Application of miR528 in regulation and control of included angle of corn leaves

Abstract

The invention discloses an application of miR528 in regulation and control of an included angle of corn leaves. The nucleotide sequence of the miR528 is shown as SEQ ID NO: 1 in a sequence table. It is found for the first time that miR528 can regulate and control the corn leaf included angle, compared with wild type miR528, the leaf included angle of upper leaves and flag leaves of a corn ear of silent miR528 is reduced, and a foundation is laid for creation of close planting corn.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of miR528 in regulating maize leaf angle and high-density planting. Background Art

[0002] Maize is the most widely used food crop for feed and industrial conversion, and its demand is increasing year by year. The leaf angle is the main factor determining the compactness of the maize plant type, directly affecting the light transmittance in the maize population canopy and the light-receiving posture of the leaves, and further affecting the light interception ability of the maize canopy and the population light energy utilization rate. Therefore, finding key genes that regulate the maize leaf angle has important theoretical guiding significance for cultivating maize varieties with compact leaves and for the molecular design breeding of high-density and high-yield maize.

[0003] Important progress has been made in the research on the genetic regulation mechanism of the maize leaf angle size. Currently, the known key regulatory genes can be mainly divided into three major functional categories: (1) genes related to leaf morphogenesis; (2) genes related to plant hormone signal transduction pathways; (3) genes regulating the mechanical strength of the leaf midrib. Among them, the third category of genes mainly regulates the leaf angle by affecting vascular bundle development, and its mechanism of action is closely related to lignin metabolism. Lignin, as a unique secondary cell wall component of vascular plants, is a high-molecular compound formed by the oxidative polymerization of hydroxycinnamyl alcohol monomers derived from the phenylpropanoid metabolic pathway. Its basic structural units include three aromatic alcohol monomers: coniferyl alcohol (G-type monomer), sinapyl alcohol (S-type monomer), and p-coumaryl alcohol (H-type monomer). After these monomers are synthesized in the cytoplasm, they are transported across the membrane through ATP-binding cassette transporters to the cell wall and finally polymerize in specific regions of the cell wall. During the lignin biosynthesis process, plant laccase (Laccase, LAC), as a key blue copper oxidase, is responsible for catalyzing the dehydrogenation polymerization reaction between monomers. Research shows that specific regulation of LAC family members through genetic engineering technology can precisely adjust the lignin content and its monomer composition ratio in plants. This precise regulation at the molecular level not only affects the development process of the secondary cell wall of vascular bundles but also effectively changes the mechanical strength characteristics of the midrib tissue, thus providing an important technical path for the genetic improvement of the maize leaf angle.

[0004] miRNAs are a class of endogenous, single-stranded, non-coding RNA molecules, about 21-23 nt in length, and are widely present in eukaryotic cells such as animals, plants, and humans. In plants, miRNAs form RNA-induced silencing complexes with target genes and regulate the proteins of target genes at the post-transcriptional level. miRNAs in plants also participate in the regulation of lignin biosynthesis. In Arabidopsis, miR857 participates in the regulation of secondary growth of vascular tissues by acting on its target gene AtLAC7. In miR857 overexpressing plants, the expression level of AtLAC7 is down-regulated, the laccase activity is decreased by 10.8%, and the lignin content is reduced; while in miR857 knockdown plants, the laccase activity is increased by 33.3%, the lignin content is increased, and the degree of lignification of secondary xylem is increased. miR397b regulates lignin content and seed number by regulating AtLAC4. miR397b overexpressing plants show reduced lignin content and reduced secondary wall thickness. miRNA528 is a small RNA unique to specific monocotyledonous plants. In maize, miRNA528 affects the synthesis of lignin in transgenic maize by regulating the expression of ZmLAC3 and ZmLAC5, and further affects the lodging resistance of maize. However, whether miR528 regulates the leaf angle of maize needs further study. In-depth analysis of the function and mechanism of miR528 in regulating the leaf angle of maize will provide important theoretical basis and gene resources for the cultivation of compact maize materials. Summary of the Invention

[0005] The object of the present invention is to provide the application of miR528 in regulating the leaf angle of maize.

[0006] The application of miR528 in regulating the leaf angle of maize.

[0007] The nucleotide sequence of the said miR528 is shown in Sequence Listing SEQ ID NO: 1.

[0008] Silencing miR528 in maize results in a smaller leaf angle of maize leaves.

[0009] A method for improving the planting density of maize, which silences miR528 in maize.

[0010] The beneficial effects of the present invention: The present invention first discovers that miR528 can regulate the leaf angle of maize. Compared with the wild type, the leaf angles of the upper leaves and flag leaves on the maize ear with silenced miR528 are both reduced, which lays a foundation for creating dense planting maize. Brief Description of the Drawings

[0011] Figure 1 Identification of transgenic materials with silenced miR528.

[0012] Figure 2Lignin content and lignin staining in the leaf sheath of miR528 - silenced transgenic materials In the figure, A - Phloroglucinol staining in the leaf sheath of ZmmiR528 - knockdown plants, scale bar is 200 μm; B - Lignin staining signal intensity in the sclerenchyma cell layer of the cortex; C - Lignin staining signal intensity in the sclerenchyma cell layer of the cortex; D - Acetyl bromide lignin content (n = 3).

