Application of a CsABI5-5 gene in regulating resistance to citrus Huanglongbing and ulcer diseases

Abstract

The invention discloses an application of CsABI5-5 gene in regulating citrus resistance to Huanglongbing and canker, and belongs to the field of genetic engineering technology. The invention obtains transgenic Ziyang orange hairy roots by vacuum infection of rhizobacterium rhizogenes to infect citrus stem segments with Huanglongbing, and finds that CsABI5-5 overexpressed transgenic hairy roots are resistant to the spread and proliferation of Huanglongbing bacteria CLas. In addition, by evaluating the canker resistance of overexpressed CsABI5-5 transgenic late Jin orange, it is found that the susceptible area of ​​transgenic late Jin orange is significantly smaller than that of late Jin orange of non-transgenic control group (WT) after inoculation with citrus canker bacteria Xcc for 10d, and its lesion area and disease index are significantly lower than those of WT group plants, indicating that overexpression of CsABI5-5 can improve the resistance of plants to citrus canker and significantly reduce the infection symptoms of Xcc. Lay a foundation for understanding the function of CsABI5-5 in citrus Huanglongbing and canker.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an application of a CsABI5-5 gene in regulating citrus resistance to Huanglongbing and ulcer diseases. Background Art

[0002] Citrus Huanglongbing is currently the most devastating disease in the citrus industry, of which Candidatus Liberibacter asiaticus (CLas) is the main pathogenic bacteria. The difficulty of in vitro culture of CLas and its long incubation period are the main obstacles to studying the pathogenic mechanism of Huanglongbing, which leads to the rapid spread and serious damage of Huanglongbing. In addition, citrus lacks natural resistance to Huanglongbing, and no citrus varieties resistant to Huanglongbing have been found so far. Therefore, it is urgent to explore resistance genes and cultivate resistant varieties.

[0003] Citrus canker is another bacterial disease caused by Xanthomonas citri subsp. Citri (Xcc). It causes severe yield losses to the global citrus industry due to its wide range of spread, multiple pathways of harm, rapid spread, obvious symptoms, and difficulty in prevention and control. Currently, improving plant disease resistance through molecular breeding is a promising method to control the spread of citrus canker.

[0004] In plants, ABI5 is not only involved in multiple plant hormone signaling pathways in response to abiotic stresses, but also in biological processes in response to biotic stresses. Recently, it was found that ABI5 can directly activate the CsCalS11 promoter in citrus, and after citrus is infected with Huanglongbing, it enhances the expression of CsABI5-CsCalS11 through the ABA signaling pathway, causing abnormal deposition of callose in the phloem, laying a solid foundation for further exploration of the formation of Huanglongbing symptoms and the cultivation of resistant varieties. Summary of the invention

[0005] The technical problem to be solved by the present invention is: to provide an application of a CsABI5-5 gene in regulating citrus resistance to Huanglongbing and ulcer diseases, so as to solve the technical problem that Huanglongbing and ulcer diseases in citrus cannot be effectively controlled.

[0006] To achieve the above object, the technical solution adopted by the present invention is: to provide an application of a CsABI5-5 gene in regulating plant resistance to Huanglongbing and ulcer disease, and the nucleotide sequence of the CsABI5-5 gene is shown in SEQ ID NO: 1.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows:

[0008] Furthermore, the application is to improve the plant's resistance to Huanglongbing and ulcer diseases by overexpressing the CsABI5-5 gene.

[0009] Furthermore, the plant is citrus.

[0010] The invention also discloses a preparation containing CsABI5-5 gene for improving the resistance of plants to Huanglongbing and ulcer diseases.

