Method for regulating and controlling bud-stage root length salt-tolerant heterosis of hybrid rice grain by using exogenous melatonin

Abstract

The invention provides a method for regulating and controlling the salt-tolerant heterosis of the root length in the germination stage of hybrid rice by using exogenous melatonin, which comprises the following steps: sowing hybrid rice, spraying melatonin, and regulating and controlling the salt-tolerant heterosis of the hybrid rice through the synergistic effect of melatonin and hormone. According to the method, the effect of regulating and controlling the root length salt-tolerant heterosis in the bud stage of the hybrid rice is achieved by applying the exogenous melatonin for the first time, and the scientific theory that plant hormones participate in formation of the stress-tolerant heterosis is expected to be enriched; through implementation of the project, a novel method for regulating and identifying the root length salt-tolerant heterosis in the germination stage of the hybrid rice is expected to be developed, and technical and theoretical support is provided for breeding of saline-alkali soil rice varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of rice genetic breeding, and particularly relates to a method for regulating the salt tolerance heterosis of hybrid rice root length at the germination stage by using exogenous melatonin. Background Art

[0002] China is the third largest country with saline-alkali soil distribution in the world. More than 90% of it is inland saline-alkali land, with about 6.67 million hm 2 of developable rice planting potential. However, rice seedlings are highly sensitive to salt at the seedling stage, and seed germination and root development are easily inhibited by salt stress, which limits the direct utilization of saline-alkali land. At present, mainly improved measures such as transplanting after freshwater seedling raising or direct seeding with dynamic flowing water irrigation are adopted, but these methods increase labor and time costs. Research shows that alleviating salt stress during the seed germination period through chemical regulation means such as plant growth regulators can not only promote the root development of rice, but also provide a new way to explore the direct seeding and seedling raising mode for saline-alkali land, thereby improving planting efficiency.

[0003] Seed germination is a crucial stage in plant growth, development and morphogenesis. The rapid elongation of seminal roots plays an important role in ensuring water and nutrient absorption, promoting the early nutrition uptake of hybrids and the formation of heterosis at the seedling stage. However, root development is easily affected by abiotic stresses such as temperature, light and soil salinity. Salt stress can lead to stress responses such as shortened root length and thinner diameter, involving multiple signaling pathways such as endogenous hormones, redox balance and ion homeostasis. Endogenous hormones play a key role in the root stress response. For example, abscisic acid (ABA), as an important stress hormone, its level significantly increases under salt stress, inhibiting root cell proliferation and shortening root length. Auxin (IAA) regulates the plasticity of root architecture through receptor-mediated signal transduction, thereby responding to salt stress. In addition, ABA and hormones such as IAA and gibberellin (GA) coordinately regulate root development under salt stress through crosstalk mechanisms.

[0004] Melatonin (MT) is a highly conserved biomolecule. Since its first discovery in 1995, its endogenous content in plants is generally known to be low. Its structure and metabolic pathway are similar to those of auxin (IAA), and both can promote plant growth and development under normal physiological conditions. As an important exogenous plant growth regulator, melatonin has a significant regulatory effect on root development, seed germination, and leaf senescence under stress conditions. Research shows that melatonin has strong antioxidant properties. Exogenous application can increase the root dry weight and fresh weight of rice seedlings under salt stress by enhancing the activity of antioxidant enzymes, significantly inhibit the accumulation of reactive oxygen species (ROS), improve the antioxidant defense ability, and promote the growth of seedling root length and shoot length. In addition, melatonin can interact with a variety of endogenous hormones to jointly regulate root development under stress. For example, exogenous melatonin significantly increases the primary root length and lateral root number of melons under copper stress by inhibiting jasmonic acid biosynthesis; under salt stress, melatonin promotes cotton seed germination and root elongation by regulating the expression of ABA and GA signal transduction genes.

[0005] However, the mechanism of action of melatonin in the formation of heterosis in rice, especially its regulatory mode in the salt tolerance trait of root length at the bud stage, has not been systematically revealed and applied. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for regulating the salt tolerance heterosis of root length at the bud stage of hybrid rice by using exogenous melatonin, and achieve the effect of regulating the salt tolerance heterosis of root length at the bud stage of hybrid rice by applying exogenous melatonin.

[0007] To solve the above technical problems, the present invention provides a method for regulating the salt tolerance heterosis of root length at the bud stage of hybrid rice. After sowing the hybrid rice, melatonin is sprayed.

[0008] In the above method, further, the concentration of the melatonin is 50 μM to 150 μM.

[0009] In the above method, further, the concentration of the melatonin is 100 μM.

