Application of tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in regulation and control of plant anthracnose resistance

Abstract

The invention discloses application of a tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in regulation and control of plant anthracnose resistance. The biological function of the CsCAD1L gene is verified for the first time through an overexpression experiment. Through instantaneous overexpression of the CsCAD1L gene in tea tree leaves, the resistance of tea trees to anthracnose can be significantly improved; meanwhile, the CsCAD1L gene is over-expressed in the tobacco, so that the resistance of the tobacco to the anthracnose can be obviously improved. The functional verification of the cinnamyl alcohol dehydrogenase CsCAD1L gene of the tea tree can provide a gene resource for the breeding of a new variety of the tea tree. The invention provides an important gene resource which possibly has universality and is used for regulating the anthracnose resistance of the plants, and an excellent candidate gene is provided for cultivating anthracnose-resistant plant varieties.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of the tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in regulating plant resistance to anthracnose. (II) Background Technology

[0002] tea tree[ Camellia sinensis *L.) O. Kuntze* is an evergreen shrub or small tree belonging to the genus *Lemon* in the family Theaceae. Widely distributed in many countries, it is one of the oldest tree species. As a vital leaf crop in my country, tea is susceptible to leaf diseases that directly reduce tea yield and quality. Tea trees thrive in warm, humid climates, which also provide suitable conditions for anthracnose. Anthracnose is one of the most serious fungal leaf diseases affecting tea trees. Reports indicate that anthracnose causes 30-50% yield losses annually in China, severely impacting and even hindering the sustainable development of the tea industry. Therefore, selecting and cultivating highly resistant tea varieties to reduce the damage caused by anthracnose and improve tea quality and yield is of great significance.

[0003] Lignin, an important structural component of plant cell walls, constitutes approximately 25%-40% of the cell wall, second only to cellulose, and is a significant phenolic polymer. Lignin deposition in cell walls not only enhances the overall mechanical support of plants but also plays a crucial protective role against pathogen invasion, drought stress, and other biotic and abiotic stresses. Lignin formation is achieved through the phenylpropanoid metabolic pathway, a process requiring the synergistic action of multiple enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in the lignin biosynthesis pathway, catalyzing the synthesis of hydroxycinnamaldehyde (…). p The NADPH-dependent reduction of coumarin, sinigrin, and coniferaldehyde yields the corresponding alcohols. p -Coumaryl alcohol, sinigrin, and coniferyl alcohol promote the formation of lignin monomers (syringyl lignin, S-lignin; guaiacyl lignin, G-lignin; para-hydroxy-phenyl lignin, H-lignin). Tea plants are perennial woody plants, and conventional breeding methods such as hybridization and radiation-induced mutagenesis are characterized by complex procedures, long cycles, and low efficiency. Due to the conservation of genes, different genes have different effects on lignin synthesis. Therefore, it is necessary to find genes that effectively regulate lignin synthesis in tea plants to improve their resistance to anthracnose. (III) Summary of the Invention

[0004] The purpose of this invention is to provide an application of the tea plant cinnamyl alcohol dehydrogenase CsCAD1L gene in regulating the plant's resistance to anthracnose. This invention utilizes transient overexpression of this gene in tea plants and stable overexpression in tobacco to study its biological function. The results demonstrate that the CsCAD1L gene enhances the tea plant's resistance to anthracnose by regulating lignin synthesis, providing a genetic resource for breeding highly anthracnose-resistant tea varieties and possessing excellent potential application value.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides an application of the tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in regulating plant resistance to anthracnose.

[0007] Furthermore, the plants include woody plants and herbaceous plants.

[0008] Furthermore, the plants mentioned include tea trees and tobacco.

[0009] Furthermore, the nucleotide sequence of the tea plant cinnamyl alcohol dehydrogenase CsCAD1L gene is shown in SEQ ID NO. 1, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 2. The CsCAD1L gene involved in this invention encodes a protein of 144 amino acids, containing an alcohol dehydrogenase domain ADH_N, and its CDS sequence has been submitted to NCBI (GenBank accession number: PX609947).

