Lotus japonicus pphl5 gene and application thereof in improving drought resistance of plants

Abstract

The application discloses a Lotus japonicus PHL5 gene and application thereof in improving drought resistance of plants. The application provides a PHL class transcription factor gene LjPHL5 isolated from Lotus japonicus and having a nucleotide sequence shown in SEQ ID No. 1, and a transcription factor amino acid sequence encoded by the gene and shown in SEQ ID No. 2. The application also provides a recombinant expression vector of the gene, an engineering bacterium, and application of the gene in cultivating drought-resistant leguminous plants. It is proved by the application that the Lotus japonicus PHL family gene LjPHL5 regulates drought response, provides a drought-resistant gene resource, and can be used in plant drought-resistant molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and forage molecular breeding technology, specifically involving the LjPHL5 gene, a PHL-type transcription factor derived from Lobelia chinensis, a recombinant vector, an engineered strain, and the application of this gene in enhancing the drought stress resistance of Lobelia chinensis. Background Technology

[0002] Drought stress is a core abiotic stress that restricts pasture growth and the development of grassland animal husbandry. Global warming has led to frequent seasonal droughts. In water-scarce environments, leguminous pastures experience stunted growth, reduced photosynthetic efficiency, and deterioration in nutritional quality, resulting in large-scale grassland yield reductions and severe economic and ecological losses. Discovering highly efficient drought-resistant genes and cultivating drought-resistant transgenic pastures are the most fundamental technical means to alleviate the harm of drought stress.

[0003] The classic function of the PHR (Phosphate Starvation Response) transcription factor family is to regulate plant responses to low phosphorus stress. Recent studies have confirmed the involvement of PHR / PHL members in drought and salt stress responses: overexpression of ZmPHR1 in maize enhances drought resistance, and PHR1 and PHL1 in Arabidopsis regulate stomatal opening and closing and seed germination through the ABA pathway. However, existing research has significant limitations: current PHR / PHL stress resistance studies mostly focus on phosphorus signaling cross-pathways, with very few direct analyses of independent ABA drought resistance regulatory modules; furthermore, the drought regulatory function and molecular mechanisms of PHL-like transcription factors in the legume model forage grass *Lobelia chinensis* have not been reported.

[0004] Lotus japonicus L. is a perennial, high-quality leguminous forage grass. It is tolerant of poor soil and has a high protein content, making it an ideal forage species for widespread cultivation in arid mountainous areas. However, drought significantly inhibits its growth, limiting its planting and production. Identifying key drought-resistant genes in the Lotus japonicus PHL family has significant theoretical and industrial application value for the genetic improvement of forage stress resistance. Summary of the Invention

[0005] The objectives of this invention include: providing a PHL-type transcription factor LjPHL5 gene derived from the root of *Lotus styrax* that can significantly enhance plant drought resistance; providing a recombinant plant expression vector and genetically engineered bacteria containing the LjPHL5 gene; and providing new materials for cultivating drought-resistant transgenic plants using the LjPHL5 gene.

[0006] Based on the first principal aspect of the present invention, a PHL-type transcription factor encoding gene LjPHL5 is provided, the nucleotide sequence of which is shown in SEQ ID No. 1. This gene was isolated from the legume forage grass Lobelia chinensis, and its full-length CDS is 948 bp, encoding 315 amino acids (SEQ ID No. 2).

[0007] The LjPHL5 protein contains a typical MYB-DNA binding domain (containing the SHAQKYF motif) and a coiled-coil domain typical of the PHR family, classifying it as a MYB-CC transcription factor. Subcellular localization experiments confirmed that the LjPHL5 protein is located in the cell nucleus.

[0008] The promoter region of the LjPHL5 gene contains multiple cis-acting elements related to drought stress response, such as DRE, DRE core, MYB, and MYC.

