CN115927330A - Application of peach MIR6288b in regulation of plant branch number - Google Patents

Abstract

The invention discloses application of peach MIR6288b in regulation and control of plant branch number, wherein a precursor sequence of MIR6288b gene is shown as SEQ ID NO.1, a mature sequence miR6288b-5p is shown as SEQ ID NO.2, and a mature sequence miR6288b-3p is shown as SEQ ID NO. 3. According to the invention, an overexpression vector is constructed by cloning peach MIR6288b, and the function of MIR6288b is verified by transferring the peach MIR6288b into arabidopsis through an agrobacterium-mediated genetic transformation method, compared with a wild plant, the number of branches of a transgenic plant is remarkably increased, and the molecular mechanism related to the flowering quantity of peach trees is favorably clarified on the molecular level, so that the method can assist in breeding a new peach variety with less branches, is suitable for labor-saving cultivation, and can be applied to research on genetic improvement of the plant branch quantity.

Description

Application of peach MIR6288b in regulation of plant branch number

Technical Field

The invention belongs to the technical field of biology, and particularly relates to peach MIR6288b and application thereof in regulation of plant branch number.

Background

The main cultivated species in peach production is a common type, the tree shape of the peach is open, the branches are many, the branch angles are large, the bearing age is prolonged, the nutrition growth is vigorous, the branches of the tree body are luxuriant, the tree crown is finally shielded, the light transmittance and the fruit quality are improved, the occurrence probability of plant diseases and insect pests is increased, and the labor cost of summer pruning and winter pruning is greatly increased. The statistical results of the national peach industry technical system cultivation research laboratory show that the labor cost accounts for more than half of the production cost of peaches, and manual pruning is the most important part of manual investment. Therefore, in order to realize labor-saving cultivation of peaches, the analysis of the tree type regulation mechanism of the peach trees is urgent.

The plant microRNA is a neck ring structure coded by an endogenous MIR gene, is a small molecule single-stranded non-coding RNA with the length of about 19-24 nucleotides formed under the shearing action of DICER-LIKE1 (DCL 1) enzyme, can identify target gene mRNA in a base complementary pairing mode, and regulates and controls the expression of the gene at the transcription level or the post-transcription level. mirnas play an important regulatory role in the process of branch formation in plants. miR319 targets and degrades AtTCP4 to ensure normal growth of Arabidopsis roots; the cotton miR164 regulates the content of endogenous ABA and regulates the formation of cotton branches by targeting a gene GhCUC 2; the rice miR167a regulates the redistribution of auxin and the tillering angle of rice in a mode of targeting an auxin response factor OsARF 12/17/25. However, the current miRNA for regulating the branch number is rarely reported.

Disclosure of Invention

The invention aims to provide peach (Prunus persica L.) MIR6288b and application thereof in regulating and controlling the branch number of plants.

In order to achieve the above purpose, the technical scheme of the invention is summarized as follows:

the precursor sequence of peach MIR6288b is shown in SEQ ID NO.1, the mature sequence miR6288b-5p is shown in SEQ ID NO.2, and the mature sequence miR6288b-3p is shown in SEQ ID NO. 3.

The main object of the present invention is to provide the use of the peach MIR6288b or the biomaterial related to MIR6288b described above. Specifically, the MIR6288b of peach can increase the number of plant branches, so that the novel peach variety which has less number of branches and is suitable for labor-saving cultivation can be selected and bred in an auxiliary way by inhibiting the expression of the MIR6288b in a target plant, and plays an important role in the aspect of genetic improvement of the number of plant branches.

The expression of MIR6288b in a target plant can be inhibited by knocking out MIR6288b, and specifically, a MIR6288 b-deleted mutant plant can be obtained by means of gene editing. Mutant plants with MIR6288b deletion can be obtained by regenerating the plant cells into plants. And the recombinant plasmid is transferred into the plant by utilizing an agrobacterium transformation method.

The biological material can be an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a tissue or an organ containing the MIR6288b, and can also be a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a tissue or an organ containing the expression cassette.

