Application of PLC2 gene in regulating growth and development of castor

Abstract

The application discloses application of a PLC2 gene in regulating growth and development of castor-oil plant and belongs to the technical field of plant genetic engineering. The application discloses application of the PLC2 gene in regulating plant height, seed quality, pollen vitality, flowering period or silique of the castor-oil plant. It is proved that, compared with wild type plants, the plant height of the PLC2 gene overexpression mutant plants is shorter, the leaf abscission is faster, the main stem ear is longer and the flowering is earlier, the number of pistils and stamens is increased, the pistil stigma is longer, the fruits and seeds are fuller, the glossiness is higher, and the mature seed germination rate is higher. It is shown that the PLC2 gene can be applied to genetic engineering and breeding of the castor-oil plant and has important utilization value in cultivating castor-oil plant varieties with excellent traits.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the PLC2 gene in regulating the growth and development of castor beans. Background Technology

[0002] Castor bean (Ricimus communis L.), also known as castor bean, is an annual or perennial herbaceous plant belonging to the Euphorbiaceae family and the Ricinus genus. It can also grow into a perennial shrub or small tree, commonly found in tropical or southern regions. Castor bean has extremely high comprehensive utilization value. The entire plant of colored castor beans can be used for ornamental purposes; the roots can be used medicinally, having the effects of dispelling wind, promoting blood circulation, relieving pain, and calming the nerves; the stems can be used to make newsprint and cigarette paper, and the stem fibers can be used for hemp weaving; the leaves can reduce swelling, detoxify, relieve itching, feed castor silkworms, and make pesticides; the long flowering period of castor bean makes it suitable for beekeeping; the seeds can be pressed to obtain oil cake, which can be used as feed, fertilizer, for brewing soy sauce, producing protein powder and amino acid powder, preparing biological pesticides, and extracting inositol, calcium phytate, pigments, flavonoids, etc. Furthermore, ricin exhibits insecticidal activity against various vegetable pests through antifeedant, stomach poison, contact, repellent, and growth-inhibiting effects. Therefore, castor oil, as a biological pesticide, possesses unique advantages and has a very promising development prospect. In short, with the development of science and technology, the economic and practical value of castor oil has entered a golden age.

[0003] Phospholipase C (PLC) is an important regulatory enzyme involved in the processing of various lipids and calcium. 2+ Dependent signaling pathway. In animals, PLC selectively catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) on the glycerol side of the phosphodiester bond. Plant PLC genes are mainly involved in cell growth and differentiation, hormone signal transduction, responses to biotic and abiotic stresses, and regulation of polar growth. 2+ Ions play an important role as second messengers in various signal transduction pathways. However, there is currently very little research on the impact of the PLC gene on castor bean phenotypic development. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the PLC2 gene in regulating the growth and development of castor beans, so as to solve the problems existing in the prior art. This invention confirms that overexpression of the PLC2 gene promotes the growth and development of castor beans, and can be applied to the genetic engineering breeding of castor beans. It has important utilization value in the cultivation of castor bean varieties with excellent traits.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the PLC2 gene in any of the following:

[0007] (1) Application in regulating the growth rate of castor bean plants;

[0008] (2) Application in regulating castor bean seed quality;

[0009] (3) Application in regulating castor pollen viability;

[0010] (4) Application in regulating the flowering period of castor beans;

[0011] (5) Application in regulating the quality of castor bean capsules;

[0012] The reference sequence of the PLC2 gene is accessed by NCBI under accession number XM_025158535.2.

[0013] This invention also provides the use of the protein encoded by the PLC2 gene in any of the following:

[0014] (1) Application in regulating the growth rate of castor bean plants;

[0015] (2) Application in regulating castor bean seed quality;

[0016] (3) Application in regulating castor pollen viability;

[0017] (4) Application in regulating the flowering period of castor beans;

[0018] (5) Application in regulating the quality of castor bean capsules;

[0019] The reference sequence of the PLC2 gene is accessed by NCBI under accession number XM_025158535.2.

[0020] The present invention also provides the use of a recombinant vector containing the PLC2 gene in any of the following:

[0021] (1) Application in regulating the growth rate of castor bean plants;

[0022] (2) Application in regulating castor bean seed quality;

[0023] (3) Application in regulating castor pollen viability;

[0024] (4) Application in regulating the flowering period of castor beans;

[0025] (5) Application in regulating the quality of castor bean capsules;

[0026] The reference sequence of the PLC2 gene is accessed by NCBI under accession number XM_025158535.2.

[0027] The present invention also provides the use of recombinant bacteria comprising the recombinant vector in any of the following:

[0028] (1) Application in regulating the growth rate of castor bean plants;

[0029] (2) Application in regulating castor bean seed quality;

[0030] (3) Application in regulating castor pollen viability;

[0031] (4) Application in regulating the flowering period of castor beans;

[0032] (5) Application in regulating the quality of castor bean capsules.

