Application of miR395l and its precursors in promoting sorghum sugar accumulation and biomass formation

Abstract

This invention discloses the application of miR395l and its precursor in promoting sugar accumulation and biomass formation in sorghum, belonging to the field of plant genetic engineering technology. The nucleotide sequence of miR395l is shown in SEQ ID NO.1, and the nucleotide sequence of the miR395l precursor is shown in SEQ ID NO.2. This invention reveals for the first time the function of miR395l, a member of the miR395 family, in sugar accumulation and biomass formation in sorghum, and utilizes genetic engineering techniques to improve the content of soluble sugars, biomass yield, and grain size in sorghum stalks, providing a new technical means for molecular design breeding of sorghum.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of miR395l and its precursors in promoting sorghum sugar accumulation and biomass formation. Background Technology

[0002] sorghum ( Sorghum bicolor Sorghum is an important food and forage crop, possessing advantages such as salt and alkali tolerance, drought and flood resistance, tolerance to poor soil conditions, high photosynthetic efficiency, and large biomass. Based on the difference in soluble sugar content in the stems, sorghum can be divided into two main types: grain sorghum and sweet sorghum. Sweet sorghum stems are rich in fermentable sugars such as sucrose, glucose, and fructose, which can be directly used as raw materials for sugar production and fuel ethanol after juicing. Further increasing the sugar content in sweet sorghum stems has become one of the important goals of sorghum genetic improvement. For grain sorghum, sugars can act as important signaling molecules to promote biomass accumulation and yield. Therefore, optimizing and improving the efficiency of sugar accumulation and distribution has significant practical production value for sorghum genetic improvement.

[0003] Significant progress has been made in improving the sugar content and yield of sorghum using traditional hybridization breeding methods. However, these methods still suffer from problems such as long breeding cycles, significant environmental impact, and low screening efficiency, making it difficult to achieve rapid and precise improvement of target traits. With the development of plant molecular biology, researchers have successively identified several key protein factors in sugar metabolism and sugar transport processes, such as sucrose phosphate synthase (SPS), trehalose-6-phosphate phosphatase (TPP), sugar transporter (SWEET), and some transcription factors. They have also used genetic engineering techniques to confirm that these factors can regulate the accumulation of sugar and biomass.

[0004] Recent studies have shown that using non-coding RNA technology to improve important agronomic traits in crops has significant application prospects, but research in sorghum is still in its early stages. MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs approximately 20–24 nt in length. They mediate the degradation or translational repression of target mRNAs by recognizing and binding to complementary sites on target mRNAs, playing important regulatory roles in biological processes such as plant growth and development, nutrient metabolism, and stress responses. Existing studies have shown that increasing or decreasing miRNA expression levels can improve plant architecture, flowering time, and yield in important crops such as rice and wheat.

[0005] The miR395 family is a widely distributed class of conserved miRNAs in plants, comprising multiple members. They primarily regulate plant sulfur metabolism by targeting transcripts of genes such as ATP sulfurylase (APS) and sulfate transporter (SULTR), playing crucial roles in sulfur uptake, transport, and assimilation. However, the regulatory roles of the miR395 family in sugar metabolism and biomass formation remain poorly understood. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned limitations of existing technologies by providing the application of miR395l and its precursors in promoting sugar accumulation and biomass formation in sorghum. This invention reveals for the first time the function of miR395l, a member of the miR395 family, in sugar accumulation and biomass formation in sorghum, and utilizes genetic engineering techniques to improve the content of soluble sugars, biomass yield, and grain size in sorghum stalks, providing a new technical means for molecular design breeding of sorghum.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the application of miR395l in promoting sorghum sugar accumulation and biomass formation, wherein the nucleotide sequence of miR395l is shown in SEQ ID NO.1; specifically as follows: GUGAAGUGCUUGGGGGAACUC. (SEQ ID NO.1) Note: According to WIPO ST.26, the "U" in the RNA sequence is represented by "T" in the sequence listing.

[0008] In the above application, promoting sorghum sugar accumulation specifically means increasing the content of soluble sugars in sorghum stalks.

[0009] In the above applications, sorghum biomass formation is promoted through at least one of the following pathways (1)-(3): (1) Increase the plant height of sorghum; (2) Increase the chlorophyll content in sorghum leaves; (3) Increase the weight of 100 grains of sorghum.

[0010] This invention has found that overexpression of the mature miR395l in sorghum can significantly increase the soluble sugar content in sorghum stems; it can also increase the plant height, leaf chlorophyll content and 100-grain weight of sorghum, and promote the formation of sorghum biomass.