[0011] The beneficial effects of the present invention are as follows: the present invention obtains transgenic Ziyang orange hairy roots by vacuum infection of rhizobacterium rhizogenes to infect citrus stem segments with Huanglongbing, and the experimental results show that the transgenic hairy roots overexpressing CsABI5-5 are resistant to the spread and proliferation of Huanglongbing bacteria CLas. The present invention also evaluates the resistance to canker disease of transgenic late Jin oranges overexpressing CsABI5-5, and finds that the susceptible area of ​​transgenic late Jin oranges is significantly smaller than that of non-transgenic control late Jin oranges 10 days after inoculation with citrus canker bacteria Xcc, and its lesion area and disease index are significantly lower than those of non-transgenic plants, indicating that overexpressing CsABI5-5 can improve the resistance of plants to citrus canker disease and significantly reduce the infection symptoms of Xcc. This lays a foundation for understanding the function of CsABI5-5 in citrus Huanglongbing and canker diseases, and provides excellent gene resources for breeding of citrus Huanglongbing and canker resistance. It also shows that the root-generating Agrobacterium transformation technology will help solve the problem of the long evaluation cycle of citrus materials' Huanglongbing resistance and facilitate further analysis of gene functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic diagram of the pLGN-CsABI5-5 vector;

[0013] Figure 2 This is the electrophoresis diagram of the amplification of the pLGN-CsABI5-5 vector;

[0014] Figure 3 This is the plasmid restriction enzyme cutting verification diagram of pLGN-CsABI5-5 vector;

[0015] Figure 4 The figures are the non-transgenic control and transgenic hairy roots;

[0016] Figure 5 PCR detection of non-transgenic control group (CK) and transgenic hairy roots;

[0017] Figure 6 is the content of CLas in the midrib of leaves infected with Huanglongbing and in transgenic hairy roots;

[0018] Figure 7 The pictures are of the non-transgenic control group (WT) and the CsABI5-5 transgenic late orange.

[0019] Figure 8 The PCR test results of non-transgenic control group (WT) and CsABI5-5 transgenic Wanjin orange;

[0020] Fig. 9 is the expression level of CsABI5-5 gene in non-transgenic control group (WT) and CsABI5-5 transgenic Wanjin orange;

[0021] Fig.10 The leaves of CsABI5-5 Wanjincheng overexpressing were inoculated with Xcc for 10 days;

[0022] Fig.11 Comparison of lesion area between non-transgenic control group (WT) and CsABI5-5 transgenic Wanjin orange;

[0023] Fig.12 These are the disease indexes of the non-transgenic control group (WT) and the CsABI5-5 transgenic late orange. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0025]

[0026] The amino acid sequence of the protein encoded by the CsABI5-5 gene (SEQ ID NO: 2) is as follows: MGSQMNFKNVGDTSYGDGKQAGNFPLARQPSVYSLTFEEFQNTWGGLGKDFGSMNMDELLKNIWTAEENHAMNSSASAAGESNAPGGNLQRQGSLTLPRTLSQKTVDEVWRDLMKEGSGGAAGGGGGGSNVPQRQQTLGEMTLEEFLVRAGVVREDAQQIGGSLNNDGFYANNNTSLALGFQQPSRNNGLIGNRIMGDGSSVPNQPPSLALNVNGVRSSQQP QQQPQQHQYQLQQQQQQQQQPQLQQHPYQQQQRQQQQAPLFPKQATVAFASPMNLVNTTQLSSPGARGQVVTVTNSSMNANLVQAGGLQGGGMGMVGLGAGGVTLATGSQVAQVS PDMITKSTADVSSPSPVPPYVFGRGRKSGALEKVVERRHRRMIKNRESAARSRARKQAYTLELEAEVAKLKELNQELERKQAEKIEMEKNKIIEKMKYRWGGKILCLRRTLTGPW.

[0027] The primer information used in the examples of the present invention is shown in Table 1:

[0028] Table 1 Primer information

[0029]

