Application of MeMYB2 transcription factor in improving cassava bacterial blight resistance

Abstract

The application belongs to the field of biology and particularly relates to application of MeMYB2 transcription factor in improving cassava bacterial wilt resistance. MeMYB2 The 443 bp specific DNA fragment from position 491 to 933 in the cDNA is used to form a hairpin RNA structure in a p18TRNAi vector, and transgenic cassava plants obtained by transforming cassava protocorm-like bodies are planted in a field, so that the incidence of bacterial wilt is reduced, and the resistance of the cassava plants to bacterial wilt is significantly improved. The above results provide a new gene resource for prevention and control of cassava bacterial wilt and provide a new idea for prevention and control of cassava bacterial wilt resistance and cultivation of cassava varieties resistant to bacterial wilt.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to the application of the MeMYB2 transcription factor in improving resistance to bacterial wilt of cassava. Background Technology

[0002] MYB transcription factors constitute the largest family of transcription factors in plants, characterized by a MYB domain consisting of at least 1-4 imperfect tandem repeats, each containing 50-53 amino acid residues. These repeats can recognize and directly bind to specific DNA motifs. The MYB transcription factor family is divided into four subgroups based on the number of repeats in the MYB domain: 4R-MYB, 3R-MYB (R1R2R3-MYB), R2R3-MYB, and R1-MYB, with R2R3-MYB being the most prevalent. Currently, in plants, MYB participates in various processes, including primary and secondary metabolism, cell fate and property determination, development, and responses to biotic and abiotic stresses.

[0003] Cassava bacterial wilt (pathogen Latin name: *Xanthomonas manihotis* (Artheodet Bey.) Stars, synonym *Xanthomonas campestris* pv. *manihotis* Starr.) is a pathogen that primarily affects leaves and stems, and is a systemic disease affecting the entire plant. Infected leaves develop moist, angular spots; under high humidity, a sap initially appears as a white, sticky substance, later turning yellowish-brown, causing plant wilting and sap exudation. Infected stems often result in stem depressions and the secretion of large amounts of mucilage, accelerating leaf wilting. Infected tubers turn the vascular bundles yellowish-brown, and the roots and vascular bundles develop dry rot, leading to complete plant death in severe cases. This disease is characterized by its wide spread, rapid onset, and severe damage; it is a systemic disease that primarily affects leaves and stems, causing yield losses exceeding 50%. Therefore, developing new cassava varieties resistant to cassava bacterial wilt is of significant importance for the development of the cassava industry. Summary of the Invention

[0004] This invention provides the application of the MeMYB2 transcription factor in improving resistance to bacterial wilt of cassava.

[0005] The technical solution of this invention is implemented as follows:

[0006] The application of the MeMYB2 transcription factor, or the MeMYB2 transcription factor gene, or a vector, expression cassette, or host bacterium containing the MeMYB2 transcription factor gene in the prevention and control of bacterial wilt of cassava and / or the enhancement of resistance to bacterial wilt of cassava, wherein the nucleotide sequence of the MeMYB2 transcription factor gene is shown in SEQ ID NO: 1.

[0007] The application of the MeMYB2 transcription factor, or the MeMYB2 transcription factor gene, or a vector, expression cassette, or host bacterium containing the MeMYB2 transcription factor gene in the prevention and control of bacterial wilt of cassava and / or the improvement of resistance to bacterial wilt of cassava without affecting cassava yield, wherein the nucleotide sequence of the MeMYB2 transcription factor gene is shown in SEQ ID NO: 1.

[0008] Furthermore, in the aforementioned application, silencing the MeMYB2 transcription factor gene delays the onset of bacterial wilt in cassava plants, slows the progression of bacterial wilt in cassava plants, reduces the incidence of bacterial wilt in cassava plants, and / or reduces the mortality rate of bacterial wilt in cassava plants.