[0009] Based on a second key aspect of the present invention, a recombinant expression vector comprising the aforementioned LjPHL5 gene is provided. The recombinant expression vector is preferably a recombinant plant expression vector, more preferably a constitutive expression vector pSH-35S-LjPHL5. This vector contains a 35S promoter, a GUS reporter gene, and a kanamycin resistance selection marker, and the LjPHL5 gene coding sequence is inserted via XbaI and EcoRI double restriction enzyme sites.

[0010] The present invention also provides a genetically engineered bacterium containing the aforementioned recombinant expression vector, preferably Agrobacterium GV3101 engineered strain.

[0011] Based on the third main aspect of the present invention, the application of the aforementioned LjPHL5 gene, the aforementioned LjPHL5 protein, the aforementioned recombinant vector, or the aforementioned genetically engineered bacteria in improving plant drought stress resistance is provided.

[0012] The application includes the following steps:

[0013] 1) Construct a recombinant plant expression vector carrying the LjPHL5 gene;

[0014] 2) Transform the recombinant plant expression vector into Agrobacterium to obtain genetically engineered bacteria;

[0015] 3) Plant explants were transformed using the genetically engineered bacteria, and positive transgenic plants were obtained through resistance screening;

[0016] 4) Molecular identification was performed on the positive transgenic plants, and their drought stress resistance was identified. Transgenic plants with enhanced drought resistance compared with wild-type control plants were screened.

[0017] The molecular identification includes GUS histochemical staining, GUS gene PCR amplification, and qRT-PCR expression level detection.

[0018] Based on a fourth key aspect of the present invention, the present invention provides a method for cultivating new varieties of drought-resistant transgenic plants, comprising the aforementioned operational steps. The drought-resistant transgenic plant is a dicotyledonous plant, such as *Lobelia chinensis*.

[0019] In this method, the plant explants are segments of the rhizome of *Louphinella*, which are infected with *Agrobacterium*, co-cultured, subjected to resistance screening, rooting induction, and hardening-off transplanting to obtain transgenic regenerated plants. Kanamycin is used as the screening agent for resistance screening, and IBA is added to the rooting medium to induce adventitious root formation.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects:

[0021] 1. Provide novel drought-resistant gene resources derived from Piper nigrum.

[0022] This invention cloned the LjPHL5 gene from *Lobelia chinensis*, whose CDS sequence is shown in SEQ ID No. 1, encoding a 315-amino acid MYB-CC transcription factor (SEQ ID No. 2). The promoter region of this gene contains drought-related cis-regulatory elements such as DRE, MYB, and MYC. Its expression level in leaves was significantly higher than in roots and stems (p<0.001), and it was upregulated by drought stress and exogenous ABA induction. These characteristics indicate that LjPHL5 possesses the structural basis and expression regulatory features to be a candidate gene for drought resistance, providing a new gene resource for molecular breeding of drought-resistant leguminous forage crops.

[0023] 2. The drought resistance phenotype and physiological indicators of LjPHL5-overexpressing transgenic *Spiraea japonica* were significantly superior to those of the wild type.

[0024] The LjPHL5 overexpressing transgenic Lobelia chinensis (TP4, TP10) obtained in this invention exhibits the following quantifiable advantages compared to wild-type (WT) after 6 days of simulated drought treatment with 25% PEG6000:

[0025] (1) Growth and phenotype: The wilting degree of transgenic lines was significantly less than that of WT, and the chlorosis of leaves was delayed; the height growth of TP4 and TP10 was 2.06 times and 2.02 times that of WT, respectively (p<0.05), and the chlorophyll accumulation was significantly higher than that of WT (p<0.01 or p<0.05).

[0026] (2) Water retention and membrane stability: The relative water content of TP4 and TP10 leaves increased by 22.97% and 15.74% respectively (p<0.001); the relative electrical conductivity decreased by 19% (p<0.001) and 9.41% (p<0.05) respectively; the MDA accumulation of TP4 was only 0.75 times that of WT (p<0.001).

