Loquat EjHB7 gene, protein and its application in regulating plant drought tolerance
By constructing and overexpressing the loquat EjHB7 gene or silencing the EjHB7 gene, the physiological response of plants under drought stress was regulated, and the activities of POD and SOD enzymes were increased. This solved the problem of loquat's sensitivity to drought stress, enhanced the drought resistance of Arabidopsis thaliana, and reduced the drought resistance of loquat.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU TAIHU EVERGREEN FRUIT TREE TECH PROMOTION CENT
- Filing Date
- 2024-12-06
- Publication Date
- 2026-05-26
Smart Images
Figure CN119591683B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the loquat EjHB7 gene, protein, and its application in regulating plant drought resistance. Background Technology
[0002] Loquat (Eriobotrya japonica Lindl.) belongs to the genus Eriobotrya in the subfamily Maloideae of the family Rosaceae. It is a typical subtropical evergreen fruit tree. Loquats are sweet and sour, rich in nutrients, and highly favored by consumers. Among them, the white-fleshed loquat is considered the finest variety, with excellent fresh-eating quality. However, loquat has a shallow root system with few fibrous roots, and the proportion of fibrous roots to total root mass is lower than that of most fruit trees. Therefore, it has a higher water requirement, and loquat orchards are mostly built on hillsides with poor irrigation conditions, making them particularly vulnerable to drought. Currently, research on the drought tolerance of loquat is relatively limited. Therefore, conducting research on the mechanism of loquat's response to drought stress has significant theoretical and practical value.
[0003] Transcription factors play a crucial role in the regulation of plant growth, development, and stress responses. Among them, HD-Zip (Homeodomain–leucine Zipper) proteins are plant-specific transcription factors involved in plant growth, embryogenesis, and vascular tissue formation. Members of the HD-Zip I subfamily play a vital role in plant stress signal transduction and resistance regulation. Current research on HD-Zip I subfamily members mainly focuses on model plants, with almost no research on loquat. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides the loquat EjHB7 gene, protein, and their application in regulating plant drought resistance. It utilizes genetic engineering and molecular biology techniques to study the role of EjHB7, a member of the loquat HD-Zip I subfamily, under drought stress.
[0005] The technical solution provided by this invention is as follows:
[0006] A loquat EjHB7 protein, which is either a) or b) of the following:
[0007] a) A protein consisting of the amino acid sequence shown in SEQ ID No. 2;
[0008] b) Proteins derived from SEQ ID No. 2 that are associated with the root development of loquat and involve substitution and / or addition and / or deletion of one or more amino acid residues of the sequence shown in SEQ ID No. 2.
[0009] The present invention also provides a loquat EjHB7 gene, which encodes the protein described above.
[0010] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No. 1.
[0011] The present invention also provides the application of the above-mentioned loquat EjHB7 protein or the above-mentioned loquat EjHB7 gene in regulating plant drought resistance.
[0012] Furthermore, the regulation of plant drought resistance includes: regulating the degree of leaf wilting in plants under drought stress conditions.
[0013] Furthermore, the regulation of plant drought resistance includes: regulating the content of MDA, proline, and reactive oxygen species in plants under drought stress conditions.
[0014] Furthermore, the content of reactive oxygen species was regulated by controlling the activity of POD and SOD enzymes in plants under drought stress.
[0015] The present invention also provides a method for regulating plant drought resistance, comprising the following steps: constructing the CDS sequence of the EjHB7 gene into a plant expression vector, transforming it into a target plant, and obtaining a transgenic plant.
[0016] Furthermore, the plant is Arabidopsis thaliana or loquat.
[0017] Furthermore, the primer sequences used for vector construction are shown in SEQ ID NO.5 and 6.