[0009] A MeMYB2 transcription factor gene fragment for the control of bacterial wilt disease in cassava, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0010] A construct containing the MeMYB2 transcription factor gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0011] Furthermore, the construction method of the aforementioned construct includes the following steps:

[0012] KpnI was added to the 5' end and ClaI to the 3' end of the MeMYB2 transcription factor gene fragment to construct a sense fragment; simultaneously, BamHI was added to the 5' end and XhoI to the 3' end to construct an antisense fragment. These two fragments were linked by an intron (ClaI and XhoI) to form a hairpin RNA construct on the p18TRNAi vector.

[0013] Vectors, expression cassettes, or recombinant bacteria containing the construct.

[0014] The application of the MeMYB2 transcription factor gene fragment, or construct, or vector, expression cassette, or recombinant bacteria in the prevention and control of bacterial wilt of cassava and the improvement of resistance to bacterial wilt of cassava.

[0015] The application of the MeMYB2 transcription factor gene fragment, or construct, or vector, expression cassette, or recombinant bacteria in the prevention and control of bacterial wilt of cassava and / or the improvement of resistance to bacterial wilt of cassava without affecting cassava yield.

[0016] Furthermore, in the aforementioned application, silencing the MeMYB2 transcription factor gene fragment delays the onset time of bacterial wilt in cassava plants, slows the progression of bacterial wilt in cassava plants, reduces the incidence of bacterial wilt in cassava plants, and / or reduces the mortality rate of bacterial wilt in cassava plants.

[0017] The application of the MeMYB2 transcription factor, or the MeMYB2 transcription factor gene, or a vector, expression cassette, or host bacterium containing the MeMYB2 transcription factor gene, or the MeMYB2 transcription factor gene fragment, or construct, or vector, expression cassette, or recombinant bacteria in the cultivation of cassava varieties resistant to bacterial wilt, wherein the nucleotide sequence of the MeMYB2 transcription factor gene is shown in SEQ ID NO: 1, the nucleotide sequence of the MeMYB2 transcription factor gene fragment is shown in SEQ ID NO: 2, and the nucleotide sequence of the construct is shown in SEQ ID NO: 3.

[0018] A type of cassava obtained by transferring an RNAi vector containing the described construct into a cassava plant.

[0019] Beneficial effects:

[0020] This invention isolates a 443 bp specific DNA fragment from positions 491 to 933 of the MeMYB2 cDNA using PCR. This fragment is then used to form a hairpin RNA construct on the p18TRNAi vector. Transgenic cassava plants obtained by transforming cassava callus tissue exhibit these characteristics when planted in the field. These results delay the onset of bacterial wilt, slow disease progression, significantly reduce symptoms after infection, and substantially decrease the incidence and mortality of bacterial wilt, thus significantly improving the resistance of cassava plants to bacterial wilt. These results provide new genetic resources for the control of bacterial wilt in cassava and offer new insights into the control of bacterial wilt in cassava and the breeding of resistant cassava varieties. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a diagram of the nucleotide sequence of the hairpin DNA construct.

[0023] Figure 2 This is a structural diagram of the MeMYB2-RNAi vector.

[0024] Figure 3 This is a PCR identification diagram of genetically modified cassava.

[0025] Figure 4 Southern Blot identification of genetically modified cassava.

[0026] Figure 5 To determine the silencing efficiency of the MeMYB2 gene in transgenic cassava using RT-qPCR.

[0027] Figure 6 This represents the field yield of genetically modified cassava.

[0028] Figure 7 This is a report on the incidence of bacterial wilt in genetically modified cassava fields.