In the present invention, there is no particular limitation on the plant suitable for use in the present invention, as long as it is suitable for carrying out a gene transformation operation, such as various crops, flowering plants, forestry plants, or the like. Said plant may for example (without limitation): dicotyledonous plants, monocotyledonous plants, woody plants, plants of the order Rosales, plants of the family Rosaceae, peach genus, plants of the family Brassicaceae, plants of the genus Arabidopsis, arabidopsis thaliana, etc.

As used herein, "plant" includes whole plants, parent and progeny plants thereof, and different parts of the plant, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, all of which have the gene or nucleic acid of interest. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises a gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by the methods therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also relates to other post-harvest derivatives of the plant, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to food products or food additives obtained from the relevant plants.

The invention also discloses a method for changing the number of plant branches, which controls the expression of the plant to MIR6288b by a transgenic, gene knockout, hybridization, backcross, selfing or asexual propagation method, wherein the precursor sequence of the MIR6288b is shown as sEQ ID No. 1.

Wherein, controlling the expression of MIR6288b by the plant means that the MIR6288b gene is over-expressed or reduced in the plant.

The transgene comprises the step of introducing a recombinant expression vector containing the MIR6288b sequence into a plant by using a Ti plasmid, a plant virus vector, direct DNA transformation, microinjection, a gene gun, conductance or an agrobacterium-mediated method to obtain a transgenic plant strain.

The gene knockout comprises the step of knocking out the MIR6288b by using a DNA homologous recombination technology, a Cre/LoxP technology or a CRISPR/Cas9 technology to obtain a transgenic plant strain.

The invention has the advantages that:

according to the invention, an overexpression vector is constructed by cloning peach MIR6288b, and the function of MIR6288b is verified by transferring the peach MIR6288b into arabidopsis through an agrobacterium-mediated genetic transformation method, compared with a wild plant, the number of branches of a transgenic plant is remarkably increased, and the molecular mechanism related to the flowering quantity of peach trees is favorably clarified on the molecular level, so that the method can assist in breeding a new peach variety with less branches, is suitable for labor-saving cultivation, and can be applied to research on genetic improvement of the plant branch quantity.

Drawings

FIG. 1 is that the general peach has a significantly higher number of branches for long time preservation than the column peach shinning hand red;

FIG. 2 is an amplification electrophoretogram of peach MIR6288 b;

FIG. 3 shows the expression level of MIR6288b in the column type peaches according to hand red and the general type peaches;

FIG. 4 is identification of peach MIR6288b transgenic Arabidopsis positive plants;

FIG. 5 is the relative expression analysis of peach MIR6288b transgenic Arabidopsis;

FIG. 6 is a peach MIR6288b transgenic Arabidopsis phenotype analysis.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. However, the specific experimental procedures referred to in the following examples were carried out in a conventional manner or under the conditions recommended by the manufacturer's instructions unless otherwise specified.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials used are commercially available, unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Moreover, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, either single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences for non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons as in genomic sequences, and/or include coding sequences as in cDNA, and/or include cDNA and its regulatory sequences. In particular embodiments, e.g., with respect to an isolated nucleic acid sequence, it is preferred to default to cDNA.

The test materials adopted by the invention are planted in the villa scientific and educational park of Henan university of agriculture according to the standard of hand-Red and Dajubao.

1. Isolation of MIR6288b

By observation, the branches of the regular peach type chakuo (O) were significantly higher than the pillar type peaches shined hand red (Z) (fig. 1).Extracting leaf bud RNA of DALIBAOJIAN and HONGHUATAO with plant total RNA extraction kit (Beijing Huayuyo Biotech limited), and reverse transcription with reverse transcription kit (Prime Script) ^TM RT Reagent Kit with gDNA Eraser, perfect Real Time) and using it as template and the following sequence as primer to amplify, thus obtaining the MIR6288b precursor sequence (as shown in FIG. 2). The gene MIR6288b precursor sequence is shown as SEQ ID NO.1 and accounts for 92nt, the mature sequence miR6288b-5p is shown as SEQ ID NO.2, and the mature sequence miR6288b-3p is shown as SEQ ID NO.3 and accounts for 21m.