[0018] Beneficial effects
[0019] This invention obtained EjHB7 transgenic Arabidopsis plants through genetic transformation technology. Under normal conditions, there were no significant differences in growth status and physiological indicators between wild-type Arabidopsis and EjHB7 transgenic Arabidopsis. However, under drought stress, the leaves of the overexpressing transgenic Arabidopsis showed less wilting than those of the wild type. EjHB7 reduces reactive oxygen species (H2O2 and O2) by increasing the activity of POD and SOD enzymes. - The accumulation of EjHB7 in Arabidopsis thaliana, H2O2 and O2 - The levels of these enzymes were significantly lower than in the wild type, decreasing by 79.26% and 59.3% respectively. The activities of POD and SOD enzymes increased by 82.95% and 2 times respectively compared to the wild type, indicating that the EjHB7 gene can regulate the drought tolerance of transgenic Arabidopsis thaliana. Furthermore, the drought tolerance of transgenic loquat plants silenced with EjHB7 decreased, suggesting that EjHB7 plays an important role in regulating plant drought tolerance. Attached Figure Description
[0020] Figure 1 Subcellular localization of EjHB7 in tobacco epidermal cells;
[0021] Figure 2This study aimed to identify EjHB7 transgenic Arabidopsis thaliana lines and analyze their phenotypic characteristics after short-term drought treatment. A: RT-PCR identification of EjHB7 transgenic Arabidopsis thaliana. M: DNA marker (DL2000); WT, negative control (wild-type plant); V, positive control (plasmid DNA); B: qRT-PCR analysis of the relative expression level of EjHB7 in EjHB7 transgenic Arabidopsis thaliana lines; C: Phenotypes of WT and EjHB7 transgenic Arabidopsis thaliana after drought treatment. Figures show the phenotypes before drought treatment (control); 10 days after drought treatment; 15 days after drought treatment; scale bar, 1 cm.
[0022] Figure 3 The changes in physiological parameters of WT and EjHB7 transgenic Arabidopsis thaliana before and after drought were shown; among them, A, malondialdehyde (MDA) content; B, proline content; different letters represented significant differences (p<0.05);
[0023] Figure 4 Analysis of hydrogen peroxide and superoxide anion content in WT and EjHB7 transgenic Arabidopsis thaliana under drought stress; A) Nitrotetrazole (NBT) staining in wild-type and transgenic Arabidopsis thaliana after 10 days of normal and drought treatment; B) Diaminobenzidine (DAB) staining in wild-type and transgenic Arabidopsis thaliana after 10 days of normal and drought treatment; C) Hydrogen peroxide (H2O2) content; D) Superoxide anion (O2) content. - The content of different letters indicates significant differences (p<0.05).
[0024] Figure 5 Analysis of POD and SOD enzyme activities in WT and EjHB7 transgenic Arabidopsis thaliana under drought stress; A represents superoxide dismutase (SOD) activity; B represents peroxidase (POD) activity. Different letters indicate significant differences (p<0.05).
[0025] Figure 6 Transient silencing of the EjHB7 gene was used to reduce drought tolerance in loquat. The study included: A) Phenotype of loquat lines with silenced EjHB7 gene after drought stress; B) qRT-PCR analysis of the relative expression level of EjHB7 in transiently silenced lines; C) MDA content; D) Hydrogen peroxide (H2O2) content; and E) Superoxide anion (O2) content. - The content of different letters indicates significant differences (p<0.05). Detailed Implementation
[0026] Example 1
[0027] Cloning analysis of the EjHB7 gene sequence
[0028] Based on the Arabidopsis thaliana AtHB7 protein sequence, a comparison was made with loquat genome data to identify the gene sequence with the highest homology, designated EVM0026197. Primers were designed based on the predicted sequence, and a loquat EjHB7 sequence was cloned using 'Baiyu' loquat cDNA as a template, named EjHB7. The complete open reading frame of this gene is 732 bp, encoding 243 amino acids, with an isoelectric point of 4.9 and a predicted molecular weight of 27.76 KD.
[0029] The nucleic acid sequence of EjHB7:
[0030] (SEQ ID NO.1).
