Application of wheat gene TaALD1 in improving wheat stripe rust resistance

Abstract

The invention discloses application of a wheat gene TaALD1 in improving wheat stripe rust resistance, relates to the technical field of biology, and has the technical key points that the nucleotide sequence of the TaALD1 is shown as SEQ ID No.1. According to the invention, TaALD1 is subjected to up-regulation expression after being infected by stripe rust, and virus-induced gene silencing is adopted to determine that TaALD1 plays a positive regulation role in wheat stripe rust resistance; and meanwhile, the transgenic ALD1 plant can obviously improve the stripe rust resistance of the wheat, has a better application prospect in the cultivation of disease-resistant wheat varieties, and lays an important foundation for the disease-resistant breeding of the wheat.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of wheat gene TaALD1 in improving wheat stripe rust resistance. Background Art

[0002] Wheat stripe rust is a worldwide airborne disease caused by the wheat-specific form of the stripe rust fungus Pucciania striiformis f.sp.tritici. It has the characteristics of wide distribution, rapid epidemic speed and serious damage. It is one of the three major diseases affecting the safe production of wheat. The main areas where wheat stripe rust occurs in my country are 11 provinces (municipalities and autonomous regions), including Gansu, Shaanxi, Sichuan, Chongqing, Yunnan and Guizhou. Among them, Gansu is one of the important annual cycle occurrence areas of wheat stripe rust in China and an important source base of the fungus. In the process of preventing and controlling wheat stripe rust, breeding and rational planting of disease-resistant varieties are the most economical and effective measures. However, due to climate change, unreasonable human cultivation measures and frequent mutations of toxic species of stripe rust, the resistance of disease-resistant varieties is gradually lost.

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

[0004] In the disease resistance response mediated by the SA signaling pathway, AGD2-like DEFENSE RESPONSES PROTEIN 1 (ALD1) is a key regulatory factor that is essential for initiating basal defense responses and SAR. ALD1 was first isolated and identified in Arabidopsis as a homologous gene of ABERRANT GROWTH AND DEATH2 (AGD2). It is an aminotransferase that catalyzes L-lysine to produce pipecolic acid (Pip). So far, no related research reports on the wheat TaALD1 gene have been reported.

[0005] To this end, the present invention aims to provide an application of the wheat gene TaALD1 in improving wheat stripe rust resistance to solve the above problems. Summary of the invention

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

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

[0008] The present invention provides application of wheat gene TaALD1 in improving resistance to wheat stripe rust. 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, and the amino acid sequence of the wheat aminotransferase TaALD1 is shown in SEQ ID NO:2.

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

[0011] The invention relates to overexpressing wheat aminotransferase gene TaALD1 in wheat plants, comprising constructing an overexpression vector pCambia1300-TaALD1 and transferring the overexpression vector into wheat immature embryos by Agrobacterium-mediated method.

[0012] SEQ ID NO:1

[0013]

[0014] SEQ ID NO:2

[0015] MMTGTTASPATKATAMPFLSSSSSFSLRASKSSNLWPTRRLPVNVRCVRSSPAAAYTTKVSRNANIAKLQAGYLFPEVARRRAAHLLKYPDAKIISLGVGDTSEPIPEVITNAMAE RAHALSTVDGYSGYGAEQGEKKLRAIAAATYYADLGIEETDIFVSDGAKCDISRLQLLFGSNVKIAVQDPSYPVYVDSSVIMGQTDLYQQDVQKYGNIEYMRCSPENGFFPDLSSV PRTDIIFFCSPNNPTGTAASRDQLTQLVKFAKDNGSIIFYDSAYAMYISDDSPKSIFEIPGAKEVAMETASFSKYAGFTGVRLGWTVVPKELLFSDGHSVAKDFNRIVCTSFNGA STISQAGGLGCLSPEGLEAMQDVVGFYKENTKIIVDTFTSLGFDVYGAKNAPYVWVHFPGRNSWDVFAEILEKAHVVTTPGIGFGPSGEGFVRVSAFGHRETIIEAARRLKQLYK.

[0016] The TaALD1 is induced to express 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 a transgenic plant can be obtained by transferring TaALD1 into wheat.

[0017] Compared with the prior art, this solution has the following beneficial effects:

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

[0019] 2. The TaALD1 gene was linked to an overexpression vector and the recombinant vector was transferred into wheat embryos using the Agrobacterium-mediated method. The transgenic wheat plants obtained significantly enhanced the resistance of wheat to stripe rust. Therefore, the TaALD1 gene can be used to create wheat lines resistant to stripe rust. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0022] Figure 3 Schematic diagram of histological observation of TaALD1 silenced leaves inoculated with stripe rust CYR23 in an embodiment of the present invention, wherein A: histological observation of TaALD1 silenced leaves (left) and non-silenced leaves (right) after stripe rust inoculation; the scale represents 50 μm; B: statistics of hyphae length after 48h and 120h of stripe rust infection; SV, substomatal capsule; HMC, inspiratory mother cell; IH, primary hyphae; C: H produced near the infection point of TaALD1 silenced leaves 48h and 120h after stripe rust infection 2 O 2 Area statistics; Asterisks indicate hyphal length or H in non-silenced leaves and TaALD1-silenced leaves at each time point 2 O 2 There was a significant difference in the production area (*P<0.05);

