Application of white birch BpSPL2 gene in improving resistance of white birch to black spot disease

Abstract

The present application relates to the technical field of plant genetic engineering, and provides application of a white birch BpSPL2 gene in improving the black spot disease resistance of white birch, improves the black spot disease resistance of white birch by inhibiting the expression of the white birch BpSPL2 gene, and further promotes the response of forest plants to biological stress, and ensures the cultivation of high-quality forest plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the birch BpSPL2 gene in improving the birch's resistance to black spot disease. Background Technology

[0002] SPL (Squamosa promoter-binding protein-like) transcription factors are a class of plant-specific transcription factors found in all green plants, including unicellular algae, mosses, gymnosperms, and angiosperms. SPL transcription factors were first cloned by Huijser et al. from snapdragon (Antirrhinum majus), where they discovered two closely related genes, AmSBP1 and AmSBP2, which bind to the promoter of the floral meristem characteristic gene SQUAMOSA (SQUA), and were named Squamosa promoter-binding proteins (SBP). Subsequently, cDNA cloning of the SBP transcription factor family was performed in Arabidopsis and mosses, revealing multiple copies of SBP transcription factor genes in these two plants. In recent years, similar genes have also been found in birch, maize, tomato, and strawberry. Studies have shown that SPL participates in regulating several important biological processes, such as developmental stage transitions, flower development, leaf growth, plant height and tillering, plant stress responses, and the maintenance of copper ion homeostasis, among other developmental and physiological and biochemical processes. Studies have shown that overexpression of AtSPL3 can advance flowering in Arabidopsis thaliana, resulting in an early-flowering phenotype. Most miR156-targeted SPL genes directly activate MADS-box genes that promote flower development, including SOC1 and AP1, thus advancing flowering. Arabidopsis AtSPL9 and AtSPL10 can upregulate miR172 expression, subsequently suppressing the miR172 target gene—TOE1 / 2—and promoting the transition from juvenile to adult stages. Arabidopsis thaliana spl8 mutants have smaller anthers and reduced fertility; further research has found that miR156-targeted SPL genes can co-regulate early anther cell division and differentiation with SPL8. Rice OsSPL14 and OsSPL16 play a decisive role in regulating grain size, shape, and color. Arabidopsis thaliana SPL8 plays a local regulatory role in GA-dependent development; overexpression of AtSPL8 in transgenic plants alters the GA response, leading to anther dehiscence and reduced fertility. Furthermore, overexpression of the AtSPL8 gene showed insensitivity to GA treatment; for example, GA treatment reduced seed germination rate and inhibited seedling root elongation. Copper ions are an important element for maintaining plant growth and development. Under copper deficiency, the AtSPL7 gene plays a positive role in maintaining copper homeostasis in plants. Regarding biotic stress, in rice, OsSPL9 can specifically bind to the miR528 promoter and activate its expression. Loss of OsSPL9 function leads to a significant reduction in miR528 accumulation and an accumulation of ascorbic acid oxidase gene AO ​​transcription levels, thereby enhancing plant resistance to rice stripe virus (RSV). Overexpression of OsSPL9, however, exhibits the opposite phenotype, indicating that OsSPL9 transcriptional activation of miR528-AO regulates RSV resistance in rice.OsSPL4 positively regulates rice blast resistance. The NtabSPL6-2 gene plays a role in resisting pathogens. After inoculation with Pseudomonas syringae, NtabSPL6-2 transgenic Arabidopsis thaliana showed milder symptoms of disease and exhibited a certain degree of resistance. The same phenomenon was observed after inoculation with Botrytis cinerea.

[0003] Currently, research on SPL genes mainly focuses on herbaceous plants such as Arabidopsis thaliana, rice, and alfalfa. There are few reports on the function of SPL genes in birch, which has a longer growth cycle, and there is no information on the function of the birch BpSPL2 gene in improving birch resistance to black spot disease. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the birch BpSPL2 gene in improving the birch's resistance to black spot disease. By inhibiting the expression of the birch BpSPL2 gene, the birch's resistance to black spot disease is improved, thereby promoting the response of forest plants to biological stress and ensuring the cultivation of high-quality forest trees.

