase / reduction of active oxygen content. Therefore, the IbbHLH118 gene provided by the present invention and the protein encoded by it play an important role in the process of plant drought resistance. Through RNAi or Crispr / Cas9 technology, it has important application value in improving plant drought resistance, and has broad application space and market prospects in the field of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 : is the PCR amplification result of transgenic sweet potato plants; wherein, M is DNA molecule Marker, W is negative control water, P is positive control (pCAMBIA1300-IbbHLH118-GFP and pCAMBIA1300-35SI-X-IbbHLH118), WT is genomic DNA of wild-type sweet potato plants, OEX1-OEX15 are overexpression lines, and RiX1-RiX5 are RNAi-silenced IbbHLH118 sweet potato positive plants.

[0084] Figure 2 The expression of IbbHLH118 gene in transgenic sweet potato positive plants and wild-type sweet potato plants; among them, WT is the cDNA of wild-type sweet potato, OEX1-OEX15 are overexpression lines, and RiX1-RiX5 are RNAi-silenced IbbHLH118 sweet potato positive plants.

[0085] Figure 3 The present invention is to identify the drought resistance of IbbHLH118 transgenic sweet potato plants and wild-type sweet potato plants in vitro; among them, WT is the wild-type sweet potato, OE-X4, OE-X6 and OE-X9 are overexpression lines, and RiX2, RiX3 and RiX5 are RNAi-silenced IbbHLH118 sweet potato lines.

[0086] Figure 4 To identify the drought resistance of transgenic sweet potato plants overexpressing IbbHLH118 and wild-type sweet potato plants in hydroponics; among them, WT is the wild type of sweet potato, and OE-X4, OE-X6 and OE-X9 are sweet potato lines overexpressing IbbHLH118.

[0087] Figure 5To identify the drought resistance of transgenic sweet potato plants overexpressing IbbHLH118 and wild-type sweet potato plants in potted plants; among them, WT is the wild type of sweet potato, and OE-X4, OE-X6 and OE-X9 are sweet potato lines overexpressing IbbHLH118.

[0088] Figure 6 This is a potted identification of drought resistance of RNAi-silenced IbbHLH118 transgenic sweet potato plants and wild-type sweet potato plants; among them, WT is the wild type of sweet potato, and Ri-X2, Ri-X3 and Ri-X4 are RNAi-silenced IbbHLH118 sweet potato lines.

[0089] Figure 7 To identify the drought resistance of transgenic sweet potato plants overexpressing IbbHLH118 and wild-type sweet potato plants; among them, WT is the wild type of sweet potato, and OE-X4, OE-X6 and OE-X9 are transgenic sweet potato lines overexpressing IbbHLH118.

[0090] Figure 8 The hormone and physiological and biochemical indexes of IbbHLH118 transgenic sweet potato plants and wild-type sweet potato plants were measured. A: ABA content measurement; B and C: ABA sensitivity measurement; DH: reactive oxygen accumulation measurement; I and J: POD and SOD activity measurement; K: proline measurement. WT is wild-type sweet potato, OE-X4, OE-X6 and OE-X9 are overexpression plants, and Ri-X2, Ri-X3 and Ri-X4 are RNAi silenced IbbHLH118 sweet potato lines. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0092] 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.

[0093] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0094] The sweet potato line 'Xushu 55-2' is preserved in our laboratory and is recorded in the following document: "Zhu Hong. Analysis of sweet potato drought transcriptome and cloning and functional identification of drought resistance-related genes IbWRKY2, IbGATA24 and IbSDT, PhD dissertation, China Agricultural University, 2018." The public can obtain it from the Sweet Potato Genetics and Breeding Laboratory of China Agricultural University to repeat this experiment, but it cannot be used for other purposes.

[0095] The sweet potato variety “Lizixiang” was preserved for this experiment and is recorded in the following document: “Zhou Yuanyuan. Cloning and functional identification of drought resistance-related genes IbAITR5, IbEGF, IbMYB116 and IbBT4 in sweet potato, PhD dissertation, China Agricultural University, 2020”. The public can obtain it from the Key Laboratory of Sweet Potato Biology and Biotechnology of the Ministry of Agriculture and Rural Affairs, China Agricultural University, to repeat this experiment, but it cannot be used for other purposes.

[0096] The cloning vector pMD19-T is a product of Takara Biotechnology (Dalian) Co., Ltd., with the product catalog number being 6013.

[0097] The vector pCAMBIA1300-GFP was kindly donated by Professor Jin Weiwei from the College of Agronomy, China Agricultural University, and is recorded in the following literature: Luo HS, Meng DX, Liu HB, Xie MJ, Yin CF, Liu F, Dong ZB, Jin WW. Ectopic Expression of the Transcriptional Regulator silky3 Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences. Plant Cell. 2020, 32: 3750-3773; it is referred to as pCAMBIA1300 in the following examples. The public can obtain the above biological materials from the applicant to repeat this experiment, but they cannot be used for other purposes.