[0013] Figure 3 Silencing miR528 reduces the leaf angle of maize In the figure, A - Phenotype of miR528 - silenced transgenic plants, scale bar is 20 cm; B - Leaf angle of the ear - positioned leaf of miR528 - silenced transgenic plants (n = 15); C - Leaf angle of the flag leaf of miR528 - silenced transgenic plants (n = 15).

[0014] Figure 4 Changes in the leaf angle of miR528 - silenced transgenic plants under different planting densities in the field Detailed implementation methods

[0015] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0016] The experimental materials used in the following examples: Maize inbred line material: C01; Strains: Escherichia coli strain DH5α, Agrobacterium tumefaciens strain EHA105; Expression vector: pCUB. Example 1

[0017] The maize miR528 family contains two precursors (miR528a and miR528b), but the mature miR528 sequences produced by these two precursors are identical, both being 5’-TGGAAGGGGCATGCAGAGGAG-3’ (SEQ ID NO: 1). Therefore, the Target Minicry method was used to construct miR528-silenced plants. Using Arabidopsis genomic DNA as a template, PCR amplifications were performed with the primers 5’-AAGAAAAATGGCCATCCCCTAGC-3’ (SEQ ID NO: 2) and 5’-CTGATTCCGAGGGGAACCGAAGCTAGGAAGGGGCTAGAAAGCAGAGGAG-3’, (SEQ ID NO: 3), as well as 5’-CTCCTCTGCTTTCTAGCCCCTTCCTAGCTTCGGTTCCCCTCGGAATCAG-3’ (SEQ ID NO: 4) and 5’-AAGAGGAATTCACTATAAAGAG-3’ (SEQ ID NO: 5); then, with the primers 5’-AAGAAAAATGGCCATCCCCTAGC-3’ (SEQ ID NO: 6) and 5’-AAGAGGAATTCACTATAAAGAG-3’ (SEQ ID NO: 7), and using the PCR product from the previous step as a template, a bridging PCR amplification was carried out to obtain an IPS1 fragment with a small bulge at the 11th position complementary to the mature miR528 sequence; the pCUB vector was digested with the restriction endonuclease Bam HI, and the target fragment was ligated to the linearized vector through In-Fusion homologous recombination to obtain the recombinant vector pCUB-MIM528, which was then transformed into Escherichia coli DH5α. Single colonies were picked for sequencing, and plasmids were extracted; the recombinant vector was transformed into Agrobacterium tumefaciens EHA105, and through Agrobacterium-mediated maize immature embryo transgenic technology, it was introduced into the recipient material C01 to obtain T 0 -generation transgenic plants. The T 0 -generation transgenic plants were continuously self-crossed to obtain homozygous plants with silenced miR528.

[0018] Leaves of the transgenic plants were taken to extract small RNAs, and Northern blotting experiments were conducted. The miR528 probe (5’-CTCCTCTGCATGCCCCTTCCA-3’ SEQ ID NO: 8) was used to detect the expression level of the mature miR528 sequence. The U6 probe (5’-AGGGGCCATGCTAATCTTCTC-3’ SEQ ID NO: 9) was used as an internal reference.

[0019] Small RNA extraction and Northern blotting: (1)Total RNA extraction: Take 1.5 g of fresh corn leaves, thoroughly grind them after using liquid nitrogen, and transfer them to a 2 ml RNase-free centrifuge tube. Add 1 ml of Trizol; vortex for 50 s until homogenized, and let it stand on ice for 5 min; add 300 μl of chloroform, vortex for 60 s, and mix well; centrifuge at 12,000 rpm for 15 min at 4°C; pipette the supernatant into a new 1.5 ml centrifuge tube, add an equal volume of pre-cooled isopropanol, invert to mix, and place at -20°C for 30 min; centrifuge at 12,000 rpm for 15 min at 4°C; pour out the supernatant, add 1 ml of 75% ethanol, invert to mix, centrifuge at 12,000 rpm for 10 min at 4°C; discard the supernatant, place it in a laminar flow hood to dry the precipitate, add 50 μl of RNase-free water to completely dissolve the precipitate, and store at -80°C; (2)Small RNA isolation: Take 20 μg of total RNA, add DEPC water to 100 μl, add an equal volume of 8 M LiCl, mix well, and place at 4°C for 2 hours; centrifuge at 12,000 rpm for 10 min, pipette the supernatant into a 1.5 ml centrifuge tube, add 1 / 10 volume of 3 M NaAc and 2.5 times volume of absolute ethanol, place at -80°C, and precipitate for more than 4 hours; centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash the precipitate once with 75% ethanol, air dry at room temperature, and then add 30 μl of DEPC water to fully dissolve it.