The primer sequence is as follows:

forward MIR6288b-F: ATTCTGGCTTAGGATCGAGTAG,

reverse MIR6288b-R: gatgccttagagaggaacctatttg;

the PCR amplification conditions were: 30s at 98 ℃ for 1 cycle; 1 cycle at 98 ℃ for 10 s; 58 ℃ for 5s,34 cycles; 5s at 72 ℃ for 1 cycle; 60s at 72 ℃ for 1 cycle.

2. Expression analysis of peach MIR6288b

Fluorescent quantitative PCR experiment steps: extracting total RNA of DABUBAO and ZUOHUANGYEBAI with total plant RNA extraction kit, and reverse transcription with reverse transcription kit (Prime Script) ^TM RT Reagent Kit with gDNA Eraser, perfect Real Time) to obtain single stranded cDNA. RT-qPCR reactions were carried out according to the kit SYBR Select Master Mix (Applied Biosystems, mardrid, calif., USA) using the ABI PRISM 7500FAST Instrument detection System (Applied Biosystems, mardrid, calif., USA); detecting and analyzing the expression quantity of the genes by using a SYBR green fluorescent dye method, wherein the peach GAPDH gene is an internal reference gene, and each sample has 3 biological repeats. The fluorescent quantitative PCR reaction system was 20. Mu.L, and included 200ng cDNA (1 uL), SYBR Select Master Mix (10 uL), 0.5 umol. L ^-1 Upstream and downstream primers (1 uL) and RNase-free water (7 uL).

Reaction procedures are as follows: pre-denaturation at 95 ℃,2min: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 42 cycles. Each sample was replicated 3 times. Quantitative primers were designed using Primer Premier 5.0 software.

The primer sequence is as follows:

MIR6288b-F：GTCGGGACTTTGCAATTCTGG，

MIR6288b-R：ATGACGTTTGGCGATTGCG；

as can be seen from FIG. 3, the expression level of the gene MIR6288b in the general peach tree is significantly higher than that in the column peach tree, indicating that the MIR6288b may be involved in the regulation of peach branch formation.

3. Functional characterization of MIR6288b

The effect of MIR6288b on shoot formation was identified by transgenic arabidopsis.

3.1 recombinant vector construction

MIR6288b amplified from Rekistrodon was used as a template, and the over-expression vector pSAK-277 was ligated by using a one-step cloning kit (Beijing Jiang, international Biogene technology Co., ltd.). The primer sequences are as follows:

forward MIR6288b-F:

reverse MIR6288b-R:

and (5) carrying out Shanghai biological sequencing after positive clones are identified.

3.2 Arabidopsis Positive plant identification

As a stable and efficient genetic transformation system is not established in peaches. Therefore, the function of MIR6288b was verified using Arabidopsis thaliana as a material. The over-expression recombinant vector pSAK-277-MIR6288b was transformed into Agrobacterium GV3101. Adjusting the concentration of the bacterial liquid to OD after the amplification culture ₆₀₀ =0.8, infecting wild type arabidopsis by dipping flower method, harvesting arabidopsis T after maturation ₀ Seeds were generated and positive plants were screened initially on kanamycin-resistant MS medium. When the Arabidopsis seedlings grow to 4-6 leaves, extracting the DNA of the leaves, further identifying the transgenic Arabidopsis strains by a PCR method (figure 4), and harvesting T ₁ And generating Arabidopsis seeds. Broadcast T ₁ And (4) generating Arabidopsis seeds, and using the obtained Arabidopsis T2 generation seedlings for phenotype observation and identification. The following experimental materials are all transgenicArabidopsis thaliana T ₂ And generation and progeny homozygous lines thereof.