[0033] Furthermore, by overexpressing the PLC2 gene, castor bean plants were shortened, the number of stem nodes was reduced, the number of male and female flowers increased, the flowering period was advanced, and the pollen viability, seed and capsule quality of castor beans were improved.

[0034] Furthermore, overexpression of the PLC2 gene significantly downregulated the expression of the PLC6 gene, while significantly upregulated the expression of the PLC2M, PLC2N, and PLC4 genes, resulting in shorter castor bean plant height, fewer stem nodes, more male and female flowers, earlier flowering, and improved pollen viability, seed, and capsule quality.

[0035] The present invention also provides a method for promoting the growth and development of castor beans, including the step of upregulating the expression level of the PLC2 gene;

[0036] The reference sequence of the PLC2 gene is accessed by NCBI under accession number XM_025158535.2.

[0037] Furthermore, the PLC2 gene was inserted into an overexpression vector and transformed into castor bean recipient material to improve pollen viability and germination rate, seed and capsule quality, increase the number of male and female flowers, and promote early flowering of castor beans.

[0038] The present invention discloses the following technical effects:

[0039] This invention involves homologous overexpression and gene editing of the PLC2 gene in castor beans. Phenotypic differences between overexpression mutant plants, gene-edited mutant plants, and wild-type plants were observed to determine the effect of the PLC2 gene on the development of female castor bean lines. The results are as follows: Compared with the wild-type WT plant of the same inflorescence type, the overexpression mutant plant G2 was shorter, had faster leaf drop, a longer main stem spike, and earlier flowering. The number of pistils and stamens increased, and the stigma of the pistils was longer. The fruits and seeds were fuller, had higher gloss, and a higher germination rate of mature seeds. Compared with the wild-type WT plant of the same inflorescence type, the PLC2 gene-edited mutant plants Q2-1 and Q2-2 had shorter main stem spikes, delayed flowering, a reduced number of pistils and stamens, shorter stigmas of female flowers, reduced pollen bioactivity of male flowers after flowering, lower fruit maturity rate in the later stages of inflorescence development, reduced seed gloss, less full seeds, and a lower germination rate of mature seeds. RT-qPCR results showed that when the PLC2 gene was overexpressed, the expression level of PLC6 was significantly downregulated, while the expression levels of PLC2M, PLC2N, and PLC4 were significantly upregulated, and the expression level of PLC4X2 gene did not change significantly.

[0040] The above results indicate that the PLC2 gene provided by this invention can be applied to the genetic engineering breeding of castor beans and has important utilization value in cultivating castor bean varieties with superior traits. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 PCR identification results of PLC2 gene ligation product transformed into E. coli; M: DL5000 Marker; 1-7: PCR identification results of PLC2 gene in bacterial culture;

[0043] Figure 2 PCR identification results of Agrobacterium after transformation with the overexpression vector pBI121-PLC2; M: DL 5000 Marker; 1-4: PCR identification results of PLC2 gene in bacterial culture;

[0044] Figure 3 RT-qPCR results of PLC2 gene overexpression in resistant plant G2; ***: 0 <P≤0.001;

[0045] Figure 4Comparison of PLC2 gene overexpression resistant plants and wild-type plants at 120 days; WT: wild-type control plants; G2: overexpression resistant plants; A: male flowers; B: female flowers; C: fruits;

[0046] Figure 5 The results of observation of flowers of PLC2 gene overexpression resistant plant G2 and wild-type plant WT; A1, A2, A3: male flower buds, mature male flowers, and female flowers of wild-type plant; B1, B2, B3: male flower buds, mature male flowers, and female flowers of G2 plant; Scale bar: 500.00 μm;

[0047] Figure 6 Pollen viability assay results for PLC2 gene overexpression resistant plant G2 and wild-type plant WT; Scale bar: 50 μm;

[0048] Figure 7 The pollen germination observation results of the PLC2 gene overexpression resistant plant G2 and the wild-type plant WT; the scale bar of the top two images: 50μm, the scale bar of the bottom two images: 25μm;

[0049] Figure 8 Comparison of mature capsules of PLC2 gene overexpression resistant plant G2 and wild-type plant WT;

[0050] Figure 9 Comparison of mature seeds of PLC2 gene overexpression resistant plant G2 and wild-type plant WT;

[0051] Figure 10 Comparison of seed germination between PLC2 gene overexpression resistant plant G2 and wild-type plant WT;

[0052] Figure 11 This is the alignment result between the gene editing target sequence and the original sequence; Note: CCC: proline in the original sequence; TCC: serine in the gene editing sequence; GTC: valine in the original sequence; GTT represents valine in the gene editing sequence;

[0053] Figure 12 Comparison of PLC2 gene-edited resistant plants and wild-type plants at 120 days of development; WT: wild-type control plants; Q2-1, Q2-2: edited resistant plants; A: male flower; B: female flower; C: fruit;

