A method for identifying plants for flood tolerance

Abstract

This invention relates to the field of plant breeding technology, and more particularly to a method for identifying the flood tolerance of plants. The method includes: subjecting a mulberry plant to flood stress treatment, then detecting the expression level of the MnFedA protein or MnFedA gene in the mulberry plant after treatment, and determining the flood tolerance of the mulberry plant based on the detection results; the flood stress treatment includes: placing the mulberry plant in water at a depth of 4-6 cm for 2-4 days. This invention has discovered a correlation between the MnFedA protein and its encoding gene and the flood stress tolerance of mulberry plants, and that the difference in expression levels is only observed under specific flood stress treatments, which has significant application value in the field of plant breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a method for identifying the flood tolerance of plants. Background Technology

[0002] Mulberry (Morus alba L.) is a perennial deciduous woody plant and an indispensable part of the sericulture industry. In recent years, given its high protein content, comprehensive amino acid profile, low cellulose content, and abundance of various physiologically active substances, mulberry has received widespread attention for its applications in food and pharmaceuticals, feed, and ecological protection. Mulberry trees planted on slopes, tidal flats, and drawdown zones not only have important ecological functions but also bring significant economic benefits. Water is a key factor affecting plant distribution and growth. Drought has a relatively slow and short-lived impact, and plants can recover quickly after rehydration; conversely, flooding is highly destructive and difficult to recover from, and even short-term flooding can cause serious damage to plants. Therefore, breeding new mulberry varieties that are resistant to flooding and stress is of significant theoretical and practical importance for establishing ecological restoration systems in ecologically fragile areas such as reservoir drawdown zones.

[0003] Traditional methods for screening mulberry varieties to withstand flooding rely heavily on phenotypic observation and physiological indicator testing, which suffer from problems such as long cycles (requiring several months), high costs (requiring large-scale field trials), and low accuracy (susceptible to environmental interference). Existing research shows that the MnFedA gene significantly enhances mulberry's flood tolerance by regulating reactive oxygen species (ROS) metabolism and photosynthetic system protection mechanisms, but a high-throughput screening system based on the expression level of this gene has not yet been established. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for identifying the flood tolerance of plants.

[0005] In a first aspect, the present invention provides a method for identifying the flood tolerance of mulberry plants, comprising: subjecting the mulberry plants to be tested to water flood stress treatment, detecting the expression level of MnFedA protein or MnFedA gene in the mulberry plants to be tested after treatment, and determining the flood tolerance of the mulberry plants to be tested based on the detection results;

[0006] The waterlogging stress treatment includes placing the mulberry plants in water at a depth of 4-6 cm for 2-4 days.

[0007] Furthermore, the waterlogging stress treatment includes: placing the mulberry plants in water at a depth of 4.5-5.5 cm for 2.5-3.5 days.

[0008] Furthermore, the MnFedA protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0009] The amino acid sequence shown in SEQ ID NO.1:

[0010] MATTTAAALSGTMVSTSFARRHAVSSLRALPNTGQALFGLKASRGGRVTAMASYKVKLITPDGEKEFDCPDDVYILDHAEEVGIDLPYSCRAGSCSSCAGKVVSGTLDQSDQSFLDDEQIDAGWVLTCVAYPQCDVTIETHKEEELTA.

[0011] Furthermore, the MnFedA gene comprises the nucleotide sequence shown in SEQ ID NO.2.

[0012] The nucleotide sequence shown in SEQ ID NO.2:

[0013] ATGGCAACAACAACAGCAGCAGCTCTCTCTGGCACGATGGTGAGCACCTCCTTCGCGAGAAGACATGCAGTGAGCAGCCTGAGGGCACTCCCCAACACGGGGCAGGCTCTCTTCGGCCTCAAGGCGAGCCGCGGAGGGCGTGTCACTGCCATGGCATACAAGGTGAAGCTGATAACACCCGACGGAGAAGGAGTTCGATTGCCCCGATGACGTCTACAT TCTCGACCACGCCGAGGAGGTTGGTATCGACCTGCCATACTCCTGCAGGGCTGGCTCTTGCTCTTCATGTGCTGGGAAGGTTGTGTCAGGAACACTCGACCAGTCTGACCAGAGCTTCCTTGATGATGAACAGATTGATGCAGGTTGGGTCCTCACTTGTGTTGCGTATCCTCAGTGTGATGTTACCATTGAGACCCACAAGGAGGAGGAACTCACTGCTTAA.

