A gene PtoCXE06 for poplar resistance to bark rot and its application

Abstract

The present invention provides a gene PtoCXE06 for poplar resistance to bark rot and its application, and relates to the field of molecular breeding technology. The present invention provides a gene PtoCXE06 for poplar resistance to bark rot, and by overexpressing the PtoCXE06 gene in poplar, the resistance of poplar to bark rot can be significantly improved. Experiments have confirmed that overexpressing the PtoCXE06 gene in white poplar can create a new poplar disease-resistant germplasm material with significantly enhanced resistance to bark rot, laying a foundation for the cultivation of new poplar varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular breeding, and in particular to a poplar bark rot resistance gene PtoCXE06 and an application thereof. Background Art

[0002] Poplars (Populus spp.) are important ecological and economic tree species, playing an important role in national economic construction and ecological environment construction. However, the occurrence and harm of poplar diseases, especially fungal diseases, are becoming increasingly serious, greatly threatening the further development of poplar plantations in my country and also having a huge impact on the world's forest resources and environmental security. Therefore, creating new disease-resistant poplar germplasm materials is of great significance for cultivating new forest varieties that meet the needs of national economic development.

[0003] The creation of new disease-resistant poplar germplasm materials based on traditional hybrid breeding and mutagenesis breeding methods has disadvantages such as low efficiency and difficulty in controlling the direction of induced mutations. It is difficult to obtain new germplasm materials with significantly changed traits in the perennial woody plant poplar. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a gene PtoCXE06 for poplar resistance to poplar bark rot fungi. Overexpression of the PtoCXE06 gene in poplars can significantly improve the resistance of poplars to poplar bark rot fungi.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a poplar bark rot resistance gene PtoCXE06, and the nucleotide sequence of the gene PtoCXE06 is shown in SEQ ID NO.1.

[0007] The invention provides a poplar anti-bark rot fungus protein, and the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0008] The present invention provides an overexpression vector comprising the gene PtoCXE06, wherein the basic vector of the overexpression vector is a plant binary expression vector ΔpCAMBIA1302.

[0009] The present invention also provides the use of the gene PtoCXE06, the protein or the overexpression vector in creating new disease-resistant germplasm materials of poplar.

[0010] The present invention also provides the use of the gene PtoCXE06, the protein or the overexpression vector in enhancing the resistance of poplar to poplar bark rot pathogen.

[0011] Preferably, the resistance of poplars to poplar bark rot pathogen is enhanced by overexpressing the gene PtoCXE06 in poplars.

[0012] The present invention also provides a method for improving the resistance of poplars to poplar bark rot pathogens, the method comprising the following steps:

[0013] The gene PtoCXE06 is constructed into the plant binary expression vector ΔpCAMBIA1302 to obtain the overexpression vector PtoCXE06-OE; the overexpression vector PtoCXE06-OE is transformed into poplars to overexpress the gene PtoCXE06, thereby improving the resistance of poplars to poplar bark rot pathogens.

[0014] Preferably, the gene PtoCXE06 is extracted and amplified from root, stem and leaf tissues of Populus tomentosa.

[0015] Preferably, after screening and cultivation of transgenic PtoCXE06 poplars, new disease-resistant poplar germplasm materials with enhanced resistance to poplar bark rot pathogens can be obtained.

[0016] Preferably, the screening is hygromycin screening.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a poplar resistance gene PtoCXE06, which can significantly improve the resistance of poplars to poplar bark rot by overexpressing the PtoCXE06 gene in poplars. Experiments have confirmed that overexpressing the PtoCXE06 gene in white poplar can create a new poplar disease-resistant germplasm material with significantly enhanced resistance to poplar bark rot, laying a foundation for the cultivation of new poplar varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression patterns of PtoCXE06 under normal conditions and stress treatment conditions.

[0020] Figure 2 Workflow for constructing PtoCXE06 overexpression vector.

[0021] Figure 3 The PCR test results of the transgenic plants DNA level, where M is marker (DL2000), WT is the wild type 741 Populus tomentosa, PC is the pCAMBIA1302-PtoCXE6 plasmid, BC is ddH2O, 1-43 is PtoCXE6-OE, and 44 / 45 is the group without sample addition.

[0022] Figure 4 RT-qPCR detection of mRNA levels in transgenic plants.

[0023] Figure 5 Comparison of infection rates between control plants and PtoCXE06 overexpressing plants. DETAILED DESCRIPTION

[0024] The present invention provides a poplar bark rot resistance gene PtoCXE06, and the nucleotide sequence of the gene PtoCXE06 is shown in SEQ ID NO.1.

