Application of wheat gene taald1 in improving resistance to stripe rust in wheat

Abstract

This invention discloses the application of the wheat gene TaALD1 in improving wheat stripe rust resistance, belonging to the field of biotechnology. The key technical points are: the nucleotide sequence of TaALD1 is shown in SEQ ID No. 1. In this invention, TaALD1 is upregulated after infection with stripe rust fungus, and virus-induced gene silencing confirms that TaALD1 plays a positive regulatory role in wheat stripe rust resistance. Simultaneously, transgenic plants of the ALD1 gene significantly enhance wheat's resistance to stripe rust, showing promising application prospects in breeding disease-resistant wheat varieties and laying an important foundation for wheat disease-resistant breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the wheat gene TaALD1 in improving wheat stripe rust resistance. Background Technology

[0002] In the process of preventing and controlling wheat stripe rust, the most economical and effective measures are to select and cultivate disease-resistant varieties in a reasonable manner. However, due to climate change, unreasonable cultivation practices, and frequent mutations of the virulence races of stripe rust fungus, the resistance of disease-resistant varieties has been gradually lost.

[0003] As an important signaling molecule, salicylic acid (SA) plays a crucial role in plant defense against live and semi-live pathogens. Studies have shown that SA participates in plant pathogen-associated molecule-triggered immunity (PTI) and effector-triggered immunity (ETI), and can stimulate systemic acquired resistance (SAR) in plants by producing a large number of pathogenesis-related proteins.

[0004] In the SA signaling pathway-mediated disease resistance response, AGD2-like DEFENSE RESPONSE PROTEIN 1 (ALD1) is a key regulator, crucial for triggering the basal defense response and SAR. ALD1 was first isolated and identified in Arabidopsis as a homolog of ABERRANT GROWTH AND DEATH2 (AGD2). It is an aminotransferase that catalyzes the production of pipecolic acid (Pip) from L-lysine. To date, no related research on the wheat TaALD1 gene has been reported.

[0005] Therefore, the present invention aims to provide the application of the wheat gene TaALD1 in improving wheat stripe rust resistance, in order to solve the above-mentioned problems. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems and provide the application of the wheat gene TaALD1 in improving wheat stripe rust resistance.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides the application of the wheat gene TaALD1 in improving wheat stripe rust resistance, and the cDNA nucleotide sequence of the TaALD1 gene is shown in SEQ ID NO:1.

[0009] The present invention also provides a wheat aminotransferase TaALD1, the amino acid sequence of which is shown in SEQ ID NO:2.

[0010] The transcription level of the TaALD1 gene is induced by stripe rust infection. TaALD1 enhances wheat's resistance to stripe rust by inhibiting the spread of stripe rust and strengthening the host's immune response. Overexpression of TaALD1 can significantly enhance wheat's resistance to stripe rust.

[0011] Overexpression of the wheat aminotransferase gene TaALD1 in wheat plants involved constructing the overexpression vector pCambia1300-TaALD1 and transferring the overexpression vector into wheat embryos using Agrobacterium-mediated transformation.

[0012] SEQ ID NO:1

[0013]

[0014] SEQ ID NO:2

[0015] MMTGTTASPATKATAMPFLSSSSSFSLRASKSSNLWPTRRLPVNVRCVRSSPAAAYTTKVSRNANIAKLQAGYLFPEVARRRAAHLLKYPDAKIISLGVGDTSEPIPEVITNAMAE RAHALSTVDGYSGYGAEQGEKKLRAIAAATYYADLGIEETDIFVSDGAKCDISRLQLLFGSNVKIAVQDPSYPVYVDSSVIMGQTDLYQQDVQKYGNIEYMRCSPENGFFPDLSSV PRTDIIFFCSPNNPTGTAASRDQLTQLVKFAKDNGSIIFYDSAYAMYISDDSPKSIFEIPGAKEVAMETASFSKYAGFTGVRLGWTVVPKELLFSDGHSVAKDFNRIVCTSFNGA STISQAGGLGCLSPEGLEAMQDVVGFYKENTKIIVDTFTSLGFDVYGAKNAPYVWVHFPGRNSWDVFAEILEKAHVVTTPGIGFGPSGEGFVRVSAFGHRETIIEAARRLKQLYK.