Arabidopsis positive seedling identification PCR primers:

MIR6288b-F：CATCGAAAGGACAGTAGAAAAGG，

MIR6288b-R：

3.3 quantitative analysis of wild type and transgenic Arabidopsis

Fluorescent quantitative PCR experiment steps: the RNA of transgenic Arabidopsis thaliana leaf was extracted using the kit of Biotech, inc., of Beijing Huayuyo, and the reverse transcription kit (Prime Scripff) was used ^M R Treagent Kitwithg DNA Eraser, perfect Real Time) to obtain single-stranded cDNA. The expression level of the gene is detected and analyzed by a SYBR green fluorescent dye method, the arabidopsis AtACT2 gene is an internal reference gene, and each sample has 3 biological repeats. Fluorescent quantitation experiments were performed with an ABI PRISM 7500FAST Sequence Detection System (Applied Biosystems, mardrid, calif., USA) instrument.

Fluorescent quantitative PCR reaction system:

green PCR Master Mix 5.0. Mu.L; forward Primer 0.3. Mu.L; reverse Primer 0.3 μ L; cDNA template 1.O.mu.L; RNase Free H ₂ O3.4μL；Total 10μL。

Reaction procedures are as follows: the qRT-PCR reaction program was: 20s at 50 ℃ and 1 cycle; 2 s at 95 ℃ for 1 cycle; 30s at 95 deg.C, 30s at 60 deg.C, and 42 cycles.

Quantitative primer:

MIR6288b-F：GTCGGGACTTTGCAATTCTGG，

MIR6288b-R：ATGACGTTTGGCGATTGCG；

as shown in FIG. 5, compared with wild type Arabidopsis, the expression level of MIR6288b in 6 MIR6288b overexpression transgenic lines is significantly improved, wherein the expression levels of lines #1, #3 and #5 are 33717, 23360 and 20174 times of wild type Arabidopsis respectively.

3.4 MIR6288b transgenic Arabidopsis thaliana phenotypic analysis

Synchronously sowing Wild Type (WT) and transgenic arabidopsis thaliana on an MS culture medium, transplanting the MS culture medium into a culture pot (10 multiplied by 10 cm) when two leaves grow out, and carrying out phenotype observation on the wild type and transgenic arabidopsis thaliana 3 weeks after bolting. As shown in FIG. 6, compared with the wild type, the number of branches of the MIR6288b overexpression strain Arabidopsis is significantly increased, the average bolting number of the wild type Arabidopsis is 3.7, and the average bolting number of the transgenic strain is 8.9, 9.6 and 9.1 respectively. It can be seen that MIR6288b plays an important role in regulating the number of branches in plants.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

Claims (9)

1. The application of peach MIR6288b or biological materials related to MIR6288b in regulating and controlling the branch number of plants, wherein the precursor sequence of the MIR6288b gene is shown in SEQ ID NO. 1.

2. Application of peach MIR6288b or biological materials related to MIR6288b in breeding transgenic plants with small branch number, wherein the precursor sequence of MIR6288b is shown in SEQ ID NO. 1.

3. Use according to claim 1 or 2, wherein the plant is peach, arabidopsis.

4. The use of claim 1 or 2, wherein the mature sequence MIR6288b-5p of MIR6288b is as shown in SEQ ID No.2, and the mature sequence MIR6288b-3p is as shown in SEQ ID No. 3.

5. Use according to claim 1 or 2, wherein the biological material is any of: the MIR6288 b-containing expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, tissue or organ, or the recombinant vector, recombinant microorganism, transgenic plant cell line, tissue or organ containing the expression cassette.

6. A method for changing the number of branches of a plant is characterized in that the expression of MIR6288b by the plant is controlled by a transgenic, gene knockout, hybridization, backcross, selfing or asexual propagation method, and the precursor sequence of the MIR6288b is shown in SEQ ID NO. 1.

7. The method according to claim 6, wherein controlling the expression of MIR6288b in a plant is performed by overexpressing or reducing the expression of the MIR6288b gene in the plant.

8. The method according to claim 6 or 7, wherein said transgene comprises introducing a recombinant expression vector comprising said MIR6288b sequence into a plant using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance or agrobacterium-mediated methods to obtain a transgenic plant line.

9. The method according to claim 6 or 7, wherein the gene knockout comprises knocking out the MIR6288b by using a DNA homologous recombination technology, a Cre/LoxP technology or a CRISPR/cas9 technology to obtain a transgenic plant line.

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