[0020] (3)Northern blotting: Prepare a 15% polyacrylamide gel, pre-electrophorese at 180 V for 30 min; the loading amount of small RNA is 5 μg, add an equal volume of 2×loading buffer, denature at 95°C for 5 min, and quickly place on ice; electrophorese at 220 V for about 2 h; transfer the small RNA to a nylon membrane using the semi-dry method; after the transfer is completed, irradiate the front and back of the membrane with ultraviolet light for 10 min to promote the formation of a cross-linked structure between the small RNA bases and the positive charges on the surface of the nylon membrane; place the membrane in a hybridization tube, add 5 ml of pre-hybridization buffer, and pre-hybridize at 37°C for 1 hour; take 2.5 μl of the denatured probe, add it to the hybridization solution, and hybridize at 37°C for more than 15 hours; wash the membrane successively with 2×SSC / 0.1% SDS, 1×SSC / 0.1% SDS, blocking solution, AP, Washing buffer, and Detection buffer, and finally wash the membrane with CDP-Star developer, and take a picture using a Tannon 5200 imager.

[0021] The results are as Figure 1 shown. Compared with the wild-type plants, TM-3 and TM-7The expression abundance of miR528 in the plant lines decreased by about 0.4 times, and these two plant lines were used in subsequent experiments.

[0022] Example 2 The lignin content in the pulvinus of miR528-silenced transgenic materials increased Lignin staining: At the silking stage of maize, take the pulvinus part of the first leaf on the ear position of wild-type and transgenic plants, cut the materials to a length of about 5 mm with a blade, and embed the materials with 3% agarose. The embedded materials were sectioned with a Leica VT1000s vibratome, and the thickness was set to 50 μm. Pick up the complete section materials and place them on a glass slide, add 100 μl of 5% phloroglucinol and stain for 2 min, then add an equal volume of concentrated hydrochloric acid and react for 1 min, add 40% glycerol to seal the slide, and observe and photograph with a Leica DM4B microscope. The ImageJ software was used for gray-scale calculation to quantitatively analyze the relative signal intensity of lignin staining in the vascular bundles of the measured layer of nuclei.

[0023] Under acidic conditions, phloroglucinol can react with total lignin to form pink or dark red substances, and this method can intuitively and quickly reflect the change of lignin content. The results showed that compared with the wild type, the lignin staining color in the pulvinus of the first leaf on the ear of the miR528-silenced transgenic lines (TM-3 and TM-7) deepened; the relative signal intensity of lignin staining was statistically analyzed, and the staining intensity of the abaxial hypodermal sclerenchyma cell layer ( Figure 2 B) and the vascular bundle sclerenchyma cell layer ( Figure 2 C) in the pulvinus band of TM-3 and TM-7 plants was higher than that of the wild type. Among them, the staining signal intensity of TM-3 plants was the highest, indicating that the lignin content in the pulvinus of the miR528-silenced transgenic lines increased. Further, the acetyl bromide method was used to determine the lignin content in the pulvinus of the first leaf on the ear of wild-type and miR528-silenced transgenic materials. The results showed that compared with the wild type, the lignin content in the pulvinus of the miR528-silenced transgenic lines increased significantly, by about 20% and 40% respectively ( Figure 2 D).

[0024] Example 3 The leaf angle of miR528-silenced transgenic materials decreased To clarify the phenotypes of the miR528 - silenced transgenic lines, the leaf angle was measured for the miR528 transgenic materials planted in the field. Field planting conditions: Planting materials: C01, miR528 - silenced transgenic plants; Normal density: 5336 plants per mu (80,000 plants per hectare); Row length of 3 meters, row spacing of 50 cm, plant spacing of 25 cm, 13 plants per row; Increased density 1: 8000 plants per mu (150,000 plants per hectare); Row length of 3 meters, row spacing of 55 cm, plant spacing of 15 cm, 21 plants per row; Increased density 2: 12000 plants per mu (180,000 plants per hectare); Row length of 3 meters, row spacing of 55 cm, plant spacing of 10 cm, 31 plants per row; Measured traits: Plant height, internode length, leaf angle.

[0025] The results showed that compared with the wild type, the leaf angles of TM3 and TM7 were reduced to varying degrees ( Figure 3 A), and the leaf angles of the leaves above the ear were reduced by 4.9° and 2.3° respectively ( Figure 3 B), and the leaf angles of the flag leaves were reduced by 5.6° and 3.4° respectively ( Figure 3 C).

[0026] To further study the relationship between the miR528 gene expression level and the leaf angle in the field, three planting densities were set in the field: normal density (5336 plants), increased density 1 (8000 plants), and increased density 2 (12000 plants). Compared with the wild - type C01, the leaf angles of the flag leaves of the miR528 - silenced transgenic plants were significantly reduced at the three planting densities, but there were no significant changes in plant height and average internode length at different densities (Figure 4).

[0027] The above - described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. Application of miR528 in regulating corn leaf angle.

2. The use of miR528 in regulating the angle of corn leaves according to claim 1, characterized in that: The nucleotide sequence of miR528 is shown in the sequence listing as SEQ ID NO:

1.

3. The use of miR528 in regulating the angle of corn leaves according to claim 1, characterized in that: Silencing miR528 in maize reduces the angle of maize leaves.

4. A method for improving corn planting density, characterized in that: Silencing miR528 in maize.

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