[0014] Furthermore, the detection is performed by PCR amplification, and the primer pairs used include the following:

[0015] FedA-F: ​​TGGTTGGTTGGATGGATTA;

[0016] FedA-R: GAAGTTGTATTAGTGTGGTTGA.

[0017] Furthermore, the PCR amplification procedure includes:

[0018] Treat at 93~98℃ for 2~5 minutes;

[0019] Treat at 93~98℃ for 8~15 seconds, at 55~65℃ for 25~40 seconds, at 70~75℃ for 25~40 seconds, for 35~50 cycles.

[0020] Furthermore, the determination of the flood tolerance of the tested mulberry plant based on the test results includes:

[0021] When the detection result of the MnFedA gene expression level is higher than the preset value, it is determined that the tested mulberry plant has high flood tolerance, and / or,

[0022] When the detection result of the MnFedA gene expression level shows no significant change, the tested mulberry plant is judged to have low flood tolerance.

[0023] Furthermore, the preset value can be 1.5 times, 2 times, 2.5 times, or 3 times the value before flooding treatment.

[0024] The invention has the following beneficial effects:

[0025] This invention has revealed a link between the mulberry ferredoxin gene (MnFedA) and the flood tolerance of mulberry plants under specific flood stress treatments. The gene showed an abnormal increase in expression level only under the specific treatment identified in this invention, while no significant changes were observed under other treatments. The method provided by this invention can be applied to the breeding of highly flood-tolerant plant varieties and has significant application value in the field of plant breeding. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a comparison of the water-submerged phenotypes of MnFedA low-expression and high-expression germplasm provided in Example 2 of the present invention; wherein the upper left is the low-expression germplasm submerged for 0 days; the upper right is the low-expression germplasm submerged for 3 days; the lower left is the high-expression germplasm submerged for 0 days; and the lower right is the high-expression germplasm submerged for 3 days.

[0028] Figure 2 This is the qPCR verification data of the *Morus* genus plant variety *Salvia splendens* (SG) provided in Example 2 of this invention; wherein, FEDA is the MnFedA gene provided in this invention.

[0029] Figure 3 This is the result of the expression level change of the MnFedA gene in the high flood tolerance strain under different flooding depths and durations of treatment provided in Example 3 of the present invention.

[0030] Figure 4 This is the result of the expression level change of the MnFedA gene in the low flood tolerance strain under different flooding depths and durations of treatment provided in Example 3 of the present invention.

[0031] Figure 5 This is the comparison result of chlorophyll content and POD activity of Shuguo (SG) and Pearl White (ZZ) plants under the conditions of 5cm water depth and 3 days of treatment provided in Example 3 of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0034] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0035] The Shuguo (SG) and Zhenzhubai (ZZ) varieties in the following examples were collected and bred by the present invention and can be obtained from the Mulberry Germplasm Resource Bank of the Institute of Economic Crops, Hubei Academy of Agricultural Sciences.

[0036] Example 1

[0037] In this embodiment, a method for identifying a plant's tolerance to flooding stress includes:

[0038] 1. Planting of mulberry germplasm resources.

[0039] Planting substrate: peat moss: vermiculite: perlite in a 5:3:2 ratio, with 5 kg / m² of organic fertilizer added (N:P:K = 15:15:15). 3 Fumigate with 1% formaldehyde and then air dry for later use.

[0040] The planting pots are white plastic pots, with an upper diameter of 36cm and a height of 26cm. Each pot contains 18kg of planting substrate.

[0041] For large, flooded planting, use plastic pots with a top diameter of 48cm and a height of 28cm. Disinfect the pots with 75% alcohol for 15 minutes before transplanting.

[0042] Before the mulberry trees sprout in March or April, transplant healthy mulberry germplasm resources that are free from obvious diseases and pests and have basically the same growth condition. After that, manage them normally. Once the transplanted mulberry trees have recovered their growth, they should be flooded.

