Method for identifying cold resistance of wheat at seedling stage by utilizing autophagy protein ATG8

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for identifying cold resistance of wheat in a seedling stage by utilizing autophagy protein ATG8. The method comprises the following steps: constructing a vector of EGFP (enhanced green fluorescent protein)-ATG8, carrying out transient expression on leaves of different wheat varieties, and observing subcellular localization change of the EGFP-ATG8 when the EGFP-ATG8 is subjected to cold stress, so as to identify the cold resistance of the wheat; compared with an existing scheme, subcellular localization of EGFP-ATG8 can be observed only under the low-temperature stress condition of 4 DEG C, the cold resistance of EGFP-ATG8 can be known, and the method has the advantages of being low in cost, easy to operate and high in precision.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a method for identifying cold resistance of wheat seedlings by using autophagy protein ATG8. BACKGROUND

[0002] Low temperature in winter is one of the main abiotic stress factors for wheat growth, which directly affects the yield and quality of wheat. Through cold resistance identification, wheat varieties suitable for low temperature environment can be screened and cultivated, and the survival ability and production potential of wheat under cold conditions can be improved. This not only has important significance for guaranteeing food security, but also helps to optimize the planting structure and improve the stability and sustainability of agricultural production. In addition, cold resistance identification is also the basis for studying the resistance mechanism of wheat, which provides a scientific basis for revealing the molecular mechanism of plant cold resistance and developing new cold resistance gene resources.

[0003] Currently, there are mainly two methods for screening wheat cold resistance: 1. Winter field seedling cold resistance identification, when the temperature drops below-10℃, the necrosis of seedling leaves is observed and counted; the smaller the necrosis area, the better the cold resistance; this method has a long cycle and is limited by winter weather conditions, and if it is a warm winter, the identification cannot be carried out; 2. Low temperature stress in artificial climate chamber, the necrosis of seedling leaves is observed; this method has high requirements for the conditions of artificial climate chamber, needs to be set below-10℃, and needs sufficient light, which consumes a lot of cost.

[0004] Therefore, whether a more simple and feasible method for identifying the cold resistance of wheat seedlings can be provided has become the main direction of the inventors' research. SUMMARY

[0005] In view of the problems of long screening cycle, harsh conditions, high cost, complex operation and low precision of cold-resistant wheat in the prior art, the application provides a method for identifying the cold resistance of wheat seedlings by using autophagy protein ATG8, which constructs an EGFP (enhancing GFP, enhanced GFP)-ATG8 vector, transiently expresses in the leaves of different wheat varieties, and identifies the cold resistance of wheat by observing the subcellular localization changes of EGFP-ATG8 under cold stress; compared with the existing scheme, the present experiment scheme only needs 4℃ low temperature stress condition, and the subcellular localization of EGFP-ATG8 is observed, so that the cold resistance can be known, which has the advantages of low cost, simple operation and high precision.

[0006] Autophagy is an important defense mechanism for wheat to adapt to low temperature environment; when wheat is subjected to low temperature stress, damaged cells will trigger the expression of autophagy-related genes (such as ATG family genes), and promote the formation of autophagosomes to degrade damaged cells to enhance the adaptability of wheat to low temperature stress; wherein the autophagy protein ATG8 is involved in the initiation, extension and completion of autophagy, and is a marker protein for detecting autophagy, the inventors construct a vector of EGFP (enhancing GFP, enhanced GFP)-ATG8, and express it in the leaves of different wheat varieties, if EGFP-ATG8 is mainly located in the nucleus under cold stress conditions, it indicates that the cold resistance of the variety is relatively poor; if EGFP-ATG8 is mainly located in the cytoplasm under cold stress conditions, it indicates that the cold resistance of the variety is relatively good. This is because the cold-resistant wheat is not sensitive to cold stress, and the cold stress has little effect on the location of EGFP-ATG8; and the cold-resistant wheat is sensitive to cold stress, and the corresponding EGFP-ATG8 will enter the nucleus for response.

[0007] The traditional wheat seedling stage cold resistance identification mainly judges the leaf necrosis under low temperature conditions, and the contents of soluble sugar, proline, POD and SOD and other antioxidant enzyme activities; compared with the traditional method, the method is simple and accurate.

