Loquat EjHB7 gene, protein and application of EjHB7 gene in regulation and control of plant drought tolerance

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a loquat EjHB7 gene, a protein and application of the EjHB7 gene and the protein in regulation and control of plant drought tolerance, EjHB7 transgenic arabidopsis plants are obtained through a genetic transformation technology, and under drought stress treatment, the leaf wilting degree of overexpressed transgenic arabidopsis is lower than that of wild arabidopsis. The EjHB7 reduces the accumulation of active oxygen H2O2 and O2 <-> by improving the activity of POD and SOD enzymes, and improves the drought tolerance of transgenic arabidopsis thaliana. In addition, the drought tolerance of the transgenic loquat plant after EjHB7 silencing is reduced, which indicates that EjHB7 plays an important role in regulating and controlling the drought tolerance of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to loquat EjHB7 gene, protein and application thereof in regulating plant drought resistance. Background Art

[0002] Loquat (Eriobotrya japonicaLindl.) belongs to the genus Eriobotrya of the subfamily Maloideae of the family Rosaceae. It is a typical subtropical evergreen fruit tree. Loquat is sweet and sour, rich in nutrients, and is deeply favored by consumers. Among them, white-fleshed loquat is the best among loquats, with good fresh food quality. However, the root system of loquat is shallow, the fibrous roots are scarce, and the proportion of fibrous roots to the total root volume is lower than that of general fruit trees. Therefore, it has a higher requirement for water. Most loquat gardens are built on hillsides with poor irrigation conditions and are particularly susceptible to drought damage. At present, there are relatively few studies on the drought tolerance of loquat. Therefore, it is of great theoretical significance and practical value to carry out research on the mechanism of loquat's response to drought stress.

[0003] Transcription factors play a key role in regulating plant growth and development as well as stress responses. HD-Zip (Homeodomain–leucine Zipper) protein is a plant-specific transcription factor that participates in plant growth, embryogenesis, vascular tissue formation, etc. HD-Zip I subfamily members play an important role in plant stress signal transduction and resistance regulation. Currently, research on HD-Zip I subfamily members is mainly focused on model plants, and research on loquat is almost blank. Summary of the invention

[0004] To solve the above technical problems, the present invention provides loquat EjHB7 gene, protein and application thereof in regulating plant drought tolerance, and uses genetic engineering molecular biology methods to study the role of loquat HD-Zip I subfamily member EjHB7 under drought stress.

[0005] The technical solution provided by the present invention is as follows: A loquat EjHB7 protein is the protein of a) or b) below: a) a protein consisting of the amino acid sequence shown in SEQ ID No.2; b) A protein derived from SEQ ID No. 2 which is related to the root development of loquat and in which one or more amino acid residues are substituted and / or added and / or deleted.

[0006] The present invention also provides a loquat EjHB7 gene, which encodes the above protein.

[0007] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No.1.

[0008] The present invention also provides the use of the loquat EjHB7 protein or the loquat EjHB7 gene in regulating plant drought resistance.

[0009] Furthermore, regulating the drought tolerance of plants includes: regulating the degree of wilting of leaves of plants under drought stress conditions.

[0010] Furthermore, regulating the drought tolerance of plants includes: regulating the content of MDA, proline and active oxygen in plants under drought stress conditions.

[0011] Furthermore, the content of reactive oxygen species is regulated by regulating the activities of POD and SOD enzymes of plants under drought stress conditions.

[0012] The present invention also provides a method for regulating plant drought tolerance, 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.

[0013] Furthermore, the plant is Arabidopsis thaliana or loquat.

[0014] Furthermore, the primer sequences used for vector construction are shown in SEQ ID NOs. 5 and 6. Beneficial Effects