[0054] Figure 13 Observation results of flowers of PLC2 gene-edited resistant plants and wild-type plants; A1, A2, A3: male flower buds, mature male flowers, and female flowers of wild-type plants; B1, B2, B3: male flower buds, mature male flowers, and female flowers of Q2-1 plants; C1, C2, C3: male flower buds, mature male flowers, and female flowers of Q2-2 plants; Scale bar: 500.00 μm;

[0055] Figure 14 Results of pollen viability assay for PLC2 gene-edited resistant plants; WT: Staining results of pollen viability in wild-type plants; Q2-1, Q2-2: Staining results of pollen viability in gene-edited resistant plants; Scale bar: 50 μm;

[0056] Figure 15 The results of pollen germination observation of PLC2 gene-edited resistant plants; WT: germination results of mature pollen from wild-type plants; Q2-1, Q2-2: germination results of mature pollen from gene-edited resistant plants; scale bar for the top three images: 50μm; scale bar for the bottom three images: 25μm;

[0057] Figure 16 A comparison of mature capsules of PLC2 gene-edited resistant plants Q2-1 and Q2-2 with wild-type plant WT;

[0058] Figure 17 Comparison of mature castor bean seeds from PLC2 gene-edited resistant plants Q2-1 and Q2-2 with wild-type WT plants;

[0059] Figure 18 Comparison of seed germination between PLC2 gene-edited resistant plants Q2-1 and Q2-2 and wild-type plant WT;

[0060] Figure 19 The results of RT-qPCR detection of other genes in PLCs after overexpression of the castor bean PLC2 gene; Note: **: 0.001 <P≤0.01,***:0<P≤0.001。 Detailed Implementation

[0061] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0062] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0063] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0064] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0065] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0066] Example 1

[0067] 1. Materials and Methods

[0068] 1.1 Materials

[0069] The castor bean material used in this invention is the aLmAB5 strain of the Lm type female line, provided by the Key Laboratory of Castor Bean Breeding of the State Ethnic Affairs Commission. The strains, vectors, reagents required for vector construction experiments, and culture media used in the research process all referenced the literature "Functional Study of Castor Bean PIP5K11 Gene in Lm Type Female Line".

[0070] 1.2 Methods

[0071] 1.2.1 Study on overexpression of PLC2 gene in castor bean

[0072] 1.2.1.1 PLC2 gene cloning

[0073] Download the PLC2 gene sequence (the reference sequence for the PLC2 gene is accessed in NCBI under accession number XM_025158535.2), and design primers using CE Design V1.03 software. The primer sequences are: GPLC2-F: 5′-ACGGGGGACTCTAGAGGATCCATGTCCGACTCTAAGGGACTCAAC-3′; GPLC2-R: 5′-AGTCCGTACCCCCGGGGATCCTCAAACAATCTCAAATTCCATTAGAAG-3′. The PCR reaction system is: 5× primer star buffer (Mg... 2+The reaction mixture consisted of 10.0 μL of dNTP Mixture (2.5 μM), 4.0 μL of GPLC2-F, 1.0 μL of GPLC2-R, 4.0 μL of cDNA, 0.5 μL of Primerstar HSDNA polymerase (2.5 U / μL), and 29.5 μL of ddH2O, for a total of 50.0 μL. The reaction program was as follows: 94℃ for 5 min, 94℃ for 30 s, 56℃ for 45 s, 72℃ for 1 min, and 72℃ for 10 min.

[0074] 1.2.1.2 Single enzyme digestion of overexpression vector plasmid

[0075] The pBI121 vector plasmid was digested with BamHI. The reaction mixture consisted of 5.0 μL 10×K Buffer, 5.0 μL BamHI, 14.0 μL pBI121 plasmid, and 76.0 μL ddH2O, for a total of 100.0 μL. The reaction conditions were 37℃ for 3 h. After digestion, the digestion products were further verified by agarose gel electrophoresis. Correct bands were recovered and purified.

[0076] 1.2.1.3 Ligation of the PLC2 gene with the pBI121 vector plasmid

[0077] After ligating the PLC2 gene to the pBI121 vector using a seamless cloning method, the ligation was carried out at 50°C for 1 hour. The ligation system was as follows: 0.72 μL of PLC2 gene, 0.28 μL of pBI121 vector plasmid purified by single enzyme digestion, 5.00 μL of Super Fusion CloningMix (2x), 4.00 μL of ddH2O, and a total of 10.00 μL.

[0078] 1.2.1.4 Transformation of Escherichia coli with the product of ligation of PLC2 gene and pBI121

[0079] The recovered PLC2 gene was ligated into the pBI121 vector plasmid and transformed into competent E. coli DH5α cells. The obtained E. coli culture was activated and identified by PCR. The PLC2 gene overexpression vector pBI121-PLC2 was thus obtained.