[0031] The amino acid sequence of EjHB7:
[0032] MLDKIEYSSPSAEDHETFSCMSPLGAATTRRKNKKNNKRFSDEQIRSLESIFESESRLEPRKKMQLAKELGLQPRQVAIWFQNKRARWKSKQLERDYSKLRANYNNLASKFEALKKEKQALVV QVQKLNNMMMMRREDCGEDVVMNNGIDGESDNGDATVSESDKVKLDFSVEKSEQGGLGVLSDDDSRIKAEYFGLEDEPNLANLVESADGSLTSAEDWGKLNSDGLFDVTSGDYQWWDFWS(SEQ ID NO.2).
[0033] Upstream primer: EjHB7-F:5'-ATGCTCGATAAAATCGAATATTC-3' (SEQ ID NO.3);
[0034] Downstream primer: EjHB7-R:5'-TCAAGACCAGAAGTCCCACCACTG-3' (SEQ ID NO.4).
[0035] Example 2
[0036] EjHB7 Subcellular Localization
[0037] The open reading frame (ORF) of the EjHB7 gene, with the stop codon removed, was ligated into the pGWB405 vector to construct the EjHB7-GFP fusion protein expression vector. The empty pGWB405 vector was used as a negative control. The vector was transformed into Agrobacterium GV3101 and plated on LB agar plates containing rifampin and kanamycin. After incubation at 28°C for 3 days, positive colonies were reactivated using LB liquid medium containing rifampin and kanamycin. The activated bacterial suspension was centrifuged and then resuspended in a resuspension solution (10 mM MES, 10 mM MgCl2, 200 μM acetylsyl syringone) to an OD600 of 0.5. After standing for 2-3 hours, the bacterial suspension was injected into tobacco leaves using a syringe. Three days after injection, the leaves were placed under a laser confocal microscope to observe the subcellular localization of EjHB7.
[0038] like Figure 1 As shown, fluorescence of the 35S-driven GFP protein (control) could be detected throughout the cell, but fluorescence of the EjHB7-GFP fusion protein could only be detected in the cell nucleus, indicating that EjHB7 is located in the cell nucleus.
[0039] Example 3
[0040] Carrier construction
[0041] The full-length coding region of EjHB7 was cloned into the pCambia2300 vector using seamless DNA cloning technology to create an EjHB7 overexpression vector, which was then transformed into Agrobacterium tumefaciens strain GV3101.
[0042] Forward primer: 5'-CGAGCTCGGTACCCGGGGATCCATGCTCGATAAAATC-3' (SEQ ID NO.5);
[0043] Reverse primer: 5'-CCTTGCTCACCATGGTGTCGACAGACCAGAAGTCCCA-3' (SEQ ID NO.6).
[0044] A 300bp fragment specific to the nonconserved domain of the EjHB7 open reading frame was amplified using a homologous recombination kit. The IIOne Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing) was used to ligate the pTRV2 vector to create the transient silencing vector pTRV2-EjHB7 for EjHB7. The constructed interference vector was then transformed into Agrobacterium tumefaciens strain GV3101.
[0045] Forward primer: 5'-TGAGTAAGGTTACCGAATTCCAACTTGGCATCAAA-3'
[0046] (SEQ ID NO.7);
[0047] Reverse primer: 5'-GTGAGCTCGGTACCGGATCCTTCGCAAGGTTTGGT-3'
[0048] (SEQ ID NO.8).
[0049] Example 4
[0050] Genetic transformation of EjHB7
[0051] The inflorescences of Arabidopsis thaliana were dipped in Agrobacterium tumefaciens solution carrying an overexpression vector of the EjHB7 gene using a flower-dipping method. The obtained transgenic Arabidopsis seeds were continuously screened on MS medium containing 50 mg / L kanamycin until T3 generation seeds were obtained. After growing the T3 generation seeds on the screening medium for 10 days, they were transplanted into soil to obtain homozygous positive seedlings. Four weeks later, the positive seedlings were validated at both the DNA (PCR) and RNA (qRT-PCR) levels.