[0011] A second aspect of the present invention provides the use of a miR395l precursor (pre-miR395l) in promoting sorghum sugar accumulation and biomass formation, the nucleotide sequence of which is shown in SEQ ID NO.2; specifically as follows: guucccuucaagcacuucacauggagcauuauuugucuuggagaaagcuuaauuugaugcauuGUGAAGUGCUUGGGGGAACUC. (SEQ ID NO.2) Note: According to WIPO ST.26, the "U" in the RNA sequence is represented by "T" in the sequence listing.

[0012] In the above applications, overexpression of the miR395l precursor was used to promote sorghum sugar accumulation and biomass formation.

[0013] Preferably, the miR395l precursor is overexpressed using any of the following substances: C1) An expression cassette containing a nucleic acid molecule encoding the miR395l precursor; C2) A recombinant expression vector containing a nucleic acid molecule encoding the miR395l precursor, or a recombinant expression vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing nucleic acid molecules encoding miR395l precursors, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant expression vector described in C2); C4) A transgenic plant cell line containing a nucleic acid molecule encoding the miR395l precursor, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing a nucleic acid molecule encoding the miR395l precursor, or transgenic plant tissue containing the expression cassette described in C1); C6) A transgenic plant organ containing a nucleic acid molecule encoding the miR395l precursor, or a transgenic plant organ containing the expression cassette described in C1).

[0014] In this invention, recombinant expression vectors containing nucleic acid molecules encoding the miR395l precursor can be constructed using plant expression vectors conventional in the art. The type of plant expression vector is not particularly limited, as long as it can carry and drive the transcription of the miR395l precursor in plant cells or tissues. The plant expression vectors include, but are not limited to, binary Agrobacterium expression vectors, plasmid vectors suitable for plant genetic transformation, and expression vectors suitable for plant microparticle bombardment transformation.

[0015] In some embodiments, the recombinant expression vector includes a promoter operatively linked to the coding sequence of the miR395l precursor. The promoter may be a constitutive promoter, an inducible promoter, a tissue-specific promoter, or an enhancing promoter. Preferably, the promoter is selected from the cauliflower mosaic virus CaMV 35S promoter, the maize ubiquitin promoter, or a functionally equivalent promoter. The promoter can be used alone or in combination with plant expression regulatory elements to regulate the transcriptional level and / or spatiotemporal specificity of the coding sequence of the miR395l precursor in the plant.

[0016] Furthermore, the recombinant expression vector may also include elements commonly used in plant expression vectors, such as transcription termination sequences, selection marker genes, screening marker genes, enhancers, origin of replication, or multiple cloning sites. By introducing the recombinant expression vector into plant cells, plant tissues, callus tissues, immature embryos, mature embryos, immature embryos, seedlings, or plants, transgenic plant materials with overexpression of miR395l precursor, heterologous expression, or for functional verification can be obtained.

[0017] In the above application, promoting sorghum sugar accumulation specifically means increasing the content of soluble sugars in sorghum stalks.

[0018] In the above applications, promoting biomass formation specifically means increasing sorghum plant height, leaf chlorophyll content, and / or increasing the 100-grain weight.

[0019] The miR395l precursor can be processed into mature miR395l within plant cells via the endogenous RNA processing system. Therefore, overexpression of the miR395l precursor in sorghum can correspondingly increase the relative expression level of mature miR395l in sorghum, thereby promoting the formation of sugar content and biomass in sorghum.

[0020] A third aspect of the present invention provides a method for promoting sorghum sugar accumulation and biomass formation, comprising the step of overexpressing miR395l or miR395l precursor in sorghum.

[0021] The beneficial effects of this invention are: This invention, by overexpressing the mature miR395l in sorghum, significantly increases the soluble sugar content in sorghum stems, resulting in higher sugar accumulation levels in the stems. This enhances the utilization value of sorghum as an energy crop, forage crop, or sugar source crop. Compared to sorghum materials without miR395l overexpression, the sorghum plants obtained by this invention exhibit a significant advantage in soluble sugar accumulation in the stems, indicating that miR395l can positively regulate sorghum sugar metabolism or sugar accumulation processes.

[0022] Meanwhile, overexpression of miR395l in this invention can also increase sorghum plant height, leaf chlorophyll content, and 100-grain weight. Increased plant height is beneficial for increasing stem yield and vegetative biomass; increased leaf chlorophyll content enhances photosynthetic capacity, providing a basis for sugar synthesis and dry matter accumulation; and increased 100-grain weight indicates that miR395l not only promotes vegetative growth but also has a positive effect on grain filling and yield traits.