[0080] 1.2.1.5 Transformation of Agrobacterium tumefaciens with overexpression of pBI121-PLC2 vector plasmid

[0081] Plasmids were extracted from correctly sequenced *E. coli* bacterial cultures, and the resulting overexpression vector plasmid pBI121-PLC2 was transformed into *Agrobacterium* GV3101 competent cells. The obtained *Agrobacterium* bacterial cultures were then activated and identified by PCR.

[0082] 1.2.1.6 Genetic transformation of castor beans using PLC2 gene overexpression vector

[0083] Using the Lm-type female castor bean variety aLmAB5 as material, the constructed overexpression vector pBI121-PLC2 containing the PLC2 gene was transformed into the cotyledonary nodes of castor beans via Agrobacterium-mediated transformation. The specific process is detailed in the reference "Functional Study of the PIP5K11 Gene in Lm-type Female Castor Bean Lines". Plants resistant to PLC2 gene overexpression were screened, domesticated, and transferred to other plants to obtain PLC2 gene overexpression resistant plants.

[0084] 1.2.1.7 Molecular identification of PLC2 gene overexpression resistant plants

[0085] Leaves from six plants overexpressing the PLC2 gene to be tested for resistance were prepared. Genomic DNA was extracted from the six plants and the control wild-type castor bean (WT), following the instructions of the kit from Beijing Zhuangmeng Biotechnology Co., Ltd. The extracted PLC2 gene DNA from the plants to be tested for resistance was used as a template for RT-qPCR. The specific primers for quantitative real-time PCR used 18S as an internal reference gene; the primer sequences are shown in Table 1 and were synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized cDNA sample was used as a template for RT-qPCR. RT-qPCR reactions were performed using qPCR and RT-qPCR systems. The reaction mixture consisted of 10.0 μL of SYBR Premix Ex Taq, 0.2 μL of Rox Reference Dye, and 18s-F (or PLC2-F).

[0086] 0.4 μL of 18s-R (or PLC2-R), 2.0 μL of template, and 7.0 μL of ddH2O were added. The reaction procedure was based on the reference "Functional Study of Castor PIP5K11 Gene in Lm Type Female Lines," with a total of 40 cycles. Each experimental material was subjected to 6 biological replicates, and C0.05 was collected. T Average value, using 2 -ΔΔCт The relative expression level of the PLC2 gene in castor bean inflorescence was calculated using a method.

[0087] Table 1 Primers for RT-qPCR identification of PLC2 gene overexpression resistant plants

[0088]

[0089] 1.2.1.8 Biological identification of PLC2 gene overexpression resistant plants

[0090] Phenotypic observations, including leaf characteristics, plant height, and flowers, were performed on the PLC2 gene overexpressing resistant plant G2 and 30 wild-type control (WT) plants at 90 and 120 days of development. The methods were based on the literature "Functional study of castor bean PIP5K11 gene in Lm-type female lines".

[0091] Pollen viability was detected using the TTC method as follows: pollen from mature, open G2 plants and 30 wild-type control (WT) plants was placed on a glass slide, and the number of viable pollen was observed and counted under a microscope.

[0092] Prepare a 15% sucrose solution, add a small amount of agar powder and 1% boric acid solution to make a culture medium. This culture medium can be used to simulate the mucus secreted on the stigma of the pistil to promote pollen germination and pollen tube formation. Use tweezers to remove pollen from G2 and 30 WT plants, scatter it evenly on a glass slide, add 15% sucrose solution and 1% boric acid solution (1-2 drops each), and incubate at 37℃ for 1 hour. Then observe the pollen germination.

[0093] One hundred seeds from 30 wild-type control plants (WT) and seeds from the overexpressing resistant plant G2 were placed in germination vermiculite and cultured at 28℃ for 72 h. The germination status of the seeds was observed, and the germination rate of WT and G2 seeds was counted.

[0094] 1.2.2 CRISPR / Cas9 gene editing study of PLC2 gene in castor bean

[0095] 1.2.2.1 Design of sgRNA target sequence for PLC2 gene

[0096] Based on the gRNA design principles, and using the DNA and RNA sequences of the PLC2 gene, sgRNA sequences were selected through a gRNA design website to design dual-target sgRNAs, as shown in Table 2. The target PAM site was selected as a 5′-NGG-3′ sgRNA sequence with a length of approximately 20 bp, a GC content of 40%-60%, and preferably free of higher-order structures.

[0097] Table 2 Editing targets of the PLC2 gene

[0098]

[0099] Note: "+" indicates a positive chain; "-" indicates a negative chain.

