Plant promoters induced by sphaerotheca fuliginea and uses thereof

Abstract

This invention belongs to the field of plant genetic engineering technology and relates to plant promoters induced by *Sclerotinia sclerotiorum* and their applications. Through gene expression analysis of the rapeseed genome, this invention identified promoters of some members of the BnSABATH gene family that are strongly induced by *Sclerotinia sclerotiorum*, with significantly upregulated gene expression levels. Furthermore, through transformation and verification using an expression vector, it was confirmed that the promoter BnSABATH1p specifically drives gene upregulation under *Sclerotinia sclerotiorum* infection, significantly enhancing plant resistance to *Sclerotinia sclerotiorum* disease. This invention provides a highly efficient and specific inducible regulatory element for improving *Sclerotinia sclerotiorum* resistance in cruciferous crops such as rapeseed, and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to plant promoters induced by Sclerotinia sclerotiorum and their applications. Background Technology

[0002] Rapeseed (Brassica napus) is one of the important oilseed crops in the Brassicaceae family, and it is a major oilseed crop widely planted worldwide. It is also an important winter oilseed crop in China. Brassica napus varieties are characterized by strong resistance, high yield, wide adaptability, and strong tolerance to adverse conditions, making them a major cultivated variety in China.

[0003] Sclerotinia sclerotiorum is a fungal plant disease caused by the necrotic pathogen Sclerotinia sclerotiorum, and it is one of the major threats to rapeseed production in China. This disease can reduce the oil content of rapeseed seeds and alter their fatty acid composition, thus affecting the quality of rapeseed oil. Sclerotinia sclerotiorum has a very wide host range, infecting more than 600 plant species across over 70 families.

[0004] In existing plant disease control technologies, using constitutive promoters to activate the expression of disease resistance genes in disease resistance breeding disrupts the inherent resource allocation balance between plant disease resistance response and growth and development. This leads to a waste of energy and resources generated by photosynthesis, and even the accumulation of toxic metabolites, affecting crop growth and development, and consequently impacting crop yield and quality. Furthermore, complex quantitative traits such as resistance to sclerotinia stem rot, controlled by multiple genes, require the aggregation of multiple resistance genes to achieve better resistance. If these resistance genes are expressed constitutively, they can have a greater negative impact on crop growth and development, even causing plant death. Therefore, identifying and cloning plant promoters induced by Sclerotinia stem rot can provide gene elements for breeding resistance-improved rapeseed varieties, and has significant application prospects. Summary of the Invention

[0005] In view of this, the present invention provides plant promoters induced by Sclerotinia sclerotiorum and their applications, with the aim of solving or at least partially solving the problems in the prior art, and providing efficient and specific inducible regulatory elements for improving the resistance of cruciferous crops such as rapeseed to Sclerotinia sclerotiorum.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a plant promoter induced by Sclerotinia sclerotiorum, the promoter being selected from at least one of BnSABATH1p, BnSABATH2p, BnSABATH3p, BnSABATH4p, BnSABATH5p and BnSABATH6p;

[0008] The nucleotide sequence of the promoter BnSABATH1p is shown in SEQ ID NO.1;

[0009] The nucleotide sequence of the promoter BnSABATH2p is shown in SEQ ID NO.2;

[0010] The nucleotide sequence of the promoter BnSABATH3p is shown in SEQ ID NO.3;

[0011] The nucleotide sequence of the promoter BnSABATH4p is shown in SEQ ID NO.4;

[0012] The nucleotide sequence of the promoter BnSABATH5p is shown in SEQ ID NO.5;

[0013] The nucleotide sequence of the promoter BnSABATH6p is shown in SEQ ID NO.6.

[0014] Preferably, the plant is a cruciferous crop.

[0015] Secondly, the present invention also provides the application of the plant promoter in regulating plant resistance to sclerotinia stem rot.

