Wheat miRNA168a and application of wheat miRNA168a in regulation and control of wheat ear development

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses wheat miRNA168a and application thereof in regulation and control of wheat ear development. A transgenic plant with miRNA168a function inhibition or overexpression is obtained in wheat through a target simulation technology and an overexpression technology respectively. By inhibiting the miRNA plant, the ear length, the spikelet number per ear and the grain number per ear can be remarkably increased, and the ear length, the spikelet number per ear and the grain number of the miRNA plant are remarkably reduced by overexpressing the miRNA plant. Therefore, the wheat miRNA168a has the functions of regulating and controlling the ear length, the spikelet number and the ear grain number of the wheat. The miRNA168a and the function of the miRNA168a have good application value, and an important gene resource and a new method are provided for increasing the ear length, the spikelet number and the grain number per ear of the wheat.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to wheat miRNA168a and its application in regulating wheat ear development. Background Art

[0002] wheat( Triticum aestivum L.) is one of the most important food crops in the world, providing energy intake for about 40% of the population. my country is one of the countries with the highest wheat production and consumption. Improving wheat yield is an important measure to ensure people's living standards and national food security. miRNA is a class of small non-coding RNAs with a length of 18-25 nucleotides, which are widely involved in the fine regulation of plant gene expression. They specifically bind to the 3'UTR or coding region of the target mRNA, promote the degradation of the target mRNA or inhibit its translation, thus playing a key role in multiple biological processes of plants (such as growth and development, morphological construction and environmental adaptation). In addition, miRNA also helps plants enhance their resistance by mediating responses to adverse stresses (such as drought, salt stress, low temperature and pathogen infection). Plant miRNA is closely linked to hormone signaling pathways, regulating the fine dynamic changes of gene networks, thereby affecting the overall development and adaptability of plants. The three factors of spike number, spike number and thousand-grain weight jointly determine the final yield of wheat. miRNA can regulate genes related to ear development, grain formation and stress resistance, thereby affecting the number of ears, number of grains per ear and thousand-grain weight of wheat. Specific miRNA can optimize the growth cycle of wheat by regulating hormone signal transduction pathways, such as abscisic acid and gibberellins, and enhance resistance to adverse conditions such as drought and salt stress, thereby increasing yield. Therefore, studying the regulatory mechanism of miRNA not only helps to deeply understand the formation of wheat yield, but also provides new molecular tools for breeding high-yield and stress-resistant varieties. Summary of the invention

[0003] The purpose of the present invention is to provide wheat miRNA168a and its application in regulating wheat ear development.

[0004] To achieve the purpose of the present invention, in a first aspect, the present invention provides a wheat miRNA168a (miR168a), whose nucleotide sequence is shown in SEQ ID NO:1.

[0005] In a second aspect, the present invention provides a precursor sequence of wheat miRNA168a, whose sequence is shown in any one of SEQ ID NOs: 2-4 or a DNA sequence having at least 70% homology with the sequence shown in any one of SEQ ID NOs: 2-4.

[0006] In a third aspect, the present invention provides biological materials containing the wheat miRNA168a or the precursor sequence, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0007] In a fourth aspect, the present invention provides the use of the wheat miRNA168a or the precursor sequence or the biological material in regulating plant traits.

[0008] Furthermore, the plant includes wheat.

[0009] The plant traits include, but are not limited to, ear length, number of spikelets or number of grains per ear.

[0010] In a fifth aspect, the present invention provides a method for increasing wheat spike length, spikelet number and / or grain number per spike, the method comprising: weakening or knocking out wheat miRNA168a by genetic engineering means.

[0011] Furthermore, the method includes: designing and synthesizing a complementary sequence or a partial complementary sequence of wheat miRNA168a, forming a double-stranded DNA based on the complementary sequence, and introducing the double-stranded DNA into wheat via a plasmid or integrating it into a wheat chromosome via genetic engineering.

[0012] Preferably, the complementary sequence or partial complementary sequence of wheat miRNA168a is as shown in SEQ ID NO:10.

[0013] In a sixth aspect, the present invention provides a method for reducing wheat spike length, spikelet number and / or grain number per spike, the method comprising: overexpressing miRNA168a in wheat by genetic engineering.

[0014] The overexpression method may be selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) by introducing enhancers; In a seventh aspect, the present invention provides the use of the transgenic plants obtained according to the method in plant breeding.

[0015] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing, or asexual reproduction.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides wheat miRNA168a and its coding gene for use in regulating spike development. Transgenic plants with miRNA168a function inhibition or overexpression were obtained in wheat by target mimicry (MIM) technology and overexpression (OE) technology, respectively. Plants inhibiting the miRNA can significantly increase spike length, the number of spikelets per spike and the number of grains per spike, while plants overexpressing the miRNA have significantly reduced spike length, the number of spikelets per spike and the number of grains. This indicates that wheat miRNA168a has the function of regulating wheat spike length, spikelet number and spikelet number.

