Application of birch BpPIF4 gene in improving drought resistance of birch

Abstract

The application provides application of a white birch BpPIF4 gene in improving drought resistance of the white birch and belongs to the technical field of molecular biology.The application constructs an overexpression vector of the BpPIF4 gene, carries out genetic transformation on the white birch by using an agrobacterium infection method, screens a transgenic line overexpressing the BpPIF4 gene, and realizes overexpression of the white birch BpPIF4 gene.The application enhances the drought resistance of the white birch by overexpressing the white birch BpPIF4 gene, and can be used for cultivating forest trees with excellent quality.The BpPIF4 gene provided by the application can participate in drought resistance of the white birch, can be used for research on biological stress response of forest plants, and provides a reference resource for research on functions of a plant bHLH gene family.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to the application of the birch BpPIF4 gene in improving the drought resistance of birch. Background Technology

[0002] Phytochrome-interacting factors (PIFs) were first identified in Arabidopsis thaliana in 1998, and members of the PIF transcription factor family have since been isolated from various plants, including rice, maize, tomato, and apple. Studies have found that PIF transcription factors participate in multiple biological pathways. Besides regulating photomorphogenesis, responding to stress environments, and participating in chlorophyll metabolism, PIFs can also respond to hormonal signals. For example, in the model crop Arabidopsis thaliana, AtPIF1 participates in light-induced seed germination, inhibits hypocotyl elongation, and negatively regulates chlorophyll synthesis. AtPIF3 has been reported to participate in light response, seed germination, chlorophyll and anthocyanin biosynthesis, and frost resistance. AtPIF4 positively regulates stomatal development. AtPIF4, AtPIF5, and AtPIF7 influence hypocotyl elongation under photoperiod regulation. PIF4 and PIF5 play a positive regulatory role in the shade avoidance response by directly controlling auxin biosynthesis and auxin signaling components, but negatively regulate anthocyanin accumulation by inhibiting the transcription of anthocyanin biosynthesis genes or upstream transcriptional regulators. Recently, PIF4 has been reported to participate in the regulation of mediator components Mediator25 (MED25) and HISTONEDEACETYLASE9 (HDA9) of thermomorphogenesis in Arabidopsis, demonstrating its key role in the thermo-response network. Furthermore, PIF homologs have been identified and functionally analyzed in other plants. MdPIF1 has been shown to negatively regulate Phy-mediated inhibition of seed germination and hypocotyl elongation. In pepper, CaPIF8 plays different regulatory roles in low-temperature and salt stress responses by promoting CBF1 expression and ABA biosynthesis, respectively. Genome-wide identification of the sweet potato PIF family revealed that IbPIF3.1 is involved in both drought and drought stress resistance to Fusarium wilt. ZmPIF1 and ZmPIF3 have been found to play important roles in ABA-mediated drought resistance in maize. PIF4 interacts with BZR to mediate the interaction between light and brassinolide signals, participating in plant light response. In tobacco, NtPIF1 negatively regulates drought resistance by inhibiting ABA and carotenoid biosynthesis. PIF4 belongs to the basic Helix-Loop-Helix (bHLH) subfamily 15 and is an important plant photomorphogenesis inhibitor, mainly binding to PHYB to mediate light signal transduction. It can respond to changes in light quality and regulate plant growth and development by modulating plant hormone synthesis, metabolism, and signal transduction. BpPIF4 is a gibberellin-related gene selected from the birch transcriptome. Physiological data analysis based on drought treatment of this gene showed that it has a positive regulatory function on drought resistance in birch.

[0003] Currently, research on PIF genes mainly focuses on herbaceous plants such as Arabidopsis thaliana, rice, and tobacco. There are few reports on the functions of PIF genes in birch, which has a longer growth cycle, and there is no information on the function of the birch BpPIF4 gene in improving the drought resistance of birch. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an application of the birch BpPIF4 gene in improving the drought resistance of birch.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an application of the birch BpPIF4 gene in altering the drought resistance of plants. Overexpression of the birch BpPIF4 gene in plants enhances their drought resistance; or inhibition of the birch BpPIF4 gene expression in plants reduces their drought resistance.