[0052] The constructed Agrobacterium strain carrying pTRV2-EjHB7 was extensively propagated, and the OD of the bacterial culture was increased. 600 Adjust the pH to 0.8-1.0, mix Agrobacterium carrying pTRV1 and pTRV2-EjHB7 at a 1:1 ratio, resuspend in a resuspension solution (10 mM MME, 10 mM MgCl2, and 200 μM acetylsyl syringone), and let stand at room temperature for 3 hours. Inject the induced bacterial suspension into the leaves of 60-day-old *Eriobotrya japonica* seedlings using Agrobacterium-mediated transformation to silence the EjHB7 gene. The wild-type control group should be injected with the pTRV2 empty vector, followed by recovery treatment at room temperature in the dark for 2-3 days.
[0053] Analysis of EjHB7 gene silencing in loquat: Transgenic lines were identified at the mRNA level. Total RNA was extracted from loquat leaves using the RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (TIANGEN). RNA was extracted from EjHB7 transiently silenced plants and loquat seedlings injected with the pTRV2 empty vector (positive control), and then reverse transcribed into cDNA. The reaction system and procedure for reverse transcription were based on... III. The RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing) was set up according to the manufacturer's instructions. The expression status of EjHB7 in transiently silenced plants was detected by qRT-PCR. The instrument used was a Bio-Rad CFX Opus 96 (Bio-Rad), and the qRT-PCR reaction system and procedure were set up according to the manufacturer's instructions for the real-time quantitative dye Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing). EjActin from loquat was selected as the internal control gene. The relative expression levels of the target gene were calculated. The identified EjHB7 transiently silenced plants and loquat seedlings injected with the pTRV2 empty vector were used for subsequent experimental treatments.
[0054] Example 5
[0055] Transgenic Arabidopsis thaliana testing
[0056] EjHB7 transgenic Arabidopsis lines were identified at the DNA level. DNA was extracted from wild-type and EjHB7 transgenic Arabidopsis plants using the Wolact Plant Genomic DNA Purification Kit (Wolact, Vicband Life Sciences Company (Hk) Limited). PCR amplification was performed using cloning primers for the EjHB7 gene. RNA was extracted from wild-type and EjHB7 transgenic Arabidopsis using the Wolact Plant RNA Isolation Kit (Wolact, Vicband Life Sciences Company (Hk) Limited) and then reverse transcribed into cDNA. The expression status of EjHB7 in transgenic Arabidopsis plants was detected by qRT-PCR, with Arabidopsis AtActin used as an internal control gene. The identified EjHB7 transgenic Arabidopsis and wild-type Arabidopsis were used for subsequent experimental treatments.
[0057] Example 6
[0058] Plant materials and treatment
[0059] After sterilization, seeds of wild-type and transgenic Arabidopsis thaliana were sown in nutrient pots filled with mixed substrate and grown in a light-controlled incubator for 4 weeks for experimental treatment. Natural drought treatment began after a single saturated irrigation, followed by cessation of watering, and the phenotypes of wild-type and transgenic Arabidopsis thaliana plants after drought treatment were observed.
[0060] Four groups were set up: three transgenic silent lines (pTRV-1, pTRV-2 and pTRV-3) with a seedling age of 60 days and wild-type loquat (WT) transformed with an empty vector (pTRV2). The seedlings were then subjected to drought stress treatment to determine the drought resistance of loquat seedlings after EjHB7 gene silencing.
[0061] Example 7
[0062] Histochemical staining and physiological parameter determination:
[0063] The Arabidopsis thaliana in the control group and the drought-treated group were stained using nitrotetrazole (NBT) staining and diaminobenzidine (DAB) staining methods to observe the superoxide anion (O2) concentration in the leaves. - The accumulation of hydrogen peroxide (H2O2).
[0064] NBT staining method for detecting O2 - :
[0065] NBT working solution: Dissolve 1 mg / mL in Hepes buffer (100 mM, pH 7.5) and add 1 / 1000 Triton X-100.
[0066] Immerse the material in NBT working solution and stain under light for 2 hours. Rinse thoroughly with distilled water, decolorize in 80% ethanol at 60°C for 2 hours, continue decolorization with 80% ethanol, and finally store in 10% (v / v) glycerol for observation and photography.