[0015] The present invention obtains EjHB7 transgenic Arabidopsis plants through genetic transformation technology. Under normal conditions, there is no significant difference in growth conditions and physiological indicators between wild-type Arabidopsis and EjHB7 transgenic Arabidopsis. However, under drought stress treatment, the wilting degree of the leaves of overexpressed transgenic Arabidopsis is lighter than that of the wild type. EjHB7 reduces the active oxygen H2O2 and O2 by increasing the activity of POD and SOD enzymes. - The accumulation of H2O2 and O2 in EjHB7 overexpressed Arabidopsis - The content was much lower than that of the wild type, decreasing by 79.26% and 59.3% respectively, and the activities of POD and SOD enzymes increased by 82.95% and 2 times respectively compared with the wild type, indicating that the EjHB7 gene can regulate the drought tolerance of transgenic Arabidopsis. In addition, the drought tolerance of transgenic loquat plants after EjHB7 silencing decreased, indicating that EjHB7 plays an important role in regulating plant drought tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The subcellular localization of EjHB7 in tobacco epidermal cells; Figure 2Identification of EjHB7 transgenic Arabidopsis lines and phenotypic analysis of transgenic Arabidopsis after short-term drought treatment; A: RT-PCR identification of EjHB7 transgenic Arabidopsis. M: DNA marker (DL2000); WT, negative control (wild-type plants); V, positive control (plasmid DNA); B, qRT-PCR analysis of the relative expression of EjHB7 in EjHB7 transgenic Arabidopsis lines; C: Phenotypes of WT and EjHB7 transgenic Arabidopsis after drought treatment. Respectively, before drought treatment (control); 10 days of drought treatment; 15 days of drought treatment; scale bar, 1 cm; Figure 3 The changes of physiological indicators of WT and EjHB7 transgenic Arabidopsis before and after drought; A, malondialdehyde (MDA) content; B, proline content; different letters indicate significant differences (p<0.05); Figure 4 Analysis of hydrogen peroxide and superoxide anion content in WT and EjHB7 transgenic Arabidopsis under drought stress; A, nitro tetrazolium (NBT) staining of wild-type and transgenic Arabidopsis under normal conditions and 10 days of drought treatment; B, diaminobenzidine (DAB) staining of wild-type and transgenic Arabidopsis under normal conditions and 10 days of drought treatment; C, hydrogen peroxide (H2O2) content; D, superoxide anion (O2 - ) content; Different letters indicate significant differences (p<0.05); Figure 5 Analysis of POD and SOD enzyme activities in WT and EjHB7 transgenic Arabidopsis under drought stress; A, superoxide dismutase (SOD) activity; B, peroxidase (POD) activity. Different letters indicate significant differences (p<0.05); Figure 6 Transient silencing of the EjHB7 gene reduced the drought tolerance of loquat; A, phenotype of loquat EjHB7 gene silencing strains after drought stress; B, qRT-PCR analysis of the relative expression of EjHB7 in the EjHB7 transient silencing strains; C, MDA content; D, hydrogen peroxide (H2O2) content; E, superoxide anion (O2 - ) content; Different letters indicate significant differences (p<0.05). DETAILED DESCRIPTION Example 1

[0017] Cloning and Analysis of EjHB7 Gene Sequence Based on the Arabidopsis AtHB7 protein sequence, we searched and compared it with the loquat genome data, and found a gene sequence with the highest homology, numbered EVM0026197. Primers were designed based on the predicted sequence, and a loquat EjHB7 sequence was cloned using the cDNA of 'Bai Yu' loquat as a template, named EjHB7. The complete open reading frame of the gene is 732bp, encoding 243 amino acids, with an isoelectric point of 4.9 and a predicted molecular weight of 27.76KD.

[0018] Nucleotide sequence of EjHB7: (SEQ ID NO.1).

[0019] Amino acid sequence of EjHB7: MLDKIEYSSPSAEDHETFSCMSPLGAATTRRKNKKNNKRFSDEQIRSLESIFESESRLEPRKKMQLAKELGLQPRQVAIWFQNKRARWKSKQLERDYSKLRANYNNLASKFEALKKEKQALVV QVQKLNNMMMMRREDCGEDVVMNNGIDGESDNGDATVSESDKVKLDFSVEKSEQGGLGVLSDDDSRIKAEYFGLEDEPNLANLVESADGSLTSAEDWGKLNSDGLFDVTSGDYQWWDFWS (SEQ ID NO.2).

[0020] Upstream primer: EjHB7-F: 5'-ATGCTCGATAAAATCGAATATTC-3' (SEQ ID NO. 3); Downstream primer: EjHB7-R: 5'-TCAAGACCAGAAGTCCCACCACTG-3' (SEQ ID NO.4). Example 2

[0021] EjHB7 subcellular localization The open reading frame (ORF) of the EjHB7 gene without the stop codon was connected to the pGWB405 vector to construct the EjHB7-GFP fusion protein expression vector. The pGWB405 empty vector was used as a negative control to transform Agrobacterium GV3101 and spread on LB plate medium containing rifampicin and kanamycin. The positive colonies were reactivated with LB liquid medium containing rifampicin and kanamycin in a 28°C incubator for 3 days. The activated bacterial solution was centrifuged and then resuspended with a resuspension solution (10 mMMES, 10 mM MgCl2, 200 µM acetosyringone) to OD600 = 0.5. After standing for 2-3 hours, the bacterial solution was injected into tobacco leaves with a syringe. After the tobacco leaves were injected, the leaves were placed under a laser confocal microscope to observe the subcellular localization of EjHB7 3 days later.