[0007] Preferably, the method for overexpressing the birch BpPIF4 gene includes the following steps:

[0008] An overexpression vector for the BpPIF4 gene was constructed, and the birch was genetically transformed using the Agrobacterium infection method. Transgenic lines that overexpress the BpPIF4 gene were screened to achieve overexpression of the BpPIF4 gene in birch.

[0009] Preferably, the overexpression vector of the BpPIF4 gene is pBI121-BpPIF4-GFP, wherein the pBI121-BpPIF4-GFP uses pBI121-GFP as the initial vector and clones the BpPIF4 gene fragment between the XbaI and XmaI enzyme restriction sites of pBI121-GFP.

[0010] Preferably, the method for inhibiting the expression of the birch BpPIF4 gene includes the following steps:

[0011] A vector to suppress BpPIF4 gene expression was constructed, and birch was genetically transformed using Agrobacterium infection. Transgenic lines that suppress BpPIF4 gene expression were screened, thus achieving the suppression of BpPIF4 gene expression in birch.

[0012] Preferably, the vector for inhibiting the expression of the birch BpPIF4 gene is pBI121-BpPIF4-SRDX. The pBI121-BpPIF4-SRDX uses pBI121-GFP as the initial vector and clones the BpPIF4 gene fragment and the added SRDX fragment between the SpeI and XmaI enzyme restriction sites of pBI121-GFP.

[0013] The present invention also provides the application of the overexpression vector or the vector that inhibits the expression of the birch BpPIF4 gene in altering the drought resistance of plants.

[0014] Preferably, the plant is birch.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention provides overexpression and repression vectors for the birch BpPIF4 gene. After genetic transformation of birch using Agrobacterium infection, these vectors are applied to the regulation of drought resistance in the plants. This invention enhances the drought resistance of birch by overexpressing the birch BpPIF4 gene, which can be used to cultivate high-quality forest trees. The BpPIF4 gene provided by this invention can participate in the drought resistance of birch and can be used in research on forest plants' response to biotic stress, and provides a reference resource for the functional study of the plant bHLH gene family. Attached Figure Description

[0017] Figure 1 The growth status of BpPIF4 overexpressing and suppressed transgenic plants and wild-type birch after drought stress treatment;

[0018] Figure 2 It is the activity of superoxide dismutase (SOD) after drought stress treatment;

[0019] Figure 3 It refers to the peroxidase (POD) activity after drought stress treatment;

[0020] Figure 4 It refers to the catalase (CAT) activity after drought stress treatment;

[0021] Figure 5 It is the endogenous ABA content after drought treatment;

[0022] Figure 6 It is superoxide anion (O) after drought treatment 2- )content;

[0023] Figure 7 It refers to the malondialdehyde (MDA) content after drought treatment. Detailed Implementation

[0024] This invention provides an application of the birch BpPIF4 gene in altering the drought resistance of plants. Overexpression of the birch BpPIF4 gene in plants enhances their drought resistance; alternatively, inhibition of the birch BpPIF4 gene expression reduces its drought resistance. The plant is birch. The nucleotide and amino acid sequences of the birch BpPIF4 gene are described in patent literature (CN114875043B).

[0025] In this invention, the method for overexpressing the birch BpPIF4 gene includes the following steps:

[0026] An overexpression vector for the BpPIF4 gene was constructed, and *Birchia speciosa* was genetically transformed using the *Agrobacterium* infection method. Transgenic lines overexpressing the BpPIF4 gene were screened, thus achieving overexpression of the BpPIF4 gene in *Birchia speciosa*. The overexpression vector for the BpPIF4 gene was pBI121-BpPIF4-GFP, which used pBI121-GFP as the initial vector and cloned the BpPIF4 gene fragment between the XbaI and XmaI restriction enzyme sites of pBI121-GFP.

