Method for identifying rice grain filling stage process based on starch particle surface lipid

Abstract

The invention discloses a method for identifying a rice grain filling stage process based on starch particle surface lipid. The method specifically comprises the following steps: taking rice starch at different stages of a filling stage as a research model, extracting surface lipids of starch particles at normal temperature by using an organic reagent (n-propyl alcohol: water), and detecting and analyzing the relative content of the surface lipids of the starch particles by using lipidomics. The identified symbolic lipid shows a remarkable dynamic change trend in the rice grain filling stage, and the increase of the relative content of the symbolic lipid can accurately indicate the stage progress of rice grain filling. Through quantitative analysis on the symbolic lipids, the filling state of the rice can be accurately evaluated from the molecular level, so that a scientific basis is provided for determining the duration of the filling period of the rice grains.

Description

Technical Field

[0001] This invention relates to the field of rice cultivation, and more specifically, to a method for identifying the grain-filling process of rice based on lipids on the surface of starch granules. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is a staple food for more than half the world's population and one of the world's most important cereal crops. Its grain-filling stage is a crucial period determining yield and quality. The grain-filling stage refers to the process from the time of pollination, when rice grains begin to accumulate starch and other nutrients, until maturity. During this stage, rice grain development involves complex physiological and biochemical reactions, mainly including the following aspects: First, carbohydrates (such as sucrose) produced by photosynthesis are transported from the leaves to the grains, providing raw materials for starch synthesis; second, starch biosynthesis takes place in the starch maker cells in the endosperm, converting sucrose into starch through a series of enzymatic reactions; finally, cell division and expansion gradually increase the grain volume, ultimately forming a plump grain. These processes coordinate with each other, jointly determining the final shape and nutritional value of the grain. The progress of the grain-filling stage directly affects the final grain plumpness, starch content, and quality; therefore, accurately identifying the progress of the grain-filling stage is of great significance for optimizing rice breeding and cultivation management.

[0003] Currently, methods for identifying the grain-filling stage of rice mainly include the measurement of physiological indicators. For example, CN201911143595.5 discloses the determination of rice grain-filling dynamics based on grain weight. However, this type of method can only provide a general division of the grain-filling stage and cannot reflect the "day-level" precision required for precision cultivation. Another example is CN202510160976.3, which discloses the identification of rice grain-filling progress based on marker gene expression levels. However, this method suffers from operational complexity and other issues, making it difficult to meet the needs of large-scale field applications. Therefore, existing technologies all face the technical bottleneck of being unable to simultaneously achieve "high precision, high throughput, and low cost."

[0004] Starch granules are the most important component of rice endosperm, and their synthesis mainly takes place within the starch granule. The starch granule is a specialized plastid responsible for starch biosynthesis and accumulation. In the early stages of rice grain development, the starch granule membrane is intact, and starch synthases and related enzymes catalyze starch synthesis within the membrane. As the grain matures, the starch granule membrane begins to degrade, leading to the accumulation of membrane lipids on the starch granule surface. The content and composition of these lipids dynamically change during the grain-filling stage, closely related to the starch synthesis rate, grain development stage, and environmental conditions. The dynamic changes in starch granule surface lipid content during grain filling theoretically possess the potential to serve as an "endogenous clock." However, to date, no method for identifying the rice grain-filling process based on starch granule surface lipids has been found. In other words, "how to obtain the changes in starch granule surface lipids in a repeatable and traceable manner, and establish a grain-filling prediction model accordingly" remains an unsolved technical challenge. In recent years, with the development of biotechnology, research methods based on lipidomics have provided new opportunities for rice grain-filling research. This method has the advantages of being easy to operate and highly sensitive, and is expected to be widely used in rice production and scientific research. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for identifying the grain-filling stage of rice based on lipids on the surface of starch granules. For the first time, a method for identifying the grain-filling stage of rice is obtained by extracting surface lipids from starch granules and then testing their types and contents using lipidomics.

[0006] The objective of this invention is achieved through the following technical solutions: The first aspect of the present invention is to provide a set of starch granule surface marker lipids for identifying the grain-filling process of rice grains, including the following lipids: fatty acid FA (18:1), and / or FA (18:2), and / or phosphatidylcholine PA (5:0-16:0), and / or lysophosphatidylcholine LPC (14:0), and / or LPC (P-14:1), and / or LPC (14:1), and / or phosphatidylcholine PC (18:1), and / or PC (32:2).