[0029] Figure 8 This is a statistical count of the number of plants infected with bacterial wilt. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0031] Example 1

[0032] MeMYB2-RNAi DNA Construction and Cassava Transformation

[0033] The nucleotide sequence of the MeMYB2 gene is shown in SEQ ID NO: 1. A 443 bp specific DNA fragment from position 491 to 933 was isolated from the MeMYB2 cDNA by PCR. The nucleotide sequence of the sense fragment (5'-3') is shown in SEQ ID NO: 2. KpnI was added to the 5' end and ClaI to the 3' end of the sense fragment to construct a sense fragment with restriction enzyme sites. Simultaneously, BamHI was added to the 5' end and XhoI to the 3' end of the antisense fragment to construct an antisense fragment with restriction enzyme sites. These two fragments were linked by an intron (ClaI and XhoI) to form a hairpin DNA construct on the p18T-RNAi vector, the nucleotide sequence of which is shown in SEQ ID NO: 3. Figure 1 Then, the hairpin DNA construct was recovered by enzyme digestion (KpnI and BamHI) and cloned downstream of the 35S promoter of the pCAMBIA1300 vector to construct the MeMYB2-RNAi vector. Figure 2 ).

[0034] PCR primers for the positive fragment: Primer F: 5'-TCAGGTACCTGAGCCCAAATTCA-3'

[0035] primer R: 5'-GTCATCGATAAATGGTGGATTAC-3'

[0036] Antisense fragment PCR primer: Primer F: 5'-TCACTCGAGAAATGGTGGATTAC-3'

[0037] primer R: 5'-GTAGGATCCTGAGCCCAAATTCA-3'

[0038] PCR reaction system:

[0039]

[0040] PCR reaction procedure:

[0041]

[0042] The constructed MeMYB2-RNAi vector plasmid was transformed into Agrobacterium tumefaciens strain LBA4404 using a freeze-thaw method. After activation, the Agrobacterium tumefaciens strain LBA4404 carrying pCAMBIA1300:35S:MeMYB2-hpRNA was cultured extensively until the OD600 value reached 0.5-1.0. The cells were then resuspended in GD liquid medium (with AS added, final concentration 200 μM) and transformed into embryogenic callus of the cassava model variety cv.60444. The specific transformation procedure is as follows: (1) Add an appropriate amount of cassava embryogenic callus to the suspension and culture for 40 min; (2) Remove the bacterial solution, spread the infected cassava callus cells on a nylon membrane, and absorb excess water; (3) Place the callus cells on GD+100μM AS medium and culture in the dark at 20℃ for 72 h; (4) Wash the callus cells 5-6 times with GD liquid medium (Carb 500 mg / L); (5) Spread the cassava callus cells on a nylon membrane, absorb excess water, and place them on an MSN (10mg / L Hyg+ 500 mg / L Carb) plate to induce resistant embryos. Following the method of Zainuddin et al. (Zainuddin IM, Schlegel K, Gruissem W, Vanderschuren H. Robust transformation procedure for the production of transgenic farmer-preferred cassava landraces. PlantMethods. 2012;8(1):24.), transgenic cassava resistant plants were obtained through resistance induction screening (COM+10 mg / L Hyg+500 mg / L Carb).

[0043] Example 2

[0044] MeMYB2-RNAi transgenic cassava showed significantly enhanced resistance to bacterial wilt.

[0045] The MeMYB2-RNAi transgenic cassava obtained through transformation was selected and identified by PCR. Figure 3 Southern blot analysis Figure 4 ) and RT-qPCR ( Figure 5 Two independent single-copy MeMYB2 gene silencing lines, MeMYB2-RNAi-17 and MeMYB2-RNAi-202, were created. The specific steps and results are as follows.

[0046] Transgenic cassava was identified by PCR using primers for the screening marker hygromycin gene (hptII), and seven positive lines were obtained: 2i-1A, 2i-17, 2i-202, 2i-203, 2i-204, 2i-26 and 2i-32.

[0047] To further determine the copy number of PCR-positive MeMYB2-RNAi transgenic lines, leaves from both transgenic and wild-type cassava were collected, and DNA was extracted using a plant genomic DNA rapid extraction kit. 70 mg of DNA from each of the PCR-positive transgenic lines and wild-type cassava was digested overnight with BamHI, and the copy number was determined by Southern blot using the hptII gene fragment as a probe. The results are as follows: Figure 3 As shown, 2i-17 and 2i-202 are two independent conversion events involving single copies.