[0017] The miRNA168a and its function provided by the present invention have great application value and provide important gene resources and new methods for increasing wheat spike length, spikelet number and grain number per spike. 。 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison analysis of the precursor sequence and mature sequence of wheat miR168a in the present invention and other species (barley, Brachypodium, rice and corn).

[0019] Figure 2 It is the expression level of miR168a in transgenic wheat in a preferred embodiment of the present invention.

[0020] Figure 3 This is the ear phenotype analysis of transgenic wheat in a preferred embodiment of the present invention.

[0021] Figure 4 This is the grain phenotype analysis of transgenic wheat in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0022] The present invention aims to provide a miRNA168a and an application thereof.

[0023] The present invention adopts the following technical solution: The present invention provides a miRNA168a, wherein the nucleotide sequence of the miRNA168a is as shown in SEQ ID NO:1.

[0024] The present invention further provides a precursor of wheat miRNA168a, wherein the nucleotide sequence of the precursor sequence of miRNA168a is as shown in any one of SEQ ID NOs: 2-4 or a DNA sequence having at least 70% homology with the sequence shown in any one of SEQ ID NOs: 2-4.

[0025] The present invention further provides a biomaterial, which comprises the miRNA168a or the precursor sequence of miRNA168a; the biomaterial is an expression cassette, a vector or a transgenic cell.

[0026] The present invention further provides the use of the miRNA168a or the precursor sequence of miRNA168a or the biological material in increasing the spike length, spikelet number or grain number per spike of a plant.

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] Example 1 Expression analysis of TamiR168a 1. Extraction and purification of wheat RNA 1) Place fresh leaves of the wheat variety Fielder in an enzyme-free centrifuge tube, freeze them in liquid nitrogen, and grind them into powder. Add 1 mL of Trizol to the centrifuge tube, shake and mix, and let stand at room temperature for 5 minutes to fully lyse them. 2) Centrifuge at 12,000 rpm, 4°C for 10 minutes, collect the supernatant and add it to a new centrifuge tube, add 200 µL of chloroform, shake and mix, and leave at room temperature for 3 minutes; 3) Centrifuge at 12,000 rpm and 4°C for 10 minutes, aspirate the top liquid phase into a new centrifuge tube, add an equal volume of isopropanol, gently invert to mix thoroughly, and place at -20°C for 30 minutes; 4) Centrifuge at 12,000 rpm, 4°C for 10 minutes and carefully remove the supernatant; 5) Add 1 mL of pre-cooled 70% ethanol aqueous solution, centrifuge at 12,000 rpm, 4°C for 10 minutes, carefully remove all the ethanol, repeat once, and dry the white precipitate at room temperature for 5 minutes; 6) Add 25 µL DEPC water and dissolve at room temperature for 10 minutes to obtain total RNA; 7) Detect the concentration and purity of RNA samples by agarose electrophoresis combined with a spectrophotometer. After confirming that the RNA quality is qualified, store it at -80℃.

[0029] 2. qRT-PCR detection of miRNA 1) miRNA is detected by the stem-loop quantitative method. The specific stem-loop structure RT primer used in the reverse transcription step is generally a sequence that can form a stem-loop structure and then add a reverse complementary sequence of 6-8 bases at the 3′ end of the miRNA. The stem-loop sequence is mostly fixed. The specific stem-loop reverse transcription primer sequence of miRNA-P81 in this embodiment is shown in SEQ ID NO: 5; 2) Design of the forward primer during the PCR reaction, remove the remaining sequence after removing the 6 bases at the 3' end that are reverse complementary to the RT primer. And add a few bases (G or C) at the 5' end of the sequence to balance the GC content in the primer and stabilize the Tm value. The reverse primer is designed for a sequence on the stem loop. In this embodiment, the upstream primer sequence of miRNA168a q-PCR is shown in SEQ ID NO:6, and the downstream primer sequence is shown in SEQ ID NO:7; 3) According to the instruction manual of the reverse transcription kit (Takara), total RNA was reverse transcribed into cDNA and the mature sequence of miR168a was specifically reverse transcribed; Each reaction system is shown in Table 1 to Table 3: Table 1 DNA removal reaction system

[0030] Reaction conditions: 42°C, 2 min, 4°C, permanent.

[0031] Table 2 Reverse transcription reaction system

[0032] Reaction conditions: 42°C, 15 min, 85°C, 5 s, 4°C, permanent.

[0033] 4) Detect the accumulation of miR168a mature sequence according to the instructions of the fluorescence quantitative kit (Takara); Table 3 Fluorescence quantitative reaction system

[0034] Reaction conditions: 95°C, 30 s; 95°C, 10 s, 60°C, 1 min, 40 cycles.