[0007] Furthermore, as the rice grain filling process continues, the relative content of the aforementioned lipids gradually increases; or, as the rice grain filling process continues, the relative content of the marker lipids increases monotonically.

[0008] As one embodiment of the present invention, the marker lipids include FA (18:1), FA (18:2), PA (5:0-16:0), LPC (14:0), LPC (P-14:1), LPC (14:1), PC (18:1) and PC (32:2).

[0009] The second aspect of the present invention is to provide a method for extracting the marker lipids described in the first aspect of the present invention: mixing starch with an organic reagent, centrifuging after extraction, collecting the supernatant, repeatedly adding the organic reagent, and repeatedly extracting; after a final centrifugation, rotary evaporation and nitrogen blowing to constant weight to obtain the lipids on the surface of starch granules.

[0010] A third aspect of the present invention is to provide a lipidomics method for identifying the marker lipids described in the first aspect of the present invention, comprising: reconstitution with dichloromethane and methanol solution, analysis using a UPLC-Orbitrap-MS system, acquisition of high-resolution mass spectrometry data using a Q Exactive HFX hybrid quadrupole-orbitrap mass spectrometer equipped with a heated electrospray ionization source, analysis using a Full-ms-ddMS2 acquisition mode, and obtaining lipid identification results.

[0011] Furthermore, the higher the relative content of FA (18:1), and / or FA (18:2), and / or PA (5:0-16:0), and / or LPC (14:0), and / or LPC (P-14:1), and / or LPC (14:1), and / or PC (18:1), and / or PC (32:2), the longer the rice grain filling process lasts.

[0012] A fourth aspect of the present invention is to provide a method for identifying the grain-filling stage of rice, comprising the following steps: S1. Collect rice grain starch samples at different stages of the rice grain filling process; S2. Extract lipids from the surface of starch granules and use lipidomics methods to detect and analyze the relative content of the marker lipids described in the first aspect of this invention. S3. The grain-filling process of rice is determined based on the relative content of marker lipids. This invention is applicable to several rice varieties with unknown grain-filling times, enabling accurate sequencing of their grain-filling stages. Furthermore, because this invention selects multiple grain-filling time points that are close together and identifies lipids with significant and obvious changes, the lipid changes can be mapped to time points approximately one week apart.

[0013] Furthermore, the relative contents of FA (18:1), and / or FA (18:2), and / or PA (5:0-16:0), and / or LPC (14:0), and / or LPC (P-14:1), and / or LPC (14:1), and / or PC (18:1), and / or PC (32:2) are calculated, and the grain filling process of rice is determined based on the relative contents.

[0014] Furthermore, the higher the relative content of FA (18:1), and / or FA (18:2), and / or PA (5:0-16:0), and / or LPC (14:0), and / or LPC (P-14:1), and / or LPC (14:1), and / or PC (18:1), and / or PC (32:2), the longer the rice grain filling process lasts.

[0015] In one embodiment of the present invention, in step S2, the extraction of lipids on the surface of starch granules is carried out by mixing starch with organic reagents, centrifuging after extraction, collecting the supernatant, adding organic reagents repeatedly, and extracting repeatedly; after the last centrifugation, rotary evaporation and nitrogen blowing are performed until constant weight is obtained to obtain lipids on the surface of starch granules.

[0016] As one embodiment of the present invention, the organic reagent is a mixture of n-propanol and water in a volume ratio of 2:1 to 5:1.

[0017] As one embodiment of the present invention, starch and organic reagent are mixed at a mass ratio of 1:9 to 1:12.

[0018] As one embodiment of the present invention, the extraction is performed at 20-30℃ for 2-4 hours.

[0019] As one embodiment of the present invention, the centrifugation speed is 2000-4000 r / min and the time is 5-20 min.

[0020] As one embodiment of the present invention, the extraction is repeated 3-5 times.

[0021] As one embodiment of the present invention, in step S2, the detection includes: analyzing the surface lipids of starch granules obtained by reconstitution extraction using a UPLC-Orbitrap-MS system, and then acquiring high-resolution mass spectrometry data using a Q Exactive HFX hybrid quadrupole-orbitrap mass spectrometer equipped with a heated electrospray ionization source, and analyzing the lipid identification results using Full-ms-ddMS2 acquisition mode.

[0022] As one embodiment of the present invention, the resolution is performed using 100-200 μL of dichloromethane:methanol (1:1-1:1.5, v / v).

