Application of NtbHLH128 gene in improving plant resistance to waterlogging stress

Abstract

The invention discloses the application of NtbHLH128 gene in improving the waterlogging stress resistance of plants, and belongs to the technical field of plant genetic engineering. The invention clones a bHLH transcription factor family gene NtbHLH128 from tobacco, and the coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1. By constructing a NtbHLH128 gene knockout vector and transforming tobacco, a homozygous NtbHLH128 gene knockout mutant plant is obtained. It is found through hydroponic treatment that the homozygous NtbHLH128 gene knockout mutant shows stronger water stress resistance than the control. The invention provides an important target gene for the directional improvement and cultivation of high-quality new varieties of water stress-resistant crops, lays a theoretical foundation for later molecular breeding work, and has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of NtbHLH128 gene in improving plant waterlogging stress resistance. Background Art

[0002] Water stress is one of the important bottleneck factors restricting the sustainable development of the crop industry in my country and even the world. With the change of global climate, extreme weather such as floods and droughts are becoming more and more frequent, posing a major threat and test to the safety of crops. Under flooding conditions, aerobic respiration of plants is inhibited, resulting in root hypoxia, which in turn leads to root rot, yellowing and withering of seedlings, wilting and poor growth and development, and other phenomena in which plant growth is significantly inhibited. When plants are hypoxic, they will perform anaerobic respiration, and most plants will produce alcohol. Excessive accumulation of alcohol will cause plant rot. Hypoxia causes insufficient energy supply for plants, especially the roots. When hypoxia occurs, the roots will absorb less inorganic salts (absorbing inorganic salts requires energy), which may cause the plants to turn yellow. Therefore, it is an important need for the sustainable and healthy development of the agricultural economy to study the resistance mechanism of crops, isolate and identify the key genes that enable plants to resist water stress, improve the drought resistance of crops through molecular breeding methods, cultivate resistant crops, and effectively improve crop yield and quality.

[0003] The bHLH transcription factor family is one of the widely existing transcription factor families in eukaryotes. The regulation of the expression of its related genes is mainly through the interaction of specific amino acid residues with target genes. The results of phylogenetic relationships show that plant bHLH transcription factors are monophyletic. bHLH transcription factors play a very important role in the normal growth and development of plants and in the response of plants to various stresses. bHLH transcription factors are not only generally involved in regulating a variety of physiological and biochemical processes, such as the conduction of light and hormone signals, branching, the development of stomata and roots, and various stress responses. In animals, bHLH transcription factors are mainly related to the perception of the external environment, the regulation of the cell cycle, and tissue differentiation. In plants, the number of bHLH family members is relatively large, second only to MYB plant transcription factors. So far, 158 and 173 bHLH transcription factors have been isolated and identified in Arabidopsis and rice using genomic and bioinformatics analysis methods, and bHLH genes have also been isolated and identified in other plants. For example, the genomes of tobacco, carrot, salvia miltiorrhiza and tomato contain 190, 146, 127 and 159 bHLH genes respectively. However, only a few bHLH proteins in plants have been analyzed. Among them, the bHLH128 gene exists in the genomes of tomato, potato, pepper, tobacco and other Solanaceae plants, and the function of the bHLH128 gene is still unclear. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the NtbHLH128 gene in improving plant resistance to waterlogging stress, so as to solve the problems existing in the above-mentioned prior art. The NtbHLH128 gene provided by the present invention provides an important target gene for the directional improvement and cultivation of new varieties of high-quality water stress-resistant crops, lays a theoretical foundation for subsequent molecular breeding work, and has important application value.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of the NtbHLH128 gene or a protein encoded by the NtbHLH128 gene in regulating plant resistance to waterlogging stress. The coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0007] Furthermore, by inhibiting the expression of the NtbHLH128 gene, the resistance of plants to waterlogging stress is enhanced.

[0008] Furthermore, enhancing the resistance of a plant to waterlogging stress includes alleviating the root hypoxia symptoms of the plant.

[0009] Furthermore, the plant includes tobacco.

[0010] The present invention also provides a method for enhancing the resistance of plants to waterlogging stress, comprising the step of inhibiting the expression of the plant NtbHLH128 gene, wherein the coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0011] Furthermore, inhibiting the expression of the plant NtbHLH128 gene includes knocking out the NtbHLH128 gene.