[0008] The method for identifying the cold resistance of wheat seedlings by using the autophagy protein ATG8 provided by the application comprises the following specific steps: 1) 35S-EGFP-ATG8 reporter plasmid construction: homologous recombination of EGFP-ATG8 gene into CCDB-35S vector to form 35S-EGFP-ATG8 reporter plasmid; More specifically, the EGFP-ATG8 is recombined between the PST1 and XBAL1 enzyme cutting sites of the CCDB-35S vector; The sequence of EGFP is shown in SEQ ID NO. 1, the sequence of ATG8 is shown in SEQ ID NO. 2, and the sequence of the 35S-EGFP-ATG8 reporter plasmid is shown in SEQ ID NO. 4, wherein the terminal "TAA" in SEQ ID NO. 1 is a stop codon, so it is deleted in the sequence of 35S-EGFP-ATG8 shown in SEQ ID NO. 4; The gene homologous recombination adopts the CU201-02 homologous recombination kit of Beijing Quanshi Gold Biotechnology Co., Ltd.; The sequence of the EGFP-ATG8 gene is shown in SEQ ID NO. 3, wherein EGFP is upstream and ATG8 is downstream; 2) Wheat seedling culture: culture the wheat in a light incubator to the two-leaf one-stamen stage; 3) 35S-EGFP-ATG8 wheat leaf infection: transform 35S-EGFP-ATG8 reporter plasmid into Agrobacterium EHA105 competent cells, and then cultivate the Agrobacterium EHA105 competent cells containing the 35S-EGFP-ATG8 reporter plasmid in a constant temperature incubator at 28°C to an OD value of 0.6-0.8, and then infect the wheat leaves to mark the infiltration area; 4) Cold stress treatment: after 48-72 h of infection in the light incubator, cut the leaf infiltration area and place it at 4°C for about 48-72 h; 5) Preparing a sample for microscopic examination: cut part of the leaf infiltration area and place it on a glass slide, drop 100-500 μL tap water on the upper part of the cut leaf, cover it with a cover glass and gently squeeze it to complete the sample preparation; Place the sample under a 100x fluorescence microscope and observe the localization of EGFP-ATG8.

[0009] If EGFP-ATG8 is mainly localized in the nucleus under cold stress conditions, it indicates that the variety has relatively poor cold resistance; if EGFP-ATG8 is mainly localized in the cytoplasm under cold stress conditions, it indicates that the variety has relatively good cold resistance.

[0010] The main localization in the nucleus and the main localization in the cytoplasm can be determined according to the actual observation, for example, if the cells with EGFP-ATG8 localized in the nucleus account for more than a certain proportion of the total cells in the field of view, it is determined that the main localization is in the nucleus, and if it is less than a certain proportion, it is determined that the main localization is in the cytoplasm. The proportion can be flexibly adjusted according to the screening target, and is not rigidly limited in the present application.

[0011] As a preferred, the EGFP-ATG8 fragment synthesis method in step 1) is: splice and design EGFP sequence and wheat ATG8 open reading frame sequence to form EGFP-ATG8 sequence, and then generate EGFP-ATG8 gene by gene chemical synthesis method; the EGFP sequence is shown as SEQ ID NO. 1, and the wheat ATG8 open reading frame sequence is shown as SEQ ID NO. 2; The gene ID of the wheat ATG is TraesCS2B02G491200; The method of gene chemical synthesis generates EGFP-ATG8 gene, which is described in detail in “1.1 Oligonucleotide column synthesis technology” and its Figure 1 .

[0012] The enhancing GFP sequence (EGFP) is selected, which has obvious difference with the common GFP sequence. The EGFP has C, G and T at the 193, 195 and 695 positions, while the common GFP has T, C and A. The expression signal of the EGFP in wheat is more stable, strong and easy to observe. The common GFP sequence is shown as SEQ ID NO. 5.

[0013] Preferably, the culture condition of the light incubator in step 2) is: 22℃, light for 16 h; 20℃, dark for 8 h.

[0014] In some embodiments of the present application, the wheat variety in step 2) is cold-resistant wheat or non-cold-resistant wheat. The cold-resistant wheat includes one or more of Jimai 70, Jimai 22 and Jimai 379; the non-cold-resistant wheat includes one or more of Zhongdongchun, Feilder and Chuanmai 107; 9 seedlings per variety, 3 repeats, 3 seedlings per repeat.

[0015] Jimai 70, Jimai 22 and Jimai 379 are approved varieties in the Huanghuai North Winter Wheat Region, and the approval standards clearly stipulate that the cold resistance should be good, otherwise they will not be approved. The approval standards are detailed in (Guopinshen

[2024] 1 document).