[0016] The TaALD1 is induced to be expressed by wheat stripe rust pathogen; the TaALD1 has a positive regulatory effect on wheat resistance to stripe rust; the TaALD1 can enhance wheat resistance to stripe rust pathogen; and transgenic plants can be obtained by transferring TaALD1 into wheat.

[0017] Compared with existing technologies, the beneficial effects of this solution are:

[0018] 1. TaALD1 was induced to express by stripe rust fungus, and it was determined that TaALD1 positively regulates wheat resistance to stripe rust by inhibiting the spread of stripe rust fungus and enhancing the host's immune response (i.e., increasing the production of reactive oxygen species);

[0019] 2. The TaALD1 gene was ligated into an overexpression vector, and the recombinant vector was transferred into wheat embryos using Agrobacterium-mediated transformation. The resulting transgenic wheat plants showed significantly enhanced resistance to stripe rust. Therefore, the TaALD1 gene can be used to create wheat lines resistant to stripe rust. Attached Figure Description

[0020] Figure 1This is a schematic diagram illustrating the transcriptional level analysis of TaALD1 after wheat water source 11 was inoculated with stripe rust physiological races CYR23 (non-toxic) and CYR31 (toxic) in an embodiment of the present invention.

[0021] Figure 2 This is a schematic diagram showing the phenotypic observation results of TaALD1 silent leaves inoculated with stripe rust fungi CYR23 and CYR31, respectively, in an embodiment of the present invention;

[0022] Figure 3 This is a schematic diagram of histological observation of TaALD1 silent leaves inoculated with stripe rust fungus CYR23 in an embodiment of the present invention. A: Histological observation of TaALD1 silent leaves (left) and non-silenced leaves (right) after stripe rust inoculation; the scale bar represents 50 μm; B: Statistical analysis of hyphal length after 48 h and 120 h of stripe rust infection; SV, substomatal sac; HMC, asspiratory mother cells; IH, primary hyphae; C: Statistical analysis of H2O2 area produced near the infection point in TaALD1 silent leaves after 48 h and 120 h of stripe rust infection; asterisks indicate significant differences in hyphal length or H2O2 production area between non-silenced leaves and silent TaALD1 leaves at each time point (*P < 0.05).

[0023] Figure 4 This is a schematic diagram of the expression level detection and phenotypic observation results of T1 generation transgenic TaALD1 wheat plants in an embodiment of the present invention, wherein A: phenotypic identification of T1 generation transgenic TaALD1 plants after inoculation with CYR32; B: expression level detection of T1 generation transgenic TaALD1 plants. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0026] Example 1: Analysis of TaALD1 expression after stripe rust infection

[0027] Wheat seed 11 showed an incompatible interaction with stripe rust fungus patho CYR23 (reactive type 0) and an affinity interaction with CYR31 (reactive type 4). After the first wheat leaf was fully expanded, stripe rust fungus was applied to both sides of the first wheat leaf using a toothpick. After inoculation, the leaves were placed at 10±1℃ in the dark and moist for 24 hours, and then transferred to a light incubator for normal culture. Total RNA was extracted from wheat leaves at 0, 12, 24, 48, 72, and 120 hours after stripe rust inoculation. The transcriptional level of TaALD1 was analyzed using real-time quantitative PCR, with wheat elongation factor as an internal reference gene. The TaALD1 expression level in wheat leaves inoculated with CYR23 at 0 hpi (hours post-inoculation) was set as 1. An asterisk indicates a significant difference in TaALD1 expression between wheat leaves inoculated with CYR23 and CYR31 at each time point (*P<0.05).

[0028] The results are as follows Figure 1 As shown, after inoculation with stripe rust fungus CYR23, the expression level of TaALD1 increased significantly, reaching a maximum at 48 h, and the expression level was significantly higher than that after inoculation with CYR31 from 24 to 72 h.