[0010] The present invention carried out melatonin treatment on different varieties of rice at gradient concentrations (50 μM, 75 μM, 100 μM, 125 μM, 150 μM) and found that 100 μM melatonin has the best effect on alleviating the root development of rice under salt stress.

[0011] Compared with the prior art, the advantages of the present invention are as follows:

[0012] The present invention provides a method for regulating the salt tolerance heterosis of root length at the germination stage of hybrid rice by using exogenous melatonin. For the first time, melatonin is associated with the analysis of salt tolerance heterosis of root length at the germination stage of hybrid rice. By clarifying the molecular mechanism of melatonin-hormone co-regulation of salt tolerance heterosis, it is expected to enrich the scientific theory of plant hormones participating in the formation of stress tolerance heterosis; through the implementation of the project, it is expected to develop new methods for regulating and identifying the salt tolerance heterosis of root length at the germination stage of hybrid rice, providing technical and theoretical support for the breeding of rice varieties suitable for saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0014] Figure 1 It shows the results of the effects of different treatments on the root lengths of the two-line hybrid rice Y Liangyou 900 and its parents R900 and Y58S in Experiment 1 of the present invention.

[0015] Figure 2 It shows the microscope imaging results in Experiment 2 of the present invention.

[0016] Figure 3 It shows the qPCR detection results in Experiment 3 of the present invention.

[0017] Figure 4 It shows the results of the determination of the content of + Na + and K

[0018] Figure 5 It shows the results of the determination of the physiological and biochemical indexes of root length at the germination stage in Experiment 4 of the present invention.

[0019] Figure 6 It shows the analysis results of the heterosis over high parent and the heterosis over mid-parent in Experiment 4 of the present invention.

[0020] Figure 7 It shows the detection results of the expression levels of OsAKT1, OsHAK1, OsGR2, and OsGPX1 in Experiment 4 of the present invention.

[0021] Figure 8 It shows the results of the determination of the content of endogenous hormones in Experiment 5 of the present invention.

[0022] Figure 9 It shows the correlation analysis between root length and the content of endogenous hormones in Experiment 5 of the present invention.

[0023] Figure 10 It shows the analysis results of the heterosis rate over high parent and the heterosis rate over mid-parent in Experiment 5 of the present invention.

[0024] Figure 11Statistical results of the expression levels of key genes for melatonin synthesis in Experiment 5 of the present invention.

[0025] Figure 12 Results of the phenotypic analysis of rice in Experiment 6 of the present invention.

[0026] Figure 13 Results of the salt tolerance phenotypic identification of the root lengths of different hybrid rice and their parents at the germination stage in Experiment 7 of the present invention. Detailed implementation manners

[0027] The present invention will be further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0028] The materials, reagents, and instruments used in the following examples can all be obtained from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0029] Example 1

[0030] A method for regulating the heterosis of salt tolerance of hybrid rice root length at the germination stage by using exogenous melatonin (MT) includes the following steps:

[0031] (1) Select plump seeds of two-line hybrid rice Y Liangyou 900 (Y900) and its parents R900 (male parent) and Y58S (female parent), sterilize them with 5% NaClO for 15 min, rinse them 5 times with sterile water, and soak the seeds at 37°C for 48 h.

[0032] (2) After taking out the seeds, incubate them in the dark at 37°C for 24 h.

[0033] (3) Lay three layers of filter paper on the culture dish, place the seeds on the filter paper, 20 seeds per dish. After mixing and shaking the 50 mM NaCl and 100 μM MT treatment solutions, immerse the seeds. Blank control (CK): Immerse the seeds with 8 mL of water. [[ID=३३]]

[0034] (4) Treat the culture dishes in an environment of 28°C, 80% RH, and a 12 h photoperiod for 96 h. Measure the root length, quickly freeze it in liquid nitrogen, and store it at -80°C. Set 3 biological replicates.

[0035] Comparative Example 1

[0036] According to the method of Example 1, the difference is that in step (3), the seeds are sprayed with 100 μM of MT in soil with a salt concentration of 50 mM.

[0037] Experiment 1: Phenotypic analysis.

[0038] After 96 h of treatment, measure the root length (n = 50), and the data are expressed as mean ± SD. Take root samples, fix them with FAA, embed them in paraffin, and observe and image them with a Nikon E100 microscope. Set 5 replicates.