[0012] Furthermore, enhancing the resistance of a plant to waterlogging stress includes alleviating the root hypoxia symptoms of the plant.

[0013] Furthermore, the plant includes tobacco.

[0014] The present invention also provides the use of NtbHLH128 gene or the protein encoded by it in regulating the flavonoid content of plants under waterlogging stress. The coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1, and the flavonoids include rutin and kaempferol-3-O-rutin.

[0015] Furthermore, by inhibiting the expression of the NtbHLH128 gene, the content of the flavonoids in plants under waterlogging stress is increased.

[0016] The present invention discloses the following technical effects:

[0017] The present invention cloned a bHLH transcription factor NtbHLH128 from tobacco, constructed a NtbHLH128 CRISPR-Cas9 gene editing knockout vector, and transformed tobacco to obtain a homozygous NtbHLH128 gene knockout mutant plant bhl01-3. The present invention further found through hydroponic treatment that the homozygous NtbHLH128 gene editing knockout mutant bhl01-3 showed stronger water stress resistance than the control (WT).

[0018] The present invention provides an important target gene for the directional improvement and cultivation of new varieties of high-quality water stress-resistant crops, lays a theoretical foundation for subsequent molecular breeding work, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The editing site and mutation information detection results of the bhl01-3 knockout mutant material gene;

[0021] Figure 2 The amino acid information detection results of the bhl01-3 knockout mutant material after mutation;

[0022] Figure 3 The results of determination of chlorogenic acid (A), rutin (B) and kaempferol-3-O-rutin (C) contents in leaves of bhl01-3 knockout mutant materials, where CK1 and CK2 represent two replicates of NtbHLH128 gene non-knockout materials, and bhl01-3-1 and bhl01-3-2 represent two replicates of knockout mutant material bhl01-3;

[0023] Figure 4 This is the phenotype of transgenic material after hydroponic treatment. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example

[0030] 1. Cloning of the tobacco NtbHLH128 gene

[0031] The nucleotide coding sequence of NtbHLH128 gene was amplified by homologous cloning method with the cDNA of common tobacco safflower Dajinyuan as template. The nucleotide coding sequence is 1113 bp in length, as shown in SEQ ID NO.1, encoding 370 amino acids. The sequence of the amino acids encoded by it is shown in SEQ ID NO.2.

[0032] >NtbHLH128 CDS sequence (SEQ ID NO.1):

[0033]

[0034] >NtbHLH128 amino acid sequence (SEQ ID NO.2):

[0035] MYPSSTSSSSQNSMSHSTTAGGGSNGGGGLTRYGSAPGAFLTTAVESVIGANNHDFNLHGSHHQHLGPSRYFSPNMTSSNSLNSESTSKAKEQSSLQRSIGFNDLTIGSGGGGGGGGGCGVMPAASSSTTPLVRHSSSPARFLNQLAAAAGDTGFSVSMGRGSYNSKGVADSSRGITRLNSQLSFT RQEALSQIAEENEDVEGTSTDNGHRKSTHSYATASSFAMGSWEDNNSIMFSVTPSKRAKHISNDIVNGLDDGETQFQFGLSQTALEMASMDRLLHIPEDSVPCKIRAKRGCATHPRSIAERERRTRISGKLKKLQDLVPNMDKQTSYADMLDLAVQHIRTLQDQVQNLNTELENCKCGCKKSSQ*.

[0036] The sequences of the primer combinations NtbHLH128-F and NtbHLH128-R used to amplify the nucleotide coding sequence of the NtbHLH128 gene are as follows:

[0037] NtbHLH128-F: 5'-ATGTATCCTTCCTCAACTTC-3' (SEQ ID NO.3);

[0038] NtbHLH128-R: 5'-TTATTGACTGGATTTCTTACAT-3' (SEQ ID NO. 4).