[0023] Therefore, miR395l and its precursor provided by this invention can simultaneously improve the sugar accumulation trait and biomass-related traits of sorghum, achieving a synergistic improvement in soluble sugar content in stems, plant growth potential, and grain yield traits. This overcomes the problem of difficulty in simultaneously improving sugar accumulation and biomass formation in existing sorghum breeding, and provides an effective molecular target and technical approach for cultivating new sorghum materials with high sugar content, high biomass, and high grain yield potential. Attached Figure Description

[0024] Figure 1 The results of stalk sugar content determination of grain sorghum BTx623 and sweet sorghum M-81E at different growth stages; in the figure, Stage 1 is the three-leaf stage, Stage 2 is the flag leaf visible stage, and Stage 3 is the booting stage; DAF (Day after flowering) indicates the number of days after flowering, 0DAF, 5DAF, 10DAF, 15DAF, 25DAF, and 35DAF represent the day of flowering and the 5th, 10th, 15th, 25th, and 35th days after flowering, respectively.

[0025] Figure 2 The relative expression levels of miR395l and its precursor miRNA in grain sorghum BTx623 and sweet sorghum M-81E at different growth stages; in the figure, A represents the relative expression level of miR395l; B represents the relative expression level of miR395l precursor miRNA.

[0026] Figure 3 The relative expression levels of miR395l in transgenic sorghum plants and wild-type plants created in Example 2.

[0027] Figure 4 Example 3: Growth phenotypes of transgenic sorghum plants and wild-type plants at maturity.

[0028] Figure 5 Results of the determination of soluble sugar content in the stems of transgenic sorghum plants and wild-type plants in Example 3.

[0029] Figure 6 The results of plant height, chlorophyll content, and flowering time of transgenic and wild-type sorghum plants in Example 3 are shown in the figure. In the figure, A is the plant height measurement result, B is the chlorophyll content measurement result, and C is the flowering time statistical result.

[0030] Figure 7 Example 3: Grain traits of transgenic sorghum plants and wild-type plants; In the figure, A is the grain phenotype; B is the statistical result of 100-grain weight. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] As mentioned earlier, optimizing and improving the efficiency of sugar accumulation and distribution has important practical production value for the genetic improvement of sorghum.

[0033] The miR395 family is a widely distributed class of conserved miRNAs in plants, comprising multiple members. They primarily regulate plant sulfur metabolism by targeting transcripts of genes such as ATP sulfurylase (APS) and sulfate transporter (SULTR), playing crucial roles in sulfur uptake, transport, and assimilation. Current research on the miR395 family mainly focuses on sulfur metabolism regulation, while its biological functions in sugar metabolism and biomass formation lack systematic study and application.

[0034] This invention reveals for the first time the function of miR395l, a member of the miR395 family, in sugar accumulation and biomass formation in sorghum, and utilizes genetic engineering techniques to improve the content of soluble sugars, biomass yield, and grain size in sorghum stalks, providing a new technical means for molecular design breeding of sorghum.

[0035] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0036] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, conventional conditions are generally followed. Where: The sorghum materials BTx623, M-81E, and P184 are all known sorghum materials in the prior art. BTx623 is a grain sorghum, described in the journal article "Paterson AH, Bowers JE, Bruggmann R, et al. The Sorghumbicolor genome and the diversification of grasses. Nature, 2009, 457:551–556." M-81E is a sweet sorghum, described in the journal article "Sun X, Zheng H, Li J, Liu L, Zhang X, Sui N. Comparative Transcriptome Analysis Reveals New lncRNAs Responding to Salt Stress in Sweet Sorghum. Frontiers in Bioengineering and Biotechnology, 2020, 8:331". P184 is a conventional grain sorghum variety, described in the journal article "Zhou J, Li R, Wang Z, et al. Engineering herbicide-resistant sorghum with CRISPR / Cas9-mediated The above-mentioned sorghum material is available from the applicant for use in replicating this invention. (Journal of Integrative Plant Biology, 2026).