[0027] In this invention, the method for inhibiting the expression of the birch BpPIF4 gene includes the following steps:

[0028] A vector to suppress BpPIF4 gene expression was constructed, and *Birchia speciosa* was genetically transformed using the *Agrobacterium* infection method. Transgenic lines suppressing the BpPIF4 gene were screened to achieve suppressed expression of the *Birchia speciosa* BpPIF4 gene. The vector suppressing *Birchia speciosa* BpPIF4 gene expression was pBI121-BpPIF4-SRDX. pBI121-BpPIF4-SRDX used pBI121-GFP as the initial vector, and a BpPIF4 gene fragment and an added SRDX fragment were cloned between the Spe I and XmaI restriction sites of pBI121-GFP. The amino acid sequence of the SRDX fragment was DLDLELRLG (SEQ ID NO:1).

[0029] The present invention also provides the application of the overexpression vector or the vector that inhibits the expression of the birch BpPIF4 gene in altering the drought resistance of plants; the plant is preferably birch.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] Construction of transgenic birch plants:

[0033] The constructed BpPIF4 gene overexpression vector pBI121-BpPIF4-GFP and repressor expression vector pBI121-BpPIF4-SRDX were transformed into birch using the leaf disc transformation method, resulting in pBI121-BpPIF4-GFP transgenic birch plants (35S::BpPIF4) and pBI121–BpPIF4-SRDX transgenic birch plants (35S::BpPIF4-SRDX). (For the construction methods of the overexpression vector pBI121-BpPIF4-GFP and the repressor expression vector pBI12-BpPIF4-SRDX, as well as the methods for transforming birch to obtain pBI121-BpPIF4-GFP and pBI121-BpPIF4-SRDX transgenic birch plants, please refer to patent document CN114875043B).

[0034] Example 2

[0035] Cultivation of transgenic birch:

[0036] 1. Transgenic birch was subcultured using WPM rooting medium (growth conditions were approximately 24±2℃, relative humidity 65-75%, and light intensity and photoperiod set at 46 μmol·m). -2 ·s -1 (with 16h light / 8h darkness), the culture medium system is as follows: WPM powder 2.14g / L, sucrose 20g / L, calcium salt 0.56g / L, IBA 0.4mg / L, plant agar 8g / L, pH adjusted to 5.8 with 5M NaOH, and dispensed into 100mL / bottle.

[0037] 2. Cut stem segments with apical buds, about 3 cm long, from wild-type birch, 35S::BpPIF4-23, 35S::BpPIF4-25, 35S::BpPIF4-SRDX-A, and 35S::BpPIF4-SRDX-D (the strains identified in previously authorized patents, see patent document CN114875043B), transgenic birch tissue culture seedlings, and insert them into WPM rooting medium for 40 days.

[0038] 3. Transplant transgenic seedlings grown under normal culture conditions (subculture conditions) for 40 days into a soil substrate of vermiculite:potting soil = 1:1 (Note: the culture medium on the roots of the seedlings must be thoroughly rinsed before transplanting). Indoor soil conditions were maintained (light intensity and photoperiod were 46 μmol·m²). -2 ·s -1 Cultivate the seedlings in a soil substrate (16 hours of light / 8 hours of darkness; vermiculite:potting soil = 1:1) until they stabilize (they survive and grow normally after being transplanted from the culture medium to the soil substrate). Once the birch seedlings are stable, transplant them into larger soil pots for further cultivation.

[0039] Example 3

[0040] Drought stress treatment of transgenic lines:

[0041] Three-month-old wild-type birch, 35S::BpPIF4-23, 35S::BpPIF4-25, 35S::BpPIF4-SRDX-A, and 35S::BpPIF4-SRDX-D transgenic birch seedlings with good growth were selected for drought treatment. All seedlings were thoroughly watered the day before the drought stress. Afterward, the control group was watered normally every day, while the drought stress group was not watered. Significant changes in the plants were observed after 23 days of treatment.