[0010] Furthermore, the application includes the following methods: 1) making the plant contain the CsCAD1L gene; or 2) making the plant overexpress the CsCAD1L gene.

[0011] Furthermore, in method 2), the CsCAD1L gene is transferred into the plant genome using Agrobacterium-mediated transformation to obtain plants with transient overexpression of the CsCAD1L gene.

[0012] Furthermore, in method 2), the CsCAD1L gene is transferred into the plant genome using the leaf disc method to obtain plants with stable overexpression of the CsCAD1L gene.

[0013] Furthermore, the CsCAD1L gene is inserted into the plant genome by inserting it into the BamHⅠ and SalⅠ sites of the plant expression vector pCAMBIA2300 with a 35S promoter or the XbaⅠ and SacⅠ sites of pCAMBIAsuper1300, thereby enhancing the plant's resistance to anthracnose.

[0014] This invention provides an application of the CsCAD1L gene, a tea plant cinnamyl alcohol dehydrogenase, in breeding plants to improve their anthracnose resistance. The application involves overexpressing the CsCAD1L gene in the plant genome.

[0015] The transgenic technology system for tea is still immature, while tobacco is a model plant in the field of plant biology research. Therefore, Agrobacterium-mediated transgenic technology can be used to overexpress the CsCAD1L gene in tobacco, thereby studying the biological function of the CsCAD1L gene in plant resistance to anthracnose. Simultaneously, transient overexpression of the CsCAD1L gene in tea leaves using Agrobacterium-mediated transformation can further verify the biological function of the CsCAD1L gene under anthracnose stress. Cloning and functional studies of the cinnamyl alcohol dehydrogenase CsCAD1L gene provide valuable genetic resources for the breeding of superior new tea varieties.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] This invention validates the biological function of the CsCAD1L gene for the first time through overexpression experiments. Transient overexpression of the CsCAD1L gene in tea leaves significantly enhances the resistance of tea plants to anthracnose; similarly, overexpression of the CsCAD1L gene in tobacco significantly enhances the resistance of tobacco to anthracnose. Functional validation of the tea plant cinnamyl alcohol dehydrogenase CsCAD1L gene can provide gene resources for the breeding of new tea varieties. This invention provides an important and potentially universal gene resource for regulating the ability of plants to resist anthracnose, offering an excellent candidate gene for cultivating anthracnose-resistant plant varieties. (iv) Description of the attached drawings

[0018] Figure 1 This is a bar graph showing the expression level of the CsCAD1L gene in tea leaves in Example 2.

[0019] Figure 2 The image shows the identification of anthracnose resistance in tea leaves after transient overexpression in Example 2. A. Lesions on tea leaves after inoculation with Camellia anthracnose; B. Statistical analysis of lesion area.

[0020] Figure 3 This is a diagram showing the expression of the CsCAD1L gene in the OE-4 and OE-14 tobacco strains overexpressing Benzodiaceae in Example 3.

[0021] Figure 4 The following are images illustrating the anthracnose resistance identification of the overexpressing Tobacco Benzoinus lines OE-4 and OE-14 in Example 3: A. Lesions on tobacco leaves after inoculation with Camellia anthracnose; B. Statistical analysis of lesion area.

[0022] Figure 5The images show the lignin content in tea leaves and tobacco plants overexpressing Benzoinus 'Ben' in Examples 2 and 3; A. Total lignin content in tea leaves overexpressing Benzoinus 'Ben'; B. Total lignin content in tobacco plants OE-4 and OE-14 overexpressing Benzoinus 'Ben'. (V) Detailed Implementation Methods

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: unless otherwise specified, the embodiments are carried out in accordance with conventional experimental methods or the manufacturer's instructions.