[0023] Figure 4 Schematic diagram of the expression level detection and phenotypic observation results of T1 generation TaALD1 gene transgenic wheat plants in an embodiment of the present invention, wherein A: phenotypic identification of T1 generation TaALD1 gene transgenic plants after inoculation with CYR32; B: expression level detection of T1 generation TaALD1 gene transgenic plants. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

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

[0027] Wheat water source 11 has a non-affinity interaction with the physiological race CYR23 of stripe rust (reaction type, type 0), and an affinity interaction with CYR31 (reaction type, type 4). After the first leaf of wheat is fully expanded, use a toothpick to dip the stripe rust and smear it on the front and back of the first leaf of wheat. After inoculation, place it in a dark and moisturizing environment at 10±1℃ for 24 hours, and then transfer it to a light incubator for normal culture. Total RNA of wheat leaves was extracted at 0, 12, 24, 48, 72 and 120 hours after inoculation with stripe rust, and the transcription level of TaALD1 was analyzed by real-time fluorescence quantitative PCR technology, with wheat elongation factor as the internal reference gene. The expression level of TaALD1 in wheat leaves inoculated with CYR23 at 0hpi (hours post inoculation) after stripe rust inoculation was set to 1, and the asterisk indicates that there is a significant difference in the expression level of TaALD1 in 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 the maximum value at 48h, and the expression level was significantly higher than that after inoculation with CYR31 at 24-72h.

[0029] Example 2: Analysis of resistance of TaALD1 to stripe rust

[0030] Plant wheat seeds from water source 11 in a substrate and culture them at 15±1°C and 16h light / 8h dark. When the wheat plants grow to the three-leaf stage, inoculate the second leaf with BSMV (barley stripe mosaic virus). After the inoculation is completed, use a spray bottle to spray a small amount of water on the wheat leaves from a high altitude, and place the wheat at 25°C in the dark with 100% moisture retention for 24 hours, and then culture them normally with 16 / 8h light / darkness. After the virus-inoculated wheat plants show mosaic symptoms, they are inoculated with stripe rust fungi CYR23 and CYR31 respectively. The results are as follows: Figure 2 As shown in the figure, after wheat plants with TaALD1 silenced were inoculated with stripe rust CYR23, necrotic spots appeared on the leaves, and a large number of summer spores appeared near the necrosis. However, after inoculation with CYR31, there was no significant difference in the phenotype between leaves with TaALD1 silenced and leaves without silence, indicating that TaALD1 is involved in wheat resistance to stripe rust and plays a positive regulatory role.

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

[0032] Wheat plants after docking with bacteria (including plants with silenced TaALD1 gene and plants without silenced TaALD1 gene) were sampled at 48hpi and 120hpi, and the leaf segments were placed in a centrifuge tube with decolorizing solution (0.75g trichloroacetic acid + 500mL anhydrous ethanol + 100mL chloroform) for decolorization. The decolorizing solution needs to be replaced during the process until the leaves completely turn green and the leaves in the centrifuge tube become colorless. After that, the decolorizing solution was aspirated, and a transparent solution (250g chloral hydrate + 90mL water) was added to make the leaf segments transparent. Finally, DAB and WGA dyes were used for staining to observe the spread of stripe rust and the production of active oxygen by the host, and the data were statistically analyzed. The results are as follows: Figure 3 As shown in the figure, after wheat plants with TaALD1 silenced were inoculated with the avirulent stripe rust race CYR23, the hyphae length increased significantly compared with the unsilenced wheat plants, and the H 2 O 2 The area has been significantly reduced.

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

[0034] The expression vector pCambia1300-TaALD1 was transformed into the susceptible variety Fielder by Agrobacterium-mediated method to further study the function of TaALD1 in wheat stripe rust resistance. Ten positive strains of TaALD1-T1-L5 were inoculated with the stripe rust epidemic race CYR32, and the phenotype was observed about 14 days after inoculation. The T1-L1 strain was used as a negative control. The results showed that compared with the wild-type Fielder wheat, the gene expression level of the positive transgenic TaALD1 wheat plants was significantly increased ( Figure 4 A), after inoculation with the stripe rust epidemic race CYR32, the stripe rust spores on the leaves were significantly reduced, and the stripe rust resistance of wheat was significantly increased ( Figure 4 B).

[0035] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Application of 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 use of the wheat gene TaALD1 in improving wheat stripe rust resistance as claimed in claim 1, characterized in that: The TaALD1 is induced to express by wheat stripe rust.

3. The use of the wheat gene TaALD1 in improving wheat stripe rust resistance as claimed in claim 1, characterized in that: The TaALD1 has a positive regulatory effect on wheat resistance to stripe rust.

4. The use of the wheat gene TaALD1 in improving wheat stripe rust resistance as claimed in claim 1, characterized in that: The TaALD1 inhibits the spread of stripe rust fungi, enhances the host immune response, and improves the resistance of wheat to stripe rust fungi.

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

6. A wheat aminotransferase TaALD1, characterized in that: The amino acid sequence of the wheat aminotransferase TaALD1 is shown in SEQ ID NO:2.

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