Application of calmodulin in regulating seed number in arabidopsis siliques

Abstract

The application provides application of calmodulin in regulation of seed quantity in Arabidopsis thaliana siliques, and the seed quantity in the siliques of Arabidopsis thaliana is increased by applying calmodulin to the culture environment outside Arabidopsis thaliana cells.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biotechnology, and particularly relates to application of calmodulin in regulating seed quantity in Arabidopsis thaliana. BACKGROUND

[0002] Arabidopsis thaliana (L.) Heynh. is an annual weak herb of Brassicaceae, with a height of 20-35 cm, covered with simple and branchy hairs. The stem is not branched or branched from the middle and upper part, and the lower part is sometimes light purple white, and the stem is often longitudinally grooved, and the lower part is covered with simple hairs and occasionally mixed with 2-forked hairs. The basal leaves are rosette-shaped, inversely ovate or spoon-shaped, 1-5 cm long and 3-15 mm wide, with a blunt or slightly acute tip, a gradually narrowed base into a stalk, and a few indistinct teeth on the edge, and 2-3 forked hairs on both surfaces; the stem leaves are stalkless, needle-shaped, strip-shaped, long circular or elliptical, 5-15 (-50) mm long and 1-2 (-10) mm wide.

[0003] The inflorescence is a loose raceme, and can be elongated to 20 cm during fruiting; the sepals are long ovate, about 1.5 mm long, with a blunt tip, a capsule-shaped base of the outer circle, and no hair or a few simple hairs on the outer surface; the petals are white, long circular strip-shaped, 2-3 mm long, with a blunt tip, and a linear base. The silique is 10-14 mm long and less than 1 mm wide, with blunt or bluntly rounded two ends, a midrib and sparse reticulate veins, and is mostly orange yellow or light purple; the fruiting pedicel is stretched, 3-6 mm long. There is one row of seeds in each chamber, and the seeds are ovate, small and reddish brown. The flowering period is from April to June.

[0004] Calmodulin (calmodulin) is a kind of calcium ion receptor protein widely existing in eukaryotic cells and highly conserved in evolution. Calmodulin is a single-chain protein composed of 148 amino acids, with a relative molecular mass of 16.7 kDa. The isoelectric point is about 4.0, and it is an acid-soluble heat-stable protein that can maintain good biological activity even at very high external temperatures. The amino acid levels of calmodulin from different biological sources are extremely similar, the homology is also very high, and the similarity of physical and chemical properties and biological activity is also high, which is very important for maintaining the interaction of a variety of calmodulin binding protein families, but the control of biological traits by calmodulin remains to be studied. SUMMARY

[0005] Therefore, the application provides application of calmodulin in regulating seed quantity in Arabidopsis thaliana, which can greatly increase the seed quantity in the stem silique of Arabidopsis thaliana by applying calmodulin to the culture environment outside Arabidopsis thaliana cells.

[0006] The application of a calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana, by applying a calmodulin to the culture environment outside the cells of Arabidopsis thaliana to increase the number of seeds in a silique of Arabidopsis thaliana.

[0007] Further, the method of applying a calmodulin to the culture environment outside the cells of Arabidopsis thaliana comprises adding a calmodulin to the seed culture medium of Arabidopsis thaliana; or genetically modifying Arabidopsis thaliana to overexpress a calmodulin and transport the calmodulin to the culture environment outside the cells through a signal peptide.

[0008] Further, the application of a calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana comprises the following steps:

[0009] The seeds of Arabidopsis thaliana are sterilized and placed in a liquid MS medium containing a calmodulin, and then placed in a dark place at 4℃ for 2 days; the seeds of Arabidopsis thaliana and the culture medium are transferred to a transparent tube and subjected to shake culture in a light culture room at a temperature of 22℃; after the culture, the seeds are transplanted and the application of the calmodulin is continued.

[0010] Further, the concentration of the calmodulin in the liquid MS medium containing the calmodulin is 1×10 -7 M.

[0011] Further, the light intensity during the shake culture is 120-130 μM.m -2 .s -1 .

[0012] Further, the genetic modification of Arabidopsis thaliana comprises the following steps:

[0013] A vector containing a calmodulin gene is constructed; the vector containing the calmodulin gene is introduced into a plant of Arabidopsis thaliana to perform genetic transformation of Arabidopsis thaliana; the transformed plant of Arabidopsis thaliana is cultivated, the seeds of Arabidopsis thaliana are harvested, and multiple generations of breeding are performed to screen a T3 generation homozygous plant.