[0016] Preferably, the regulation is positive regulation; the regulation includes: promoting the expression of plant promoters and improving plant resistance to sclerotinia stem rot.

[0017] More preferably, the plant promoter is promoter BnSABATH1p, whose nucleotide sequence is shown in SEQ ID NO.1.

[0018] More preferably, the promotion of plant promoter expression includes the following steps: preparing a reagent for promoting promoter BnSABATH1p expression, transforming plant plants, and culturing to obtain transgenic plants with increased promoter BnSABATH1p expression.

[0019] More preferably, the reagent used to promote the expression of promoter BnSABATH1p is selected from one of the following (A)-(B):

[0020] (A) Recombinant vector used to induce expression of promoter BnSABATH1p;

[0021] (B) Recombinant microorganisms containing the recombinant vector described in (A).

[0022] More preferably, constructing the recombinant vector includes the following steps:

[0023] (1) Using rapeseed gDNA as a template, PCR amplification was performed using primer pairs BnSABATH1p-F and BnSABATH1p-R to obtain amplified fragment I;

[0024] The nucleotide sequences of BnSABATH1p-F and BnSABATH1p-R are shown in SEQ ID NO.9-10, respectively.

[0025] (2) Mix the amplified fragment I and the coding sequence of gene BnSABATH1 in equal volumes to obtain a gene fragment mixture;

[0026] (3) Using the gene fragment mixture as a template, PCR amplification was performed using primer pairs BnSABATH1p-121H-F and BnSABATH1p-121H-R to obtain amplified fragment II;

[0027] The nucleotide sequences of BnSABATH1p-121H-F and BnSABATH1p-121H-R are shown in SEQ ID NO. 11-12, respectively.

[0028] (4) The amplified fragment II was ligated into the PBI121S linearized vector, which had its 35S promoter sequence removed by double digestion with HindIII and BamHI, via homologous recombination to obtain the recombinant vector.

[0029] More preferably, the transformed plant includes the following steps: transferring the reagent used to promote the expression of promoter BnSABATH1p into the plant via Agrobacterium-mediated transformation.

[0030] Preferably, the plant is a cruciferous crop.

[0031] Thirdly, the present invention also provides a method for cultivating plants with high resistance to sclerotinia stem rot, comprising the following steps: preparing a reagent for promoting the expression of promoter BnSABATH1p, transforming plant plants, and culturing to obtain transgenic plants with increased expression of promoter BnSABATH1p.

[0032] Preferably, the nucleotide sequence of the promoter BnSABATH1p is shown in SEQ ID NO.1.

[0033] Preferably, the reagent used to promote the expression of promoter BnSABATH1p is selected from one of the following (A)-(B):

[0034] (A) Recombinant vector used to induce expression of promoter BnSABATH1p;

[0035] (B) Recombinant microorganisms containing the recombinant vector described in (A).

[0036] More preferably, constructing the recombinant vector includes the following steps:

[0037] (1) Using rapeseed gDNA as a template, PCR amplification was performed using primer pairs BnSABATH1p-F and BnSABATH1p-R to obtain amplified fragment I;

[0038] The nucleotide sequences of BnSABATH1p-F and BnSABATH1p-R are shown in SEQ ID NO.9-10, respectively.

[0039] (2) Mix the amplified fragment I and the coding sequence of gene BnSABATH1 in equal volumes to obtain a gene fragment mixture;

[0040] (3) Using the gene fragment mixture as a template, PCR amplification was performed using primer pairs BnSABATH1p-121H-F and BnSABATH1p-121H-R to obtain amplified fragment II;

[0041] The nucleotide sequences of BnSABATH1p-121H-F and BnSABATH1p-121H-R are shown in SEQ ID NO. 11-12, respectively.

[0042] (4) The amplified fragment II was ligated into the PBI121S linearized vector, which had its 35S promoter sequence removed by double digestion with HindIII and BamHI, via homologous recombination to obtain the recombinant vector.