[0048] To further determine the silencing efficiency of the MeMYB2 gene in transgenic cassava, the relative expression levels of the MeMYB2 gene in leaves of wild-type and transgenic cassava (2i-17 and 2i-202) were detected using RT-qPCR. Total RNA was extracted from leaves of wild-type and the two transgenic lines, and reverse transcribed into cDNA first strand. The expression level of MeMYB2 was then detected by RT-qPCR. The results are as follows: Figure 4 As shown, the expression level of 2i-17 was downregulated by 78% (relative expression level 0.22), and the expression level of 2i-202 was downregulated by 45% (relative expression level 0.55). This indicates that the two lines are not only independent single-copy transformation events, but also have high MeMYB2 gene silencing efficiency, which can be used for subsequent functional studies.

[0049] Using cassava variety cv.60444 as the experimental variety, transgenic cassava and wild-type cassava tubers with similar growth vigor and thickness were selected and planted at the Wenchang Transgenic Biology Experiment Base. Field yield measurements at the Wenchang base in February 2024 showed that the MeMYB2-RNAi transgenic cassava tubers were robust, and the yield was not significantly different from the wild type. Figure 6In November 2024, a bacterial wilt outbreak occurred in the field. Wild-type cv.60444 plants quickly developed obvious symptoms. After infection, the disease progressed rapidly, with leaves turning yellow and wilting. Half a month after the onset of the disease, 100% of wild-type plants showed obvious symptoms. At this time, no obvious symptoms were found in the two MeMYB2-RNAi transgenic lines, which remained green. Figure 7 One month after the wild-type plants developed symptoms, the incidence rate (number of plants with obvious symptoms) of the transgenic plants was counted. The incidence rates of the two MeMYB2-RNAi transgenic lines were 10% and 7.41%, respectively. Figure 8 Not only was the infection rate significantly lower than that of the wild type, but the symptoms of the transgenic cassava plants were also significantly milder. The mortality rate of wild-type cassava was 100%, while the mortality rates of transgenic cassava were 5% and 7.41%, respectively, showing a significant reduction in mortality.

[0050] The above results indicate that, compared with wild-type cassava, the transgenic lines exhibited symptoms of bacterial wilt later, with a significantly lower incidence, slower disease progression, milder symptoms, and a significantly lower mortality rate, demonstrating a significantly enhanced resistance to bacterial wilt.

[0051] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of an RNAi construct silencing the MeMYB2 transcription factor, a vector containing the RNAi construct, an expression cassette containing the RNAi construct, or a recombinant bacterium containing the RNAi construct in the prevention and control of bacterial wilt of cassava and / or the enhancement of resistance to bacterial wilt of cassava, characterized in that, The nucleotide sequence of the RNAi construct is shown in SEQ ID NO:

3.

2. The application as described in claim 1, characterized in that, The RNAi construct reduces the incidence and / or mortality rate of bacterial wilt in cassava plants.

3. The application of an RNAi construct silencing the MeMYB2 transcription factor, a vector containing the RNAi construct, an expression cassette containing the RNAi construct, or a recombinant bacterium containing the RNAi construct in controlling cassava bacterial wilt and / or improving cassava bacterial wilt resistance without affecting cassava yield, characterized in that, The nucleotide sequence of the RNAi construct is shown in SEQ ID NO:

3.

4. The application as described in claim 3, characterized in that, The RNAi construct reduces the incidence and / or mortality rate of bacterial wilt in cassava plants.

5. The application of an RNAi construct silencing the MeMYB2 transcription factor, a vector containing the RNAi construct, an expression cassette containing the RNAi construct, or a recombinant bacterium containing the RNAi construct in the cultivation of cassava varieties resistant to bacterial wilt, characterized in that, The nucleotide sequence of the RNAi construct is shown in SEQ ID NO:

3.

6. The application as described in claim 5, characterized in that, The RNAi construct reduces the incidence and / or mortality rate of bacterial wilt in cassava plants.

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