[0022] like Figure 1 As shown, the fluorescence of 35S-driven GFP protein (control) was detected throughout the cells, but the fluorescence of EjHB7-GFP fusion protein was only detected in the nucleus, indicating that EjHB7 was localized in the nucleus. Example 3

[0023] Vector construction The full-length coding region of EjHB7 was cloned into the pCambia2300 vector using DNA seamless cloning technology to create an EjHB7 overexpression vector, which was then transformed into the Agrobacterium tumefaciens strain GV3101.

[0024] Forward primer: 5′-CGAGCTCGGTACCCGGGGATCCATGCTCGATAAAATC-3′ (SEQ ID NO. 5); Reverse primer: 5′-CCTTGCTCACCATGGTGTCGACAGACCAGAAGTCCCA-3′ (SEQ ID NO. 6).

[0025] A specific 300 bp fragment of the non-conserved domain of the EjHB7 open reading frame was amplified and ligated into the pTRV2 vector using the homologous recombination kit ClonExpress® II One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing) to create the EjHB7 transient silencing vector pTRV2-EjHB7. The constructed interference vector was transformed into the Agrobacterium tumefaciens strain GV3101.

[0026] Forward primer: 5′-TGAGTAAGGTTACCGAATTCCAACTTGGCATCAAA-3′ (SEQ ID NO. 7); Reverse primer: 5′-GTGAGCTCGGTACCGGATCCTTCGCAAGGTTTGGT-3′ (SEQ ID NO. 8). Example 4

[0027] Genetic transformation of EjHB7 The flower dipping method was used to soak the inflorescence of Arabidopsis thaliana with Agrobacterium carrying the overexpression vector of the EjHB7 gene. The transgenic Arabidopsis thaliana seeds were continuously screened on MS medium containing 50 mg / L kanamycin until T3 generation seeds were obtained. After growing on the screening medium for 10 days, the T3 generation seeds were transplanted into the soil to obtain homozygous positive seedlings, which were verified at the DNA level (PCR) and RNA level (qRT-PCR) after 4 weeks.

[0028] The constructed Agrobacterium strain carrying pTRV2-EjHB7 was propagated in large quantities and the OD of the bacterial solution was 600Adjust to 0.8-1.0 respectively, mix the Agrobacterium carrying pTRV1 and pTRV2-EjHB7 in a ratio of 1:1, resuspend in resuspension solution (10 mMMES, 10 mM MgCl2 and 200 μM acetosyringone), and let stand at room temperature for 3 h. Use Agrobacterium-mediated method to inject the induced bacterial solution into the leaves of Baiyu loquat seedlings that have grown for 60 days to silence the EjHB7 gene. The wild-type control group should be injected with pTRV2 empty vector, and then recover at room temperature in dark for 2-3 days.

[0029] Analysis of EjHB7 expression in gene-silenced loquat: Transgenic lines were identified at the mRNA level, and total RNA from loquat leaves was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN). RNA was extracted from EjHB7 transiently silenced plants and loquat seedlings injected with pTRV2 empty vector (positive control), and then reverse transcribed into cDNA. The reaction system and reaction procedure of reverse transcription were set according to the instructions of HiScript® III RT SuperMix for qPCR (+gDNA wiper) (Vazyme BiotechCo., Ltd, Nanjing). The expression of EjHB7 in transiently silenced plants was detected by qRT-PCR. The instrument used was Bio-Rad CFX Opus 96 (Bio-Rad), and the reaction system and reaction procedure of qRT-PCR were set according to the instructions of the fluorescent real-time quantitative dye Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing). EjActin from loquat was selected as the internal reference gene, and 2 -△△Ct Methods The relative expression of the target gene was calculated. The identified EjHB7 transiently silenced plants and loquat seedlings injected with pTRV2 empty vector were used for subsequent experimental treatments. Example 5

