CN116478994A - Application of miR6478 in enhancing plant disease resistance - Google Patents

Application of miR6478 in enhancing plant disease resistance Download PDF

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CN116478994A
CN116478994A CN202310485020.1A CN202310485020A CN116478994A CN 116478994 A CN116478994 A CN 116478994A CN 202310485020 A CN202310485020 A CN 202310485020A CN 116478994 A CN116478994 A CN 116478994A
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mir6478
cucumber
sequence
disease resistance
plant
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梁超琼
李彩虹
张瑞丽
程敏
曹佩佩
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Shaanxi Academy of Forestry
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Shaanxi Academy of Forestry
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Abstract

The invention discloses application of miR6478 in enhancing plant disease resistance. According to the invention, the cucumber transformed with miR6478 is taken as a study object, the influence of miR6478 on the damage degree of the infected cucumber is studied from the aspect of molecular biology, and the regulation and control of miR6478 on the disease resistance pathway of the cucumber are proved. The method has great significance in revealing functions of miR6478 and cultivating new cucumber varieties.

Description

Application of miR6478 in enhancing plant disease resistance
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of miR6478 in plant disease resistance enhancement.
Background
Cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMCCV) seriously affects quality and yield of cucurbitaceous crops such as cucumber, watermelon and melon. Plant endogenous mircoRNA (miRNA) can be involved in a number of life processes such as leaf morphogenesis, flower and fruit development, response to biotic and abiotic stresses, etc. by regulating downstream target genes. The research shows that under the condition of CGMCCV stress, a plurality of miRNAs can participate in the antiviral reaction of the cucumber by regulating and controlling target genes. For example, miR159 and csa-miRn6-3p can regulate a number of genes associated with plant immune responses at the posttranscriptional level, such as the potential disease resistance gene serine/threonine protein kinase PBS1, the positive regulator of apoptosis GAMYB, and the gene H2B involved in salicylic acid-mediated defensive responses, among others. Therefore, the key miRNA involved in the interaction process of plants and viruses is screened to obtain the miRNA-mediated transgenic cucumber resisting CGMCCH, so that scientific basis can be provided for screening new cucumber antiviral genes, a new thought is provided for comprehensively preventing and controlling cucumber green mottle mosaic virus diseases, and further, a foundation and guidance are provided for cultivating antiviral crops.
Disclosure of Invention
The invention aims to provide miRNA related to plant disease resistance and application thereof.
The cucumber endogenous miRNA provided by the invention is named miR6478, and the nucleotide sequence of the cucumber endogenous miRNA is SEQ ID No.1, wherein the SEQ ID No.1 consists of 21 nucleotides.
Biological materials related to miR6478 also fall within the scope of the invention.
The biological material associated with miR6478 is a short tandem target mimetic (Short tandem target mimic, STTM) sequence that encodes a nucleic acid molecule that inhibits the miRNA molecule.
The biological material associated with miR6478 is an expression cassette comprising a nucleic acid molecule encoding a nucleic acid molecule that inhibits the miRNA molecule.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
In one embodiment of the invention, the nucleic acid molecule encoding the miRNA molecule is shown as SEQ ID No.2.
The biological material associated with miR6478 is a recombinant vector comprising a nucleic acid molecule encoding a nucleic acid molecule that inhibits the miRNA molecule, or the expression cassette.
The biological material associated with miR6478 is a recombinant microorganism comprising a nucleic acid molecule encoding an inhibitor of the miRNA molecule, or the expression cassette, or the recombinant vector.
The biological material associated with miR6478 is a transgenic plant cell line comprising a nucleic acid molecule encoding a nucleic acid molecule that inhibits the miRNA molecule, or the expression cassette, or the recombinant vector.
The miRNA molecule, the short tandem target mimetic sequence, the expression cassette, or the recombinant vector, or the recombinant microorganism, or the transgenic plant cell line can be used to regulate plant disease resistance.
In embodiments of the invention, the vector may be a plasmid, phage, or viral vector.
In a specific embodiment of the invention, the recombinant vector can be an expression vector that expresses a nucleic acid molecule that inhibits expression of miR6478. Specifically, it may be a recombinant vector containing SEQ ID No.2. The recombinant vector containing SEQ ID No.2 refers to a vector pTRV2:STTM-miR6478 obtained by integrating a short tandem target mimic (Short tandem target mimic, STTM) sequence (DNA fragment shown in SEQ ID No. 2) of miR6478 with a length of 126bp into a pTRV2 vector through homologous recombination.
Wherein, STTM is a specific sequence (GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAA T) with a length of 48 bases, two TM (Target mimic) are connected, a protruding structure consisting of 3 bases (cta) is arranged at the miRNA cutting sites on the two TMs, and miRNA can be combined with the protruding structure but cannot be really cut by the protruding structure, so that the function of inhibiting miRNA is further played. Thus, the vector can be used to express a target mimetic sequence of a miRNA (shown in fig. 1) to silence a plant endogenous miRNA.
