RsLBD11 gene and application of RsLBD11 gene in regulation and control of fleshy roots of cherry radishes

Abstract

The invention discloses an RsLBD11 gene and application of the RsLBD11 gene in regulation and control of fleshy roots of cherry radishes. The RsLBD11 gene is identified in a radish genome, RsLBD11 gene expression is regulated and controlled through a CRISPR / Cas9 gene editing technology, it is found that the RsLBD11 gene can regulate and control the size of the fleshy roots of the cherry radishes and the number and size of root cambium cells, a foundation is laid for improvement of the yield and quality of the cherry radishes, a new germplasm resource is provided for breeding of the cherry radishes, the breeding efficiency is greatly improved, and the breeding cost is reduced. The breeding process is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering breeding technology and specifically relates to RsLBD11 Genes and their application in regulating fleshy roots of cherry radish. Background Art

[0002] Cherry radish( Raphanus sativus L. var. radculus pers ), a genus of the family Cruciferae, an annual or biennial plant, belonging to the herbaceous class, is a new type of mini vegetable, named after its edible fleshy root bark, which is red and round in shape like a cherry. The fleshy root is the main storage organ and edible organ of cherry radish. The size and shape of the fleshy root directly affect the yield and quality of radish, and is an important target shape for radish breeding. The formation of the swelling organ of root / stem crops is a typical metamorphosis organ development process, and the formation and development of the metamorphosis organ directly affects the yield of root and tuber crops.

[0003] Traditional breeding methods are difficult to effectively promote the growth and development of cherry radish fleshy roots. Traditional breeding methods improve the growth and development of cherry radish fleshy roots through hybridization and breeding. This method is inefficient, takes a long time to breed, has a large workload, and is easily affected by environmental factors. In addition, traditional breeding methods are difficult to target and regulate key genes related to the growth and development of cherry radish fleshy roots. However, genetic engineering technology can target and regulate key genes related to the growth and development of cherry radish fleshy roots, thereby effectively improving the growth and development of cherry radish fleshy roots. For example, by overexpressing genes that promote fleshy root expansion or knocking out genes that inhibit fleshy root expansion, the growth and development traits of cherry radish fleshy roots can be significantly improved.

[0004] As a member of the LBD transcription factor family, LBD11 is mainly involved in the development and differentiation of plant organs such as roots, stems, and leaves. Studies have shown that LBD11 has a significant regulatory effect on the secondary growth of plants. However, research on the effect of RsLBD11 on the fleshy roots of cherry radish has not been reported. CRISPR / Cas9 gene editing technology can precisely modify genes by targeting specific DNA sequences. This technology has broad application prospects in crop breeding and provides new opportunities for crop breeding. Summary of the invention

[0005] The object of the present invention is to provide RsLBD11 Genes and their application in regulating the fleshy roots of cherry radish lay the foundation for improving the yield and quality of cherry radish and provide new germplasm resources for cherry radish breeding.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: RsLBD11 Gene, the RsLBD11The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] The above RsLBD11 The application of genes in regulating plant growth and development.

[0008] Furthermore, the plant is cherry radish.

[0009] Furthermore, the growth and development site is a fleshy root.

[0010] Further, reduce RsLBD11 Gene expression reduced fleshy root length, fleshy root diameter, fleshy root cambium cell number and fleshy root size.

[0011] A method for regulating the growth and development of cherry radish, comprising the steps of regulating the growth and development of cherry radish by CRISPR / Cas9 RsLBD11 Gene expression level.

[0012] The beneficial effects of the present invention are: (1) Improving the growth and development of cherry radish: knocking out RsLBD11 Gene, can significantly reduce the length of cherry radish fleshy roots, fleshy root diameter, fleshy root cambium cell number and size.

[0013] (2) Improving the yield and quality of cherry radish: Regulation through CRISPR / Cas9 gene editing technology RsLBD11 Gene expression is not affected by the environment, thereby regulating the growth and development of the fleshy roots of cherry radish, and also laying the foundation for improving the yield and quality of cherry radish.

[0014] (3) The present invention analyzes RsLBD11 The function of the gene provides new germplasm resources for cherry radish breeding, while improving breeding efficiency and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For pYAO- RsLBD11 PCR electrophoresis verification results of bacterial solution after transformation of recombinant vector into Agrobacterium; M: DL2000; 1-3: pYAO- RsLBD11 Target fragment of PCR in Agrobacterium culture.