[0025] The invention provides a poplar anti-bark rot fungus protein, and the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0026] The present invention provides an overexpression vector comprising the gene PtoCXE06, wherein the basic vector of the overexpression vector is a plant binary expression vector ΔpCAMBIA1302.

[0027] In the present invention, the plant binary expression vector ΔpCAMBIA1302 is a binary plant expression vector transformed from the traditional plant expression vector pCAMBIA1302. The present invention selects the plant binary expression vector ΔpCAMBIA1302 as the basic vector to avoid the influence of the GFP tag contained in the pCAMBIA1302 vector on the activity of the target gene encoding protein.

[0028] The present invention also provides the use of the gene PtoCXE06, the protein or the overexpression vector in creating new disease-resistant germplasm materials of poplar.

[0029] The present invention also provides the use of the gene PtoCXE06, the protein or the overexpression vector in enhancing the resistance of poplars to poplar bark rot. In the present invention, the resistance of poplars to poplar bark rot is enhanced by overexpressing the gene PtoCXE06 in poplars.

[0030] The present invention also provides a method for improving the resistance of poplars to poplar bark rot pathogens, the method comprising the following steps:

[0031] The gene PtoCXE06 is constructed into the plant binary expression vector ΔpCAMBIA1302 to obtain the overexpression vector PtoCXE06-OE; the overexpression vector PtoCXE06-OE is transformed into poplars to overexpress the gene PtoCXE06, thereby improving the resistance of poplars to poplar bark rot pathogens.

[0032] In the present invention, the gene PtoCXE06 is preferably extracted and amplified from the root, stem and leaf tissues of Populus tomentosa; the extraction and amplification preferably include the following steps: extracting the total RNA from the root, stem and leaf tissues of Populus tomentosa respectively, mixing them in equal amounts and then reverse transcribing them into cDNA; using the cDNA as a template, the PtoCXE06 gene can be obtained after primer amplification. In the present invention, the transformation is preferably Agrobacterium tumefaciens-mediated transformation. In the present invention, the poplars with genetically modified PtoCXE06 can be screened and cultivated to obtain new disease-resistant poplar germplasm materials with enhanced resistance to poplar bark rot fungi; the screening is preferably hygromycin screening, and the purpose of the screening is to obtain transgenic positive plants.

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] Expression pattern analysis of PtoCXE06

[0038] The transcriptome data of wild type and biotic and abiotic treatments of Populus trichocarpa were downloaded from the GEO database (Gene Expression Omnibus, https: / / www.ncbi.nlm.nih.gov / geo / ), with the accession number GSE109609. Through data analysis, the TPM values ​​of the PtCXE06 gene of Populus trichocarpa under normal conditions (WT) and different treatment conditions were screened. Among them, the different treatments include: salicylic acid treatment (SA), methyl jasmonate treatment (MeJA), Marssonina brunnea treatment (MB), cold treatment (cold), salt treatment (SN), and mechanical wounding (WD). The log2(TPM+1) value was used to generate a heat map of the PtoCXE06 gene expression pattern, and the results are shown in Tables 1 and 2. Figure 1 shown.

[0039] Table 1 Expression of PtoCXE06 under normal conditions and stress treatments

[0040] Group WT SA MeJA MB cold SN WD PtoCXE06 gene 0.197 1.833 3.308 1.513 0.391 1.947 0.837

[0041] It can be seen that PtoCXE06 has a low expression under normal growth conditions, while its expression level increased significantly after stress treatment, especially under methyl jasmonate treatment, where the expression level was the highest, indicating that PtoCXE06 may play an important role in poplar stress response.

[0042] Example 2

[0043] Cloning of the PtoCXE06 coding sequence

[0044] Total RNA from roots, stems, and leaves of Populus tomentosa was extracted using RNAprep Pure Plant Kit (Tiangen, Beijing), and then the total RNA from each tissue was mixed in equal amounts, and the mixed RNA was reverse transcribed into cDNA using PrimeScriptTM RT reagent Kit (TakaRa);

[0045] The above cDNA was used as a template to amplify the PtoCXE06 gene using specific primers. Among them, the upstream primer is 5'-CATACTGTTCTTCATCTCCCTTAG-3' (SEQ ID NO.3), and the downstream primer is 5'-GCCAAAGTTCATTTCAGTCC-3' (SEQ ID NO.4). The amplified product was recovered using a DNA rapid recovery / purification kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.); the recovered DNA fragments were connected to the pEASY-Blunt vector (Quanshijin, Beijing), and the connection system and reaction conditions were: PCR product, 4μl, pEASY-Blunt vector 1μl; room temperature reaction for 5min. Then the connection product was transformed into Escherichia coli DH10b. After resistance screening, a single clone was selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the gene sequence of PtoCXE06.