[0024] In the following examples, pCAMBIA2300 and pCAMBIAsuper1300 are both expression vectors and are commercially available; tea tree ( Camellia sinensis The variety is 'Longjing 43' ( Longjing43 ); tobacco is Benedictine tobacco ( Nicotiana benthamiana Camellia anthrax ( ); Camellia anthrax bacteria ( Colletotrichum camelliae ) strain is LS_19, Differences in the Characteristics and Pathogenicity of Colletotrichum camelliae and C. fructicola Isolated from the Tea Plant Camellia sinensis [(L.) O. Kuntze]; Destroys anthrax bacteria ( Colletotrichum destructivum The strain used was NX1-1, sourced from: Jiang Chaoyang et al., Identification and biological characteristics of the pathogen causing grape anthracnose in Ningxia, Northwest Agriculture Journal, 2024. Agrobacterium GV3101 is a commonly used strain and is commercially available.

[0025] The main reagents used in the following examples were: lignin content assay kit, kanamycin, rifampin, etc., purchased from Sangon Biotech (Shanghai) Co., Ltd.; RNA extraction kit, reverse transcriptase, ChamQ Universal SYBRqPCR Master Mix, Phanta Max Super-Fidelity DNA Polymerase and 2×Rapid Tap Master Mix for PCR reactions, ClonExpress II One Step Cloning Kit and ClonExpress MultiS One Step Cloning Kit for gene cloning, plasmid extraction kit, gel extraction kit, etc., all purchased from Nanjing Novizan Biotechnology Co., Ltd.; Hoagland nutrient solution for tea branch culture was purchased from Beijing Coollab Technology Co., Ltd. All other chemical reagents used in the examples were imported or domestically produced analytical grade reagents.

[0026] In the examples, the primers to be synthesized and the vectors to be sequenced were both provided by Zhejiang Shangya Biotechnology Co., Ltd.

[0027] Table 1. Composition of Hogland nutrient solution (mg / L)

[0028]

[0029] Example 1: Cloning and overexpression vector construction of the CsCAD1L gene of tea plant cinnamyl alcohol dehydrogenase

[0030] 1. Primer design

[0031] Based on the CsCAD1L sequence in the TPIA database (https: / / tpia.teaplants.cn / ), its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence encoding the protein is shown in SEQ ID NO.2. Two pairs of primers were designed: upstream primer F1 and downstream primer R1, and upstream primer F2 and downstream primer R2.

[0032] F1: 5'-CGAGCTGTACAAGGGATCCATGGGTAGCTTGGAGGACGAA-3';

[0033] R1: 5'-GGCATGCCTGCAGGTCGACTTACTTTTGATCAGTGA-3'.

[0034] F2: 5'-CGATACACCAAATCGACTCTAGAATGGGTAGCTTGGAGGACG-3';

[0035] R2: 5'-AACGATCGGGGAAAATTCGAGCTCTTACTTTTGATCAGTGACCA-3'.

[0036] SEQ ID NO.1

[0037] ATGGGTAGCTTGGAGGACGAAAGAACAACAACAGGATGGGCTGCAACAGACCCTTCTGGGGTACTCTCTCCTTATACTTACACTCTCAGGAACACTGGTCCGGAAGATGTTTTCATCAAGGTGATTTGTTGTGGAATCTGCCATTCTGATATTCATCAGATCAAGAATGATCTTGGCATGTCCAATTACCCCATGGTTCCTGGGCATGAAGTGGTTG GTGAAGTGATAGAGGTGGGATCCAATGTGAGCAAGTTCAAGATAGGAGACACAGTGGGAGTTGGATGCATCGTTGGATGCTGCAGAAACTGTAGACCTTGCAAATCAGACAAAGAGCAATACTGCAACAAGAAGATCTGGTCCTACAATGATATCTACACCGATGGTAAACCCACCCAAGGTGGCTTTGCTGGCTCCATGGTCACTGATCAAAAGTAA

[0038] SEQ ID NO.2

[0039] MGSLEDERTTTGWAATDPSGVLSPYTYTLRNTGPEDVFIKVICCGICHSDIHQIKNDLGMSNYPMVPGHEVVGEVIEVGSNVSKFKIGDTVGVGCIVGCCRNCRPCKSDKEQYCNKKIWSYNDIYTDGKPTQGGFAGSMVTDQK

[0040] 2. Construction of overexpression vector and Agrobacterium-mediated transformation

[0041] RNA was extracted from tea leaves using an RNA extraction kit, and then reverse transcribed into cDNA using reverse transcriptase.