[0014] Further, the vector comprises a CAMV35 promoter, a signal peptide of a pollen coat binding protein SLR1-BR of Brassica napus, an AtCaM2 calmodulin gene, and a nopaline synthase terminator.

[0015] The application of a calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana, by applying a calmodulin to the culture environment outside the cells of Arabidopsis thaliana, can greatly increase the number of seeds in a silique of Arabidopsis thaliana, and the shape of the silique is fuller than that when no calmodulin is applied. BRIEF DESCRIPTION OF DRAWINGS

[0016] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and serve as an aid in explaining the exemplary embodiments of the present application and their descriptions, and do not constitute an improper limitation on the present application. In the drawings:

[0017] Figure 1 : Comparison of siliqua shape between wild type Arabidopsis thaliana with and without application of calmodulin;

[0018] Figure 2 : Schematic diagram of each gene fragment in the vector of Example 2;

[0019] Figure 3 : Comparison of protein expression localization of the fusion gene in Arabidopsis thaliana cells of Example 2;

[0020] Figure 4 : Photos of siliquae and seeds of the L1 overexpression strain of Example 2;

[0021] Figure 5 : Photos of siliquae and seeds of the L2 overexpression strain of Example 2;

[0022] Figure 6 : Photos of siliquae and seeds of the L3 overexpression strain of Example 2;

[0023] Figure 7 : Photos of siliquae and seeds of the wild type strain;

[0024] Figure 8 : Comparison of siliqua shape between the L2 overexpression strain and the wild type strain of Example 2. DETAILED DESCRIPTION

[0025] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. In addition, except for the special explanations in the present examples, the terms and processes involved in the present examples are understood according to the general cognition and conventional methods in the prior art.

[0027] The following examples are useful for better understanding the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. The siliqua length is measured by using ImageJ software. The experimental data is the average value ± standard deviation of 3 repeated experiments. The data of siliqua length and seed number are analyzed by using Graphad Prism, and the difference analysis is performed by using t-test method.

[0028] Example 1

[0029] This embodiment relates to adding calmodulin in Arabidopsis seed culture medium, and observing the influence of exerting calmodulin on the culture environment outside Arabidopsis cells on the seed quantity in Arabidopsis siliques.

[0030] The prepared materials of the present application include:

[0031] Arabidopsis thaliana ecotype Landsberg erecta (Ler); bacterial strains DH5α and BL21, Agrobacterium strain GV3101; vector pET28b(+).

[0032] Using the method of genetic engineering, the calmodulin expression gene vector pET28b-AtCaM2 containing the nucleotide sequence shown in SEQ ID NO. 1 is constructed by using DH5α, the vector pET28b-AtCaM2 is introduced into the competent cells of Escherichia coli BL21, a single colony containing the recombinant plasmid is picked and inoculated in 5ml LB medium containing Kan, and cultured at 37℃ overnight. According to the inoculation amount of 2%, it is inoculated in 20ml LB, and cultured at 37℃ until OD600=0.6. IPTG (final concentration 1mM) is added for induction, and the culture is continued for 2h. 1.5ml of bacterial liquid is taken, centrifuged at 12,000g and 4℃ for 5min, and the bacterial body precipitate is collected.

[0033] The bacterial body precipitate is resuspended in 100μl SDS-PAGE (SDS-polyacrylamide gel electrophoresis) 2x sample buffer, 100℃ metal bath for 5min, and cooled on ice. 12,000g, 4℃ centrifugation for 5min, take 15μl supernatant electrophoresis. Concentration gel 4%, separation gel 12%, voltage 120 volts; Coomassie brilliant blue R-250 staining (0.1% Coomassie brilliant blue R-250, 40% methanol, 10% glacial acetic acid); then decolorized in the decolorizing solution (40% methanol and 10% glacial acetic acid). SDS-PAGE detection shows that there is an obvious induced target band at a molecular weight of 17KD compared with the sample before induction, which can be used for CaM2 purification experiment.