[0027] (3) Osmotic regulation and antioxidant: The proline content of TP4 and TP10 was 1.37 times (p<0.001) and 1.14 times (p<0.05) of that of WT, respectively; DAB and NBT staining showed that the accumulation of H2O2 and O2 in transgenic leaves was significantly less than that of WT; the activities of three antioxidant enzymes, SOD, POD and CAT, were significantly higher than those of WT (p<0.01 or p<0.05), among which the POD activity of TP4 was 2.03 times that of WT (p<0.01).

[0028] The above results indicate that overexpression of LjPHL5 synergistically enhances the drought resistance of Lobelia chinensis from multiple levels, including water retention, membrane protection, osmotic regulation, and antioxidant defense.

[0029] 3. Effects of LjPHL5 overexpression on the expression of genes related to the ABA signaling pathway

[0030] This invention, by detecting changes in the expression of key genes in the ABA pathway and the content of endogenous ABA under drought stress, preliminarily reveals the action pathway of LjPHL5:

[0031] After 6 days of drought treatment, the endogenous ABA content in transgenic lines increased significantly, with TP4 increasing by 25.51% compared to WT (p<0.001) and TP10 increasing by 13.26% (p<0.01). The key ABA synthesis gene NCED3 was significantly upregulated in TP4 (p<0.05). The ABA receptor gene PYL1 was 2.21 times and 1.87 times higher in TP4 and TP10 than in WT, respectively (p<0.01). The negative regulator ABI1 was significantly downregulated in transgenic lines (p<0.01). The positive regulator kinase gene SnRK2.6 was extremely significantly upregulated in TP4 (p<0.01).

[0032] The above results provide experimental evidence for understanding the molecular mechanism by which LjPHL5 participates in drought resistance. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0034] Figure 1 The image shows the PCR amplification electrophoresis diagram of the CDS sequence of the LjPHL5 gene cloned from Lophatherum gracile in this invention; M is the DL2000 DNA Marker, the target fragment size is 948 bp (SEQ ID No. 1), and this fragment was used for the subsequent construction of plant expression vectors after being verified by sequencing.

[0035] Figure 2 This diagram shows a predicted conserved domain of the LjPHL5 protein in one embodiment of the present invention; it contains the PHR family characteristic MYB-DNA binding domain (SHAQKYF motif) and coiled-coil domain.

[0036] Figure 3 The image shows a laser confocal image of the subcellular localization of the LjPHL5 protein in one embodiment of the present invention; the fluorescence signal completely overlaps with the cell nuclear marker, proving that the protein is localized in the cell nucleus.

[0037] Figure 4 The spatiotemporal expression pattern of the LjPHL5 gene in one embodiment of the present invention is shown; A: Root, stem and leaf tissue-specific expression; B: 100 μMABA-induced expression; C: 25% PEG6000 drought-induced expression; D: ABA+PEG combined-induced expression.

[0038] Figure 5 The image shows a map of the recombinant overexpression vector pSH-35S-LjPHL5 plasmid in one embodiment of the present invention; labeled with the 35S promoter, LjPHL5 insert, GUS selection marker, resistance gene, and restriction enzyme sites XbaI / EcoRI.

[0039] Figure 6 The following is an example of the identification of transgenic Lophatherum gracile positive plants in one embodiment of the present invention: A: GUS histochemical staining; B: GUS marker gene PCR electrophoresis identification; C: qRT-PCR detection results of the relative expression level of LjPHL5 in each resistant line.

[0040] Figure 7 This invention illustrates a comparison of the phenotypes of wild-type WT and transgenic lines TP4 and TP10 after 6 days of drought treatment with 25% PEG6000; the wilting phenotype was observed continuously at 0d, 2d, 4d, and 6d.

[0041] Figure 8 The following is an example of the detection of physiological indicators of WT and transgenic lines under drought stress in one embodiment of the present invention: A: relative water content of leaves; B: relative electrical conductivity; C: malondialdehyde (MDA) content; D: proline content.

[0042] Figure 9 The following is an example of the detection of antioxidant enzyme activity under drought stress in one embodiment of the present invention; A: POD enzyme activity; B: SOD enzyme activity; C: CAT enzyme activity.