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

[0006] This invention provides an application of the birch BpSPL2 gene in improving the birch's resistance to black spot disease.

[0007] As a preferred method, inhibiting the expression of the BpSPL2 gene in birch can enhance its resistance to black spot disease.

[0008] Preferably, the application includes the following steps:

[0009] (1) Construct the BpSPL2 gene repression expression vector pROKII-BpSPL2-SRDX;

[0010] (2) The vector pROKII-BpSPL2-SRDX obtained in step (1) was transformed into birch to obtain pROKII-BpSPL2-SRDX transgenic birch.

[0011] (3) Transgenic birch was cultivated, and the stem segment with the terminal bud at the top of the transgenic birch tissue culture seedling was inserted into WPM rooting medium for culture.

[0012] Preferably, the stem segment is 2-4.5 cm in length.

[0013] Preferably, the WPM rooting medium uses distilled water as a solvent and comprises the following final concentration components: WPM powder 1.8-2.5 g / L, sucrose 17-23 g / L, calcium salt 0.3-0.8 g / L, indolebutyric acid (IBA) 0.2-0.7 mg / L, and plant agar 5-10 g / L.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects:

[0015] This invention improves the resistance of birch to black spot disease by inhibiting the expression of the BpSPL2 gene. The invention also provides transgenic birch plants with overexpression and inhibition of the BpSPL2 gene, which are involved in birch leaf disease resistance. These transgenic plants can also be used to discover downstream regulatory genes and enrich the regulatory pathways of plants in response to biotic stress. The BpSPL2 gene-inhibited birch plant 35S::BpSPL2-SRDX obtained in this invention exhibits enhanced leaf disease resistance and can be used to cultivate high-quality forest trees. Furthermore, the examples also show that the BpSPL2 gene participates in the physiological process of birch disease resistance and negatively regulates birch bud germination; compared with wild-type plants, the overexpression line has weaker leaf disease resistance, while the inhibited expression line has stronger leaf disease resistance. Attached Figure Description

[0016] Figure 1 Classification of birch black spot disease severity;

[0017] Figure 2 The overall growth status of BpSPL2 overexpressing, inhibited expression transgenic plants and wild-type plants;

[0018] Figure 3 The lateral branch and leaf status of BpSPL2 overexpressing, suppressed expression transgenic plants and wild-type plants;

[0019] Figure 4 Statistics on the number of diseased leaves in BpSPL2 overexpressing, suppressed-expressing transgenic plants and wild-type plants;

[0020] Figure 5 Comparison of leaf diseases between BpSPL2 overexpression, suppressed expression transgenic plants and wild-type plants;

[0021] Figure 6 The number of diseased leaf spots in transgenic plants with BpSPL2 overexpression, suppressed expression, and wild-type plants was statistically analyzed. Detailed Implementation

[0022] This invention provides an application of the birch BpSPL2 gene in improving the birch's resistance to black spot disease.

[0023] In this invention, the ability of birch to resist black spot disease is improved by inhibiting the expression of the birch BpSPL2 gene.

[0024] In this invention, the application includes the following steps:

[0025] (1) Construct the BpSPL2 gene repression expression vector pROKII-BpSPL2-SRDX;

[0026] (2) The vector pROKII-BpSPL2-SRDX obtained in step (1) was transformed into birch to obtain pROKII-BpSPL2-SRDX transgenic birch.

[0027] (3) Transgenic birch was cultivated, and the stem segment with the terminal bud at the top of the transgenic birch tissue culture seedling was inserted into WPM rooting medium for culture.