[0098] pCAMBIA1300-35SI-X was purchased from Wuhan Transduction Laboratory Co., Ltd. and was modified based on pCAMBIA1300. 35S promoter + Intro + NOS terminator were added to the MCS, and the forward and reverse sequences were inserted respectively to form a hairpin structure.

[0099] The plant total RNA extraction kit was the Transzol Plant Total RNA Extraction Kit (Catalog No.: ET111) from TransGen Biotech, Beijing.

[0100] PrimeScriptTM RT reagent Kit with gDNA Eraser Kit (Takara, PR047A) is a product of Takara Biotechnology (Dalian) Co., Ltd.

[0101] The following examples were processed using SPSS 19.0 statistical software, and the experimental results were expressed as mean ± standard deviation, and Student's t-test was used for the test. * indicates a significant difference (P < 0.05), ** indicates a very significant difference (P < 0.01), and *** indicates a very significant difference (P < 0.001). In the following examples, unless otherwise specified, three replicates were set for each treatment group.

[0102] Example 1. Acquisition of IbbHLH118 gene

[0103] 1. Obtaining cDNA template

[0104] Total RNA from the test tube seedlings of the sweet potato line Xushu 55-2 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.

[0105] 2. Using the cDNA obtained in step 1 as a template, Sweetpotato GARDEN(kazusa.or.jp) The sequences provided on the website were used to design primers IbbHLH118-F and IbbHLH118-R. PCR amplification was performed to obtain a PCR amplification product of about 897 bp and sequenced. The primer sequences are as follows:

[0106] IbbHLH118-F: 5′-ATGCGAATGGATAACTTTCAT-3′

[0107] IbbHLH118-R: 5′-TCAGTACAGTTGATTAATAGAATCACT-3′

[0108] The results showed that the PCR amplification product was a DNA molecule with a nucleotide sequence of SEQ ID NO.2. The gene shown in the sequence was named IbbHLH118 gene, the protein encoded by it was named IbbHLH118 protein or protein IbbHLH118, and the amino acid sequence was shown in SEQ ID NO.1.

[0109] Example 2: Application of IbbHLH118 protein in improving plant drought resistance.

[0110] 1. Construction of recombinant plasmid pCAMBIA 1300-IbbHLH118-GFP

[0111] 1. Artificially synthesize a double-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 2. Use the double-stranded DNA molecule as a template and OE-F-Xba I and OE-R-Pst I as primers to perform PCR amplification to obtain a double-stranded DNA molecule containing restriction endonuclease Xba I at the N-terminus and restriction endonuclease Pst I at the C-terminus.

[0112] OE-F-Xba I: 5'- GCTCTAGA ATGCGAATGGATAACTTTCAT-3' (underlined is the recognition sequence of restriction endonuclease Xba I);

[0113] OE-R-Pst I: 5'- AACTGCAG PCR amplification was performed using GTACAGTTGATTAATAGAATCACT-3' (the underline is the recognition sequence of restriction endonuclease Pst I) as a primer to obtain a double-stranded DNA molecule containing restriction endonuclease XbaI at the N-terminus and restriction endonuclease Pst I at the C-terminus.

[0114] 2. Double-digest the vector pCAMBIA 1300-GFP with restriction endonucleases Xba I and Pst I to recover the vector backbone 1 of about 10783 bp.

[0115] 3. The double-stranded DNA molecule containing restriction endonuclease Xba I at the N-terminus and restriction endonuclease Pst I at the C-terminus was double-digested with restriction endonucleases Xba I and Pst I to recover fragment 2 containing about 910 bp.

[0116] 4. Connect fragment 2 to vector backbone 1 to obtain the recombinant plasmid pCAMBIA 1300-IbbHLH118-GFP.

[0117] According to the sequencing results, the structure of the recombinant plasmid pCAMBIA 1300-IbbHLH118-GFP was described as follows: the small fragment between the restriction endonuclease Xba I and Pst I recognition sequences of the plasmid pCAMBIA1300-GFP was replaced with the DNA molecule shown in SEQ ID NO.2, and the other nucleotide sequences of the plasmid pCAMBIA1300-GFP were kept unchanged to obtain the recombinant expression vector pCAMBIA1300-IbbHLH118-GFP. The recombinant expression vector pCAMBIA 1300-IbbHLH118-GFP expressed the IbbHLH118 protein with an amino acid sequence as shown in SEQ ID NO.1.