miR6478 can be transiently silenced in cucumber by a tobacco brittle Virus (Tobacco rattle Virus, TRV) based miRNA silencing (Virus-based MicroRNA Silencing, vbMS) technique. The recombinant vector is pTRV2:STTM-miR6478 vector, and the pTRV2:STTM-miR6478 vector is obtained by introducing fragments shown in SEQ ID No.2 into the pTRV2 vector.
The present invention provides a method for growing transgenic plants with enhanced disease resistance comprising silencing said miRNA molecules in a recipient plant to obtain a transgenic plant with higher disease resistance than said recipient plant.
Specifically, vbMS techniques can be employed to transiently silence miR6478 in cucumber. In an embodiment of the invention, the recombinant microorganism containing pTRV1 and the recombinant microorganism containing pTRV2 STTM-miR6478 vector are used for co-transfection of the receptor plant.
In the method of growing a transgenic plant with enhanced disease resistance, the nucleic acid molecule expressing the miRNA molecule, the expression cassette, or the recombinant vector is introduced into the recipient plant by using the recombinant microorganism.
In embodiments of the invention, the recombinant microorganism may be a yeast, bacterium, algae, or fungus. The bacteria may be agrobacterium; the agrobacterium may specifically be GV3101 agrobacterium.
In a specific embodiment of the invention, the recombinant microorganism is GV3101 Agrobacterium containing pTRV1 and pTRV2:STTM-miR6478 in the method for cultivating transgenic plants with enhanced disease resistance.
In the above method, the transgenic plant has a disease resistance higher than that of the recipient plant, which is embodied in all or part of the following A1) -A2):
a1 The accumulation of CGMV RNA in said transgenic plant is lower than that of said recipient plant;
a2 Less chlorosis and yellowing spots on the leaves of the transgenic plant than on the recipient plant.
In an embodiment of the invention, the transgenic plant cell line does not comprise propagation material of the plant.
In an embodiment of the invention, the modulation of plant disease resistance is an increase in plant disease resistance, embodied in all or part of B1) -B2) as follows:
b1 When the expression level of miR6478 in a plant is decreased, the accumulation level of CGMMV RNA in the plant is decreased;
b2 When the expression level of miR6478 in a plant is reduced, the chlorosis and yellowing spots on leaves of the plant are reduced.
In the method, the plant is dicotyledonous plant; the dicotyledonous plant may be cucumber; the cucumber can be a Xintaimi thorn cucumber.
In the above methods, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the plant of interest with the gene, but also its progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, whole plants, and cells.
The invention adopts various biological means such as bioinformatics prediction, molecular cloning technology, agrobacterium-mediated transformation, miRNA silencing (Virus-based MicroRNA Silencing, VBMS) based on tobacco brittle Virus, real-time fluorescence quantitative PCR and the like, takes cucumber for silencing miR6478 as a research object, takes cucumber transferred into an empty vector pTRV1+pTRV2 as a contrast, firstly silences miR6478, and observes chlorosis and yellowing spots on leaves after the occurrence of the diseases by measuring the accumulation of viral RNA after CGMCCV inoculation, thus researching the influence of miR6478 on the damage degree of infected plants from the aspect of molecular biology. Compared with the plants in the control group, the transgenic plants silencing miR6478 are proved to have higher disease resistance than the plants in the control group, so that miR6478 is a micro RNA related to plant disease resistance and can be used for regulating and controlling the disease resistance of target plants.
Drawings
Fig. 1 is a schematic diagram of a short tandem target simulation sequence of cucumber miR6478
FIG. 2 is a quantitative analysis of miR6478 in miR 6478-silenced cucumber
FIG. 3 is an analysis of CGMCC RNA accumulation in a miR 6478-silenced cucumber
In FIGS. 2 to 3, TRV:00 represents a blank group (cucumber seedlings were immersed after being mixed in a ratio of 1:1 using pTRV1+pTRV2 mixed bacterial liquid)
TS in FIGS. 2-3 represents transient silencing (Transient silencing)
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Agrobacterium competent cells GV3101 in the examples described below are available from Shanghai Biotechnology Inc.
The cucumber inbred line "Xintaimi thorn" in the following examples was purchased from Xintaimi thorn cucumber stock farms in Xintai city, shandong province.
Vectors pTRV1, pTRV2 in the examples described below are commercially available from the biotechnology company of henna.
The miR6478 mature sequence in the invention is an RNA molecule shown as SEQ ID No.1 in a sequence table.
SEQ ID No.1: miR6478 mature sequence, 21bp, RNA;
CCGACCUUAGCUCAGUUGGUG
the nucleic acid sequence for silencing miR6478 is a DNA molecule shown in SEQ ID No.2 in a sequence table.
SEQ ID No.2: STTM sequence, 126bp, DNA of miR 6478;
aaggttaccg aattccacca actgactagc taaggtcggg ttgttgttgt tatggtctaa 60
tttaaatatg gtctaaagaa gaagaatcac caactgacta gctaaggtcg ggagctcacg 120cgtctc 126