[0042] Using tea leaf cDNA as a template, the PCR amplification products were ligated with the transient overexpression vector pCAMBIA2300 (digested with BamHI and SalI) using primers F1 / R1 and the ClonExpress II One Step Cloning Kit. The ligation product (transient overexpression vector pCAMBIA2300-CsCAD1L) was transformed into Agrobacterium GV3101 (CsCAD1L-OE) using a freeze-thaw method. Agrobacterium containing the gene of the transient overexpression vector pCAMBIA2300-CsCAD1L was obtained. Under the same conditions, Agrobacterium containing the empty plasmid pCAMBIA2300 was constructed as a control (CK).

[0043] Using tea leaf cDNA as a template, the PCR amplification products were ligated with the stably overexpressing pCAMBIAsuper1300 vector digested with XbaⅠ and SacⅠ using the ClonExpress II One Step Cloning Kit. The ligation product (stable overexpression vector pCAMBIAsuper1300-CsCAD1L) was transformed into Agrobacterium GV3101 using the freeze-thaw method. Agrobacterium containing the stable overexpression vector pCAMBIAsuper1300-CsCAD1L gene was obtained.

[0044] 3. Preparation of Agrobacterium suspension

[0045] The Agrobacterium bacteria constructed in step 2 were inoculated into LB medium and cultured at 28°C and 200 rpm until OD200. 600 The value is 0.5 ~ 0.8, and Agrobacterium suspension is obtained.

[0046] Example 2: Transient overexpression of the CsCAD1L gene in tea leaves

[0047] Tea tree branches with five leaves from the current year were selected and cultured in Hoagland's nutrient solution. Agrobacterium suspension containing the transient overexpression vector pCAMBIA2300-CsCAD1L (Example 1) was injected into the entire leaf. Leaves injected with the control Agrobacterium suspension served as a negative control. After injection, the leaves were cultured in the dark at 25°C for 12 h. Anthracnose mycelium was then inoculated onto the upper surface of the leaves by scrambling. The size of the lesions was measured and photographed at 12 h, 24 h, 48 h, and 72 h. Figure 2 As shown in Figure A), the sample was frozen in liquid nitrogen at -80°C.

[0048] Total RNA was extracted from the above samples, reverse transcribed into cDNA, and the expression level of the CsCAD1L gene in the samples was detected using qRT-PCR. Results are shown below. Figure 1It can be seen that the expression level of the CsCAD1L gene at 24 h and 48 h was significantly higher than that of the control group, and tea leaves with transient overexpression were selected. Phenotypic analysis showed that the lesions of tea leaves with transient overexpression were significantly reduced after inoculation with Camellia anthracnose. Figure 2 (A, B). The results showed that transient overexpression of the CsCAD1L gene significantly enhanced the resistance of tea plants to Camellia anthracnose.

[0049] qRT-PCR primers:

[0050] F:CTTGGAGGACGAAAGAACA

[0051] R:CTTCATGCCCAGGAACCAT

[0052] Example 3: Construction and Disease Resistance Phenotypic Analysis of Tobacco Plants Overexpressing the CsCAD1L Gene

[0053] Agrobacterium GV3101 containing the stable overexpression vector pCAMBIAsuper1300-CsCAD1L, successfully constructed in Example 1, was transformed into wild-type Nicotiana benthamiana using the leaf disc method, and seeds of the T1 generation of transgenic tobacco were harvested. The pCAMBIAsuper1300 vector carries the kanamycin selection resistance gene; therefore, using kanamycin and PCR amplification of the target gene to screen and isolate transgenic tobacco lines can quickly obtain homozygous lines in the T2 generation.