[0118] 2. Construction of recombinant plasmid pCAMBIA1300-35SI-X-IbbHLH118

[0119] 1. Artificially synthesize a double-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 2. Using the double-stranded DNA molecule as a template, select the 27-298 bp sequence of the gene as the interference sequence, use RNAi-F1-BamH I and RNAi-R1-Sal I as primers to perform PCR amplification to obtain the positive strand, and use RNAi-F2-Kpn I and RNAi-R2-Sac I as primers to perform PCR amplification to obtain the antisense strand.

[0120] RNAi-F1-BamHI: 5'- GGATCC AAATCCATTCTTTCCCTCGC-3' (the underline is the recognition sequence of restriction endonuclease BamH I);

[0121] RNAi-R1-Sal I: 5'- GTCGAC TCTGCTGCTTCTTCTTTCCCT-3' (the underline is the recognition sequence of restriction endonuclease Sal I)

[0122] RNAi-F2-Kpn I: 5'- GGTACC TCTGCTGCTTCTTCTTTCCCT-3′ (the underline is the recognition sequence of restriction endonuclease Kpn I);

[0123] RNAi-R2-Sac I: 5'- GAGCTC AAATCCATTCTTTCCCTCGC-3' (the underline is the recognition sequence of restriction endonuclease Sac I)

[0124] 2. Clone the above positive chain sequence into the multiple cloning site 1 (5'-BamHI-SalI-3') of the vector pCAMBIA1300-35SI-X in the 5'-3' direction to form pCAMBIA1300-35SI-X-IbbHLH118;

[0125] 3. After successful sequencing verification, the above successfully constructed clone was used as the target vector, and the above antisense chain sequence was cloned again into the multi-cloning site 2 (5'-KpnI-SacI-3') in the 5'-3' direction. Sequencing verified that the second insertion was successful, and the construction was completed.

[0126] The sequencing results showed that the recombinant plasmid pCAMBIA1300-35SI-X-IbbHLH118 contained the DNA molecule represented by formula (I):

[0127] SEQ forward-X-SEQ reverse (I);

[0128] The SEQ forward sequence is the 27th to 298th position of the sequence 2 in the sequence list; the SEQ reverse sequence is the reverse complement of the SEQ forward sequence; the X is the spacer sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse.

[0129] 3. Obtaining transgenic sweet potato plants

[0130] 1. Induction of embryonic callus and establishment of embryonic cell suspension line of sweet potato variety Lizixiang

[0131] The harvested chestnut-scented sweet potato tubers were used to provide sweet potato stem tips. The stem tip meristems were peeled and inoculated on MS solid medium containing 2.0 mg / L 2,4-D. Callus tissue was induced in the dark at room temperature of 27±1°C. Then, the callus was expanded and subcultured to establish an embryonic cell suspension line for transformation.

[0132] 2. Cultivation of Agrobacterium

[0133] The recombinant plasmid pCAMBIA-1300-IbbHLH118-GFP and the recombinant plasmid pCAMBIA1300-35SI-X-IbbHLH118 were used to transform Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which were named EHA105 / pCAMBIA 1300-IbbHLH66-GFP and EHA105 / pCAMBIA1300-35SI-X-IbbHLH118, respectively.

[0134] Activate Agrobacterium culture on the resistance plate, pick a single colony and inoculate it into 5 mL of LB liquid medium with the corresponding antibiotics, and culture it at 28°C with shaking at 200 rpm until the OD 600 The value is in the range of 0.4 to 0.6.

[0135] 3. Preparation of suspension cell lines and infection with Agrobacterium tumefaciens

[0136] Suspension cell lines that have grown for 8 to 12 weeks and are in good condition are selected for grinding. After subculture for 3 days, embryonic suspension cell clusters with a diameter of about 0.7-1.4 mm are taken out for Agrobacterium infection and transformation.

[0137] 4. Co-culture and delayed culture

[0138] The suspension cell lines infected with Agrobacterium EHA105 / pCAMBIA1300-IbbHLH66-GFP and EHA105 / pCAMBIA1300-35SI-X-IbbHLH118 were transferred to MS solid medium containing 30 mg / L acetosyringone (AS) and 2 mg / L 2,4-D for co-culture in a dark environment at 27±1°C. After 3 days of co-culture, the cell mass was washed once with MS liquid medium containing 200 mg / L cephalosporin (CS) and 2 mg / L 2,4-D, and then soaked in MS liquid medium containing 100 mg / L CS and 2 mg / L 2,4-D for 30 minutes, and finally delayed cultured with MS liquid medium containing 2 mg / L 2,4-D for 1 week. The culture conditions were 27±1°C, 500 Lux light (13 h light per day), and shaking at 100 rpm.