[0016] Figure 2 for RsLBD11 The sgRNA site of the gene and rslbd11 Sanger sequencing maps of mutant plants and wild-type plants (WT).

[0017] Figure 3 The wild type (WT) and rslbd11 Observation and analysis of cambium cells in mutant plants; A: wild type andrslbd11 Morphological images of cambium cells of mutant plants; B: number of cambium cells; C: length of cambium cells; D: width of cambium cells.

[0018] Figure 4 for rslbd11 Phenotypic analysis of mutant cherry radish; A: rslbd11 Analysis of plant type of mutant cherry radish; B: rslbd11 Mutant cherry radish fleshy root phenotype; C: RsLBD11 In wild type and rslbd11 Relative expression level in mutant cherry radish; D: rslbd11 Mutant cherry radish fleshy root length; E: rslbd11 Diameter of fleshy roots of mutant cherry radish. DETAILED DESCRIPTION

[0019] In order to make the technical solutions and advantages of the present invention clearer, the content of the invention of the present invention is described in detail by the following examples, but it should not be regarded as limiting the scope of the invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0020] Example 1 Construction of Radish RsLBD11 Gene Editing Vectors Use CRISPR-GE (Genome Editing)-Liu YG Lab website (http: / / skl.scau.edu.cn / ) to perform RsLBD11 The target primers were dissolved in water to a 10 μM stock solution, and 5 μL of each was added to 40 μL of 0.5×TE (pH 8.0), heated at 98°C for 3 min, cooled naturally to room temperature, and stored in a -20°C refrigerator for ligation.

[0021] pYAO- RsLBD11 Construction of recombinant vector: The pCAMBIA1300-pYAO-cas9 vector plasmid was digested with restriction endonuclease BsaI-HF (purchased from NEB) to obtain a linearized vector and then purified by gel recovery. The linearized vector was ligated with the annealed target primer using 10×T4 DNA ligase (purchased from TaKaRa), and the ligation product was transformed into DH5α Escherichia coli competent cells. Single clones were picked for bacterial liquid PCR identification and plasmids were extracted for sequencing. After successful sequencing, pYAO- RsLBD11The recombinant vector was transformed into GV3101 Agrobacterium competent cells for plant genetic transformation.

[0022] Table 1 RsLBD11 Gene sgRNA Primers Example 2 Agrobacterium-mediated genetic transformation of cherry radish Transgenic cherry radish was obtained by using the Agrobacterium in situ genetic transformation method. The specific steps are as follows: cherry radish seeds were evenly sown in a substrate containing perlite and vermiculite for cultivation, the surface was covered with plastic wrap for 2 days and then uncovered, radish seedlings that had grown for about 10 days (cotyledons were fully expanded) were selected, and the top of the seedling was cut against the growth direction of the cotyledons with a clean surgical blade, one of the cotyledons and all the stem tips were removed, leaving only one cotyledon.

[0023] Prepare the resuspension (MS liquid medium) in advance before infection, shake the Agrobacterium carrying the target gene vector to ensure that the OD600 of the bacterial solution is between 0.6-0.8, centrifuge at 4000 rpm for 5 min, and resuspend it in MS liquid medium to make the OD600 of the resuspension between 0.6-0.8. After removing the cotyledons, use a sterile syringe to inoculate the incision wound area with the pYAO- RsLBD11 Recombinant vector ( RsLBD11 The negative control was injected with MS liquid medium at the wound, and then with pYAO- RsLBD11 Wrap the incision with cotton wool soaked in sterile high-pressure water for 1 hour and mark it. Wrap the incision with cotton wool soaked in sterile high-pressure water 1 hour later and fix it with tin foil. Culture it in the dark for 2 days. Then continue to culture and grow the seedlings under a photoperiod of 16 hours / 25℃ light and 8 hours / 18℃ dark. When true leaves grow out of the incision, move them into the greenhouse for further culture. Finally, transgenic cherry radish ( rslbd11 mutants).