[0039] Figure 1 These are the results of the effects of different treatments on the root lengths of Y900 and its parents R900 and Y58S. In the figure, A shows the pictures of root lengths after different treatments of the seeds, and B shows the results of root length comparison and mid-parent heterosis statistics. It can be seen from the figure that under normal conditions, the root length of Y900 is close to that of the male parent R900 and significantly higher than that of the female parent (MPH = 20.00%). Under 50 mM NaCl stress, the root length of Y900 only decreased by 1%, while that of the parent R900 decreased by 12%; the female parent Y58S decreased by 29%, indicating that the hybrid has salt adaptability and mid-parent heterosis.

[0040] After adding 100 μM MT, the root lengths of all materials were significantly restored. Among them, the root length of R900 increased from 2.22 cm to 3.17 cm; the male parent Y900 increased from 2.47 cm to 3.37 cm; the female parent Y58S increased from 1.16 cm to 1.98 cm, and Y900 still maintained heterosis (OPH = 7%; MPH = 31%).

[0041] Experiment 2: Cytological analysis.

[0042] The roots of the two-line hybrid rice Y Liangyou 900 (P) and its parents R900 (F1) and Y58S (M) were respectively subjected to microscopic imaging. Figure 2 These are the results of microscopic imaging: In the figure, A shows the pictures of microscopic imaging, and B shows the results of cell length statistics. It can be seen from the figure that NaCl inhibits the elongation of meristematic cells, and this is significantly reversed after MT treatment. The cell length of Y900 is extremely significantly higher than that of both parents (OPH = 63.84%; MPH = 95.80%).

[0043] Experiment 3: qPCR detection.

[0044] RNA was extracted using TransZol Up and reverse-transcribed using a kit. The QuantStudio TM 3 system was used for detection, with OsUBQ5 as the internal reference, and the primers were designed by Primer 3 Plus. The 2-ΔΔCt method was used to calculate the expression level. Three technical replicates were set.

[0045] Figure 3 These are the results of qPCR detection. In the figure, A shows the statistical results of the expression level of OsCTPS1 (promoting meristematic cell proliferation), and B shows the statistical results of the expression level of OsLKRT1 (regulating the length of root tip cells). The results show that salt stress restricts root growth by inhibiting the elongation of meristematic cells, while MT promotes cell elongation by activating related genes, synergistically maintaining the heterosis of Y900.

[0046] Experiment 4: Determination of physiological and biochemical indexes.

[0047] 4.1. Determination of Na + / K + content: After acid digestion of root samples, Na + / K + content was determined by ICP-MS.

[0048] Figure 4 For the determination results of Na + and K + contents, A in the figure is the determination result of Na + ; B in the figure is the determination result of K + content; C in the figure is the determination result of K + / Na + ratio. It can be seen from the figure that salt stress significantly increased the Na + content in the roots of the three materials. Among them, Y900 was 10.99% and 5.64% lower than the parental lines R900 and Y58S, respectively. After MT treatment, the maximum decrease in Na + in Y900 reached 38.37%, and its K + / Na + ratio was the highest under salt stress, reaching 37.06%, while that of the male parent R900 was 33.38% and that of the female parent Y58S was 24.80%, indicating that Y900 has a stronger ability to regulate ion homeostasis.

[0049] 4.2. Determination of the contents of CAT, H2O2, POD, MDA, and SOD: The content of proline was detected by the AKAM003M kit, the content of MDA was detected by the AKFA013M detection kit, the content of CAT was detected by the AKAO003-2M kit, the content of POD was detected by the AKAO005M kit, the content of SOD was detected by the AKAO001M kit, and the content of H2O2 was detected by the AKAO009M kit. All were set with 3 replicates.

[0050] Figure 5 For the determination results of the physiological and biochemical indexes of root length at the bud stage, A in the figure is the content of CAT, B in the figure is the content of H2O2, C in the figure is the content of POD, D in the figure is the content of MDA, and E in the figure is the content of SOD.

[0051] Figure 6 For the analysis results of over-high-parent heterosis and over-mid-parent heterosis.

[0052] OPH (over-high-parent heterosis) = ((F1 - HP) / HP × 100%).

[0053] MPH (over-mid-parent heterosis) = ((F1 - MP) / MP × 100%).

[0054] HP and MP represent the high-parent phenotypic value and the mid-parent phenotypic value, respectively. ANOVA and t-tests (p < 0.05) were performed using SPSS 21.0, and graphs were plotted using GraphPad Prism 8.0.2. Data are presented as mean ± SD.

[0055] It can be seen from the figure that under salt stress, the accumulation of H2O2 and MDA in Y900 is the lowest, and it is further reduced after MT treatment, revealing that it alleviates salt stress by reducing oxidative damage. The activities of CAT, POD, and SOD in Y900 are significantly higher than those of its parents, and the over-parent heterosis rates reach 33.36%, 9.96%, and 15.23%, respectively. After MT treatment, the activities of POD and CAT increase most significantly, and the over-parent heterosis rates are 15.52% and 5.85%, respectively.