[0139] 5. Screening and culture of resistant cell clusters

[0140] After delayed culture, the cell mass was transferred to MS solid medium containing 5.0 mg / L hygromycin (Hyg), 100 mg / L CS and 2 mg / L 2,4-D for dark culture at 27±1°C, and the medium was replaced every 2 weeks. After 4 weeks, the resistant cell mass was transferred to MS solid medium containing 10.0 mg / L Hyg, 100 mg / L CS and 2 mg / L 2,4-D and cultured for 4 to 8 weeks, with the medium replaced every 2 weeks.

[0141] 6. Induction of somatic embryos

[0142] The resistant cell clusters with good growth status were transferred to MS medium containing 1.0 mg / L ABA and 100 mg / L CS to induce somatic embryo growth. The culture conditions were 27±1°C and 3000 Lux light (13 h light per day).

[0143] 7. Regeneration and identification of transgenic plants

[0144] The mature somatic embryos that turned green on the MS medium containing 1 mg / LABA after 2 to 4 weeks of induction were transferred together with the callus tissue to the MS solid medium and cultured until the complete plant was formed at a temperature of 27±1°C, 13 hours of light per day, and a light intensity of 3000 Lux to obtain transgenic sweet potato plants.

[0145] 8. Identification of transgenic plants:

[0146] A combination of PCR and qRT-PCR was used.

[0147] 1) The PCR detection method is as follows:

[0148] DNA of wild-type Li Zixiang and transgenic plants was extracted for PCR identification. pCAMBIA 1300-IbbHLH118 and pCAMBIA1300-35SI-X-IbbHLH118 vector plasmids were used as positive controls, water and wild-type Li Zixiang were used as negative controls, and the primers were as follows:

[0149] pCAMBIA 1300-F: 5'-GACGCACAATCCCACTATCC-3'

[0150] pCAMBIA 1300-R: 5'-TCAGTACAGTTGATTAATAGAATCACT-3'

[0151] pCAMBIA 1300-35SI-F: 5'-CGTAAGGGATGACGCACAA-3'

[0152] pCAMBIA 1300-35SI-R: 5'-GATAATCATCGCAAGACCGG-3'

[0153] The amplified PCR products were separated by electrophoresis in 1% (w / v) agarose gel. The PCR-positive plants should have a specific electrophoresis band of 998 bp and 471 bp. The strain number of the PCR-positive plants was recorded.

[0154] The results are as follows Figure 1 As shown in (A) and (B), the results showed that only the positive control and the transgenic sweet potato plants OEX1-OEX15 showed electrophoresis bands near 897 bp, and RiX1-RiX5 showed electrophoresis bands near 471 bp. The wild-type Li Zixiang and the negative control water did not show any bands. It was preliminarily determined that the present invention obtained the transgenic sweet potato positive plants OEX1-OEX15 and RiX1-RiX5.

[0155] 2) qRT-PCR

[0156] RNA was extracted from positive sweet potato plants and reverse transcribed to obtain cDNA for qRT-PCR, with the wild-type Lizixiang as the control CK.

[0157] Ibactin gene as internal reference:

[0158] IbActin-F: 5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

[0159] IbActin-R: 5′-TGGAAAATTAGAAGCACTTCCTGTGAAC-3′

[0160] The primer sequence of IbBHLH118 is:

[0161] IbBHLH118-qRT-F: 5′-AGACATGCTTGGATGGGGTT-3′

[0162] IbBHLH118-qRT-R: 5′-TGGCCTTGTTGCACCAGTAG-3′

[0163] The results are as follows Figure 2 As shown, the results showed that IbbHLH118 was significantly differentially expressed in transgenic sweet potato plants. Three transgenic sweet potato lines overexpressing IbbHLH118 with high up-regulated expression, OE-X4, OE-X6, and OE-X9, and three transgenic sweet potato lines silencing IbbHLH118 with RNAi with the lowest down-regulated expression, Ri-X2, Ri-X3, and Ri-X5, were selected for propagation to obtain the T1 generation transgenic sweet potato lines overexpressing IbbHLH118, OE-X4, OE-X6, OE-X9, Ri-X2, Ri-X3, and Ri-X5, for subsequent stress resistance tests.

[0164] IV. Stress resistance evaluation of transgenic sweet potato plants overexpressing IbBHLH118

[0165] WT is the wild type Li Zixiang, and the transgenic sweet potato is the plants of the T1 generation transgenic sweet potato lines OE-X4, OE-X6, OE-X9, Ri-X2, Ri-X3, and Ri-X5 overexpressing IbbHLH118.

[0166] 1. In vitro identification of drought resistance of IbbHLH118 transgenic sweet potato plants

[0167] Specific steps: The IbbHLH118 T1 generation transgenic sweet potato lines OE-X4, OE-X6, OE-X9, Ri-X2, Ri-X3, and Ri-X5 and the wild type chestnut fragrance (CK) were cultured on MS solid culture medium containing 30% PEG6000, respectively. The culture temperature was 27±1°C, with 13 hours of light per day and a light intensity of 3000Lux. After 4 weeks, the root length of each line was counted and its drought resistance was preliminarily identified. Three replicates were set for each line and the results were averaged.