[0016] The above-mentioned Jimai 70, Jimai 22 and Jimai 379 are cold-resistant winter wheat varieties known in the art, which are clearly stipulated in the approval standards, and are also recorded in the following published literatures: [1] Fan Qingqi, Chu Xiusheng, Sui Xin Xia, et al. High-yield, anti-down new wheat variety Jimai 70 [J]. China Seed Industry, 2021, 02: 114-115. DOI: 10.19462 / j.cnki.1671-895x.2021.02.041; [2] Zhai Jianqiang, Xu Jinbiao, Xi Xiusheng. Characteristics and high-yield cultivation points of Jimai 22 [J]. Agricultural Engineering Technology, 2020, 40 (08): 56. DOI: 10.16815 / j.cnki.11-5436 / s.2020.08.042; [3] https: / / www.chinaseed114.com / seed / 17 / seed_80726.html Different cold resistance of wheat varieties Growth point development process and physiological index difference analysis [J].

[0017] The above Zhongguochun, Feilder and Chuanmai 107 belong to the cold-intolerant varieties known in the art and are recorded in the published documents: [1] Liu Chang, Du Yuchen, Li Ninghui, et al. Difference analysis of growth point development process and physiological indexes of different cold-resistant wheat varieties [J]. Journal of Triticeae Crops, 2023, 43(06): 721-728 and [2] Yu Chunlian. New disease-resistant wheat variety Chuanmai 107 [J]. Sichuan Agricultural Science and Technology, 2001, 07: 11.

[0018] As preferred, the second leaf from the base of the wheat is selected for infection in step 3), and the main reason is that the leaf is relatively strong and easy to infect.

[0019] As preferred, the method for infecting the leaf in step 3) is to make a wound in the middle of the second leaf, absorb 1 mL of bacterial solution with a 1 mL syringe, block the wound and penetrate the bacterial solution into the leaf, and mark the penetration area on the leaf with a marker pen.

[0020] As preferred, the conditions of the light incubator in step 4) are: temperature 22℃, light 16 h, and darkness 8 h.

[0021] Compared with the prior art, the present application has the following beneficial effects: The present application adopts ATG8 construction plasmid and transfection, and observes the distribution of EGFP-ATG8 in cells under cold stress to realize the detection of wheat cold resistance, which can directly observe and identify the cold resistance from the cell level, is simple and convenient to operate, has low cost, and has high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure is the localization microscopy result of EGFP-ATG8 in Zhongguochun; EGFP-ATG8 is mainly localized in the nucleus, and the bright spots in the figure are the nucleus; Figure 2 The figure is the localization microscopy result of EGFP-ATG8 in Feilder; EGFP-ATG8 is mainly localized in the nucleus, and the bright spots in the figure are the nucleus; Figure 3 The figure is the localization microscopy result of EGFP-ATG8 in Chuanmai 107; EGFP-ATG8 is mainly localized in the nucleus, and the bright spots in the figure are the nucleus; Figure 4 The figure is the localization microscopy result of EGFP-ATG8 in Jimai 70; EGFP-ATG8 is mainly localized in the cytoplasm; Figure 5 The figure is the localization microscopy result of EGFP-ATG8 in Jimai 22; EGFP-ATG8 is mainly localized in the cytoplasm; Figure 6 The results of the localization microscopy of EGFP-ATG8 in Jimai 379; EGFP-ATG8 is mainly localized in the cytoplasm. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be further described below in combination with specific embodiments, and these embodiments are only used to more specifically describe the technical solutions of the present application, and should not be understood as limiting the protection scope of the present application.

[0024] The substances and reagents used in the following examples are commercially available products unless otherwise specified; the detection reagents or detection kits used are used according to the conventional methods in the art or according to the instructions.

[0025] Example 1 Method for constructing 35S-EGFP-ATG8 reporter plasmid EGFP-ATG8 gene is homologously recombined into CCDB-35S vector to form 35S-EGFP-ATG8 reporter plasmid; the gene homologous recombination uses CU201-02 homologous recombination kit of Beijing Quanshi Gold Biotechnology Co., Ltd.; The sequence of the EGFP-ATG8 gene is shown in SEQ ID NO. 3, in which EGFP is upstream and ATG8 is downstream.

[0026] Among them, the EGFP-ATG8 fragment synthesis method is: the EGFP sequence and the wheat ATG8 open reading frame sequence are spliced to design an EGFP-ATG8 sequence, and then the EGFP-ATG8 gene is generated by gene chemical synthesis method; the sequence of the EGFP sequence is shown in SEQ ID NO. 1, and the sequence of the wheat ATG8 open reading frame sequence is shown in SEQ ID NO. 2; More specifically, EGFP-ATG8 is recombined between the PST1 and XBAL1 enzyme cutting sites of the CCDB-35S vector. Among them, the sequence of EGFP is shown in SEQ ID NO. 1, the sequence of ATG8 is shown in SEQ ID NO. 2, and the sequence of the 35S-EGFP-ATG8 reporter plasmid is shown in SEQ ID NO. 4.