[0056] 4.3. Gene analysis.

[0057] Figure 7 This is the detection result of the expression levels of OsAKT1, OsHAK1 (K + transport), and OsGR2, OsGPX1 (antioxidant). A in the figure is OsAKT1, B in the figure is OsHAK1, C in the figure is OsGR2, and D in the figure is OsGPX1. It can be seen from the figure that under salt stress, the expression levels of OsAKT1, OsHAK1, OsGR2, and OsGPX1 in Y900 are all higher than those of its parents.

[0058] Experiment 5: Determination of MT and important endogenous hormone indexes of Y900-related materials.

[0059] The contents of 9 endogenous hormones in Y900 and its parents after 96 h of treatment with CK, NaCl, and NaCl + MT were measured. The experimental procedure was as follows: After the root samples were extracted with a mixed solution of methanol, water, and formic acid in a volume ratio of 15:4:1, the contents of 9 hormones such as GA were determined by LC-MS / MS (ABSciex 6500). Three replicates were set.

[0060] Figure 8These are the results of the determination of endogenous hormone contents. In the figure, A is MT, B is IAA, C is GA, D is CTK, E is ABA, F is ETH, G is SA, and H is JA. The results show that salt stress significantly inhibits the synthesis of endogenous MT. After exogenous MT treatment, the MT contents of all materials increase extremely significantly, and the increase amplitude of Y900 is the largest. Salt stress reduces the IAA content, which is significantly restored after exogenous MT treatment. The IAA increase amplitude of R900 is twice that of Y900 and Y58S. Under salt stress, the GA reduction of both parents exceeds 50%, while that of Y900 is only 20.53%. After MT treatment, the GA content of Y900 is significantly higher than that of the parents. Salt stress significantly increases the contents of ABA, JA, and SA, while MT treatment reverses this trend, and the reduction amplitude of R900 is the most significant.

[0061] Figure 9 This is the correlation analysis of root length and endogenous hormone contents. It can be seen from the figure that the root lengths of all materials are significantly positively correlated with the contents of MT, IAA, and GA (P<0.01). Among them, the correlations of IAA (r = 0.970) in R900 and GA (r = 0.794) in Y900 are the strongest. The root length is negatively correlated with ABA and JA, and the negative correlation of SA only exists in the parents, which may be affected by the genetic background.

[0062] Figure 10 These are the results of the analysis of over-parent heterosis rate and mid-parent heterosis rate. It can be seen from the figure that under salt stress, the over-parent heterosis rate (OPH) and mid-parent heterosis rate (MPH) of the root length of Y900 are positively correlated with the contents of MT, IAA, and GA (the correlation with MT is the highest), and negatively correlated with ABA, JA, and SA. After exogenous MT treatment: the correlation coefficients of MT with OPH / MPH increase to 88.0% and 85.1% respectively, which are significantly higher than those in the salt stress group (71.20%, 70.20%); the negative regulation of ABA on OPH turns into a positive effect. In summary, exogenous MT maintains salt tolerance heterosis by coordinately regulating hormone synthesis (promoting MT / IAA / GA, inhibiting ABA / JA), which is consistent with the results of the previous soaking and spraying experiments.

[0063] Figure 11Statistical results of the expression levels of key genes for MT synthesis. In the figure, A is OsCOMT15, B is OsCOMT7, C is OsASMT1, D is OsTDC2, E is APO2, F is OsGA20ox1, G is PCF3, and H is OsABA1. qPCR analysis showed that the expressions of key genes for MT synthesis (OsCOMT7, OsCOMT15, OsASMT1, OsTDC2) were all up-regulated, confirming that exogenous MT promoted the accumulation of endogenous MT by activating the synthesis pathway. The expression of the auxin GA synthesis gene APO2 was synchronously up-regulated. The expression of the synthesis gene OsGA20ox1 was enhanced. The expressions of ABA and JA synthesis genes (OsABA1, PCF3) were down-regulated, indicating that MT alleviated salt stress by inhibiting the synthesis of stress hormones.

[0064] Example 2

[0065] A method for regulating the salt tolerance heterosis of the root length of hybrid rice at the germination stage by using exogenous melatonin, comprising the following steps:

[0066] (1) Select plump seeds of two-line hybrid rice Y Liangyou 900 (Y900) and its parents R900 (male parent) and Y58S (female parent), sterilize them with 5% NaClO for 15 min, rinse them 5 times with sterile water, and soak them at 37 °C for 48 h.