[0028] In this invention, the preferred temperature for cultivating transgenic birch is 22-26℃, more preferably 23-25℃; the preferred relative humidity for cultivating transgenic birch is 65-75%, more preferably 68-73%. The preferred light intensity for cultivating transgenic birch in this invention is 40-50 μmol / m². 2 / s, further preferably 44-48 μmol / m 2 / s, and more preferably 46 μmol / m 2 / s; The preferred photoperiod for cultivating transgenic birch is 16h light / 8h darkness.

[0029] In this invention, the length of the stem segment is preferably 2-4.5 cm, more preferably 2.5-4 cm, and even more preferably 3 cm. The stem segment is cultured in WPM rooting medium for 30-50 days, more preferably 35-45 days, and even more preferably 40 days.

[0030] In this invention, the WPM rooting medium uses distilled water as a solvent and preferably comprises the following final concentration components: WPM powder 1.8-2.5 g / L, sucrose 17-23 g / L, calcium salt 0.3-0.8 g / L, indolebutyric acid 0.2-0.7 mg / L, and plant agar 5-10 g / L. More preferably, it comprises WPM powder 1.9-2.3 g / L, sucrose 18-22 g / L, calcium salt 0.4-0.6 g / L, indolebutyric acid 0.3-0.6 mg / L, and plant agar 6-9 g / L. Even more preferably, it comprises WPM powder 2.14 g / L, sucrose 20 g / L, calcium salt 0.56 g / L, indolebutyric acid 0.4 mg / L, and plant agar 8 g / L. The pH value of the WPM rooting medium in this invention is preferably 5.5-6, more preferably 5.6-5.9, and even more preferably 5.8. In this invention, 5M NaOH is preferably used to adjust the pH value.

[0031] In this invention, tissue culture seedlings obtained after cultivation in WPM rooting medium are transplanted into a soil substrate for further cultivation. The soil substrate is preferably a mixture of vermiculite and potting soil (purchased from the Harbin Flower Market) at a mass ratio of 1:(0.5-1.5), more preferably 1:(0.8-1.2), and even more preferably 1:1. Tissue culture seedlings cultured from stem segments with terminal buds exhibit a higher survival rate when subsequently transplanted into soil.

[0032] 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.

[0033] Example 1

[0034] The BpSPL2 gene repression expression vector pROKII-BpSPL2-SRDX and the overexpression vector pGWB5-BpSPL2-GFP were constructed. Then, the two expression vectors were transformed into birch using the leaf disc transformation method to obtain pROKII-BpSPL2-SRDX transgenic birch plants (hereinafter referred to as 35S::BpSPL2-SRDX) and pGWB5-BpSPL2-GFP transgenic birch plants (hereinafter referred to as 35S::BpSPL2). For specific operations, please refer to the patent document with publication number CN 111690664A.

[0035] In patent document CN 111690664A, OE3 and OE5 represent the overexpression of transgenic birch 35S::BpSPL2-3 and 35S::BpSPL2-5, respectively; DR1 and DR4 represent the inhibition of transgenic birch expression, hereinafter referred to as 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4.

[0036] Stem segments with apical buds, taken from the top of 35S::BpSPL2-3, 35S::BpSPL2-5, 35S::BpSPL2-SRDX-1, and 35S::BpSPL2-SRDX-4 transgenic birch tissue culture seedlings and wild-type birch (WT) tissue culture seedlings, were inserted into WPM rooting medium and cultured for 40 days. The growth conditions were: temperature 24±2℃, relative humidity 65-75%, and light intensity 46 μmol / m². 2 The photoperiod is 16 hours of light followed by 8 hours of darkness. The WPM rooting medium system uses distilled water as a solvent and includes 2.14 g / L WPM powder, 20 g / L sucrose, 0.56 g / L calcium salt, 0.4 mg / L IBA, and 8 g / L plant agar. The pH is adjusted to 5.8 with 5M NaOH, and the medium is dispensed into 100 mL bottles for later use.