[0100] 1.2.2.2 Construction of gene editing vector PKSE401-PLC2-gRNA and resistant plants

[0101] The PKSE401 vector and the complete gene were synthesized using double sgRNA target primers after digestion with a single enzyme. Ligation was performed overnight at 4°C using T4 DNA ligase. The ligation product was transformed into competent *E. coli* DH5α cells via heat shock, plated on LB agar plates containing 50 mg / L kanamycin, and incubated overnight at 37°C on a shaker at 180 rpm for transformation resistance screening. Three to four single colonies were picked and cultured in 100 μL liquid LB medium (containing 50 mg / L kanamycin sulfate) for 2 h, followed by PCR detection. The PCR products were analyzed and sequenced. The successfully sequenced bacterial culture was mixed with glycerol and stored at -80°C to obtain the complete PLC2 gene editing vector PKSE401-PLC2-gRNA. This was then transformed into *Agrobacterium* GV3101 competent cells, following the method described in the literature "Functional Study of the PIP5K11 Gene in Lm-type Female Lines of Castor Bean". The appropriate *Agrobacterium* strains were selected for preservation. Using the Lm-type female castor bean strain aLmAB5 as material, the constructed PKSE401-PLC2-gRNA gene editing expression vector was transformed into the cotyledon nodes of castor beans via Agrobacterium-mediated transformation to obtain gene-edited resistant plants Q2-1 and Q2-2.

[0102] 1.2.2.3 Biological identification of PLC2 gene-edited resistant plants

[0103] Phenotypic observations, including leaf size, plant height, and stem nodes, were performed on gene-edited resistant plants Q2-1 and Q2-2 and 30 wild-type control (WT) plants at 90 and 120 days of development. The methods followed the literature "Functional Study of Castor Bean PIP5K11 Gene in Lm-type Female Lines". Pollen viability of Q2-1, Q2-2, and 30 WT plants was measured using the same methods as for the overexpression resistant plants. Pollen germination rate of Q2-1, Q2-2, and 30 WT plants was measured using the same methods as for the overexpression resistant plants. Seed germination rate of Q2-1, Q2-2, and 30 WT plants was also measured using the same methods as for the overexpression resistant plants.

[0104] 1.2.3 Effect of PLC2 gene overexpression on expression levels of PLCs in the same family

[0105] The expression levels of PLC2 gene family members (PLC2, PLC4, PLC6, PLC4X2, PLC2M, and PLC2N) in the overexpressing mutant plant G2 were analyzed using RT-qPCR. RT-qPCR specific primers were selected from the reference "Functional Identification of PLC Family Genes Related to Inflorescence Development in Castor Bean Lm-type Female Lines," with 18S as the internal reference gene. Primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. Six biological replicates were performed for each experimental material, and C0.05 was collected. T Average value, using 2-ΔΔCт The relative expression levels of PLC gene family members were calculated using a method. SPSS 19.0 software was used for data statistics and analysis. An independent samples t-test was used to compare the significance of the means, and the results were presented as a bar chart of relative expression levels.

[0106] 2 Results and Analysis

[0107] 2.1 Results of PLC2 gene overexpression study in castor bean

[0108] 2.1.2 Results of transformation of Escherichia coli competent cells and Agrobacterium tumefaciens with overexpression vector pBI121-PLC2

[0109] The PLC2 gene fragment was ligated into the linearized overexpression vector plasmid pBI121, transformed into *E. coli* DH5α competent cells, and single colonies were picked for PCR identification. Figure 1 It was found that the PCR products from lanes 5, 6, and 7 each yielded a single band of approximately 2091 bp, similar to the expected band size. These were sent to the company for sequencing. The correctly sequenced recombinant *E. coli* culture was stored for later use, thus obtaining the overexpression vector pBI121-PLC2. The successfully constructed overexpression vector pBI121-PLC2 was transformed into *Agrobacterium* GV3101 competent cells, and single-colony PCR identification was performed. The results are as follows... Figure 2 .

[0110] 2.1.3 RT-qPCR detection results of overexpressing resistant plants

[0111] The expression level of the PLC2 gene in the overexpression resistant plants was determined, and the overexpression resistant G2 plants were identified by RT-qPCR. RNA was extracted from the inflorescence axis of the main stem of G2 plants and wild-type control plants (WT), and the obtained RNA was reverse transcribed to synthesize cDNA as a template for the next reaction. RT-qPCR detection was performed on G2 plants, and the results are as follows: Figure 3 .

[0112] 2.1.4 Results of biological level detection of PLC2 gene overexpression resistant plants

[0113] 2.1.4.1 Statistical analysis results of flowering and fruit development stages of overexpressing resistant plants

[0114] Statistical analysis of the flowering and fruit development stages of the PLC2 gene overexpression resistant plant G2 compared with wild-type (WT) plants: As shown in Table 3, 90 days after transplanting, G2 plants were shorter than WT plants; WT plants had fewer leaf drops, while G2 plants had faster basal leaf senescence; G2 plants had fewer stem nodes than WT plants; G2 plants flowered about 35 days earlier than WT plants, and had more male and female flowers.

[0115] Table 3. Phenotypic statistics of PLC2 gene overexpression resistant plants at 90 days.

[0116]

[0117] Note: Different lowercase letters indicate significant differences (P<0.05).