[0067] (2) After taking out the seeds, culture them in the dark at 37 °C for 24 h.

[0068] (3) After the soil is exposed to the sun, fill it into a seedling-raising box and divide it into three groups, which are treated respectively according to the following methods:

[0069] CK: Spray 8 mL of water (0 mM NaCl) on the seeds. Set 3 replicates.

[0070] Group 1: Pretreat with 50 mM NaCl to simulate the saline-alkali environment, sow Y900 and its parents after they show white tips (10 seeds / hole), and apply salt stress for 24 h. Set 3 replicates.

[0071] Group 2: Pretreat with 50 mM NaCl to simulate the saline-alkali environment, sow Y90, and its parents after they show white tips (10 seeds / hole), spray 100 μM MT (S20287, Yuanye Bio) after 24 h of salt stress, spray each hole 3 times (once every other day), and measure the root length 7 days after treatment. Set 3 replicates.

[0072] (4) Treat the petri dish in an environment of 28 °C, 80% RH, and 12 h photoperiod for 96 h. Measure the root length, quickly freeze it in liquid nitrogen, and store it at -80 °C. Set 3 biological replicates.

[0073] Experiment 6: Phenotypic analysis of the rice in Example 2.

[0074] Figure 12 Measurements of root lengths of two-line hybrid rice Y Liangyou 900 (Y900) and its parents R900 (male parent) and Y58S (female parent). In the figure, A shows the pictures of root lengths after different treatments of seeds, and B shows the statistical results of root length comparison. It can be seen from the figure that the results of the soil experiment are similar to those of the indoor spraying experiment.

[0075] Example 3

[0076] Select plump seeds of Chuankangyou 727 and its parents Chuankang 606A (male parent) and Shuhui 727 (female parent), and treat them according to the method of Example 2.

[0077] Example 4

[0078] Select plump seeds of Xiangliangyou Huazhan and its parents Guangxiang 24S (male parent) and Huazhan (female parent), and treat them according to the method of Example 2.

[0079] Example 5

[0080] Select plump seeds of Huiliangyou Yuennong Simiao and its parents 1892S (male parent) and Yuennong Simiao (female parent), and treat them according to the method of Example 2.

[0081] Experiment 7: Phenotypic analysis of the rice in Examples 3 to 5.

[0082] Measure the root length after 96 h of treatment (n = 50), and the data are expressed as mean ± SD. After taking root samples, fix them with FAA, embed them in paraffin and section them, and observe and image them with a Nikon E100 microscope. Set 5 replicates.

[0083] Figure 13 Results of salt tolerance phenotypic identification of root lengths at the germination stage of different hybrid rices and their parents. In the figure, A shows the statistical chart of root lengths of Chuankangyou 727 and its parents after treatment according to the method of Example 1, B shows the statistical chart of root lengths of Xiangliangyou Huazhan and its parents after treatment according to the method of Example 1, and C shows the statistical chart of root lengths of Huiliangyou Yuennong Simiao and its parents after treatment according to the method of Example 1.

[0084] It can be seen from the figure that in the CK group: under normal conditions, the root length of the hybrid is close to that of one of the parents. The mid-parent heterosis (MPH) of the root lengths of Chuankangyou 727, Xiangliangyou Huazhan, and Huiliangyou Yuennong Simiao is 25.86%, -9.70%, and -3.08% respectively.

[0085] After 50 mM NaCl stress, the root lengths of all materials decreased significantly. The over-parent heterosis (OPH) of Chuankangyou 727, Xiangliangyou Huazhan, and Huiliangyou Yuennong Simiao were 14.45%, 11.62%, and 7.32% respectively, indicating that the hybrids had salt adaptability and over-parent heterosis. After adding 100 μM MT, the root lengths of all materials recovered significantly, and the OPH of all hybrids increased significantly compared with that under salt stress (Chuankangyou 727: 43.92%; Xiangliangyou Huazhan: 12.66%; Huiliangyou Yuennong Simiao: 17.99%). This further confirmed that the hybrids had significant advantages in root length at the germination stage under salt stress, and exogenous MT could enhance this over-parent heterosis.

[0086] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for regulating the salt tolerance heterosis of root length at the bud stage of hybrid rice by using exogenous melatonin, which is characterized in that, After sowing the hybrid rice, melatonin is sprayed.

2. The method according to claim 1, characterized in that The concentration of the melatonin is 50 μM to 150 μΜ.

3. The method according to claim 1, wherein The concentration of the melatonin is 100 μM.

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