[0029] Example 2: Resistance analysis of TaALD1 to stripe rust fungi

[0030] Wheat seeds from water source 11 were sown in the substrate and cultured at 15±1℃ under a 16h light / 8h dark environment. When the wheat plants reached the three-leaf stage, the second leaf was inoculated with BSMV (barley stripe mosaic virus). After inoculation, a small amount of water was sprayed onto the wheat leaves from a height, and the wheat was placed at 25℃ in the dark with 100% humidity for 24h, followed by normal light / dark cycles of 16 / 8h. Once the inoculated wheat plants showed mosaic symptoms, they were inoculated with stripe rust fungi CYR23 and CYR31, respectively. The results are as follows... Figure 2 As shown, after wheat plants with silenced TaALD1 were inoculated with stripe rust fungus CYR23, necrotic spots appeared on the leaves, and a large number of uredinia appeared near the necrotic areas. However, after inoculation with CYR31, there was no significant difference in phenotype between the leaves with silenced TaALD1 and those without silenced TaALD1, indicating that TaALD1 participates in wheat resistance to stripe rust fungus and plays a positive regulatory role.

[0031] Example 3: Histological observation of TaALD1-silenced wheat leaves

[0032] Wheat plants inoculated with the fungus (including plants with silenced TaALD1 gene and those without) were sampled at 48 hpi and 120 hpi. Leaf segments were placed in centrifuge tubes containing a decolorizing solution (0.75 g trichloroacetic acid + 500 mL anhydrous ethanol + 100 mL chloroform) for decolorization. The decolorizing solution was changed until the leaves were completely dechloroated and colorless. The decolorizing solution was then removed, and a clearing solution (250 g chloral hydrate + 90 mL water) was added to make the leaf segments transparent. Finally, DAB and WGA dyes were used for staining to observe the spread of the stripe rust fungus and the production of reactive oxygen species by the host, and the data were statistically analyzed. Results are as follows: Figure 3 As shown, after silent wheat TaALD1 plants were inoculated with the non-toxic race CYR23 of stripe rust, the mycelial length increased significantly compared with unsilenced wheat, and the area of ​​H2O2 produced near the infection point of stripe rust was significantly reduced.

[0033] Example 4: Phenotypic observation of TaALD1 gene transgenic wheat plants

[0034] The expression vector pCambia1300-TaALD1 was transformed into the susceptible wheat variety Fielder using Agrobacterium-mediated transformation to further investigate the function of TaALD1 in wheat resistance to stripe rust. Ten positive lines of TaALD1-T1-L5 were inoculated with the prevalent stripe rust race CYR32, and phenotypes were observed approximately 14 days post-inoculation. The T1-L1 line served as a negative control. Results showed that compared to wild-type Fielder wheat, the gene expression level in positive TaALD1-transformed wheat plants was significantly increased. Figure 4 A) Inoculation with the prevalent stripe rust race CYR32 significantly reduced the number of stripe rust spores on leaves, and markedly increased wheat resistance to stripe rust. Figure 4 B).

[0035] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The application of the wheat gene TaALD1 in improving wheat stripe rust resistance, characterized by: The nucleotide sequence of TaALD1 is shown in SEQ ID NO:

1.

2. The application of the wheat gene TaALD1 as described in claim 1 in improving wheat stripe rust resistance, characterized in that: The TaALD1 was induced to express by wheat stripe rust.

3. The application of the wheat gene TaALD1 as described in claim 1 in improving wheat stripe rust resistance, characterized in that: TaALD1 plays a positive regulatory role in wheat resistance to stripe rust.

4. The application of the wheat gene TaALD1 as described in claim 1 in improving wheat stripe rust resistance, characterized in that: TaALD1 enhances the host's immune response and improves wheat's resistance to stripe rust pathogens by inhibiting the spread of stripe rust fungus.

5. The application of the wheat gene TaALD1 as described in claim 1 in improving wheat stripe rust resistance, characterized in that: Transgenic TaALD1 wheat plants significantly enhance resistance to stripe rust.

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