[0024] Example 3 RsLBD11 Detection of gene editing materials right rslbd11Mutant and wild-type radish (WT) were subjected to leaf tissue DNA extraction and DNA sequencing. DNA was extracted using the Star Spin Plant DNA Kit Star Spin column plant DNA extraction kit from Beijing Kangrun Chengye Biotechnology Co., Ltd. The extracted DNA was used as a template for PCR amplification using the 2×Hieff®Utra-Rapid Hot Start PCR Master from Yisheng Biotechnology (Shanghai) Co., Ltd. The amplification instrument was the gene amplifier (ETC821D) from Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd. The reaction system: 2×Hieff®Utra-Rapid Hot Start PCR Master Mix 10μL, Primer F and Primer R were 1μL, RNase free water 6μL, DNA template 2μL. The amplification conditions were 94℃ 3min; 94℃ 10 s, 60℃ 20 s, 72℃ 10s, 28-35 cycles; 72℃ 5min, and stored at 4℃. Specific primers containing the target DNA fragment were designed for amplification (Table 2). The PCR amplification product was recovered using the Gel Extraction Kit from Fujian Herrui Biotechnology. The gel-recovered product was sent to Fuzhou Boshang Biotechnology Co., Ltd. for sequencing. The sequencing results were compared using DNAMAN 9.0 software. RsLBD11 Plants with sgRNA mutations in the sequence are positive plants rslbd11 Mutants (such as Figure 2 The positive plants successfully identified were subsequently subjected to gene expression and phenotypic analysis.

[0025] Table 2 rslbd11 Mutant identification primers Example 4 rslbd11 Mutant material RsLBD11 Expression detection Cherry radish RNA was extracted using AG RNAex Pro RNA extraction reagent purchased from Hunan Aikerui Biotechnology Co., Ltd., and the extracted RNA was reverse transcribed into cDNA using Evo M-MLV Reverse Transcription Premix Kit (containing gDNA removal reagent for qPCR) Ver.2 and stored in a -20 °C refrigerator for later use. The purity of the sample was tested using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and the concentration of the RNA sample was tested using a Qubit® 2.0 fluorometer (Life Technologies, CA, USA).

[0026] Designed with Primer Premier 5 software RsLBD11 The qRT-PCR primers used for the gene (Table 3) were used for real-time fluorescence quantitative detection of the above sampled materials. Actin The SYBR® Green Pro Taq HS premixed qRT-PCR kit purchased from Hunan Aikerui Biotechnology Co., Ltd. was used for qRT-PCR reaction, and the CFX96 Real-Time PCR Detection System instrument was used for qRT-PCR amplification. Each sample was biologically replicated three times, and data were statistically analyzed using Excel 2019 software. The relative expression was calculated using the 2-ΔΔCt method, and significance analysis was performed using SPSS24 software. Origin 2017 software was used to make charts.

[0027] The results are as follows Figure 4 As shown in C, compared with the wild type (WT), rslbd11 In mutant of RsLBD11 The expression level decreased.

[0028] Table 3 qPCR primers Example 5 Phenotypic observation of mutant plants To make it clear RsLBD11 The role of pYAO- RsLBD11 The recombinant vector Agrobacterium solution was injected into the fleshy roots of cherry radish through in situ genetic transformation, and the RsLBD11 Plants with weakened expression. 30 days after germination, the fleshy roots of cherry radish were sliced. The results were as follows Figure 3 As shown, laser confocal microscopy showed that the number of cambium cells in the mutant plants decreased by about 30.4% compared with the wild type, the length of cambium cells decreased by about 14.7%, and the width of cambium cells decreased by about 7.2%. Analysis of the phenotype 50 days after germination revealed that (Figure 4 ), rslbd11 The root system of cherry radish plants became shorter, and the length of the fleshy roots decreased significantly, by about 23.9%; while the diameter of the fleshy roots showed a very significant difference, decreasing by about 38.1%. This shows RsLBD11 It plays an important role in regulating the radial expansion of cherry radish fleshy roots.

[0029] In summary, the present invention uses gene editing technology to discover RsLBD11 The gene can regulate the growth of cherry radish fleshy roots, providing new germplasm resources for cherry radish breeding, while improving breeding efficiency and accelerating the breeding process.

[0030] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. RsLBD11 A gene characterized by: Said RsLBD11 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The method according to claim 1 RsLBD11 The application of genes in regulating plant growth and development.

3. The use according to claim 2, characterized in that: The plant is cherry radish.

4. The use according to claim 2, characterized in that: The growth and development site is the fleshy root.

5. The use according to claim 2, characterized in that: reduce RsLBD11 Gene expression reduced fleshy root length, fleshy root diameter, fleshy root cambium cell number and fleshy root size.

6. A method for regulating the growth and development of cherry radish, characterized in that: Gene editing of radish by CRISPR / Cas9 RsLBD11 Gene expression level.

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