[0035] Example 2 Construction and transformation of miR168a transgenic vector 1. Construction of miR168a overexpression vector According to the MIR168a precursor sequence, primers MIR168aF and MIR168aR were designed to PCR amplify the upstream 400 bp and downstream 400 bp of the MIR168a sequence from wheat Fielder DNA. MIR168aF and MIR168aR are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively. The PCR product was subjected to 1.2% agarose gel electrophoresis, and the agarose gel DNA recovery kit (Axygene) was used to recover and purify the product. The PCR system is shown in Table 4: Table 4 PCR system is as follows:

[0036] The PCR program was as follows: 94°C, 5 min; 94°C, 30 sec; 60°C, 30 sec; 72°C, 45 sec; repeated 34 times; 72°C, 10 min.

[0037] The gel recovery product was connected to the pGEM®-T EASY vector (Promega), and the ligation product was transformed into E. coli DH5α and propagated therein. The positive clones were screened by sequencing. The sequence with the correct sequence was fused to the overexpression vector pCAMBIA3300-Ubi-Bar by restriction digestion and ligation.

[0038] 2. Construction of miR168a artificial mimic target overexpression vector The precursor sequences of MIR168a in barley, Brachypodium, rice and maize were compared with the wheat genome to determine the precursor sequence of wheat MIR168a. Figure 1 The complementary sequence was designed based on the mature sequence of miR168a as a template, and three CTA bases were added between the 9th and 10th bases of the complementary sequence so that it could bind to miR168a but would not be cut. The complementary sequence MIM168a was artificially synthesized, and the sequence of MIM168a is shown in SEQ ID NO: 10. The complementary sequence was annealed by primers to form double-stranded DNA, and fused to the overexpression vector pCAMBIA3300-Ubi-Bar by enzyme cutting and ligation.

[0039] 3. Transformation of genetically modified wheat The above-constructed expression vector was transformed into Agrobacterium, and then transferred into wheat Fielder by Agrobacterium-mediated transformation method, with wheat immature embryo callus as the receptor and β-lactamase as the screening marker in the plant to obtain wheat edited transformed plants.

[0040] The specific transformation was completed by the transformation platform of the Crop Science Institute of the Chinese Academy of Agricultural Sciences.

[0041] Example 3 Phenotypic statistics of wheat miR168a transgenic plants The wheat edited plants obtained in Example 2 were cultivated together with their wild-type control plants in a greenhouse (low temperature group: 18°C / 16h light, 15°C / 8h dark; high temperature group: 25°C / 16h light, 22°C / 8h dark; control group: 23°C / 16h light, 20°C / 8h dark) until the entire life cycle was completed. 2 The ear development phenotype and grain phenotype of the transgenic plants were observed and counted. Figure 2Compared with wild-type plants, the expression of miR168a in overexpressed plants increased significantly, while the expression of miR168a in functionally inhibited plants decreased significantly. Figure 3 As shown in Figure 2, the number of seeds in transgenic plants is as follows: Figure 4 The results showed that compared with the wild type, the spike length, spikelet number and grain number per spike of transgenic wheat with suppressed miR168a function increased significantly, while the spike length, spikelet number and grain number per spike of transgenic wheat with overexpressed miR168a decreased significantly. The above results indicate that wheat miR168a is involved in regulating spike length, spikelet number and grain number per spike.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Wheat miRNA168a, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The precursor sequence of wheat miRNA168a, characterized in that The sequence is as shown in any one of SEQ ID NOs: 2-4 or a DNA sequence having at least 70% homology with the sequence shown in any one of SEQ ID NOs: 2-4.

3. A biological material containing the wheat miRNA168a according to claim 1 or the precursor sequence according to claim 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

4. Use of the wheat miRNA168a according to claim 1, the precursor sequence according to claim 2, or the biological material according to claim 3 in regulating plant traits.

5. The use according to claim 4, characterized in that: The plant is wheat; The plant traits include ear length, number of spikelets or number of grains per ear.

6. A method for increasing wheat ear length, spikelet number and / or grain number per ear, characterized in that: The method comprises: using genetic engineering means to weaken or knock out wheat miRNA168a; wherein the wheat miRNA168a is the same as that described in claim 1.

7. The method according to claim 6, characterized in that include: Design and synthesize a complementary sequence or a partial complementary sequence of wheat miRNA168a, form a double-stranded DNA based on the complementary sequence, and introduce the double-stranded DNA into wheat via a plasmid or integrate it into a wheat chromosome via genetic engineering; Preferably, the complementary sequence or partial complementary sequence of wheat miRNA168a is as shown in SEQ ID NO:

10.

8. A method for reducing wheat ear length, spikelet number and / or grain number, characterized in that: The method comprises: using genetic engineering means to overexpress miRNA168a in wheat; The overexpression method is selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) by introducing enhancers; Wherein, miRNA168a is the same as described in claim 1.

9. Use of the transgenic plant obtained by the method according to any one of claims 6 to 8 in plant breeding.

10. The use according to claim 9, characterized in that: Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

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