[0043] Preferably, the plant is a cruciferous crop.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] This invention provides plant promoters induced by *Sclerotinia sclerotiorum* and their applications. It identifies promoter regions of some members of the BnSABATH gene family that are susceptible to *Sclerotinia sclerotiorum* induction. Specifically, the promoter BnSABATH1p can specifically upregulate gene expression under *Sclerotinia sclerotiorum* infection, significantly enhancing plant resistance to *Sclerotinia sclerotiorum*. This invention provides efficient and specific inducible regulatory elements for improving *Sclerotinia sclerotiorum* resistance in cruciferous crops such as rapeseed, and has significant application value. Attached Figure Description

[0046] 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, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 A statistical analysis of the expression levels of some members of the BnSABATH gene family after 24 h of Sclerotinia sclerotiorum induction;

[0048] Figure 2 A statistical analysis chart of lesion length after inoculation with Sclerotinia sclerotiorum;

[0049] Figure 3 This is a statistical analysis graph of the transcriptional level of the gene BnSABATH1. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0051] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this invention, the term "comprising" means "including but not limited to". Various embodiments of the invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0052] The following specific embodiments further illustrate the plant promoter induced by *Sclerotinia sclerotiorum* and its applications. This section further illustrates the content of the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, such as the conditions described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0053] In the following embodiments, the rapeseed plant is the commercially available variety "Zhongyou 821" rapeseed bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, which can be purchased through commercial channels.

[0054] In the following embodiments, the Sclerotinia sclerotiorum is Sclerotinia sclerotiorum strain 1980 UF-70.

[0055] In the following embodiments, the BRAD gene ID represents the gene number in the Brassicaceae Database (http: / / brassicadb.cn).

[0056] In the following embodiments, the specific steps for inducing Sclerotinia infection are described in reference ([1] Girard IJ, Tong C, Becker MG, et al. RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection[J]. Journal of Experimental Botany,2017, 68(18): 5079-5091.).

[0057] In the following embodiments, the PBI121S vector was obtained by the method described in reference ([2] Bai Z, He Y, Huang J, et al. The genetic fusion of thaumatin-like proteins with antimicrobial peptidesor receptor-like kinases at different evolutionary time points contributes to plant resistance against Sclerotinia disease[J]. Plant Physiology and Biochemistry, 2026: 111065.).

[0058] In the following examples, all data are expressed as mean ± standard deviation (Mean ± SD); statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, LLC., USA); one-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for pairwise comparisons if significant differences were found; P < 0.05 indicated statistical significance. "Indicates a significant difference (P < 0.05)" "" indicates a highly significant difference (P < 0.01).

[0059] Example 1: Screening of Sclerotinia sclerotiorum inducible promoters

[0060] To screen promoters in the rapeseed genome that are induced by Sclerotinia sclerotiorum, this invention uses transcriptome data from rapeseed plant leaf tissues 24 hours after Sclerotinia sclerotiorum infection, with transcriptome data from uninoculated plants as a control group, to systematically screen and identify promoters induced by Sclerotinia sclerotiorum. It was found that the expression of some members of the BnSABATH gene family was significantly induced by Sclerotinia sclerotiorum.

[0061] Figure 1 The figure shows a statistical analysis of the expression levels of some members of the BnSABATH gene family after 24 h of Sclerotium sclerotiorum induction. In the figure, "FPKM" represents the fragment length read per million bases per thousand bases of transcription. The results showed that the expression levels of the following genes in the BnSABATH gene family were significantly increased after infection induction: BnSABATH1 (BRAD gene ID: BnaA02g12540D), BnSABATH2 (BRAD gene ID: BnaCnng41630D), BnSABATH3 (BRAD gene ID: BnaA07g03510D), BnSABATH4 (BRAD gene ID: BDBnaA07g03730D), BnSABATH5 (BRAD gene ID: BnaA07g37560D), and BnSABATH6 (BRAD gene ID: BnaC06g12270D). This indicates that the corresponding promoters of these genes (i.e., BnSABATH1p, BnSABATH2p, BnSABATH3p, BnSABATH4p, BnSABATH5p, and BnSABATH6p, as shown in SEQ ID NO. NO.1-6 shows the Sclerotinia sclerotiorum inducible promoter.