[0027] The gene ID of the wheat ATG is TraesCS2B02G491200; The method for generating the EGFP-ATG8 gene by gene chemical synthesis is shown in detail (https: / / www.detaibio.com / topics / gene-synthesis.html); methods that can be used are as follows: (1): DMTro is a protecting group of 5' hydroxyl, which reacts with TCA (trichloroacetic acid) and the 5' end of the nucleotide pre-attached to the solid carrier (CPG) to remove DMT and free the 5' hydroxyl end; (2): Phosphoramidite-protected dNTPs and activator tetrazole are mixed to obtain a nucleoside phosphoramidite activation intermediate, at which time the 3' end is activated and the 5'-hydroxyl is protected by DMTro; (3): The products of (1) and (2) undergo condensation reaction coupling to form a new C-phosphite bond; (4): The 5'-hydroxyl of the remaining part of (1) is not involved in the reaction, and the 5'-hydroxyl is closed by acetylation; (5): The C-phosphite bond formed in (3) is unstable and easy to be hydrolyzed, so it is oxidized to a more stable phosphotriester bond by an oxidizing agent iodine. Through the above steps, a deoxyribonucleotide is attached to the solid carrier, the DMTro at the 5' end is removed, and all the required bases are synthesized by repeating the above steps. Finally, after high-temperature treatment with ammonia, the synthesized chain is cut off from the solid carrier CPG, and is stored after purification according to different downstream applications.

[0028] The 35S-EGFP-ATG8 reporter plasmid used in the following examples is the 35S-EGFP-ATG8 reporter plasmid synthesized in Example 1.

[0029] Example 2 Cold resistance identification of Zhongguochun 1) Wheat seedling culture: Zhongguochun wheat is cultured to the two-leaf one-stem stage in a light incubator, and the culture conditions are: 22°C, 16 h of light; 20°C, 8 h of darkness; 2) 35S-EGFP-ATG8 wheat leaf infection: the 35S-EGFP-ATG8 reporter plasmid is transformed into Agrobacterium EHA105 competent cells, and then the Agrobacterium EHA105 competent cells containing the 35S-EGFP-ATG8 reporter plasmid are cultured in a constant temperature incubator at 28°C to an OD value of 0.6-0.8 to obtain a bacterial solution, and the bacterial solution is infiltrated into the second leaf from the base of the wheat and marked the infiltration area; The method for infiltrating the leaf is: a wound is made in the middle of the second leaf with a needle tip, 1 mL of bacterial solution is absorbed with a 1 mL syringe, the wound is blocked and the bacterial solution is slowly infiltrated into the leaf, and the infiltration area is marked on the leaf with a marker pen; 3) Cold stress treatment: after 48 h of infiltration in the light incubator (22°C, 16 h of light, 8 h of darkness), the infiltration area of the leaf is cut off and placed in a refrigerator at 4°C for about 48 h; 4) Preparation of the slide: cut a part of the leaf penetration area, put it on the slide, drop 100 μL tap water on the upper part of the cut leaf, cover the cover glass and gently squeeze, complete the preparation of the sample; Place the sample under a 100-fold fluorescence microscope and observe, as shown in Figure 1 , to determine the location of EGFP-ATG8, wherein the bright green dots represent the nucleus.

[0030] Example 3: Cold resistance identification of Feilder The method of Example 2 was used to identify the cold resistance of wheat, except that Feilder wheat was used instead of Chinese Spring wheat, and the identification results are shown in Figure 2 .

[0031] Example 4: Cold resistance identification of Chuanmai 107 The method of Example 2 was used to identify the cold resistance of wheat, except that Chuanmai 107 wheat was used instead of Chinese Spring wheat, and the identification results are shown in Figure 3 .

[0032] Example 5: Cold resistance identification of Jimai 70 The method of Example 2 was used to identify the cold resistance of wheat, except that Jimai 70 wheat was used instead of Chinese Spring wheat, and the identification results are shown in Figure 4 .

[0033] Example 6: Cold resistance identification of Jimai 22 The method of Example 2 was used to identify the cold resistance of wheat, except that Jimai 22 wheat was used instead of Chinese Spring wheat, and the identification results are shown in Figure 5 .