[0023] Compared with the prior art, the present invention has the following beneficial effects: The progress of grain filling directly affects the final grain plumpness, starch content, and quality. The relative content of marker lipids on the surface of starch granules screened in this invention shows a monotonically increasing trend during rice grain filling, and can serve as marker lipids to accurately reflect the temporal progression of rice grain filling (reflecting the "day-level" precision required in precision cultivation). By detecting the relative content of these marker lipids, the grain filling process of rice can be identified, helping to determine the duration of the grain filling process. For example, for samples of the same variety collected at different filling stages, if the collection time sequence cannot be distinguished by conventional methods, the relative content changes of these specific lipids can be analyzed. Based on the differences in their content, the order of sample collection can be accurately determined, thereby identifying the filling stage to which the samples belong. This method provides a reliable and efficient marker for basic research on the filling process and breeding practices. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The relative content trends of the marker lipids FA (18:1), FA (18:2), PA (5:0-16:0), and LPC (14:0) on the surface of starch granules of selected rice grains during the grain-filling stage at 6, 14, 22, 30, 38, and 46 days after flowering were shown. Figure 2 The relative content trends of LPC (P-14:1), LPC (14:1), PC (18:1), and PC (32:2) on the surface of starch granules of selected rice grains during the grain-filling stage at 6, 14, 22, 30, 38, and 46 days after flowering were shown. Figure 3 The trend of 1,000-grain weight of rice grains during the grain-filling stage at 6, 14, 22, 30, 38, and 46 days after flowering. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0026] This invention provides a method for identifying the grain-filling stage of rice based on lipids on the surface of starch granules; the specific steps are as follows: 1. Rice materials Yinxiang 38, a high-quality domestic japonica rice variety.

[0027] 2. Planting Planted under standard cultivation conditions in the standard experimental field of Qingpu Modern Agricultural Park in Shanghai, and harvested in August, September and October.

[0028] 3 Sampling The rice ears are threshed to remove branches and impurities, resulting in pure rice grains.

[0029] Example Step 1: Starch Extraction Starch extraction was performed on rice grains collected at six post-flowering stages (6, 14, 22, 30, 38, and 46 days after flowering). Rice grains were placed in NaOH (4 g / L) at a 1:10 (w / v) ratio and allowed to stand overnight at 4°C, followed by multiple washes. The soaked grains were then crushed using a high-speed blender until completely broken down. The mixture was then filtered through a 200-mesh sieve, centrifuged, and the supernatant was removed to separate the protein and impurity layers from the precipitate. Distilled water was added to the precipitate, and the washing process was repeated until pure, white starch was obtained. The pure starch was suspended in anhydrous ethanol, vacuum filtered, and then dried in a 40°C oven for 48 hours. The dried starch was then ground and passed through a 200-mesh sieve to obtain rice starch.

[0030] Step 2: Extraction of lipids from the surface of starch granules Lipids on the surface of starch granules were extracted using an organic reagent method. 30-40 g of starch was mixed with an organic reagent (n-propanol:water = 3:1) at a certain ratio (1:10). The mixture was extracted at room temperature for 2 h with continuous stirring using a magnetic stirrer. After extraction, the mixture was centrifuged (3000 r / min, 15 min). The supernatant was collected and the extraction solution was added back. The extraction was repeated 4 times using the above method. After the last centrifugation, the mixture was rotary evaporated and blown with nitrogen to constant weight to obtain lipids on the surface of starch granules.

[0031] Step 3: Identification of lipids on the surface of starch granules The lipids were reconstituted using 100 μL of dichloromethane:methanol (1:1, v / v) and analyzed using a UPLC-Orbitrap-MS system. High-resolution mass spectrometry data were then acquired using a Q Exactive HFX hybrid quadrupole-orbitrap mass spectrometer equipped with a heated electrospray ionization (ESI) source, and analysis was performed in Full-ms-ddMS2 acquisition mode. The ESI source parameters were set as follows: spray voltage -2.8 kV / 3.0 kV, sheath gas pressure 60 arb, auxiliary gas pressure 10 arb, purge gas pressure 0 arb, capillary temperature 320 °C, and auxiliary gas heater temperature 350 °C. Lipid identification results were obtained based on these parameters.