[0062] Example 2: Cloning of the promoter BnSABATH1p and the coding sequence of the gene BnSABATH1

[0063] To further reveal the relationship between the promoter sequences of BnSABATH gene family members and the improvement of plant resistance to sclerotinia stem rot, this study used the BnSABATH1 gene as an example, cloning the promoter BnSABATH1p and the coding sequence of the BnSABATH1 gene for subsequent experiments.

[0064] 1. Cloning of the promoter BnSABATH1p

[0065] Young leaf tissues of rapeseed plants were collected, and gDNA was extracted using a high-efficiency plant genomic DNA extraction kit (Beijing Tiangen Biotech Co., Ltd., catalog number: DP350). Using the gDNA as a template, PCR amplification was performed using primer pairs BnSABATH1p-F (as shown in SEQ ID NO. 9) and BnSABATH1p-R (as shown in SEQ ID NO. 10) to obtain the promoter BnSABATH1p, which has an 18bp overlap at the 3' end with the 5' end of the coding sequence. The sequence of the promoter BnSABATH1p is shown in SEQ ID No. 1. The PCR amplification system consisted of: 4 μL dNTP (10 mmol / L), 5 μL 10× Taq buffer, 1 μL each of upstream and downstream primers (10 μmmol / L), 0.5 μL DNA polymerase (5 U / μL), 1 μL cDNA template, and 37.5 μL dH2O. PCR amplification conditions were: 94℃ pre-denaturation for 5 min, 94℃... The PCR products were denatured at 58 °C for 45 s, annealed at 58 °C for 45 s, extended at 72 °C for 2 min (repeated 30 times), and extended at 72 °C for 10 min. The PCR products were purified and recovered using a standard agarose gel extraction kit (Beijing Tiangen Biotech Co., Ltd., catalog number: DP209).

[0066] 2. Cloning of the coding sequence of gene BnSABATH1

[0067] Young leaf tissues from rapeseed plants were flash-frozen in liquid nitrogen (-196 °C), and total RNA was extracted using Trizol reagent (ThermoFisher Scientific Inc., catalog number: 15596026). cDNA was synthesized using Superscript™ reverse transcriptase (Promega Biotech Co., Ltd., catalog number: M1701). Using the cDNA as a template, PCR amplification was performed using primer pairs BnSABATH1-CDS-F (as shown in SEQ ID NO.7) and BnSABATH1-CDS-R (as shown in SEQ ID NO.8) to obtain the coding sequence of the gene BnSABATH1. The PCR amplification system, amplification conditions, and purification and recovery of the product were the same as in Section 1 of this example.

[0068] Example 3: Construction of BnSABATH1p::BnSABATH1 recombinant vector and transgenic plants

[0069] 1. Construction of the BnSABATH1p::BnSABATH1 recombinant vector

[0070] The coding sequence of the gene BnSABATH1 and the promoter BnSABATH1p sequence cloned in Example 2 were mixed in equal volumes. Using this mixture as a template, overlap PCR amplification was performed using primers BnSABATH1p-121H-F (as shown in SEQ ID NO. 11) and BnSABATH1p-121H-R (as shown in SEQ ID NO. 12) to obtain a DNA fragment containing the coding sequence of the gene BnSABATH1 and the promoter BnSABATH1p sequence. The DNA fragment was then ligated into a PBI121S linearized vector (which had its 35S promoter sequence removed by double digestion with HindIII and BamHI) via homologous recombination to obtain the BnSABATH1p::BnSABATH1 recombinant vector.