[0118] From Table 4, Figure 4 Analysis showed that at 120 days after transplanting, G2 plants were shorter than WT plants, with faster leaf senescence and fewer stem nodes. While wild-type WT plants were just entering the fruit development stage, most of the fruits in G2 plants had already reached maturity and abscission. G2 plants also had more capsules than WT plants. These results indicate that, compared to the wild-type control WT, overexpression of the PLC2 gene in the resistant G2 plants significantly promotes earlier flowering, a greater number of capsules, and better seed maturity in castor beans.

[0119] Table 4. Phenotypic statistics of PLC2 gene overexpression resistant plants at 120 days.

[0120]

[0121] 2.1.4.2 Results of floral structure analysis of gene-overexpressing resistant plants

[0122] The differences in flowers between the PLC2 gene-overexpressing resistant plant G2 and the wild-type control plant WT were observed using a digital microscope. The results are as follows: Figure 5 As shown. From Figure 5 Observations A1 and B1 revealed that the male flower buds of G2 plants had more stamens and developed faster than those of WT plants. From... Figure 5 Observations in A2 and B2 plants revealed that G2 plants had better mature male flowers than WT plants, with more pollen in the anthers and longer filaments. Figure 5 Observations in A3 and B3 revealed that the number of stigmas in the female flowers of G2 and WT plants was the same, both being 3 pairs, but the stigmas of the female flowers of G2 were longer than those of WT plants.

[0123] 2.1.4.3 Results of pollen viability detection in gene-overexpressing resistant plants

[0124] By measuring the viability of mature castor bean pollen, it was found that the stronger the pollen viability, the deeper the staining after TTC staining. The viability of pollen from the PLC2 gene-overexpressing resistant plant G2 and the wild-type control WT was detected, and the results are shown in Table 5. Figure 6 As shown in the figure, 100 pollen grains were observed and counted in different fields of view. Compared with WT, the pollen viability of G2 was significantly improved. This indicates that overexpression of the PLC2 gene helps to improve the pollen viability of the plant.

[0125] Table 5. Pollen viability statistics of PLC2 gene overexpression resistant plants G2 and wild-type plants WT.

[0126]

[0127] Pollen germination rate was determined in plants resistant to PLC2 gene overexpression, and the results are shown in Table 6. Figure 7 As shown in the figure, 100 pollen grains were observed and counted in different fields of view. Compared with the wild-type control WT, the mature pollen germination rate of the overexpressing resistant plant G2 was stronger in G2; compared with WT, the pollen germination rate of G2 was significantly improved. This indicates that overexpression of the PLC2 gene promotes pollen tube germination and accelerates seed development.

[0128] Table 6. Statistics on the germination of mature pollen in PLC2 gene-overexpressing resistant plants.

[0129]

[0130] 2.1.4.4 Phenotypic observation results of fruits from overexpressing resistant plants

[0131] Comparative analysis of capsules from PLC2 gene-overexpressing resistant plants G2 and wild-type plants WT: Figure 8 As shown, G2 capsules are larger and split more noticeably than WT capsules.

[0132] 2.1.4.5 Statistical Results of Seed Phenotypic Observation of Overexpression Resistant Plants

[0133] Seeds from the overexpressing resistant plant G2 were compared with those from the wild-type control plant WT, and the results were as follows: Figure 9 .Depend on Figure 9 It can be seen that G2 seeds are fuller, darker in color, and have more obvious and glossy seed coat patterns compared to WT seeds after maturity.

[0134] Statistical analysis of the 100-seed weight of mature seeds from G2 and WT plants was performed, and the results are shown in Table 7. The table shows that the 100-seed weight of mature seeds from G2 was significantly better than that from WT. This indicates that overexpression of the PLC2 gene improves the saturation and quality of castor bean seeds.

[0135] Table 7. Statistical analysis of the 100-seed weight of PLC2 gene-overexpressing resistant plants and wild-type plants.

[0136]

[0137] G2 and WT mature seeds were germinated for 72 hours, and the germination rate was recorded. The results are shown in Table 8. Figure 10 As shown, compared with WT, G2 seeds showed a significantly improved germination rate, and G2 seeds germinated faster with longer radicles.

[0138] Table 8. Statistics on seed germination of PLC2 gene overexpression resistant plants

[0139]

[0140] Molecular-level analysis was performed on six plants that overexpressed resistance, resulting in one overexpression mutant plant, namely the G2 plant. Compared with the wild-type control plant WT, the G2 plant was shorter, had faster leaf drop, more stamens in its male flower buds that developed faster, stronger pollen viability in its anthers and a higher pollen germination rate, larger capsules that split more clearly, and fuller seeds with a higher germination rate.

[0141] The results showed that overexpression of the PLC2 gene could promote the development of castor fruits in the Lm-type female line aLmAB5 and increase the number of castor seeds.