[0043] Figure 10The following is an example of ABA pathway-related index detection in one embodiment of the present invention: A: endogenous ABA content in leaves; B: NCED3 gene expression; C: PYL1 gene expression; D: ABI1 gene expression; E: SnRK2.6 gene expression. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0045] The specific meanings of the technical terms that may be involved in the following embodiments are explained below:

[0046] PHL (PHR-Like) transcription factors belong to the MYB-CC family. In this invention, LjPHL5 is a member of the PHL family derived from *Paecilomyces cerevisiae*, and its nucleotide sequence is shown in SEQ ID No. 1, while the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0047] PEG-simulated drought stress refers to the use of polyethylene glycol (PEG6000) to reduce the water potential of nutrient solution and simulate the osmotic stress experienced by plants under natural drought conditions.

[0048] All other technical terms involved, such as recombinant expression vectors, Agrobacterium-mediated transformation, GUS histochemical staining, and qRT-PCR, shall be understood according to their conventional meaning in this field.

[0049] The following examples are for illustrative purposes only and do not limit the scope of protection of the present invention. Unless otherwise specified, all biochemical reagents and kits are commercially available conventional products. Greenhouse cultivation conditions for Piper longum: 25℃, 16h light / 8h darkness; PEG concentration is 25% by mass / volume; ABA treatment concentration is 100μM, prepared with distilled water for foliar spraying.

[0050] Example 1: Cloning, sequence and structural analysis of the LjPHL5 gene from the root of *Lotus Root*

[0051] 1.1 Materials and Reagents

[0052] The roots of *Lotus rotundifolia* were grown in the greenhouse of the Key Laboratory of Agricultural Bioengineering, Guizhou University; RNA extraction kit OMEGAR6834-01; GeneStar reverse transcription kit; pMD18-T cloning vector; *E. coli* DH5α competent cells.

[0053] 1.2 RNA extraction and cDNA synthesis

[0054] Fresh leaves of *Lotus Root* were taken, quick-frozen and ground in liquid nitrogen, and total RNA was extracted using a kit. After the purity was tested and the DNA was intact, cDNA was obtained by reverse transcription.

[0055] 1.3 Full-length CDS amplification

[0056] Primers with restriction enzyme sites were designed based on the Lj1g3v4317480 sequence of the Lobelia chinensis genome:

[0057] LjPHL5-F: GAGAACACGGGGGACTCTAGAATGAATGAATGGAACTCC;

[0058] LjPHL5-R: GGTGATTTCAGCGTCACGAATTCTTAGGTTCCTTTCCTTTTGG; Amplified fragment 948bp. 20μL PCR system: 2μL cDNA, 1μL each of forward and reverse primers, 10μL 2×ExPremierMix, ddH2O added; Program: 94℃ pre-denaturation for 2 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ final extension for 4 min.

[0059] 1.4 Cloning Sequencing and Bioinformatics Analysis

[0060] PCR products were recovered from the gel, ligated into pMD18-T, transformed into DH5α, and positive clones were selected by blue-white screening and sent for sequencing. Sequencing results: LjPHL5CDS is 948 bp in length (SEQ ID No. 1), encoding 315 amino acids (SEQ ID No. 2); the protein contains a conserved MYB-DNA domain and a coiled helix; the 2000 bp promoter contains drought-responsive elements DRE, MYB, and MYC; subcellular localization predicted the cell nucleus; phylogenetic tree showed that it is most closely related to alfalfa and chickpea PHL homologous proteins.

[0061] Example 2: Spatiotemporal expression characteristics of LjPHL5 (qRT-PCR)

[0062] Internal reference gene LjUBI, quantitative real-time primers:

[0063] Q-LjPHL5-F:GCTGCCACAGACAAGAGTCC;

[0064] Q-LjPHL5-R: GCTCATGCAGATCCTTGCTC;

[0065] LjUBI-F: CTTCACCTTGTTCCTTCGTCTG;

[0066] LjUBI-R: TGGATCTTAGCCTTCACGTTG.