[0034] The BL21 single colony containing pET28b-AtCaM2 is picked in 20ml LB culture solution (100mg / L Kan), and cultured at 30℃ overnight; according to the ratio of 1:50, the culture is expanded, and cultured at 37℃ until the bacterial density OD 600=0.8, IPTG was added (final concentration 1 mM) and incubation was continued for 2 h; the cells were collected by centrifugation at 6,000 rpm for 15 min at 4°C; 1 L of bacteria was resuspended in 50 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 2 mM Na2.EDTA, 1 mM DTT, 200 μg / ml lysozyme) and incubated for 30 min on ice; 20 s of sonication with 10 s intervals were repeated 20 times; 3 min at 95°C, rapid cooling to room temperature; centrifugation at 17,000 rpm for 45 min at 4°C; the supernatant was collected and adjusted to a final concentration of 5 mM CaCl2; the solution was filtered through a 0.2 μm filter and applied to a Phenyl-Sepharose hydrophobic affinity column. 2+ The column was equilibrated with 5 column volumes of SB (50 mM Tris-HCl, 0.1 mM CaCl2pH 7.5) and the sample was applied. After sample application, the column was washed with 5 column volumes of SB and then with about 5 column volumes of WB (50 mM Tris-HCl, 0.1 mM CaCl2, 0.5 M NaCl pH 7.5). The CaM was eluted with EB (50 mM Tris-HCl, 5 mM EGTA pH 8.0) and collected. The purity of the CaM was checked by 12% SDS-PAGE. The sample was dialysed against ultrapure water, lyophilised and stored at -80°C.

[0035] Pharmacia AKTA FPLC purification of CaM: flow rate 1 ml / min, detection at 280 nm and 215 nm; the hydrophobic affinity column was equilibrated with 5 column volumes of SB (50 mM Tris-HCl, 0.1 mM CaCl2pH 7.5); after sample application, the column was washed with 5 column volumes of SB and then with about 5 column volumes of WB (50 mM Tris-HCl, 0.1 mM CaCl2, 0.5 M NaCl pH 7.5); the CaM was eluted with EB (50 mM Tris-HCl, 5 mM EGTA pH 8.0) and collected; the purity of the CaM was checked by 12% SDS-PAGE; after dialysis against ultrapure water, the sample was lyophilised and stored at -80°C.

[0036] Protein determination: the protein content was determined by the Coomassie Brilliant Blue G-250 staining method at 595 nm using a microplate reader, using bovine serum albumin (BSA) as a standard curve.

[0037] Liquid medium culture of Arabidopsis thaliana: the seeds were surface sterilised (75% ethanol, 30 s; sterile water, 3 washes) and deep sterilised (15% sodium hypochlorite, 10 min; sterile water, 3 washes) and then incubated in liquid MS medium (1% sucrose, 1 g / L MES, pH 5.8) containing 1 x 10 -7 M CaM for 2 days in the dark at 4°C; the seeds were transferred to 50 ml transparent tubes, 7 ml of liquid MS medium (1% sucrose, 1 g / L MES, pH 5.8) containing 1 x 10 -7 M CaM for 2 days in the dark at 4°C; the seeds were transferred to 50 ml transparent tubes, 7 ml of liquid MS medium (1% sucrose, 1 g / L MES, pH 5.8) containing 1 x 10 -2 -1 M CaM for 2 days in the dark at 4°C; the seeds were transferred to 50 ml transparent tubes, 7 ml of liquid MS medium (1% sucrose, 1 g / L MES, pH 5.8) containing 1 x 10 -7 ​in Hoagland nutrient solution, and spraying 1 x 10 -7 M Hoagland nutrient solution. The control group was also cultured by the above process, but without calcine in the MS medium and Hoagland nutrient solution.

[0038] The siliques shape and seed number of wild-type Arabidopsis thaliana with and without calcine were counted, as shown in Table 1 below, and the siliques shape was compared with the control as shown in Figure 1 .

[0039] Table 1

[0040]

[0041] ** indicates that the numerical value reaches a significant difference level (P < 0.01) between the transgenic line and the wild-type control

[0042] As can be seen from the above table, the seed number in the siliques of Arabidopsis thaliana with calcine is significantly higher than that of Arabidopsis thaliana without calcine, which is due to the influence of the calcine applied to the culture environment of Arabidopsis thaliana, which increases the seed number in the siliques of Arabidopsis thaliana.

[0043] Example 2

[0044] This example relates to genetically modifying Arabidopsis thaliana to overexpress calcine and transporting calcine to the culture environment outside the cell through a signal peptide.