[0142] 2.2 Results of CRISPR / Cas9 gene editing of the castor bean PLC2 gene in castor bean

[0143] 2.2.1 RT-qPCR detection of gene-edited resistant plants

[0144] Even if the target gene PLC2 is edited and mutated, it can still be transcribed into mRNA normally and will still be detected by quantitative real-time PCR. Therefore, RT-qPCR detection is not necessary. The results of comparing the gene-edited target sequence with the original sequence are as follows: Figure 11 This confirms that the target gene PLC2 was successfully edited.

[0145] 2.2.2 Results of biological level detection of PLC2 gene-edited resistant plants

[0146] 2.2.1 Statistical analysis results of flowering and fruit development stages of gene-edited resistant plants

[0147] Statistical analysis of the flowering and fruit development periods of PLC2 gene-edited resistant plants Q2-1 and Q2-2 was performed. As shown in Table 9, at 90 days after transplanting, compared with WT, Q2-1 and Q2-2 had more leaves that did not fall off; Q2-1 had more stem nodes than WT, while Q2-2 had fewer stem nodes than WT; and flowering of Q2-1 and Q2-2 was significantly delayed compared with WT.

[0148] Table 9. Phenotypic statistics of PLC2 gene-edited resistant plants at 90 days.

[0149]

[0150] From Table 10, Figure 12 Analysis shows that 120 days after transplanting, the WT inflorescence had entered the fruit development stage, while the Q2-1 flowers had not yet fully opened and pollinated, and the Q2-2 flowers had not yet fully entered the fruit development stage. Compared with WT, Q2-1 flowered about 40 days later, and Q2-2 flowered about 25 days later. After pollination, most of the female flowers of Q2-1 and Q2-2 failed to form fruits and withered.

[0151] Table 10. Phenotypic statistics of PLC2 gene-edited resistant plants at 120 days.

[0152]

[0153] 2.2.2 Results of floral structure analysis of gene-edited resistant plants

[0154] The differences in flowers between PLC2 gene-edited resistant plants Q2-1 and Q2-2 and wild-type plant WT were observed using a digital microscope. The results are as follows: Figure 13 As shown. From Figure 13 Observations at A1, B1, and C1 revealed that the male flower buds of Q2-1 and Q2-2 were smaller than those of WT. Figure 13 Observations in A2, B2, and C2 revealed that Q2-1 and Q2-2 had poorer mature male flower condition, shorter filaments, and relatively fewer pollen compared to WT plants. Figure 13 Observations in A3, B3, and C3 revealed that the stigmas of the female flowers in Q2-1 and Q2-2 were shorter than those in the WT plants.

[0155] 2.2.3 Results of pollen viability testing in gene-edited resistant plants

[0156] Pollen viability of PLC2 gene-edited resistant plants Q2-1 and Q2-2 was detected, and the results are shown in Table 11. Figure 14 As shown, when 100 pollen grains were observed in different fields of view, the pollen from Q2-1 and Q2-2 plants showed lighter staining than that from WT plants, and the pollen viability of Q2-1 and Q2-2 plants was significantly reduced. This indicates that PLC2 gene editing affects castor bean pollen viability.

[0157] Table 11. Pollen viability statistics of PLC2 gene-edited resistant plants

[0158]

[0159] Pollen germination rates of PLC2 gene-edited resistant plants Q2-1 and Q2-2 were determined, and the results are shown in Table 12. Figure 15 As shown in the figure, 100 pollen grains were observed in different fields of view. Compared with WT, the germination rate of mature pollen from Q2-1 and Q2-2 was significantly reduced. This indicates that PLC2 gene editing affects pollen germination and pollen tube formation.

[0160] Table 12 Statistics on mature pollen germination in PLC2 gene-edited resistant plants

[0161]

[0162] 2.2.4 Observation results of capsule phenotype in gene-edited resistant plants

[0163] The capsules of PLC2 gene-edited resistant plants Q2-1 and Q2-2 were observed, such as... Figure 16 As shown, the capsules of Q2-1, Q2-2, and WT all have three chambers; compared with WT, the capsules of Q2-1 and Q2-2 are smaller, lighter in color, and less prone to dehiscence. This indicates that PLC2 gene editing affects castor bean capsule development.

[0164] 2.2.5 Results of Observation on Seed Phenotypic Characteristics of Gene-Edited Resistant Plants

[0165] like Figure 17 As shown, the seeds of Q2-1 and Q2-2, after maturation, are smaller and shrunken than those of WT, with shallower seed coat patterns and no luster, indicating poorer seed quality.

[0166] The 100-seed weight of mature seeds from Q2-1, Q2-2 and WT plants was statistically analyzed, and the results are shown in Table 14.

[0167] Table 13 shows that, compared with WT, Q2-1 and Q2-2 resulted in a lower 100-seed weight, reduced seed saturation, and decreased seed quality. This indicates that PLC2 gene editing reduces the quality and grade of mature castor seeds.