[0037] Example 1: Correlation investigation between miR395l and sorghum sugar content 1. Test method: The method for determining soluble sugar content is referenced in Xu Q, Chen S, Yunjuan R, et al. Regulation of Sucrose Transporters and Phloem Loading in Response to Environmental Cues. Plant Physiology, 2017, 176(1):930-945. Soluble sugars in sorghum tissues were extracted using ethanol extraction and determined using an ion chromatography system (Thermo Fisher Scientific, USA). Stem tissues from sweet sorghum M-81E and grain sorghum BTx623 at different developmental stages were selected as experimental materials. The entire stem tissue was taken at the three-leaf stage, and stem tissues from the fourth internode were taken at the flag leaf stage, the heading stage, and different stages after flowering. 100 mg of tissue sample was taken, ground into powder in liquid nitrogen, and then soluble sugars were extracted using ethanol. The sugar extract was analyzed using an ion chromatography system equipped with a CarboPacPA10 analytical column. 200 mM NaOH was used as the mobile phase, and the flow rate was 1 mL·min. -1 An amperometric detector was used for detection, with a gold electrode as the working electrode and an Ag / AgCl electrode as the reference electrode. The total run time was 40 min. A standard curve was established using a mixture of glucose, fructose, and sucrose standards. The relative content of different sugar components was analyzed using Chromeleon software (ThermoFisher Scientific, USA) based on the peak area normalization method. The total soluble sugar content was the sum of the glucose, fructose, and sucrose contents. Three biological replicates were set up for each sample.

[0038] Total RNA was extracted from stem tissues of sweet sorghum M-81E and grain sorghum BTx623 at different developmental stages using TransZol reagent (TransGen Biotech, China) according to the manufacturer's instructions. The entire stem tissue was collected at the three-leaf stage, while the fourth internode stem tissue was collected at the flag leaf stage, booting stage, and different post-flowering stages. For the stem tissue, Plus RNAiso reagent and High-Salt Solution (TaKaRa Bio Inc., Japan) were added during RNA extraction to remove polysaccharides and polyphenols. miRNA reverse transcription was performed using stem-loop RT primers, and cDNA was synthesized according to the kit instructions (Sangon Biotech, China). RT-qPCR was then performed using SYBR Green PCR Master Mix (TransGenBiotech, China). Each sample was tested in triplicate, with SbU6 as an internal control. The relative expression levels of mature miR395l and pre-miR395l were calculated using the 2-ΔΔCt method.

[0039] 2. Test Results: The results of stalk sugar content determination of sweet sorghum M-81E and grain sorghum BTx623 at different developmental stages are as follows: Figure 1 As shown, significant differences were found in the sugar content of the stems of sweet sorghum and grain sorghum after flowering.

[0040] The results of the expression level determination of mature miR395l and miR395l precursor (pre-miR395l) are as follows: Figure 2 As shown, the expression levels of mature miR395l and its precursor (pre-miR395l) increased significantly on day 10 after flowering in sorghum, and were found to be highly abundant in sweet sorghum.

[0041] The above results indicate that changes in miR395l expression are strongly correlated with the sugar accumulation process, suggesting that miR395l is a positive regulator of sugar accumulation in sorghum.

[0042] Example 2: Construction of sorghum miR395l overexpression vector and creation of transgenic plants 1. Construction of sorghum miR395l overexpression vector: Using sorghum leaf cDNA as a template, the DNA sequence of the miR395l precursor (pre-miR395l) (SEQ ID NO. 3) was amplified, and the amplified product was cloned into the Gateway entry vector pENTR / SD / D-TOPO (Thermo Fisher Scientific, USA). After confirmation by sequencing, the target fragment was recombined into the plant binary expression vector pANIC6E containing the maize ubiquitin promoter using LR Clonase recombinase (Thermo Fisher Scientific, USA), thus constructing the miR395l overexpression vector pUBI::pre-miR395l.

[0043] 2. Creation of genetically modified plants: The constructed miR395l overexpression vector pUBI::pre-miR395l was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium. Agrobacterium-mediated transformation was then used to transform the sorghum variety P184, obtaining stable transgenic sorghum plants. MIR395l-OE ).

[0044] The expression level of mature miR395l in transgenic sorghum plants (MIR395l-OE) was identified using RT-qPCR, with wild-type (WT) plants as controls.

[0045] The results are as follows Figure 3 As shown, the results indicate that the expression levels of miR395l in both the MIR395l-OE#3 and MIR395l-OE#15 transgenic sorghum lines were significantly higher than those in the wild-type plants. This demonstrates that transgenic sorghum plants (MIR395l-OE) overexpressing miR395l have been successfully created.

[0046] Example 3: Investigation of sugar content and agronomic traits in transgenic sorghum plants overexpressing miR395l 1. Test method: The transgenic sorghum plants (MIR395l-OE#3 and MIR395l-OE#15) overexpressing miR395l created in Example 2, as well as wild-type plants (WT), were planted under the same conditions.