[0037] Transgenic seedlings grown under normal culture conditions for 40 days were transplanted into a soil substrate of vermiculite:potting soil = 1:1 (Note: the culture medium on the roots of the seedlings must be thoroughly rinsed off before transplanting). They were then cultured indoors in soil (vermiculite:potting soil = 1:1) until the seedlings stabilized (surviving and growing normally after transplanting from the culture medium to the soil substrate). Once the birch seedlings were stable, they were transferred to larger soil pots and moved outdoors (to a natural environment) for further cultivation.

[0038] Example 2

[0039] We conducted follow-up observations on BpSPL2 transgenic and wild birch plants transplanted outdoors, and observed and statistically analyzed the overall growth of different groups of plants in autumn (September). We compared leaf diseases of BpSPL2 transgenic and wild birch plants, and then classified them according to the number of lesions on the leaves (as shown in Figure 1).

[0040] (I) Analysis of the number of diseased leaves in different plants

[0041] like Figure 2 As shown, the birch plants in the suppressed expression groups 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4 were in the best overall condition, with good growth and very few diseased leaves; the wild-type birch plants were in the second best condition, with weaker growth and a relatively higher number of diseased leaves, including yellow and withered leaves; the birch plants in the overexpression groups 35S::BpSPL2-3 and 35S::BpSPL2-5 were in the worst overall condition, with weak growth, a significantly higher number of diseased leaves, and a significant increase in the number of yellow and withered leaves.

[0042] Regarding the condition of lateral branches and leaves of birch plants, the lateral branches and leaves of the suppressed expression groups 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4 were intact, with very few diseased or yellowed leaves, and mainly infected with level 1 diseases, with no level 3 or 4 diseases. The lateral branches and leaves of wild-type birch were mostly intact, with a relatively higher number of diseased and yellowed leaves, and all four levels of diseases were present, with level 1 and level 2 diseases being relatively more prevalent. The lateral branches and leaves of overexpressed 35S::BpSPL2-3 and 35S::BpSPL2-5 showed significant damage, with a large number of diseased and yellowed leaves, and all four levels of diseases were present, with level 2 and level 3 diseases being relatively more prevalent, followed by level 4 diseases (e.g., ...). Figure 3 and Figure 4 ).

[0043] (II) Analysis of the degree of disease infection in the leaves of different plants

[0044] Depend on Figure 5 and Figure 6The comparison of leaf diseases among different groups of birch plants showed that the birch plants overexpressing 35S::BpSPL2-3 and 35S::BpSPL2-5 had the most severe leaf diseases and the largest number of lesions; while the birch plants with suppressed expression of 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4 had the mildest leaf diseases and the fewest lesions.

[0045] As can be seen from the above embodiments, the present invention provides transgenic birch plants that participate in the disease resistance of birch leaves by overexpression and suppression of the BpSPL2 gene. The results show that the 35S::BpSPL2-SRDX birch plant with suppressed BpSPL2 gene expression enhances the disease resistance of the leaves and improves the resistance of birch to black spot disease by suppressing the expression of the birch BpSPL2 gene.

[0046] 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. The application of inhibiting BpSPL2 gene expression in improving the resistance of birch to black spot disease, characterized in that, The nucleic acid sequence of the birch BpSPL2 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application includes the following steps: (1) Construct the BpSPL2 gene repression expression vector pROKII-BpSPL2-SRDX; (2) The vector pROKII-BpSPL2-SRDX obtained in step (1) was transformed into birch to obtain pROKII-BpSPL2-SRDX transgenic birch. (3) Transgenic birch was cultivated, and the stem segment with the terminal bud at the top of the transgenic birch tissue culture seedling was inserted into WPM rooting medium for culture.

3. The application according to claim 2, characterized in that, The stem segment is 2-4.5 cm in length.

4. The application according to claim 2, characterized in that, The WPM rooting medium uses distilled water as a solvent and comprises the following final concentration components: 2.14 g / L WPM powder, 20 g / L sucrose, 0.56 g / L calcium salt, 0.4 mg / L indolebutyric acid, and 8 g / L plant agar.

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