[0046] Example 3

[0047] Construction of PtoCXE06 overexpression vector

[0048] The PtoCXE06 gene sequence obtained in Example 2 was constructed into the plant binary expression vector ΔpCAMBIA1302. The specific process is as follows: Figure 2 ;

[0049] The ΔpCAMBIA1302 vector and the cloning vector containing PtoCXE06 were digested with SalI and PmlI, and the PtoCXE06 gene fragment was ligated to the ΔpCAMBIA1302 vector after digestion with T4 DNA ligase, and finally the overexpression vector PtoCXE06-OE was obtained. All restriction endonucleases were purchased from NEB and operated according to the instructions.

[0050] Example 4

[0051] 1. Through Agrobacterium tumefaciens-mediated transformation, multiple candidate transgenic poplar plant lines overexpressing PtoCXE06 were obtained, and then the candidate plants were tested to screen transgenic positive plants. The specific screening steps are as follows:

[0052] 1) The genomic DNA of the candidate transgenic plants was extracted using DNAsecure Plant Kit (Tiangen, Beijing), and the extraction steps were carried out according to the kit instructions;

[0053] 2) Using the extracted genomic DNA of the candidate transgenic plants as a template, PCR amplified the HPT marker gene to detect whether the T-DNA segment carrying the PtoCXE06 gene was integrated into the poplar genome. The upstream primer was 5'-TGCTCCATACAAGCCAACC-3' (SEQ ID NO.5) and the downstream primer was 5'-TGTCCTGCGGGTAAATAGC-3' (SEQID NO.6). The PCR amplification system is shown in Table 2;

[0054] Table 2 Amplification system

[0055] Reagents Volume (μl) 10×ExTaq Reaction Buffer 2.0 dNTP(2.5mM) 2.0 Forward Primer (2.0 mM) 2.0 Reverse Primer (2.0 mM) 2.0 Template (genomic DNA or cDNA) 1.0 Ex Taq polymerase (5U / ul) 0.1 <![CDATA[ddH2O]]> 10.9

[0056] Amplification program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 55-58°C for 30 sec, extension at 72°C for 1.5 min, 35 cycles; extension at 72°C for 10 min; 15°C for 30 sec.

[0057] The results of PCR amplification of HPT marker gene are as follows Figure 3 As shown, it can be seen that the T-DNA segment carrying the PtoCXE06 gene was successfully integrated into the poplar genome.

[0058] 2. RNA was extracted from the wild-type control plant and the transgenic plant, and the RNA was reverse transcribed into cDNA using PrimeScriptTM RTreagent Kit (Perfect Real Time) (TakaRa). The reverse transcription system is shown in Table 3 below;

[0059] Table 3 Reverse transcription system

[0060] Reagents volume 5×PrimeScript Buffer(for Real Time) 2μl PrimeScript RT Enzyme Mix I 0.5μl Oligo dT Primer (50μM) 0.5μl Random 6 mers (100μM) 0.5μl Total RNA 400ng <![CDATA[Rnase Free ddH2O]]> Add to 10 μl

[0061] Using the cDNA synthesized by reverse transcription as a template, Premix Ex Taq TM II (Tli RNaseHPlus) was used for RT-qPCR detection to detect the transcription level of PtoCXE06 in poplar transgenic plants. The detection primers of RT-qPCR are shown in Table 4; the reaction system of RT-qPCR is shown in Table 5.

[0062] Table 4 Primer information for RT-qPCR detection of PtoCXE06

[0063]

[0064] Table 5 RT-qPCR reaction system

[0065] Reagents Volume (μl) SYBR Premix Ex Taq II(Tli RNaseH Plus)(2×) 10 PCR Forward Primer (10μM) 0.8 PCR Reverse Primer (10μM) 0.8 cDNA template (<100ng) 2 Sterilized water 6.4

[0066] The RT-qPCR detection process adopts a two-step method: (1) pre-denaturation: 95℃, 30sec; (2) PCR reaction: 95℃5sec, 60℃30sec, 40 cycles; (3) melting curve analysis: 95℃5sec, 60℃60sec. The RT-qPCR detection results are shown in Figure 4 .