[0067] DAB staining method for detecting H2O2:
[0068] DAB working solution: Dissolve 1 mg / mL in phosphate buffer (10 mM, pH 7.8), add 1 / 1000 Triton X-100, adjust pH to 3.8 with concentrated hydrochloric acid, and prepare fresh before use.
[0069] Staining: Immerse the material in DAB working solution for staining. When brown spots appear, decolorize. The decolorization process is the same as that of NBT.
[0070] According to the manufacturer's instructions, the contents of malondialdehyde (MDA), hydrogen peroxide, and superoxide anion in Arabidopsis thaliana and loquat were determined using a malondialdehyde (MDA) content kit, a hydrogen peroxide test kit, and a superoxide anion kit, respectively (all kits were purchased from Suzhou Keming Biotechnology Co., Ltd.). According to the manufacturer's instructions, the activities of proline, POD, and SOD enzymes in Arabidopsis thaliana were determined using a proline content assay kit, a superoxide dismutase (SOD) kit (WST-8 method), and a peroxidase (POD) test kit, respectively (all kits were purchased from Suzhou Keming Biotechnology Co., Ltd.).
[0071] EjHB7 overexpression improves the drought stress tolerance of Arabidopsis plants:
[0072] To verify the function of EjHB7 in regulating plant drought tolerance, we obtained a total of 6 Arabidopsis thaliana lines overexpressing EjHB7. Figure 1 Three transgenic lines with high EjHB7 expression levels (L2, L4, and L6) were selected for further experiments. Figure 2 B).
[0073] To assess the impact of EjHB7 on drought tolerance, we subjected transgenic Arabidopsis plants to natural drought treatment. Figure 6 A). After 10 days of natural drought, the leaves of WT Arabidopsis thaliana began to wilt, while the leaves of the three transgenic plants (L2, L4, and L6) remained vibrant. Figure 2 C) After 15 days of drought treatment, the leaves of both WT and EjHB7 transgenic plants showed severe wilting. However, compared with the leaves of WT plants, the leaves of the EjHB7-overexpressing Arabidopsis lines showed less wilting. Figure 2 C).
[0074] Given the different phenotypes of WT and EjHB7 transgenic Arabidopsis plants under drought, we further assessed their damage under drought stress using several physiological indicators. Under normal growth conditions, there was no significant difference in MDA content between WT and transgenic Arabidopsis. After drought treatment, compared with WT, the MDA content in the leaves of EjHB7-overexpressing Arabidopsis was lower, with an average reduction of 56.42%. Figure 3 A). Furthermore, under drought stress, the proline content in both WT and EjHB7 transgenic plants significantly increased. However, the increase in proline content in the leaves of EjHB7-overexpressing Arabidopsis thaliana was greater, averaging 3.08-fold (A). Figure 3 B). These results indicate that ectopic expression of EjHB7 in Arabidopsis plants enhances their tolerance to drought stress.
[0075] EjHB7 stimulates the scavenging of reactive oxygen species under drought conditions:
[0076] To evaluate reactive oxygen species (H2O2 and O2) in WT and EjHB7 transgenic Arabidopsis thaliana. - To accumulate α, after 10 days of drought treatment, Arabidopsis thaliana plants from both the control and drought-treated groups were subjected to DAB and NBT staining. Figure 4 As shown, under normal conditions, there were no significant differences between WT and EjHB7 transgenic Arabidopsis thaliana. After 10 days of drought, deeper staining was observed in the leaves of both WT and EjHB7 transgenic Arabidopsis thaliana, indicating higher levels of ROS accumulation under drought conditions. However, the degree of staining suggests higher levels of H2O2 and O2 in the EjHB7 overexpression lines. - The accumulation is far lower than WT, ( Figure 4 A and B). H2O2 and O2 - The content determination results were consistent with the staining results: EjHB7 overexpression of H2O2 and O2 in Arabidopsis thaliana - The content was much lower than WT, decreasing by 79.26% and 59.3% respectively. Figure 4 (C and D).