[0168] a Control group, i.e., no stress treatment: the plants were grown under normal culture conditions on MS solid medium for 4 weeks, and the growth of the plants was observed;

[0169] b Drought group, ie drought treatment (30% PEG6000 treatment): MS solid medium containing 30% PEG6000 was used, and plant growth was observed after drought treatment for 4 weeks.

[0170] The results are as follows Figure 3 As shown in (A) and (B), after 4 weeks of culture on normal MS medium, there was no significant difference in root length between the IbBHLH118 T1 transgenic sweet potato lines OE-X4, OE-X6, OE-X9, Ri-X2, Ri-X3, and Ri-X5 and the wild-type Li Zixiang. After 4 weeks of stress culture on MS medium containing 30% PEG6000, the overexpression transgenic sweet potato lines had poor growth and difficulty in rooting; the growth state, root length and fresh weight of the RNAi silenced transgenic sweet potato lines were better than those of the wild-type Li Zixiang and the overexpression IbBHLH118 T1 transgenic sweet potato lines.

[0171] The in vitro identification results preliminarily indicated that overexpression of the IbBHLH118 gene reduced the drought resistance of sweet potato plants.

[0172] 2. Hydroponic evaluation of drought resistance of transgenic sweet potato plants overexpressing IbBHLH118

[0173] The T1 transgenic sweet potato lines OE-X4, OE-X6 and OE-X9 overexpressing IbBHLH118 and the wild-type Li Zixiang were acclimated and transplanted to the isolated field. After growing for 2 months, 20 cm stem segments were cut for hydroponic identification. Three replicates were set for each line.

[0174] a Control group, i.e., no stress treatment: the plants were grown under normal culture conditions of Hoagland solution for 2 weeks, and the growth of the plants was observed;

[0175] b Drought group, i.e. drought treatment (15% PEG6000 treatment): using Hoagland solution containing 15% PEG6000, after drought treatment for 1 week, the plants were treated with Hoagland solution for 1 week, and the growth of the plants was observed.

[0176] After the treatment, the rooting status and biomass of each plant were counted.

[0177] The results are as follows Figure 4 As shown in (A), (B) and (C), in the control group, under the Hoagland solution culture conditions, the T1 transgenic sweet potato lines overexpressing IbbHLH118, OE-X4, OE-X6, OE-X9 and the wild-type Li Zixiang grew well, with no significant difference. After the drought group was treated with Hoagland solution containing 15% PEG6000 for 1 week, the transgenic sweet potato lines withered and died, while the wild-type Li Zixiang had a higher survival rate, and some leaves remained green ( Figure 4 The fresh weight (FW) and dry weight (DW) of wild-type Li Zi Xiang were significantly better than those of T1 transgenic sweet potato lines overexpressing IbbHLH118 ( Figure 4 (A) and (B)).

[0178] 3. Potted identification of drought resistance of transgenic sweet potato plants overexpressing IbbHLH118

[0179] The stem segments of the T1 transgenic sweet potato lines OE-X4, OE-X6, and OE-X9 overexpressing IbbHLH118 and the wild-type Li Zixiang were planted in transplanting boxes, with more than two stem nodes per stem segment, and the ratio of vermiculite to nutrient soil was 1:1. After transplantation, they were fully irrigated. Natural drought stress treatment began after the stem segments grew new leaves. After 4 weeks of treatment, the rooting and biomass of each line were counted.

[0180] a Control group, i.e., no stress treatment: the plants were grown under normal culture conditions of Hoagland solution for 4 weeks, and the growth of the plants was observed;

[0181] b Drought group, i.e. drought treatment (natural drought treatment): after stopping irrigation for 4 weeks, the growth of plants was observed.

[0182] The results are as follows Figure 5 As shown in (A), (B) and (C), there was no significant difference in dry weight and fresh weight between the wild-type Li Zixiang in the control group and the T1 transgenic sweet potato lines OE-X4, OE-X6 and OE-X9 overexpressing IbbHLH118; while the transgenic sweet potato lines in the drought group were severely withered, while the wild-type Li Zixiang plants still had more green leaves, and their dry weight (DW) and fresh weight (FW) were significantly lower than those of the wild-type Li Zixiang.

[0183] The experimental results showed that overexpression of the IbbHLH118 gene can significantly reduce sweet potato's resistance to drought stress.