[0071] 2. Construction of transgenic plants

[0072] The BnSABATH1p::BnSABATH1 recombinant vector was transformed into Agrobacterium GV3101 (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1001), and the inflorescence immersion method was used to transform Columbia-0 type Arabidopsis thaliana. gDNA was extracted from transgenic progeny plants (method as described in Section 2 of Example 2), and PCR identification was performed using the promoter upstream primer BnSABATH1p-F and the vector-specific primer 121S-R (as shown in SEQ ID NO. 13). Positive transgenic plants were self-crossed, and homozygous transgenic lines were identified based on the positive rate of the progeny plants. Two homozygous transgenic lines (#5 and #9) were obtained from the T3 generation homozygous lines for subsequent experiments.

[0073] Example 4: Identification of disease resistance in transgenic plants and analysis of promoter response to Sclerotinia sclerotiorum expression levels

[0074] 1. Disease resistance identification of transgenic plants

[0075] The homozygous transgenic lines #5 and #9 obtained in Example 3 were grown in a greenhouse at 22℃ and 70% relative humidity, with a light cycle of 16 h light + 8 h dark. After 4-5 weeks of growth, plants with uniform growth were selected for inoculation analysis of live Sclerotinia sclerotiorum mycelium. The specific steps were as follows: Sclerotinia sclerotiorum was cultured on PDA medium (Thermo Fisher Scientific Inc., catalog number: CM0139) until the mycelium expanded to about 2 / 3 of the culture dish area. A 3 mm mycelial block was taken from the edge of the mycelium for inoculation experiments. Three whooping leaves were selected from each homozygous transgenic plant and inoculated at the main vein. The inoculated plants were covered with plastic wrap to maintain humidity. A blank PDA medium was set up as a control group. The length (mm) of the lesion area was measured at 24 h, 36 h and 48 h after inoculation. At the same time, plant samples were taken at 36 h after inoculation for subsequent expression level analysis.

[0076] Figure 2 The statistical analysis of lesion length after inoculation with *Sclerotinia sclerotiorum* is shown in the figure, where "WT" represents the control group. The results show that the homozygous transgenic lines #5 and #9 exhibited significantly stronger resistance to *Sclerotinia sclerotiorum* than the control group, indicating that the promoter BnSABATH1p can act as an inducible promoter to regulate the response of other genes to *Sclerotinia sclerotiorum* infection, thereby achieving the effect of regulating plant resistance to *Sclerotinia sclerotiorum*.

[0077] 2. Analysis of promoter-responsive expression levels in *Sclerotium sclerotiorum*

[0078] Total RNA was extracted from plant samples inoculated for 36 h in Section 1 of this Example and reverse transcribed to synthesize cDNA (method as in Section 1 of Example 2); the transcription level of gene BnSABATH1 was detected by qPCR (the upstream primer BnaA02.SABATH1-qF and the downstream primer BnaA02.SABATH1-qR for detecting gene BnSABATH1 are shown in SEQ ID NO.14-15, respectively, and BnActin gene was used as internal reference gene).

[0079] Figure 3 A statistical analysis of the transcriptional level of gene BnSABATH1 is shown in the figure, with "WT" representing the control group. The results showed that the transcriptional level of gene BnSABATH1 in homozygous transgenic lines #5 and #9 was significantly higher than that in the uninoculated control group (P < 0.01). These results indicate that after inoculation with Sclerotinia sclerotiorum, the expression level of gene BnSABATH1 regulated by the promoter BnSABATH1p was significantly increased by more than 20-fold, further validating that the promoter BnSABATH1p is a plant promoter induced by Sclerotinia sclerotiorum.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A plant promoter induced by *Sclerotinia sclerotiorum*, characterized in that, The promoter is BnSABATH1p, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The plant promoter as described in claim 1, characterized in that, The plant in question is a cruciferous crop.

3. The application of the plant promoter as described in any one of claims 1-2 in regulating plant resistance to sclerotinia stem rot.

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