[0042] Experimental results: such as Figure 1 As shown. By Figure 1 It can be seen that the two 35S::BpPIF4-SRDX suppressed expression plants were the first to show leaf wilting, followed by the wild type. However, the two 35S::BpPIF4 overexpression plants did not show significant changes. After drought treatment, the degree of plant damage was 35S::BpPIF4-SRDX suppressed expression birch plants > wild type > 35S::BpPIF4 overexpression birch plants.

[0043] After the observation was completed, the materials were collected for physiological index testing.

[0044] 1. Superoxidase (SOD) activity assay. Refer to the instructions for the Superoxidase (SOD)-WST-8 Activity Assay Kit from Suzhou Greens Biotechnology Co., Ltd. for specific assay methods.

[0045] Experimental results: such as Figure 2 As shown.

[0046] 2. Peroxidase (POD) activity assay

[0047] For specific detection methods, please refer to the instructions for the Peroxidase (POD) kit from Suzhou Greens Biotechnology Co., Ltd.

[0048] Experimental results: such as Figure 3 As shown.

[0049] 3. Detection of catalase (CAT) activity

[0050] For specific detection methods, please refer to the instruction manual for the peroxidase (CAT) kit from Suzhou Greens Biotechnology Co., Ltd.

[0051] Experimental results: such as Figure 4 As shown.

[0052] 4. Detection of abscisic acid (ABA) content

[0053] For specific detection methods, please refer to the instruction manual of the Plant Hormone Abscisic Acid (ABA) Enzyme-Linked Immunosorbent Assay Kit from Shanghai Keqiao Biotechnology Co., Ltd.

[0054] Experimental results: such as Figure 5 As shown.

[0055] 5. Detection of superoxide anion (O2-) content

[0056] For specific detection methods, please refer to the instruction manual for the Oxygen-Free Radical (OFR) kit from Suzhou Greens Biotechnology Co., Ltd.

[0057] Experimental results: such as Figure 6 As shown.

[0058] 6. Detection of malondialdehyde (MDA) content

[0059] For specific testing methods, please refer to the instruction manual for the malondialdehyde (MDA) content test kit from Suzhou Greens Biotechnology Co., Ltd.

[0060] Experimental results: such as Figure 7 As shown.

[0061] Depend on Figure 2-7 The test results showed that before drought stress treatment, the activities of overexpressed, wild-type, and suppressed SOD and CAT were reduced. 2- There were no significant differences among all groups. POD activity was higher in wild-type than in overexpressed and suppressed groups. ABA content decreased sequentially from overexpressed to wild-type and then to suppressed groups. 2- The MDA content in the wild-type was lower than that in the overexpressed and suppressed types. After drought stress treatment, the SOD, POD, CAT activities and ABA content in the 35S::BpPIF4 overexpression line were higher than those in the wild-type, while MDA and O content were lower. 2- The levels of all 35S::BpPIF4 overexpressing plants were lower than those of the wild type, while inhibiting expression yielded the opposite result. This further validated the observed phenotypic changes, and the experimental results consistently demonstrated that birch plants overexpressing 35S::BpPIF4 exhibited stronger tolerance to drought stress.

[0062] As can be seen from the above examples, the BpPIF4 gene is involved in the response of birch to drought stress and positively regulates the resistance of birch to drought stress. It also provides a theoretical basis for using genetic engineering technology to regulate the response of forest trees to drought stress and has great application value.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of a birch BpPIF4 gene in improving drought resistance of birch, characterized in that, Overexpression of the birch BpPIF4 gene in birch enhances the drought resistance of birch.

2. Use according to claim 1, characterized in that, The method for overexpressing the birch BpPIF4 gene includes the following steps: An overexpression vector for the BpPIF4 gene was constructed, and the birch was genetically transformed using the Agrobacterium infection method. Transgenic lines that overexpress the BpPIF4 gene were screened to achieve overexpression of the BpPIF4 gene in birch.

3. Use according to claim 2, characterized in that, The overexpression vector of the BpPIF4 gene is pBI121-BpPIF4-GFP. The pBI121-BpPIF4-GFP uses pBI121-GFP as the initial vector and clones the BpPIF4 gene fragment between the Xba I and Xma I enzyme restriction sites of pBI121-GFP.

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