[0184] 4. Potted evaluation of drought resistance of transgenic sweet potato plants with RNAi silencing IbbHLH118

[0185] The RNAi-silenced IbbHLH118 T1 transgenic sweet potato lines Ri-X2, Ri-X3, and Ri-X5 and the stem segments of the wild-type Li Zixiang were planted in a transplanting box, with more than two stem nodes per stem segment. The ratio of vermiculite to nutrient soil was 1:1, and the plants were fully irrigated after transplantation. Natural drought stress treatment began after the stem segments grew new leaves. After 6 weeks of treatment, the rooting and biomass of each line were counted.

[0186] a Control group, i.e., no stress treatment: the plants were grown under normal culture conditions of Hoagland solution for 6 weeks, and the growth of the plants was observed;

[0187] b Drought group, i.e. drought treatment (natural drought treatment): after stopping irrigation for 6 weeks, the growth of plants was observed.

[0188] The results are as follows Figure 6As shown in (A), (B) and (C), there was no significant difference in the dry weight and fresh weight of the wild-type Li Zixiang in the control group and the RNAi-silenced IbbHLH118 T1 transgenic sweet potato lines Ri-X2, Ri-X3, and Ri-X5; while the wild-type Li Zixiang sweet potato line in the drought group was severely withered, while the transgenic plants still had more green parts of their leaves, and their dry weight (DW) and fresh weight (FW) were significantly higher than those of the wild-type Li Zixiang.

[0189] The experimental results showed that RNAi silencing of the IbbHLH118 gene can significantly improve sweet potato's resistance to drought stress.

[0190] 5. Identification of drought pools in transgenic sweet potato plants overexpressing IbbHLH118

[0191] The stem segments of the T1 transgenic sweet potato lines OE-X4, OE-X6, and OE-X9 overexpressing IbbHLH118 and the wild-type Lizixiang were planted in drought ponds, with more than two stem nodes per segment and 20 replicates per line. Natural drought stress treatment began after the stem segments grew new leaves, and after 3 months of treatment, the rooting and biomass of each line were counted.

[0192] a Control group, i.e. no stress treatment: the relative soil moisture content was maintained at 65%-75% for 3 months, and the growth of the plants was observed;

[0193] b Drought group, i.e. drought treatment (natural drought treatment): after stopping irrigation for 3 months, the growth of plants was observed.

[0194] The results are as follows Figure 7 As shown in (A), (B) and (C), there was no significant difference in the dry weight and fresh weight of the wild-type Li Zixiang in the control group and the T1 transgenic sweet potato lines OE-X4, OE-X6, and OE-X9 overexpressing IbbHLH118; while the aboveground biomass (AW) and underground biomass (BW) of the transgenic sweet potato lines in the drought group were significantly lower than those of the wild-type Li Zixiang.

[0195] The experimental results showed that overexpression of the IbbHLH118 gene can significantly reduce sweet potato's resistance to drought stress.

[0196] 6. Determination of physiological and biochemical indicators

[0197] (1) Determination of ABA content

[0198] Abscisic acid (ABA) plays an important role in plant stress response. ABA can improve the salt tolerance of plants, alleviate the osmotic stress and ion stress caused by excessive salt, maintain water balance, induce the accumulation of proline, a plant osmotic regulator, maintain the stability of cell membrane structure, and increase the activity of protective enzymes. During drought stress, ABA can significantly reduce leaf water evaporation, reduce leaf cell membrane permeability, increase the soluble protein content of leaf cells, induce the formation of protective enzymes in the biomembrane system, reduce the degree of membrane lipid peroxidation, enhance antioxidant capacity, and improve the drought resistance of plants.

[0199] The determination method refers to the reference: Yang, J., Zhang, J., Wang, Z., Zhu, Q., Wang, W. (2001) Hormonal changes in the grains of rice subjected to water stress during grainfilling. Plant Physiol. 127: 315-323.

[0200] The sweet potato plants are the sweet potato plants treated with the control group in step 1 for 4 weeks and the sweet potato plants treated with drought in the drought group in step 1 for 4 weeks. The sweet potato plants are the plants of the T1 generation transgenic sweet potato lines of overexpression and RNAi silencing IbBHLH118 and the plants of the wild type Lizixiang (CK). The experiment needs to be repeated three times, and the results are averaged.

[0201] The results are as follows Figure 8 As shown in (A), the control was no stress, and the PEG treatment was to simulate natural drought stress. The results showed that the ABA content of the plants of the T1 generation transgenic sweet potato lines with RNAi silencing IbbHLH118 was significantly higher than that of the wild-type Li Zixiang, and the ABA content of the plants of the T1 generation transgenic sweet potato lines overexpressing IbbHLH118 was significantly lower than that of the wild-type Li Zixiang.