[0067] 1. Tissue expression: The highest expression level was found in leaves, which was 5.28 times higher than that in roots (p<0.001), and in stems, it was 2.06 times higher than that in roots (p<0.01).

[0068] 2. PEG drought induction: peak expression at 12h, which was 1.98 times that of the control (p<0.01).

[0069] 3. ABA induction: peak value reached at 6h, 6.52 times that of the control (p<0.001).

[0070] 4. Combined ABA+PEG induction: The peak value at 12h was 5.10 times that of the control, and the induction amplitude was significantly higher than that of single stress.

[0071] Example 3: Subcellular localization of LjPHL5 protein

[0072] A 35S-EGFP-LjPHL5 fusion vector (digested with KpnI / BamHI) was constructed, transformed into Agrobacterium GV3101, and infected tobacco leaves. After co-culturing for 2-3 days, laser confocal microscopy observation showed that the EGFP fluorescence completely overlapped with the cell nuclear marker, confirming that LjPHL5 was located in the cell nucleus.

[0073] Example 4: Construction of recombinant plant expression vectors

[0074] 1. Constitutive vector pSH-35S-LjPHL5: pSH737-35S vector and pMD18T-LjPHL5 were double-digested with XbaI / EcoRI, seamlessly cloned and ligated, and then transformed into GV3101 after verification;

[0075] Example 5: Agrobacterium-mediated genetic transformation and transgenic identification of Lophatherum gracile

[0076] 1. Transformation process: disinfection of Piper kaempferi root and stem segments → Agrobacterium infection for 8 min → co-culture for 2-3 days → kanamycin selection medium → resistant bud differentiation → rooting medium for seedling hardening and transplanting;

[0077] 2. Positive identification: (1) GUS staining: resistant leaves are blue, wild type is not colored; (2) GUS gene PCR: primers amplify 304bp band, positive plants have band, WT has no band; GUS-F: GGTGATTGATGAAACTGCTG; GUS-R: GAACATTACATTGACGCAGG; (3) qRT-PCR quantification: a total of 10 overexpression lines were obtained, TP4 (21.66 times) and TP10 (14.51 times) are high expression lines, which are used for subsequent functional verification.

[0078] Example 6: Identification of drought resistance function of transgenic Piper longum (PEG simulated drought)

[0079] Treatment conditions: 6 days of stress with 25% PEG6000 nutrient solution, with a control group receiving normal water supply.

[0080] 1. Phenotype and growth: WT plants showed severe wilting, while TP4 and TP10 plants showed only slight wilting; the plant height increase of TP4 was 2.06 times that of WT, and that of TP10 was 2.02 times (p<0.05); the chlorophyll content was significantly higher than that of the wild type.

[0081] 2. Water retention and cell membrane damage: The relative water content of TP4 increased by 22.97%, and that of TP10 increased by 15.74% (p<0.001); the relative conductivity of TP4 decreased by 19%, and that of TP10 decreased by 9.41%; the MDA of TP4 was only 0.75 times that of WT.

[0082] 3. Osmotic regulators: TP4 proline is 1.37 times the WT, and TP10 is 1.14 times;

[0083] 4. Reactive oxygen species and antioxidant enzymes: DAB / NBT staining showed H2O2 and O2 in transgenic leaves. - Less accumulation; POD (TP4 was 2.03 times that of WT), SOD, and CAT enzyme activities were all significantly higher than those of wild type.

[0084] Example 7: LjPHL5 regulates drought resistance mechanism through ABA signaling pathway

[0085] 1. Endogenous ABA content: After 6 days of drought, TP4ABA increased by 25.51% (p<0.001) and TP10 increased by 13.26% (p<0.01).