[0077] In plants, POD and SOD enzymes remove ROS such as H2O2 and O2. - To improve the drought resistance of plants. We measured the activities of these two antioxidant enzymes and found that after drought treatment, their activities were higher in EjHB7-overexpressing Arabidopsis thaliana, increasing by 82.95% and 2-fold respectively compared to WT. Figure 5 This result indicates that EjHB7 can promote the interaction of H2O2 and O2 by regulating the activity of POD and SOD antioxidant enzymes. - The removal.
[0078] Transient silencing of the EjHB7 gene reduces drought resistance in loquat:
[0079] To further elucidate the function of EjHB7 in loquat under drought stress, virus-induced gene silencing (VIGS) was used to suppress EjHB7 expression in loquat. qRT-PCR analysis of the silenced loquat plants showed that the expression level of EjHB7 was significantly lower than that of the control group. Figure 6 B). When EjHB7 silenced plants (pTRV-1, pTRV-2, and pTRV-3) were treated with unloaded control loquat seedlings (WT) under drought stress for 20 days, the EjHB7 silenced plants showed more severe leaf wilting than the control group. Figure 6 A). MDA content indicates that, compared to WT, the cells of EjHB7 silenced plants suffered more severe damage ( Figure 6 C).
[0080] H2O2 and O2 in EjHB7 silenced loquat plants (pTRV-1, pTRV-2, and pTRV-3) and control loquat seedlings (WT) - After measurement, the results were contrary to those of Arabidopsis thaliana. H2O2 and O2 in the EjHB7 silent loquat strain - The cumulative amount is significantly higher than WT ( Figure 6 (D, E). These results indicate that, compared to the control, the silenced EjHB7 plants were more sensitive to drought stress. This material can serve as a reference system for evaluating the stability and adaptability of other breeding materials or varieties. Furthermore, it is an important experimental material in the field of gene editing, serving as a gene editing site and contributing to the progress of molecular breeding.
[0081] EjHB7 transgenic Arabidopsis plants were obtained through genetic transformation. Drought treatment revealed no significant differences in growth status and physiological indicators between WT (wild-type Arabidopsis) and EjHB7 transgenic Arabidopsis under normal conditions. However, under drought treatment, the overexpressing transgenic Arabidopsis exhibited less leaf wilting than the WT plants. EjHB7 reduces reactive oxygen species (H2O2) and O2 by increasing the activity of POD and SOD enzymes. - The accumulation of EjHB7 enhances the drought tolerance of transgenic Arabidopsis thaliana. Furthermore, the drought tolerance of transgenic loquat plants silenced with EjHB7 decreased. These results indicate that EjHB7 plays a crucial role in regulating plant drought tolerance.
Claims
1. Eriobotrya japonica EjHB7 application of the gene in regulating drought tolerance of plants, the gene EjHB7 The sequence of the gene is shown as SEQ ID No. 1, and the plants are Eriobotrya japonica or Arabidopsis thaliana.
2. Use according to claim 1, characterized in that, The regulation of plant drought resistance includes: regulating the degree of leaf wilting under drought stress conditions.
3. Use according to claim 1, characterized in that, The regulation of plant drought resistance includes: regulating the content of MDA, proline and reactive oxygen species in plants under drought stress.
4. Use according to claim 1, characterized in that, The regulation of plant drought resistance includes: regulating the activity of POD and SOD enzymes in plants under drought stress.
5. A method for regulating plant drought tolerance, characterized in that, Includes the following steps: The CDS sequence of the gene is constructed into a plant expression vector, transformed into a plant of interest, and a transgenic plant is obtained. EjHB7 The sequence of the gene is shown as SEQ ID No.
1. EjHB7 The sequence of the gene is shown as SEQ ID No.
1. The plant in question is either Arabidopsis thaliana or loquat.
6. The method for regulating drought tolerance of a plant according to claim 5, wherein, The primer sequences used for vector construction are shown in SEQ ID NO.5 and 6.