[0202] (2) ABA sensitivity assay

[0203] Three-month-old field-grown sweet potato leaves were soaked in a basic solution (50 mM KCl, 10 mM MES-KOH, and 10 mM CaCl2, pH 6.1) and illuminated for 3 h to open all the stomata. The leaves were then transferred to a 20 μM ABA treatment solution and illuminated for another 2 h. The lower epidermis of the leaves was torn off and the excess mesophyll cells were gently brushed off with a brush until about 2-3 mm 2 The epidermis of each material was sliced ​​and observed and photographed under a microscope. ImageJ software was used to measure and count the stomatal opening of each material. For each material control and treatment, 3 leaves were selected to tear off the lower epidermis for observation. 4-5 fields of view were selected for each epidermis, and about 80 stomata were measured.

[0204] The results are as follows Figure 8 As shown in (B) and (C), the results showed that the ABA sensitivity of the T1 transgenic sweet potato lines with RNAi silencing IbbHLH118 was significantly higher than that of the wild-type Li Zixiang, and the ABA sensitivity of the T1 transgenic sweet potato lines overexpressing IbbHLH118 was significantly lower than that of the wild-type Li Zixiang.

[0205] (3) DAB staining, NBT staining and H2O2 content determination

[0206] When plants are in adverse conditions or aging, H2O2 accumulates due to the enhanced metabolism of active oxygen in the body. H2O2 can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H2O2 content, the greater the degree of damage to plants caused by adverse conditions.

[0207] The H2O2 accumulation of sweet potato plants was detected by a hydrogen peroxide (H2O2) kit (Suzhou Keming Biology, catalog number: H2O2-2-Y), and DAB and NBT staining were used to further reflect the H2O2 accumulation. The sweet potato plants were sweet potato plants treated with the control group in step 1 for 4 weeks and the drought group in step 1 for 4 weeks. The sweet potato plants were IbbHLH118 T1 transgenic sweet potato plants and wild-type Li Zixiang (CK) plants. The experiment was repeated three times, and the results were averaged.

[0208] The results are as follows Figure 8 As shown in (D)-(H), the control is no stress, and PEG is simulated drought stress. The results show that the H2O2 accumulation of the T1 generation transgenic sweet potato line with RNAi silencing IbbHLH118 is significantly lower than that of the wild type Li Zixiang, and the H2O2 accumulation of the T1 generation transgenic sweet potato line overexpressing IbbHLH118 is significantly higher than that of the wild type Li Zixiang.

[0209] (4) POD activity assay

[0210] POD activity can be used as a biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of damage to the plant caused by adversity.

[0211] The POD activity of sweet potato plants was detected using a peroxidase (POD) kit (Suzhou Keming Biology, catalog number: POD-2-Y). The sweet potato plants were sweet potato plants treated with the control group in step 1 for 4 weeks and sweet potato plants treated with drought in the drought group in step 1 for 4 weeks. The sweet potato plants were plants of the IbbHLH118 T1 transgenic sweet potato line and plants of the wild-type Lizixiang (CK). The experiment was repeated three times, and the results were averaged.

[0212] The results are as follows Figure 8 As shown in (I), the control is no stress, and PEG is simulated drought stress. The results showed that the POD activity of the T1 generation transgenic sweet potato line with RNAi silencing IbbHLH118 was significantly higher than that of the wild-type Li Zixiang, and the POD activity of the T1 generation transgenic sweet potato line overexpressing IbbHLH118 was significantly lower than that of the wild-type Li Zixiang.

[0213] (5) SOD activity assay

[0214] SOD activity can be used as a biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of damage to the plant caused by adversity.

[0215] The SOD activity of sweet potato plants was detected using a superoxide dismutase (SOD) kit (Suzhou Keming Biology, catalog number: SOD-2-Y). The sweet potato plants were sweet potato plants treated with the control group in step 1 for 4 weeks and the drought group in step 1 for 4 weeks. The sweet potato plants were plants of the IbbHLH118 T1 transgenic sweet potato line and plants of the wild-type Li Zixiang (CK). The experiment was repeated three times, and the results were averaged.

[0216] The results are as follows Figure 8 As shown in (J), the control is no stress, and PEG is simulated drought stress. The results showed that the SOD activity of the T1 generation transgenic sweet potato line with RNAi silencing IbbHLH118 was significantly higher than that of the wild-type Li Zixiang, and the SOD activity of the T1 generation transgenic sweet potato line overexpressing IbbHLH118 was significantly lower than that of the wild-type Li Zixiang.

[0217] (6) Determination of proline content

[0218] Under normal conditions, the free proline content of plants is very low, but when encountering stresses such as drought, low temperature, and salt, the free amino acid will accumulate in large quantities, and the accumulation index is related to the stress resistance of the plant. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

[0219] The proline content of sweet potato plants was detected using a proline (PRO) content kit (Suzhou Keming Biology, catalog number: PRO-2-Y). The sweet potato plants were sweet potato plants treated with the control group in step 1 for 4 weeks and the drought group in step 1 for 4 weeks. The sweet potato plants were plants of the IbbHLH118 T1 transgenic sweet potato line and plants of the wild-type Li Zixiang (CK). The experiment was repeated three times and the results were averaged.