[0086] 2. Primers for qPCR detection of ABA pathway genes: NCED3-F: CGAGCTCAAAATACACCCCT; NCED3-R: ACGTCTGAGCTCAAGGACAA; PYL1-F: CTTCGACAAGCCACAGTCT; PYL1-R: GAGTCTTTCGGTGGA GGTGG; ABI1-F: CTGTGGGCCTGGAAATGGAT; ABI1-R: ATGAGCCTAGCTACCCCGAA; SnRK2.6-F: CCTCGTGCAAAAGATGACGAA; SnRK2.6-R: CTGAGGACATGGACAAGCCA;

[0087] 3. Expression results: NCED3 (ABA synthesis): significantly upregulated in transgenic lines; PYL1 (ABA receptor): TP4 was 2.21 times that of WT, and TP10 was 1.87 times (p<0.01); ABI1 (negative regulation): expression in transgenic lines was significantly lower than that of WT; SnRK2.6 (positive kinase): highly upregulated in transgenic lines.

[0088] Sequence List:

[0089] SEQ ID NO.1 LjPHL5CDS nucleotide sequence (948bp)

[0090] 。

[0091] SEQ ID NO.2 LjPHL5 protein amino acid sequence (315aa)

[0092] MMNGTPCWEVVGSELLSFEPIKSPYNFVTTTASPVFGASAKSLVSFQAQEHDHGQNQNIDVPGWCYEFPSTPMMIFQGSEDNFTNVTSTEQDRPSSQFTSSLHSVAESFISSSEDSHSCSENYGKIEPGMPLYDHLLQEQLRNNAATDKSPLEISFQR NQLGSCTKQEKQAPGLSRRSPTSKRRIRWSKDLHEPFMMIVNSLGGPEKAKPKAILEMMKSDLLSISHIKSHLQKCRSTTYMHKALQERSEGRHRTDGVTELQVQISHMQIEEESRKLQLEVRRSICLQLEMQQNLKMLIEGQRKELKVMLDRQKERT.

[0093] The acquisition, storage, and application of user personal information involved in the technical solution of this invention all comply with the provisions of relevant laws and regulations and do not violate public order and good morals.

[0094] It should be understood that the various forms of processes shown above can be used, with steps reordered, added, or deleted. For example, the steps described in this invention can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this invention can be achieved, and this invention is not limited thereto.

[0095] The technical terms, principles, or means related to the technical solutions of the present invention mentioned in the above embodiments, which are not described in detail above, are all well-known technologies or common practices that are known to those skilled in the art.

[0096] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A PHL class transcription factor-encoding gene LjPHL5, characterized in that, The LjPHL5 gene has the following nucleotide sequence: The nucleotide sequence is shown in SEQ ID No.

1.

2. The PHL class transcription factor LjPHL5 protein encoded by the LjPHL5 gene according to claim 1, characterized in that, The protein has the following amino acid sequence: The amino acid sequence is shown in SEQ ID No.

2.

3. A recombinant expression vector comprising the LjPHL5 gene as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is a recombinant plant expression vector.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant plant expression vector is a constitutive expression vector pSH-35S-LjPHL5.

6. Genetically engineered bacteria comprising the recombinant expression vector according to any one of claims 3 to 5.

7. The application of the LjPHL5 gene of claim 1, the LjPHL5 protein of claim 2, the recombinant vector of any one of claims 3 to 5, or the gene of claim 6 in improving plant drought stress resistance.

8. The application according to claim 7, characterized in that, Includes the following steps: 1) Construct a recombinant plant expression vector carrying the LjPHL5 gene; 2) Transform the recombinant plant expression vector into Agrobacterium to obtain genetically engineered bacteria; 3) Plant explants were transformed using the genetically engineered bacteria, and positive transgenic plants were obtained through resistance screening; 4) Molecular identification was performed on the positive transgenic plants, and their drought stress resistance was identified. Transgenic plants with enhanced drought resistance compared with wild-type control plants were screened.

9. A method for breeding new drought-resistant transgenic plant varieties, characterized in that, It includes all the operational steps of claim 8; Furthermore, the drought-resistant transgenic plant is a dicotyledonous plant; The dicotyledonous plant in question is a legume.

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