according to the invention, the cucumber transformed with miR6478 is used as a research object, the wild type Xintaimi thorn cucumber is used as a control, and the influence of miR6478 on the damaged degree of infected plants is researched from the aspect of molecular biology by detecting the accumulation amount of virus RNA in acceptor plants challenged to inoculate cucumber green mottle mosaic virus, so that the regulation and control of miR6478 on plant disease resistance paths are proved. The method has great significance for revealing the disease resistance function of miR6478 and cultivating disease-resistant cucumber varieties, and is beneficial to enriching vegetable breeding resources.
Application of example 1 and miR6478 in regulation and control of cucumber disease resistance
1. Influence of silencing miR6478 on disease resistance of cucumber
Short tandem target simulation (Short tandem targets mimic, STTM) sequences of miR6478 (shown in figure 1) are designed and synthesized, miRNA silencing technology (Virus-based MicroRNA Silencing, VBMS) based on tobacco brittle fracture Virus (Tobacco rattle Virus, TRV) is adopted to silence miR6478 in cucumber seedlings, and a test flow is shown in figure 2.
1. Construction of silencing miR6478 vector and cucumber obtaining
(1) Construction of silencing vector pTRV2 STTM-miR6478 and Agrobacterium transformation
The STTM fragment of miR6478 (SEQ ID No. 2) was synthesized artificially, and the target fragment was cloned into pTRV2 vector (purchased from Takara Shuzo Co., ltd.) using InFusion HD Cloning Kit (purchased from Hunan Fenghui biotechnology Co., ltd.) to obtain pTRV2: STTM-miR6478 vector, and 2.5 mu LIn-Fusion reaction solution was used to transform the pTRV2: STTM-miR6478 vector into E.coli competent cell stillar (purchased from Beijing full gold biotechnology Co., ltd.); plasmid DNA of pTRV1 (purchased from Hunan Fenghui biotechnology Co., ltd.) and pTRV2: STTM-miR6478, which were identified as positive by sequencing analysis, were transferred into Agrobacterium GV3101 competent cells (purchased from Shanghai Weidi biotechnology Co., ltd.) respectively, single colonies were picked up for colony PCR verification, and plasmid DNA was extracted for sequencing.
(2) Acquisition of silencing miR6478 cucumber
MiR6478 was transiently silenced in cucumber using Virus-based miRNA silencing (VBMS) technology. The method comprises the following specific steps:
(1) the cucumber seeds are planted in a plastic flowerpot after germination accelerating, and are placed in a plant growth incubator for growth.
(2) When the growth point of the first true leaf of the cucumber seedling is exposed (about 5-7 days), slightly causing a micro wound by a blade under a dark condition, soaking cotton by agrobacterium (mixed according to the proportion of 1:1) which contains pTRV1 and pTRV2: STTM-miR6478 vectors and resuscitated by using infiltration liquid, and placing the soaked cotton at the micro wound; treatment with pTRV1+pTRV2 mixed bacterial solution (mixed according to the ratio of 1:1) was used as a blank; agrobacterium (mixed according to a ratio of 1:1) transformed into pTRV1 and pTRV2:PDS is used as a silencing indication control group, wherein pTRV2:PDS (PDS is phytoene desaturase, phytoene desaturase, accession number ABE 99707) is specifically that a fragment of PDS is inserted between CP and Rz of pTRV2 vector to replace the original MCS, and pTRV2:PDS is obtained. Re-wetting cotton masses with corresponding bacterial liquid at intervals of 2h, repeating for 3 times, removing cotton masses with bacterial liquid after infiltration, placing cucumber seedlings in a plant growth incubator, growing under 16h illumination and 8h darkness, and watering as required during the growth period.
2. Influence of silencing miR6478 on disease resistance of cucumber
(1) Cucumber seedlings were grown for about 10 days after treatment and when the first true leaves were expanded, CGMCC was inoculated. And (3) after the cucumber plants grow for 15-20 days, sampling the 2 nd and 3 rd leaves on the plants, extracting total RNA, and respectively detecting the expression level of miR6478 and the accumulation condition of CGMCCV virus RNA by adopting a real-time fluorescence quantitative PCR method.
(2) Respectively adopting miR6478 and CGMCCV specific primers to carry out qRT-PCR detection, and adoptingThe method calculates the relative expression quantity of miR6478 and CGMCCV coat protein genes and analyzes the expression condition. The upstream primer sequence of miR6478 is 5'-CCGACCTTAGCTCAGTTGGTG-3', and the downstream general primer consists ofmiRcute enhanced miRNA fluorescent quantitative detection kit (SYBR) (FP 411, available from the digen biochemical technologies (beijing) inc); the upstream primer sequence of the internal reference gene EF-1a is 5'-ACTGGTGGTTTTGAGGCTGGT-3', and the downstream primer sequence is 5'-CTTGGAGTATTTGGGTGTGGT-3'; the upstream primer sequence of the internal reference gene Ubiquitin is 5'-CTAATGGGGAGTGGGGAAGTA-3', and the downstream primer sequence is 5'-GTCTGGATGGACAATGTTGAT-3'; the qPCR primer sequence of the CGMCC coat protein gene is upstream 5'-ACAGCCGCTAGGGCTGAGATA-3' and downstream 5'-CCAATGAGCAAACCGTTCGAT-3'.
The results show that: in the case of the cucumber transfected with Agrobacterium containing pTRV1 and with Agrobacterium containing pTRV2:STTM-miR6478 to silence miR6478 (TS-miR 6478 in FIG. 2, FIG. 3), the expression level of miR6478 was reduced to 0.004 (FIG. 2), and the accumulation amount of CGMV RNA was reduced to 0.076 (FIG. 3) relative to the control (TRV: 00 in FIG. 2, FIG. 3) of the transpiration vector (pTRV1+pTRV2). Less chlorosis and yellowing spots on cucumber leaves silencing miR6478 than in the empty vector control.
In conclusion, compared with the control group plants, the transgenic plants silencing miR6478 have higher disease resistance than the control group plants, and miR6478 is a microRNA related to plant disease resistance and can be used for regulating and controlling the disease resistance of target plants.