[0030] Transgenic Arabidopsis thaliana detection EjHB7 transgenic Arabidopsis lines were identified at the DNA level. DNA from wild-type and EjHB7 transgenic Arabidopsis plants was extracted using the Wolact Plant Genomic DNA Purification Kit (Wolact, Vicband Life Sciences Company (Hk) Limited). PCR amplification was performed using the cloning primers for the EjHB7 gene. RNA from wild-type and EjHB7 transgenic Arabidopsis was extracted using the Wolact Plant RNA Isolation Kit (Wolact, Vicband Life Sciences Company (Hk) Limited) and then reverse transcribed into cDNA. The expression of EjHB7 in transgenic Arabidopsis plants was detected by qRT-PCR, and Arabidopsis AtActin was used as an internal reference gene. The identified EjHB7 transgenic Arabidopsis and wild-type Arabidopsis were used for subsequent experimental treatments. Example 6

[0031] Plant material and treatment The seeds of wild-type and transgenic Arabidopsis were sterilized and sown in nutrient pots containing mixed substrates and grown in a light incubator for 4 weeks for experimental treatment. Natural drought treatment started with a saturated irrigation, then watering was stopped, and the phenotypes of wild-type and transgenic Arabidopsis plants after drought treatment were observed.

[0032] Three transgenic silenced strains (pTRV-1, pTRV-2 and pTRV-3) at 60 days old and wild-type loquat (WT) transformed with an empty vector (pTRV2) were selected as one group, and a total of 4 groups were set up. They were then subjected to drought stress to determine the drought resistance of loquat seedlings after EjHB7 gene silencing. Example 7

[0033] Histochemical staining and physiological index determination: The Arabidopsis thaliana in the control group and the drought-treated group were stained with nitrotetrazolium (NBT) and diaminobenzidine (DAB) to observe the superoxide anion (O2 - ) and hydrogen peroxide (H2O2) accumulation.

[0034] NBT staining method for detecting O2 - : NBT working solution: 1 mg / mL dissolved in Hepes buffer (100 mM, pH 7.5), add 1 / 1000 TritonX-100.

[0035] The material was immersed in NBT working solution and dyed with light for 2 hours. It was rinsed with distilled water, decolorized in 80% ethanol at 60℃ water bath for 2 hours, and continued to decolorize with 80% ethanol, and finally stored in 10% (v / v) glycerol for observation and photography.

[0036] DAB staining method for detecting H2O2: DAB working solution: 1 mg / mL dissolved 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 before use.

[0037] Dyeing: Immerse the material in DAB working solution for dyeing, and decolorize when brown spots appear. The decolorization is the same as NBT.

[0038] The malondialdehyde (MDA) content kit, hydrogen peroxide test kit and superoxide anion kit were used to determine the contents of malondialdehyde, hydrogen peroxide and superoxide anion in Arabidopsis and loquat according to the manufacturer's instructions (all kits were purchased from Suzhou Keming Biotechnology Co., Ltd.). The proline content determination kit, superoxide dismutase (SOD) kit instructions (WST-8 method) and peroxidase (POD) test kit were used to determine the activities of proline, POD and SOD enzymes in Arabidopsis according to the manufacturer's instructions (all kits were purchased from Suzhou Keming Biotechnology Co., Ltd.).

[0039] EjHB7 overexpression improves tolerance of Arabidopsis plants to drought stress:

[0040] To evaluate the effect of EjHB7 on drought tolerance, we subjected transgenic Arabidopsis plants to natural drought treatment (Figure 6A). After 10 days of natural drought, the leaves of WT Arabidopsis began to wilt, while the leaves of the three transgenic plants (L2, L4, and L6) remained vigorous (Figure 2C). After 15 days of drought treatment, the leaves of both WT and EjHB7 transgenic plants wilted severely. However, compared with the leaves of WT plants, the leaves of EjHB7 overexpressing Arabidopsis lines wilted less (Figure 2C).

[0041] Given the different phenotypes of WT and EjHB7 transgenic Arabidopsis plants under drought, we further evaluated their damage under drought stress with the help of several physiological indices. Under normal growth conditions, there was no significant difference in the MDA content between WT and transgenic Arabidopsis. After drought treatment, the MDA content of EjHB7-overexpressing Arabidopsis leaves was lower than that of WT, with an average decrease of 56.42% (Figure 3A). In addition, under drought stress, the proline content of both WT and EjHB7 transgenic plants increased significantly. However, the proline content in the leaves of EjHB7-overexpressing Arabidopsis increased more, with an average increase of 3.08 times (Figure 3B). These results indicate that the ectopic expression of EjHB7 in Arabidopsis plants enhances their tolerance to drought stress.