[0220] The results are as follows Figure 8As shown in (K), the control is no stress, and PEG is simulated drought stress. The results show that the proline activity of the T1 transgenic sweet potato line with RNAi silencing IbbHLH118 was significantly higher than that of the wild-type Li Zixiang, and the proline activity of the T1 transgenic sweet potato line overexpressing IbbHLH118 was significantly lower than that of the wild-type Li Zixiang.

[0221] The above results showed that RNAi silencing of IbbHLH118 gene could improve the drought resistance of sweet potato.

[0222] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of IbbHLH118 protein or the gene encoding the IbbHLH118 protein in reducing drought resistance of plants; the IbbHLH118 protein is a protein with an amino acid sequence of SEQ ID No.1; the plant is sweet potato.

2. The use according to claim 1, characterized in that: The IbbHLH118 protein is derived from sweet potato.

3. Use of a substance that inhibits the expression of the gene encoding the IbbHLH118 protein in claim 1 in improving plant drought resistance; the substance that inhibits the expression of the gene encoding the IbbHLH118 protein is a biological material, and the biological material is any one of the following: B1), an RNA molecule that inhibits the expression of the gene encoding the IbbHLH118 protein; B2), a DNA molecule expressing the RNA molecule described in B1); B3), an expression cassette containing the DNA molecule described in B2); B4), a recombinant vector containing the DNA molecule described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the DNA molecule described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6), a transgenic plant cell line containing the DNA molecule described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the DNA molecule described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8), a transgenic plant organ containing the DNA molecule described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

4. The use according to claim 3, characterized in that: B1) The RNA molecule is an RNA transcribed from a DNA molecule as shown in formula (I): SEQ forward-X-SEQ reverse (I); The SEQ forward sequence is the 27th to 298th position of the sequence 2 in the sequence list; the SEQ reverse sequence is the reverse complement of the SEQ forward sequence; the X is the spacer sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse.

5. The use according to claim 3, characterized in that: B2) The DNA molecule is as shown in formula (I): SEQ forward-X-SEQ reverse (I); The SEQ forward sequence is the 27th to 298th position of the sequence 2 in the sequence list; the SEQ reverse sequence is the reverse complement of the SEQ forward sequence; the X is the spacer sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse.

6. Use of the biological material according to any one of claims 3 to 5 in the preparation of a product for improving the drought resistance of sweet potatoes.

7. Use of the biological material according to any one of claims 3 to 5 in sweet potato breeding, wherein the purpose of the sweet potato breeding is to obtain sweet potatoes with improved drought resistance.

8. The IbbHLH118 protein of claim 1.

9. A DNA molecule encoding the IbbHLH118 protein according to claim 8.

10. The DNA molecule according to claim 9, characterized in that: The nucleotide sequence of the DNA molecule is SEQ ID No.

2.

11. An expression cassette comprising the DNA molecule of claim 9 or 10.

12. A recombinant vector comprising the DNA molecule according to claim 9 or 10 or the expression cassette according to claim 11.

13. A recombinant microorganism comprising the DNA molecule according to claim 9 or 10, the expression cassette according to claim 11 or the recombinant vector according to claim 12.

14. The RNA molecule according to B1) of any one of claims 3 to 5.

15. The coding gene described in B2) of any one of claims 3 to 5.

16. The expression cassette of any one of claims 3 to 5.

17. The recombinant vector according to any one of claims 3 to 5.

18. The recombinant microorganism according to any one of claims 3 to 5.

19. A method for improving drought resistance of sweet potatoes, characterized in that: The method comprises introducing the DNA molecule represented by formula (I) in claim 5 into a recipient sweet potato, inhibiting the expression of the IbbHLH118 protein encoding gene, and obtaining a target sweet potato having higher drought resistance than the recipient sweet potato.

20. A method for reducing drought resistance of sweet potatoes, characterized in that: The method comprises introducing the DNA molecule of claim 9 or 10 into a recipient sweet potato, increasing the expression of the IbbHLH118 protein encoding gene, and obtaining a target sweet potato having lower drought resistance than the recipient sweet potato.

21. A method for preparing drought-resistant sweet potatoes, characterized in that: The method comprises introducing the DNA molecule represented by formula (I) in claim 5 into a recipient sweet potato, inhibiting the expression of the IbbHLH118 protein encoding gene, and obtaining a target sweet potato having higher drought resistance than the recipient sweet potato.

Images