[0030] Example 1 Construction of overexpression vector

[0031] The primers pCsABI5-5-F / R (Table 1) for constructing the pLGN-CsABI5-5 overexpression vector were designed using the In-Fusion HD Cloning primer design tool on the TaKaRa official website (https: / / www.takarabio.com) with the pGEM-T-CsABI5-5 sequence. The pGEM-T-CsABI5-5 plasmid was used as a template for PCR amplification, with a size of 1356 bp. The amplified product was recovered by gel recovery and homologously recombined with the pLGN vector linearized with SalⅠ and EcoRⅠ to obtain the pLGN-CsABI5-5 overexpression vector ( Figure 1), the pLGN-CsABI5-5 vector was transformed into competent K599 and EHA105 according to the instructions of Agrobacterium tumefaciens EHA105 competent (Qingke Biotechnology) and Agrobacterium rhizogenes K599 competent (Weidi Biotechnology), and single clones were selected and the plasmid was extracted and digested with enzymes ( Figure 2 and Figure 3 ). If the target gene size band exists after electrophoresis of the enzyme digestion product, it means that the vector transformation is successful and the bacterial solution can be preserved for subsequent experiments.

[0032] Example 2 Identification of transgenic hairy roots

[0033] CLas mainly moves in citrus with phloem sap, first spreading from the invasion point to the roots, then to the newly sprouted young leaves, and finally to the mature leaves. In order to preliminarily understand the resistance of CsABI5-5 to Huanglongbing, the pLGN-CsABI5-5 vector was transferred into the stem segments of Ziyang orange infected with Huanglongbing by vacuum infection of stem segments with Agrobacterium rhizogenes to induce hairy root regeneration. After the transgenic hairy roots were cultured in the incubator for about 3 months, each hairy root on the stem segment was stained with GUS to identify its positivity. DNA was extracted from the GUS-positive transgenic hairy roots and the main veins of the corresponding stem segments according to the instructions of the DNA kit. The hairy roots of Ziyang orange infected with Huanglongbing and negative for GUS staining were used as the negative control group (CK), and the pLGN-CsABI5-5 plasmid was used as the positive control. The DNA of the hairy roots was amplified by PCR using primers 35S-F / pCsABI5-5-R (1). After electrophoresis, the target band was about 1900 bp (35S: 550 bp + CsABI5-5: 1356 bp), which was the transgenic hairy root. The statistical data of the transgenic hairy roots are shown in Table 2.

[0034] Table 2 Statistical data of transgenic hairy roots

[0035] Vector Name pLG pLGN-CsABI5-5 Number of rooting plants 13 20 Total number of plants 18 20 Rooting rate 72.22％ 100％ Number of GUS positive roots 8 6 Total hair roots 55 106 GUS positive rate 14.5％ 5.66％ PCR positive rate 100％ 100％

[0036] As shown in Table 2, the rooting rate of CsABI5-5 transgenic hairy roots was 100%, and the GUS staining positive rate was low, only 5.66%. Figure 4 As shown in Figure 2, the growth of hairy roots in the pLGN-CsABI5-5 group was similar to that in the CK group. By PCR identification, a fragment of about 1900 bp was detected in the GUS-positive hairy roots in the pLGN-CsABI5-5 group ( Figure 5 ), a total of 3 Ziyang orange stem segments (OE-3, OE-5 and OE-8) had positive hairy roots.

[0037] Example 3 Evaluation of transgenic hairy roots against citrus Huanglongbing disease

[0038] The probe method was used to detect the CLas content in the hairy roots and leaf main veins of the pLGN-CsABI5-5 transgenic strain. DNA was extracted from the GUS-positive transgenic hairy roots and leaf main veins of the corresponding stem segments in Example 2 according to the instructions of the DNA kit and diluted to 10 ng / μL. The probe HLB-P and primers HLBas-F / R (Table 1) were used to detect the CLas content in the leaf main veins and transgenic hairy roots.

[0039] The CLas content per microgram of citrus DNA (lg) was calculated according to the following formula:

[0040]

[0041] Among them, C t It is the value of real-time fluorescence quantitative PCR (qPCR).

[0042] like Figure 6 As shown in the figure, the CLas content in the hairy roots of the CK group was significantly higher than that in the main veins of the leaves, about 1.2 times that of the main veins of the leaves, while the CLas content in the hairy roots of the pLGN-CsABI5-5 transgenic line was not significantly different from that in the main veins of the leaves. This indicates that overexpression of CsABI5-5 inhibits the migration and proliferation of CLas into the hairy roots, showing an antagonistic effect on Huanglongbing bacteria.