[0032] Implementation results: such as Figure 1 , 2 As shown, the relative contents of lipids on the surface of starch granules—FA (18:1), FA (18:2), PA (5:0-16:0), LPC (14:0), LPC (P-14:1), LPC (14:1), PC (18:1), and PC (32:2)—significantly increase with the progress of rice grain filling. Therefore, the stage of rice grain filling can be accurately determined based on the changes in the relative contents of these lipids.

[0033] Comparative Example Six periods after rice flowering (6, 14, 22, 30, 38, and 46 days after flowering) were selected, and sufficient grain samples were taken to measure the thousand-grain weight using an automatic seed testing instrument.

[0034] Implementation results: such as Figure 3 As shown, the thousand-grain weight of rice first increases and then stabilizes as the grain-filling period progresses. Therefore, it is difficult to accurately pinpoint the grain-filling stage based on the thousand-grain weight, especially the stage 30 days after flowering, as it is impossible to determine the specific number of days the grains are in.

[0035] In summary, this invention uses rice starch at different stages of the grain-filling period as a research model. Surface lipids of starch granules are extracted at room temperature using an organic reagent (n-propanol:water), and the relative content of these lipids is analyzed using lipidomics. The marker lipids identified in this invention exhibit significant dynamic changes during the rice grain-filling stage, and the increase in their relative content accurately indicates the stage-specific progress of grain filling. Quantitative analysis of these marker lipids allows for precise assessment of the rice grain-filling status at the molecular level, thus providing a scientific basis for determining the duration of the rice grain-filling period.

[0036] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A group of starch granule surface marker lipids for identifying the grain-filling process of rice, including at least one of the following lipids: FA (18:1), FA (18:2), PA (5:0-16:0), LPC (14:0), LPC (P-14:1), LPC (14:1), PC (18:1), PC (32:2).

2. The starch granule surface marker lipid for identifying the grain-filling process of rice according to claim 1, characterized in that, The marker lipids include FA (18:1), FA (18:2), PA (5:0-16:0), LPC (14:0), LPC (P-14:1), LPC (14:1), PC (18:1) and PC (32:2).

3. The starch granule surface marker lipids for identifying the grain-filling process of rice according to claim 1 or 2, characterized in that, As the rice grain filling process continues, the relative content of the marker lipids increases monotonically.

4. A method for identifying the grain-filling stage of rice, characterized in that, The method includes the following steps: S1. Collect rice grain starch samples at different stages of the rice grain filling process; S2. Extract lipids from the surface of starch granules in the seed starch sample, and analyze the relative content of the marker lipids as described in claim 1 based on lipidomics. S3. Determine the grain-filling process of rice based on the relative content of marker lipids.

5. The method for identifying the grain-filling stage of rice according to claim 4, characterized in that, In step S2, the extraction of lipids from the surface of starch granules involves mixing starch with an organic reagent, centrifuging after extraction, collecting the supernatant, adding the organic reagent repeatedly, and repeating the extraction process. After the final centrifugation, the mixture is rotary evaporated and purged with nitrogen until constant weight is obtained to obtain the lipids from the surface of the starch granules.

6. The method for identifying the grain-filling stage of rice according to claim 5, characterized in that, The organic reagent is a mixture of n-propanol and water in a volume ratio of 2:1 to 5:

1. And / or, starch and organic reagents are mixed in a mass ratio of 1:9 to 1:

12.

7. The method for identifying the grain-filling stage of rice according to claim 5, characterized in that, The extraction was performed at 20-30℃ for 2-4 hours. And / or, the centrifugation speed is 2000-4000 r / min, and the time is 5-20 min; And / or, extract repeatedly 3-5 times.

8. The method for identifying the grain-filling stage of rice according to claim 4, characterized in that, In step S2, the detection includes: analyzing the surface lipids of the starch granules obtained by reconstitution extraction using a UPLC-Orbitrap-MS system, and then acquiring high-resolution mass spectrometry data using a Q Exactive HFX hybrid quadrupole-orbitrap mass spectrometer equipped with a heated electrospray ionization source. The analysis is performed in Full-ms-ddMS2 acquisition mode to obtain lipid identification results.

9. The method for identifying the grain-filling stage of rice according to claim 8, characterized in that, The resolution is performed using 100-200 μL of dichloromethane:methanol (1:1-1:1.5, v / v).

10. The method for identifying the grain-filling stage of rice according to claim 4, characterized in that, The higher the relative content of marker lipids, the longer the grain filling process of rice lasts.

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