[0043] 2. Flooding treatment.

[0044] The double-pot method was used, where the plastic planting pots containing mulberry seedlings (one seedling per pot) were placed inside a larger plastic planting pot, and then water was poured into the larger pot until the water level was 5cm above the soil in the smaller planting pot. As a control, the soil was not flooded, but normal water supply and drainage were ensured.

[0045] 3. Sample collection and qRT-PCR detection.

[0046] On day 3, one functional leaf (leaf 6) from both the flooded and control mulberry germplasm resources was collected, treated with liquid nitrogen, and used for qRT-PCR detection. Specific primers for the mulberry MnFedA gene were designed, with mulberry Actin as the internal reference gene. qRT-PCR was performed using the SYBR Green SupermixiTaq™ kit.

[0047] The specific primers for MnFedA are as follows:

[0048] FedA-F: ​​TGGTTGGTTGGATGGATTA;

[0049] FedA-R: GAAGTTGTATTAGTGTGGTTGA.

[0050] The following are mulberry actin-specific primers:

[0051] actin-F: GTTCCTGCTCGTAGTCCAA;

[0052] actin-R:CCATGCTATTCTCCGTCTC.

[0053] The reaction conditions were 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, for 40 cycles.

[0054] 4. Result determination.

[0055] Comparing the expression levels of MnFedA in each flooded and control mulberry germplasm resource, when the expression is significantly upregulated (P < 0.05) and the fold change is ≥ 2.0, it can be determined that the germplasm resource has enhanced tolerance to flooding stress.

[0056] Example 2

[0057] This invention analyzed the gene expression patterns of three mulberry varieties in response to flooding stress, as detailed below:

[0058] 1. Experimental materials

[0059] Three mulberry varieties, namely Shuguo (SG), Aoyu (AY), and Zhenzhubai (ZZ), are planted, with 20 seedlings of each variety.

[0060] 2. Experimental Methods

[0061] The same method as in Example 1.

[0062] The results are as follows Figures 1-2 As shown, combined with RNA-seq results analysis, this invention identified several key pathways at time points D3 and D10. qPCR results showed that, except for the mulberry ferrugin gene MnFedA, all other genes in the flood-tolerant mulberry material were downregulated, especially the expression level of MnFedA was significantly upregulated on the 3rd day of flooding treatment.

[0063] The expression level of MnFedA in the flood-tolerant variety 'Shuguo (SG)' increased by 3.5 times on the 3rd day of flooding (P<0.01), while that in the sensitive variety 'Zhenzhubai (ZZ)' increased by only 1.2 times (P>0.05), indicating that the expression level of MnFedA is significantly positively correlated with flood tolerance.

[0064] Example 3

[0065] 1. Experimental materials

[0066] Two mulberry species with known significant differences in flood tolerance were selected:

[0067] Highly flood-tolerant variety: Shuguo (SG).

[0068] Low flood tolerance variety: Pearl White (ZZ).

[0069] Thirty healthy seedlings of each variety with similar height (20±2cm) and number of leaves (6-8) were selected and randomly divided into three biological replicate groups, with 10 seedlings in each group.

[0070] 2. Experimental Procedure

[0071] To verify the condition-specificity of the MnFedA gene in response to flooding stress, a multifactorial treatment was set up:

[0072] Flooding depth: 5cm, 10cm, 15cm (water surface exceeds soil surface).

[0073] Flooding duration: 1 day, 3 days, 7 days, 10 days.

[0074] Each treatment combination had 3 biological replicates, with unflooded plants serving as a control (0 days).

[0075] Flooding treatment: Flooding stress was carried out using the "double-pot method" described in Example 1.

[0076] Sample collection: The 6th fully expanded functional leaf was collected at each time point, flash-frozen in liquid nitrogen, and stored at -80℃.

[0077] Gene expression detection: The expression level of MnFedA gene was detected by qRT-PCR, the method was the same as in Example 1, and the internal reference gene was mulberry Actin (primers are in Example 1).