[0047] Sugar content, plant height, chlorophyll content, flowering time, and grain size of wild-type (WT) and transgenic sorghum plants (MIR395l-OE#3 and MIR395l-OE#15) were measured and analyzed. The analytical methods are as follows: (1) Determination of sugar content: The fourth internode stem tissue of wild-type plants (WT) and miR395l-OE#3 and MIR395l-OE#15 overexpressing sorghum plants (MIR395l-OE#3 and MIR395l-OE#15) was selected 10 days (10 DAF) after flowering to determine the content of soluble sugars (including glucose, fructose, and sucrose) in the stems. The specific determination and analysis methods were the same as those for the determination of soluble sugar content in Example 1.

[0048] (2) Plant height measurement: Plant height was measured at maturity in wild-type plants (WT) and transgenic sorghum plants (MIR395l-OE#3 and MIR395l-OE#15). Plant height was measured vertically from the base of the stem to the ear of the main stem, using a ruler.

[0049] (3) Chlorophyll content determination: During the flowering period of sorghum, the chlorophyll content of leaves from wild-type plants (WT) and transgenic plants (MIR395l-OE#3 and MIR395l-OE#15) was measured. SPAD values ​​were measured using a chlorophyll meter (DP-TYS-4N, Tuopu, China), and the SPAD values ​​were used as an indicator of the relative chlorophyll content of leaves.

[0050] (4) Statistics on flowering time: Starting from the day of sowing, record the number of days required until the spike on the main stem emerges as the flowering time.

[0051] (5) Seed size statistics: Mature seeds were dried at 37℃, and the weight of 100 seeds was determined using an analytical balance (accuracy 0.001 g).

[0052] 2. Test Results: Phenotypic photographs of wild-type plants (WT) and transgenic sorghum plants (MIR395l-OE#3, MIR395l-OE#15) are shown below. Figure 4 As shown; the results of the sugar content determination in the stems are as follows. Figure 5 As shown; the results of plant height, chlorophyll content, and flowering time are as follows. Figure 6 As shown; the statistical results of grain size are as follows. Figure 7 As shown.

[0053] The results showed that the total soluble sugar content of the MIR395l overexpressing plants was significantly higher than that of the wild-type plants, indicating that overexpression of miR395l can significantly increase the sugar content in sorghum stems.

[0054] Compared with the wild type, MIR395l-OE plants showed significantly increased plant height, significantly higher chlorophyll content in leaves, shorter flowering time, and significantly larger grains, indicating that overexpression of miR395l can significantly promote the formation of sorghum biomass.

[0055] In summary, increasing the expression level of miR395l can promote the accumulation of sugar and the formation of biomass in sorghum, which has important application value for improving the quality of sorghum.

[0056] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of miR395l in promoting sorghum sugar accumulation and biomass formation, characterized in that, The nucleotide sequence of miR395l is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The promotion of sorghum sugar accumulation specifically refers to increasing the content of soluble sugars in sorghum stalks.

3. The application according to claim 1, characterized in that, Promote sorghum biomass formation through at least one of the following pathways (1)-(3): (1) Increase the plant height of sorghum; (2) Increase the chlorophyll content in sorghum leaves; (3) Increase the weight of 100 grains of sorghum.

4. The application of miR395l precursor in promoting sorghum sugar accumulation and biomass formation, characterized in that, The nucleotide sequence of the miR395l precursor is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, Promote sorghum sugar accumulation and biomass formation by overexpressing the miR395l precursor.

6. The application according to claim 5, characterized in that, Overexpression of miR395l precursor using any of the following substances: C1) An expression cassette containing a nucleic acid molecule encoding the miR395l precursor; C2) A recombinant expression vector containing a nucleic acid molecule encoding the miR395l precursor, or a recombinant expression vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing nucleic acid molecules encoding miR395l precursors, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant expression vector described in C2); C4) A transgenic plant cell line containing a nucleic acid molecule encoding the miR395l precursor, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing a nucleic acid molecule encoding the miR395l precursor, or transgenic plant tissue containing the expression cassette described in C1); C6) A transgenic plant organ containing a nucleic acid molecule encoding the miR395l precursor, or a transgenic plant organ containing the expression cassette described in C1).

7. The application according to claim 4, characterized in that, The promotion of sorghum sugar accumulation specifically refers to increasing the content of soluble sugars in sorghum stalks.

8. The application according to claim 4, characterized in that, The promotion of biomass formation specifically refers to: increasing sorghum plant height, leaf chlorophyll content, and / or increasing the 100-grain weight.

9. A method for promoting sorghum sugar accumulation and biomass formation, characterized in that, include: Steps to induce overexpression of miR395l or miR395l precursor in sorghum; The nucleotide sequence of miR395l is shown in SEQ ID NO.1; the nucleotide sequence of the miR395l precursor is shown in SEQ ID NO.2.

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