[0043] Example 4 Identification of transgenic plants

[0044] The pLGN-CsABI5-5 vector was transferred into the epicotyls of Wanjin Orange by Agrobacterium tumefaciens-mediated genetic transformation to obtain overexpression (OE) transgenic Wanjin Orange plants. Four strains (OE-4, OE-11, OE-21 and OE-37) were selected for detailed analysis ( Figure 7 ). The DNA of the transgenic strains was extracted, and the wild-type Wanjin Orange, which was also genetically transformed and GUS-negative, was used as a negative control group (WT), and the pLGN-CsABI5-5 plasmid was used as a positive control. The primers 35S-F / pCsABI5-5-R (Table 1) were used to detect the insertion of the exogenous gene. PCR detection showed that CsABI5-5 was successfully inserted into the plant genome ( Figure 8 ). RT-PCR detection was performed on strains OE-4, OE-11, OE-21, OE-37 and WT using primers qCsABI5-5-F / R (Table 1). It was found that the CsABI5-5 levels in overexpressing plants OE-4, OE-11, OE-21 and OE-37 were significantly increased, which were 5.85 times, 9.89 times, 10.5 times and 9.71 times that of WT, respectively ( Fig. 9 ).

[0045] Example 5 Evaluation of resistance of transgenic plants to citrus canker

[0046] In order to evaluate the resistance of CsABI5-5 transgenic plants to citrus canker, the detached leaves of transgenic CsABI5-5 Wanjincheng were evaluated for canker resistance by needle inoculation. The specific steps included: wiping healthy leaves with 75% ethanol and sterile water three times, and then placing the leaves in a 150mm large culture dish, while keeping the petiole wrapped in cotton wool soaked in ddH2O. Use a 0.5mm needle to gently prick the areas on both sides of the veins, requiring the size and number of the pinholes in the two areas to be the same, and then use a pipette to inoculate 1μL of Xcc resuspension at the pinholes, and seal the culture dish with paraffin tape, and culture it in a 28℃ light incubator with a photoperiod of 16h light / 8h dark. Observe the disease condition of the leaves every day and keep records, take photos on the 7th and 10th days of inoculation, and count the area of ​​canker lesions (LA, mm 2 ), the condition was graded, and the grading standards are shown in Table 3.

[0047] Table 3 Ulcer disease grading standards

[0048] Disease level <![CDATA[Lesion area LA (mm 2 )]]> Level 0 LA≤0.25 Level 1 0.25<LA≤0.5 Level 2 0.5<LA≤0.75 Level 3 0.75<LA≤1 Level 4 1<LA≤1.25 Level 5 1.25<LA≤1.5 Level 6 1.5<LA≤1.75 Level 7 LA>1.75

[0049] The disease index (DI) was calculated according to the following formula:

[0050]

[0051] Ten days after inoculation, the pustules on the leaves of the WT group were large and raised, while the lesion areas on the leaves of the different CsABI5-5 overexpressing plants OE-4, OE-11, OE-21 and OE-37 were significantly smaller than those of the WT group ( Fig.10 The statistical results of the lesion area showed that the lesion area of ​​the overexpression plants OE-4, OE-11, OE-21 and OE-37 were significantly smaller than that of the WT group plants, with a reduction of about 47.95%-65.07% ( Fig.11 ), among which the lesion area of ​​OE-21 was the smallest, only 34.93% of WT. The disease index calculation results showed that the disease index of OE-4, OE-11, OE-21 and OE-37 was significantly lower than that of WT group plants, decreasing by 41.35%, 31.46%, 34.30% and 34.09% respectively. Fig.12 The above results indicate that overexpression of CsABI5-5 can enhance the resistance of citrus to ulcer disease.

Claims

1. A CsABI5-5 The application of genes in regulating plant resistance to Huanglongbing and ulcer diseases is characterized in that: Said CsABI5-5 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; the application is to overexpress CsABI5-5 Gene to improve the plant's resistance to Huanglongbing and ulcer diseases; the plant is citrus.

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