[0078] Physiological index measurement:

[0079] Chlorophyll content: measured using a SPAD-502 chlorophyll meter;

[0080] Peroxidase (POD) activity: determined using the guaiacol method;

[0081] Root activity: determined using the TTC reduction method.

[0082] Regarding the test results, the present invention confirms the flood tolerance of the strain in the following manner:

[0083] High flood tolerance: chlorophyll content decreases by ≤20%, POD activity increases by ≥50%, and root vitality is high.

[0084] Low flood tolerance: chlorophyll content decreased by ≥40%, POD activity did not change significantly, and root vitality was low.

[0085] 3. Experimental Results

[0086] (1) Pattern specificity of MnFedA gene expression.

[0087] like Figure 3 (Shu fruit) and Figure 4 qRT-PCR results for (Pearl White) showed that the expression of the MnFedA gene responded more significantly to flooding conditions. Under a water depth of 5 cm and a treatment period of 3 days, the expression level of MnFedA in *Shuguo* (SG) was significantly upregulated (Log2FC = 3.5, approximately 11.3-fold upregulation, P < 0.01); while the expression level of MnFedA in *Pearl White* (ZZ) was only upregulated by 1.2-fold (Log2FC = 0.263, P > 0.05), showing no statistical difference. Under other flooding conditions (such as water depths of 10 cm or 15 cm, or treatments exceeding 3 days), the expression level of MnFedA in both varieties did not show a significant upregulation, mostly showing no change or downregulation (see...). Figure 3 , Figure 4 ).

[0088] (2) Consistency between physiological indicators and gene expression.

[0089] like Figure 5 The results of the flood tolerance assessment showed that under the conditions of 5 cm water depth and 3 days of treatment, *Symplocos edulis* (SG) exhibited a typical high flood tolerance physiological response: chlorophyll content decreased by only 15%, POD activity increased to 2.8 times that of the control, and root activity remained at a high level (45.2 μg TTF / g·h); *Zygodium japonicum* (ZZ) exhibited a low flood tolerance physiological response: chlorophyll content decreased by 52%, POD activity did not change significantly (only 1.1 times), and root activity decreased significantly (18.7 μg TTF / g·h).

[0090] The results above show that the MnFedA gene was significantly upregulated in flood-tolerant varieties only under the condition of a flood depth of 5 cm and a duration of 3 days. No significant changes were observed under non-flood conditions or in flood-intolerant varieties. These results demonstrate that the identification method established in this invention has clear condition dependence and varietal differentiation capabilities, and can serve as a reliable basis for the rapid identification of flood tolerance in mulberry plants.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying the flood tolerance of plants in the genus *Morus*, characterized in that, include: The mulberry species to be tested were subjected to waterlogging stress treatment. After treatment, the expression level of MnFedA protein or MnFedA gene in the mulberry species to be tested was detected. The waterlogging tolerance of the mulberry species to be tested was determined based on the detection results. The waterlogging stress treatment includes: placing the mulberry plants in water at a depth of 4-6 cm for 2-4 days; The MnFedA protein comprises the amino acid sequence shown in SEQ ID NO.1, and the MnFedA gene comprises the nucleotide sequence shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The waterlogging stress treatment includes placing the mulberry plants in water at a depth of 4.5-5.5 cm for 2.5-3.5 days.

3. The method according to claim 1 or 2, characterized in that, The detection is performed by PCR amplification, and the primer pairs used include the following: FedA-F: ​​TGGTTGGTTGGATGGATTA; FedA-R: GAAGTTGTATTAGTGTGGTTGA.

4. The method according to claim 3, characterized in that, The PCR amplification procedure includes: Treat at 93~98℃ for 2~5 minutes; Treat at 93~98℃ for 8~15 seconds, at 55~65℃ for 25~40 seconds, at 70~75℃ for 25~40 seconds, for 35~50 cycles.

5. The method according to claim 1 or 2, characterized in that, The determination of the flood tolerance of the tested mulberry plant based on the test results includes: When the detection result of the MnFedA gene expression level is higher than the preset value, it is determined that the tested mulberry plant has high flood tolerance, and / or, When the detection result of the MnFedA gene expression level shows no significant change, the tested mulberry plant is judged to have low flood tolerance.

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