[0054] Overexpression lines OE-4 and OE-14 were obtained through screening. Total RNA was extracted from wild-type, OE-4, and OE-14, and cDNA was synthesized by reverse transcription. The expression level of the CsCAD1L gene in the overexpression lines OE-4 and OE-14 was detected by qRT-PCR. Results are shown below. Figure 3 It can be seen that the CsCAD1L gene is highly expressed in both OE-4 and OE-14.

[0055] Wild-type, OE-4, and OE-14 tobacco seeds were sown in nutrient soil, and their growth status was observed after 5 weeks. Figure 4 In the A-type strain, there were no significant differences in growth phenotype between the wild-type, OE-4, and OE-14 lines. When *A. anthracnose* mycelium was inoculated onto tobacco leaves and cultured at 28°C in the dark for 3 days, significant differences in resistance were observed between the overexpression lines and the wild-type; specifically, wild-type lesions were larger, while lesions in the overexpression lines were significantly smaller. Figure 4 A and B. The results showed that overexpression of the CsCAD1L gene in tobacco could enhance the tobacco's resistance to the devastating anthrax bacterium.

[0056] Example 4: Detection of lignin content in plants overexpressing CsCAD1L

[0057] The total lignin content in tea leaves transiently overexpressing CsCAD1L (Example 2) and tobacco leaves overexpressing CsCAD1L (Example 3) was determined using a lignin content assay kit. Samples were ground using liquid nitrogen, dried at 80°C to constant weight, pulverized, passed through a 50-mesh sieve, and approximately 5 mg of sample was weighed into a 1.5 mL sterile centrifuge tube. The total lignin content in the samples was determined using ultraviolet colorimetry. The results are shown in [Figure 1]. Figure 5 The total lignin content in tea leaves after transient overexpression was significantly higher than that in the control group. Figure 5 (A), and the total lignin content in the tobacco overexpression lines OE-4 and OE-14 was significantly higher than that in the wild type ( Figure 5 (B). Therefore, increasing the expression level of the CsCAD1L gene can increase the lignin content in plant cell walls.

[0058] The combined effects of these physiological changes demonstrate that the CsCAD1L gene positively regulates plant anthracnose resistance by modulating lignin synthesis. It should be noted that the above descriptions are merely some specific embodiments of the present invention. Clearly, the present invention is not limited to the above embodiments, and modifications can be made based on it. Therefore, all modifications made by those skilled in the art without departing from the spirit of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Application of a tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in regulating plant anthracnose resistance.

2. The application as described in claim 1, characterized in that, The plants mentioned include woody plants and herbaceous plants.

3. The application as described in claim 2, characterized in that, The plants mentioned include tea trees and tobacco.

4. The application as described in claim 1, characterized in that, The nucleotide sequence of the tea plant cinnamyl alcohol dehydrogenase CsCAD1L gene is shown in SEQ ID NO.

1.

5. The application as described in claim 1, characterized in that, The applications include the following methods: 1) incorporating the CsCAD1L gene into plants; or 2) overexpressing the CsCAD1L gene in plants.

6. The application as described in claim 5, characterized in that, Method 2) uses Agrobacterium-mediated transformation to transfer the CsCAD1L gene into the plant genome, resulting in plants with transient overexpression of the CsCAD1L gene.

7. The application as described in claim 5, characterized in that, Method 2) uses the leaf disc method to transfer the CsCAD1L gene into the plant genome, resulting in plants with stable overexpression of the CsCAD1L gene.

8. The application as described in claim 1, characterized in that, The CsCAD1L gene is inserted into the plant genome via the BamHⅠ and SalⅠ sites of the plant expression vector pCAMBIA2300 with a 35S promoter or the XbaⅠ and SacⅠ sites of pCAMBIAsuper1300, thereby enhancing the plant's resistance to anthracnose.

9. Application of a tea tree cinnamyl alcohol dehydrogenase CsCAD1L gene in breeding to improve plant anthracnose resistance.

10. The application as described in claim 9, characterized in that, The application involves overexpressing the CsCAD1L gene in the plant genome.

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