[0168] Table 13 Statistical analysis of 100-seed weight of PLC2 gene-edited resistant plants and wild-type plants

[0169]

[0170] Seeds of Q2-1, Q2-2, and WT were germinated for 72 hours, and the germination rate was recorded. The results are shown in Table 14. Figure 18 As shown, Q2-1 and Q2-2 seeds germinate more slowly and have a lower germination rate compared to WT. This indicates that PLC2 gene editing reduces the germination rate of castor beans.

[0171] Table 14. Statistics on seed germination of PLC2 gene-edited resistant plants

[0172]

[0173] 2.3 Results of the effect of PLC2 gene overexpression on the expression levels of PLCs in the same family

[0174] The expression levels of other members of the PLC family in the overexpressing resistant plant G2 were analyzed, and the results are as follows: Figure 19 .Depend on Figure 19 It can be seen that when the castor bean PLC2 gene is overexpressed, compared with the wild-type control (WT) plant, the expression levels of PLC2M and PLC2N genes are significantly upregulated; the expression level of PLC4 gene is significantly increased; the expression level of PLC4X2 gene is not significantly changed; but the expression level of PLC6 is significantly reduced.

[0175] In summary, this invention involved homologous overexpression and gene editing of the PLC2 gene in castor beans. Phenotypic differences between the overexpression mutant and the gene-edited mutant plants and wild-type plants were observed to determine the effect of the PLC2 gene on the development of female castor bean lines. The results are as follows: Compared with the wild-type plant WT of the same inflorescence type, the obtained overexpression mutant plant G2 was shorter, had faster leaf drop, a longer main stem spike, and earlier flowering. It also had an increased number of pistils and stamens, with a longer stigma on the pistil, and produced more fruit and seeds. The seeds were plumper and had higher gloss, resulting in a higher germination rate of mature seeds. Compared to the wild-type WT with the same inflorescence type, the two PLC2 gene-edited mutant plants, Q2-1 and Q2-2, showed the following differences: Q2-1 was taller, while Q2-2 was shorter; the main stem spike was shorter, flowering was delayed, the number of pistils and stamens was reduced, the stigmas of female flowers were shorter, pollen bioactivity of male flowers decreased after flowering, fruit maturity was lower in the later stages of inflorescence development, seed gloss was reduced, grains were not plump, and the germination rate of mature seeds was also lower. RT-qPCR results showed that when the PLC2 gene was overexpressed, the expression level of PLC6 was significantly downregulated, while the expression levels of PLC2M, PLC2N, and PLC4 were significantly upregulated, and the expression level of PLC4X2 gene did not change significantly.

[0176] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. PLC2 The application of genes in any of the following: (1) Application in regulating the growth rate of castor bean plants; (2) Application in regulating castor bean seed quality; (3) Application in regulating castor pollen viability; (4) Application in regulating the flowering period of castor beans; (5) Application in regulating the quality of castor bean capsules; The PLC2 The reference sequence of the gene is accessed by NCBI under accession number XM_025158535.2; The regulation is achieved through overexpression. PLC2 Genes that shorten castor bean plant height, reduce the number of stem nodes, increase the number of male and female flowers, advance the flowering period, and improve castor bean pollen viability, seed weight per 100 seeds, germination rate, and the number and size of capsules.

2. Includes PLC2 The application of gene recombinant vectors in any of the following: (1) Application in regulating the growth rate of castor bean plants; (2) Application in regulating castor bean seed quality; (3) Application in regulating castor pollen viability; (4) Application in regulating the flowering period of castor beans; (5) Application in regulating the quality of castor bean capsules; The PLC2 The reference sequence of the gene is accessed by NCBI under accession number XM_025158535.2; The regulation is achieved through overexpression. PLC2 Genes that shorten castor bean plant height, reduce the number of stem nodes, increase the number of male and female flowers, advance the flowering period, and improve castor bean pollen viability, seed weight per 100 seeds, germination rate, and the number and size of capsules.

3. The use of the recombinant bacteria comprising the recombinant vector of claim 2 in any of the following: (1) Application in regulating the growth rate of castor bean plants; (2) Application in regulating castor bean seed quality; (3) Application in regulating castor pollen viability; (4) Application in regulating the flowering period of castor beans; (5) Application in regulating the quality of castor bean capsules; The regulation is achieved through overexpression. PLC2 Genes that shorten castor bean plant height, reduce the number of stem nodes, increase the number of male and female flowers, advance the flowering period, and improve castor bean pollen viability, seed weight per 100 seeds, germination rate, and the number and size of capsules.

4. A method for promoting the growth and development of castor beans, characterized in that, Will PLC2 Genes were inserted into an overexpression vector and transferred into castor bean recipient material to improve pollen viability, seed weight per 100 seeds and germination rate, as well as the number and size of capsules, increase the number of male and female flowers, and promote early flowering of castor beans. The PLC2 The reference sequence of the gene is accessed by NCBI under accession number XM_025158535.2.

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