Claims (9)

1. The miRNA molecule is characterized in that the mature sequence of the miRNA molecule is SEQ ID No.1 in a sequence table.
2. A short tandem target mimetic sequence, characterized in that it encodes a nucleic acid molecule that inhibits the miRNA molecule of claim 1, whose nucleotide sequence is SEQ ID No.2 in the sequence listing.
3. An expression cassette comprising the short tandem target mimetic sequence of claim 2.
4. A recombinant vector comprising the short tandem target mimetic sequence of claim 2 or the expression cassette of claim 3.
5. A recombinant microorganism comprising the short tandem target mimetic sequence of claim 2, or the expression cassette of claim 3, or the recombinant vector of claim 4.
6. A transgenic plant cell line comprising the short tandem target mimetic sequence of claim 2, or the expression cassette of claim 3, or the recombinant vector of claim 4, or the recombinant microorganism of claim 5.
7. Use of a miRNA molecule according to claim 1, or a short tandem target mimetic according to claim 2, or an expression cassette according to claim 3, or a recombinant vector according to claim 4, or a recombinant microorganism according to claim 5, or a transgenic plant cell line according to claim 6 for modulating plant disease resistance.
8. A method of growing a transgenic plant with increased disease resistance, characterized in that a transgenic plant with a disease resistance higher than that of a recipient plant is obtained by silencing the miRNA molecule of claim 1 in said recipient plant;
preferably, the plant is a dicot.
9. The method according to claim 8, characterized in that the miRNA molecule according to claim 1 is transiently silenced in cucumber by using the short tandem target mimetic sequence according to claim 2 using tobacco-based brittle virus miRNA silencing technology.
CN202310485020.1A 2022-08-15 2023-05-03 Application of miR6478 in enhancing plant disease resistance Pending CN116478994A (en)

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