[0042] EjHB7 stimulates the scavenging of reactive oxygen species under drought conditions: To evaluate the expression of reactive oxygen species (H2O2 and O2 - ) accumulation, after 10 days of drought treatment, the Arabidopsis plants in the control group and the drought treatment group were subjected to DAB and NBT staining. Figure 4 As shown in Figure 2, no significant difference was observed between WT and EjHB7 transgenic Arabidopsis under normal conditions. After 10 days of drought, darker staining was observed in the leaves of both WT and EjHB7 transgenic Arabidopsis, indicating that ROS accumulated to higher levels under drought conditions. However, the extent of staining indicated that H2O2 and O2 - The accumulation of Figure 4 A and B). H2O2 and O2 - The results of content determination were consistent with the staining results: H2O2 and O2 in EjHB7 overexpressed Arabidopsis - The contents were much lower than those of WT, decreasing by 79.26% and 59.3% respectively ( Figure 4 C and D).

[0043] In plants, POD and SOD enzymes scavenge ROS such as H2O2 and O2 - To improve the drought tolerance 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, increasing by 82.95% and 2 times ( Figure 5 ). This result suggests that EjHB7 can promote the degradation of H2O2 and O2 by regulating the activities of POD and SOD antioxidant enzymes. - of removal.

[0044] Transient silencing of the EjHB7 gene reduces drought tolerance in loquat: To further clarify the function of EjHB7 in loquat drought stress, virus-induced gene silencing (VIGS) was used to inhibit the expression of EjHB7 in loquat. qRT-PCR analysis of 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 empty-injected control loquat seedlings (WT) for 20 days under drought stress, leaves of EjHB7-silenced plants wilted more severely than those of the control group (Figure 6A). MDA content indicated that cells in EjHB7-silenced plants were more severely damaged compared with WT ( Figure 6 C).

[0045] Effects of H2O2 and O2 on EjHB7-silenced loquat plants (pTRV-1, pTRV-2, and pTRV-3) and control loquat seedlings (WT) - The results were opposite to those of Arabidopsis. - The accumulation of Figure 6 D, E). These results show that EjHB7 silenced plants are more sensitive to drought stress than controls. This material can be used as a reference system for evaluating the stability and adaptability of other breeding materials or varieties. In addition, it is an important experimental material in the field of gene editing and can be used as a site for gene editing, which is helpful for the process of molecular breeding.

[0046] EjHB7 transgenic Arabidopsis plants were obtained through genetic transformation technology. Drought treatment found that under normal conditions, there was no significant difference in growth conditions and physiological indicators between WT (wild-type Arabidopsis) and EjHB7 transgenic Arabidopsis. However, under drought treatment, the wilting degree of overexpressed transgenic Arabidopsis leaves was less than that of WT. EjHB7 reduces reactive oxygen species H2O2 and O2 by increasing the activity of POD and SOD enzymes. - The accumulation of EjHB7 improves the drought tolerance of transgenic Arabidopsis. In addition, the drought tolerance of transgenic loquat plants was reduced after EjHB7 was silenced. The above results indicate that EjHB7 plays an important role in regulating plant drought tolerance.

Claims

1. A loquat EjHB7 protein, characterized in that: The protein is a) or b) as follows: a) a protein consisting of the amino acid sequence shown in SEQ ID No.2; b) A protein derived from SEQ ID No. 2 which is related to the root development of loquat and in which one or more amino acid residues are substituted and / or added and / or deleted.

2. A loquat HkDJ A gene characterized by The gene encodes the protein according to claim 1.

3. The loquat according to claim 2 HkDJ A gene characterized by The nucleotide sequence of the gene is shown as SEQ ID No.

1.

4. The loquat EjHB7 protein according to claim 1 or the loquat EjHB7 protein according to claim 2 HkDJ Application of genes in regulating plant drought tolerance.

5. The use according to claim 4, characterized in that: The regulating plant drought tolerance comprises: regulating the wilting degree of leaves of the plant under drought stress conditions.

6. The use according to claim 4, characterized in that: The regulating plant drought tolerance comprises: regulating the content of MDA, proline and active oxygen in plants under drought stress conditions.

7. The use according to claim 4, characterized in that: The content of reactive oxygen species is regulated by regulating the activity of POD and SOD enzymes in plants under drought stress conditions.

8. A method for regulating plant drought tolerance, characterized in that: The following steps are involved: Will HkDJ The CDS sequence of the gene is constructed into a plant expression vector and transformed into the target plant to obtain a transgenic plant.

9. The method for regulating plant drought tolerance according to claim 8, characterized in that: The plant is Arabidopsis thaliana or loquat.

10. The method for regulating plant drought tolerance according to claim 8, characterized in that The primer sequences used for vector construction are shown in SEQ ID NOs. 5 and 6.

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