[0039] 2. Construction of NtbHLH128 CRISPR-Cas9 gene editing vector

[0040] CRISPR Multi targeter software was used to design the target site gRNA, and the designed target site + PAM region was compared with the tobacco genome database to select target sites that did not span introns and had high specificity (no off-target). The gRNA sequence was annealed to form double-stranded DNA by PCR with single-stranded Oligo DNA. The pORE-Cas9 vector was used as the backbone vector. The double-stranded DNA formed by annealing was connected to the backbone vector by enzyme digestion to construct the knockout vector. The constructed knockout vector was pORE-Cas9-128. After the knockout vector was constructed, the expression vector containing the target gene was introduced into tobacco leaves by Agrobacterium-mediated transformation, and gene editing positive materials were obtained after tissue culture, antibiotic resistance screening and target site PCR detection.

[0041] The designed gRNA sequence is as follows:

[0042] NtbHLH128-gRNA: GAAGGGACTAGTACAGACAA (SEQ ID NO. 5).

[0043] The primer sequences used for target site PCR detection are as follows:

[0044] CACGACCTCTTACGTGCTCTGAT(SEQ ID NO.6);

[0045] AAGATTACCTGAGTTTCCCCGTC (SEQ ID NO. 7).

[0046] 3. Changes in rutin content in NtbHLH128 gene knockout materials

[0047] The NtbHLH128 knockout vector pORE-Cas9 was constructed using CRISPR-Cas9 technology and transformed into tobacco, successfully obtaining the homozygous gene editing knockout material bhl01-3. Sequencing analysis revealed that the bhl01-3 knockout mutation was caused by the insertion of a T base at base 620 ( Figure 1 ), resulting in the mutation of the 208th amino acid from asparagine to glutamine and causing premature termination of translation ( Figure 2 The contents of chlorogenic acid, rutin and kaempferol-3-O-rutin in leaves of NtbHLH128 gene non-knockout material (CK) and knockout mutant material bhl01-3 were determined twice. The test results showed that after NtbHLH128 gene knockout, compared with the wild-type control, the chlorogenic acid content decreased by 22.8-27.6%; the rutin content increased by 53.9-68.2%; and the kaempferol-3-O-rutin content increased by 20.3-24.7% ( Figure 3 ).

[0048] 4. Hydroponic treatment

[0049] The test materials were the NtbHLH128 gene editing knockout mutant bhl01-3 and the wild-type material (WT) without gene editing. The hydroponic treatment was carried out in a greenhouse. Equal amounts of tobacco seeds (50 seeds) were sown in white seed trays. When the tobacco seedlings grew to 4-5 leaves, the roots were washed and transferred to the nutrient solution for hydroponic treatment. After 20 days of hydroponic treatment, the phenotypes of different materials were observed and statistically analyzed, and the treatment was repeated 3 times. The experimental results showed that the wild-type material WT showed obvious root hypoxia symptoms, including rot, yellowing, and death, after 20 days of hydroponic treatment, while the NtbHLH128 gene editing knockout mutant bhl01-3 grew more normally compared with the control, without root hypoxia symptoms such as rot and yellowing. After 30 days of hydroponic treatment, the survival rate of the knockout mutant bhl01-3 plants was 100%, which was significantly higher than the survival rate of the control material (WT) of 18%-20%. The NtbHLH128 gene-edited knockout mutant material bhl01-3 showed a strong ability to resist water stress ( Figure 4 ). Combined with the higher flavonoids (rutin and kaempferol-3-O-rutin) content in bh01-3 plants, it is speculated that the higher flavonoids in bh01-3 plants alleviated the activated oxygen damage caused by flooding stress, thereby improving the flooding stress resistance of bh01-3 plants.

[0050] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of the NtbHLH128 gene or the protein encoded by it in regulating plant resistance to flooding stress, characterized in that: The coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1; By inhibiting the expression of the NtbHLH128 gene, the resistance of the plant to waterlogging stress is enhanced; Enhancing plant resistance to waterlogging stress includes alleviating the root hypoxia symptoms of plants; The plant is tobacco.

2. A method for enhancing plant resistance to waterlogging stress, characterized in that: The method comprises the steps of inhibiting the expression of the plant NtbHLH128 gene, wherein the coding sequence of the NtbHLH128 gene is shown in SEQ ID NO.1; The plant is tobacco.

3. The method according to claim 2, characterized in that Inhibiting the expression of the plant NtbHLH128 gene includes knocking out the NtbHLH128 gene.

4. The method according to claim 2, characterized in that: Enhancing plant resistance to waterlogging stress includes alleviating the root hypoxia symptoms of the plant.

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