[0034] Example 7: Cold resistance identification of Jimai 379 The method of Example 2 was used to identify the cold resistance of wheat, except that Jimai 379 wheat was used instead of Chinese Spring wheat, and the identification results are shown in Figure 6 .

[0035] Result analysis: According to Figures 1-6 , in the non-cold-resistant wheat Chinese Spring, Feilder, and Chuanmai 107, EGFP-ATG8 was mainly located in the nucleus under cold stress; while in the cold-resistant wheat Jimai 70, Jimai 22, and Jimai 379, it was mainly located in the cytoplasm. The inventors analyzed the reason that the cold-resistant wheat was not sensitive to cold stress, and the cold stress had little effect on the location of EGFP-ATG8; while the non-cold-resistant wheat was more sensitive to cold stress, and the corresponding EGFP-ATG8 would enter the nucleus to respond. Therefore, the present application can determine the cold resistance of wheat varieties by observing the subcellular localization of EGFP-ATG8.

[0036] The existing technology can identify cold resistance by observing the leaf necrosis of field seedlings in winter, but the cycle is long and limited by winter climate conditions, and if a warm winter is encountered, the identification cannot be performed; low temperature stress can also be performed in an artificial climate chamber and leaf necrosis is observed, but this method has high requirements for the conditions of the artificial climate chamber, needs to be set below-10 DEG C and needs to maintain sufficient light, and has a high cost. Therefore, the above scheme provided in the application is more suitable for detecting the cold resistance of wheat.

[0037] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any equivalent replacement, modification, etc. made by those skilled in the art within the spirit and principle of the present application without any creative labor should be included in the protection scope of the present application.

Claims

1. A method for identifying cold resistance of wheat seedling using autophagy protein ATG8, characterized in that, The specific steps are as follows: 1) 35S-EGFP-ATG8 reporter plasmid construction: EGFP-ATG8 gene is homologously recombined into CCDB-35S vector to form 35S-EGFP-ATG8 reporter plasmid; 2) Wheat seedling culture: wheat is cultured to the two-leaf one-stem stage in a light incubator; 3) 35S-EGFP-ATG8 wheat leaf infection: 35S-EGFP-ATG8 reporter plasmid is transformed into Agrobacterium EHA105 competent cells, and then the Agrobacterium EHA105 competent cells containing 35S-EGFP-ATG8 reporter plasmid are cultured in a 28°C constant temperature incubator to an OD value of 0.6-0.8 to obtain a bacterial solution, and the bacterial solution is used to infect the wheat leaves, and the penetration area is marked; 4) Cold stress treatment: after 48-72 h of infection in the light incubator, the penetration area of the leaves is cut off, and the cut leaves are placed at 4°C for 48-72 h; 5) Sample preparation for microscopic examination: the penetration area of the leaves is cut off and placed on a glass slide, 100-500 μL of tap water is dropped on the upper part of the cut leaves, a cover glass is covered and gently squeezed to complete the sample preparation; The sample is placed under a fluorescence microscope for observation to determine the localization of EGFP-ATG8.

2. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that, The magnification of the fluorescence microscope is ≥100 times.

3. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that: EGFP-ATG8 is recombined between the PST1 and XBAL1 enzyme cutting sites of the CCDB-35S vector, wherein the sequence of EGFP is shown as SEQ ID NO. 1, the sequence of ATG8 is shown as SEQ ID NO. 2, the sequence of EGFP-ATG8 gene is shown as SEQ ID NO. 3, and the sequence of 35S-EGFP-ATG8 reporter plasmid is shown as SEQ ID NO.

4.

4. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that: The culture conditions of the light incubator in steps 2) and 4) are: 22°C, light for 16 h; 20°C, dark for 8 h.

5. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that: In step 3), the second leaf from the bottom of the wheat is selected for infection.

6. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 5, characterized in that: A wound is made in the middle of the second leaf, 1 mL of bacterial solution is absorbed with a 1 mL syringe, the wound is blocked, the bacterial solution is infiltrated into the leaf, and the infiltration area is marked on the leaf.

7. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that: The conditions of the light incubator in step 4) are: temperature 22°C, light for 16 h, and dark for 8 h.

8. The method for identifying cold resistance of wheat seedling by using autophagy protein ATG8 according to claim 1, characterized in that: EGFP-ATG8 is mainly localized in the nucleus, indicating that the cold resistance of this variety is relatively poor; EGFP-ATG8 is mainly localized in the cytoplasm, indicating that the cold resistance of this variety is relatively good.

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