[0067] It can be seen that the expression level of PtoCXE06 in transgenic plants was significantly increased compared with the control plants.

[0068] Example 5

[0069] Overexpression of PtoCXE06 enhances poplar resistance to poplar bark rot pathogen

[0070] 1. After overexpressing PtoCXE06 in poplars according to Example 4, when the plants grow to a height of 50 cm, the 3rd to 5th leaves of the plants overexpressing PtoCXE06-OE and the wild-type plants (WT) are selected from the top, respectively, and the leaves are placed face down on a culture dish covered with moist filter paper. The poplar bark rot bacteria cultured on PDA medium for four days are taken with a sampler to take a 6 mm agar block, and the bacteria-containing side is inoculated on the back of the leaves face down. After inoculation, the probability of plaques appearing in all leaves inoculated with poplar bark rot bacteria, i.e., the infection rate of poplar bark rot bacteria, is counted every other day. The results are as follows: Figure 5 .

[0071] according to Figure 5The results showed that during the six days of inoculation with poplar bark rot pathogen, the infection rate of PtoCXE06-OE plants was lower than that of the wild type.

[0072] 2. Based on the length of the lesions formed on the surface of the infected leaves, a grading standard for the severity of poplar bark rot disease was established, as shown in Table 6.

[0073] Table 6 Classification standards for poplar bark rot pathogens

[0074]

[0075] Among them, the inconspicuous lesions with a length of less than 0.7 cm are grade I, and the representative value is 0; the lesions with a length of 0.7-1.4 cm are grade II, and the representative value is 1; the lesions with a length of 1.4-2.1 cm are grade III, and the representative value is 2; the lesions with a length of 2.1-2.8 cm are grade IV, and the representative value is 3; the lesions with a length of more than 2.8 cm are grade V, and the representative value is 4. On the sixth day after inoculation according to step 1, the length of each bacterial spot on the leaves inoculated with the poplar bark rot pathogen was measured, and according to the above-mentioned disease grading standards, the number of lesions of each severity level was counted, and the disease index (DI) was calculated, and a resistance evaluation system based on the disease index (DI) was constructed. The disease index (DI) represents the severity of the disease in the plant, and its calculation formula is as follows:

[0076]

[0077] Among them: x is the representative value of each grading standard; n is the representative value of the highest grading standard 4; f is the number of lesions of each grading standard.

[0078] According to the above formula, the DI value of the wild type was 74.84, while the DI of the plant overexpressing PtoCXE06-OE was 12.97, which was significantly lower than the DI value of the wild type (p<0.001, independent sample t test). Therefore, overexpression of the PtoCXE06 gene greatly enhanced resistance to poplar bark rot.

[0079] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A gene PtoCXE06 for poplar resistance to bark rot, characterized in that: The nucleotide sequence of the gene PtoCXE06 is shown in SEQ ID NO.

1.

2. A poplar anti-bark rot fungal protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An overexpression vector comprising the gene PtoCXE06 according to claim 1, characterized in that: The basic vector of the overexpression vector is the plant binary expression vector ΔpCAMBIA1302.

4. Use of the gene PtoCXE06 according to claim 1, the protein according to claim 2 or the overexpression vector according to claim 3 in creating new germplasm materials of poplar trees resistant to poplar bark rot pathogen.

5. Use of the gene PtoCXE06 according to claim 1, the protein according to claim 2 or the overexpression vector according to claim 3 in enhancing the resistance of poplar to poplar bark rot pathogen.

6. The use according to claim 5, characterized in that: By overexpressing the gene PtoCXE06 in poplar, the resistance of poplar to poplar bark rot is enhanced.

7. A method for improving poplar resistance to poplar bark rot pathogens, characterized in that: The method comprises the following steps: The gene PtoCXE06 is constructed into the plant binary expression vector ΔpCAMBIA1302 to obtain an overexpression vector PtoCXE06-OE; the overexpression vector PtoCXE06-OE is transformed into poplars to overexpress the gene PtoCXE06, thereby improving the resistance of the poplars to poplar bark rot pathogens; The nucleotide sequence of the gene PtoCXE06 is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The gene PtoCXE06 is extracted and amplified from root, stem and leaf tissues of Populus tomentosa.

9. The method according to claim 7, characterized in that: After screening and cultivation of transgenic PtoCXE06 poplars, new disease-resistant poplar germplasm materials with enhanced resistance to poplar bark rot pathogens can be obtained.

10. The method according to claim 9, characterized in that The screening is hygromycin screening.

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