[0045] This example constructs a binary vector pBI121-35S::SP-AtCaM2-GFP (nucleotide sequence as shown in SEQ ID NO. 2) and its control vector pBI121-35S::SP-GFP (nucleotide sequence as shown in SEQ ID NO. 3), respectively, and the schematic diagram of the gene fragments of the above two vectors is shown as Figure 2 .

[0046] The nucleotide sequence and encoded amino acid sequence of the SP-AtCaM2-GFP fusion gene are 1257 bp in total, starting with 78 nucleotides encoding 26 amino acids at the N-terminus of the fusion protein (signal peptide of Brassica napus pollen coat binding protein), followed by 444 nucleotides encoding 148 amino acids (Arabidopsis thaliana subtype 2 calcine protein (AtCaM2)), and then 735 nucleotides at the C-terminus encoding green fluorescent protein GFP. The nucleotide sequence of the SP-GFP fusion gene used as a control in this experiment does not contain the 444 nucleotides encoding Arabidopsis thaliana subtype 2 calcine protein.

[0047] Arabidopsis thaliana cultivation: Arabidopsis thaliana seeds were surface-sterilized (75% ethanol, 30 s; sterile water washing 4 times) and then submerged (10% sodium hypochlorite, 10 min; sterile water washing 3 times) before being sown on sterile MS medium. The seeds were then placed in the dark at 4°C for 2 days, followed by incandescent light (120-130 μM / d) at 22°C for 16 / 8 h (L / D). -2 .s -1 Seedlings were cultured in a light-incubated incubator; after 14 days, they were transplanted into nutrient soil (soaked in Hoagland nutrient solution) for further cultivation.

[0048] Preparation of Agrobacterium competent cells: Pick a single colony of Agrobacterium GV3101 and inoculate it into 2 ml of YEB (Rif - In the culture medium, incubate overnight at 28°C with shaking; take 500 μl and inoculate into 50 ml of YEB (Rif 125 mg / L) medium for expansion, and incubate at 28°C with shaking until OD. 600 =0.6; Place the bacterial culture on ice for 30 min; Centrifuge at 5,000 rpm at room temperature for 5 min, resuspend the bacterial cells in 10 ml of 0.15 M NaCl solution; Centrifuge at 5,000 rpm at room temperature for 5 min, discard the supernatant, resuspend the bacterial cells in 1 ml of 20 mM CaCl2, add sterile glycerol to a final concentration of 20%, aliquot into 200 μl containers, and store at -80 °C.

[0049] Transformation of Agrobacterium: Add 10 μl of plasmid DNA extracted from the kit to 200 μl of Agrobacterium competent cells and mix well; incubate on ice for 30 min, freeze in liquid nitrogen for 5 min, and incubate in a metal bath at 37℃ for 5 min; add 1 ml of YEB medium (without antibiotics) and incubate at 28℃ for 4 h; centrifuge at 10,000 rpm at room temperature for 30 s, discard the supernatant, resuspend the cells in 200 μl of YEB medium (without antibiotics), plate on YEB (Kan 100 mg / L, Rif 125 mg / L) medium, and incubate at 28℃ for 2 days.

[0050] Identification of Agrobacterium using the recovery mobilization method: Agrobacterium plasmids were extracted; they were transferred into Escherichia coli DH5α, cultured overnight, and single colonies were selected for liquid culture. Plasmid DNA was extracted and enzyme digestion was performed to identify whether it was a positive clone containing the target gene.

[0051] Genetic transformation of Arabidopsis thaliana: Selection of Arabidopsis thaliana materials: Arabidopsis thaliana enters the flowering period 2 weeks after transplanting. The main stem is cut off, and the lateral stems enter the flowering period about 3 weeks later. When it has produced only 1-2 siliques, it can be used for transformation. Before transformation, the siliques and fully opened flower buds are cut off, leaving only the tender flower buds.

[0052] Agrobacterium culture: pick single colony to 5ml YEB medium (Kan 100μg / ml, Rif 125μg / ml), 30°C overnight culture, 1:100 dilution to 500ml YEB, shake culture to OD 600 =1.2, 5,000g, 4°C centrifuge 15min to collect bacteria, resuspend bacteria to OD 600 =0.8.

[0053] Vacuum infiltration method to transform Arabidopsis: pour the prepared Arabidopsis material into the transformation medium containing Agrobacterium, immerse the whole inflorescence into the transformation medium, soak for 5min, lie on a clean plastic pot, cover with plastic film to avoid light and keep moist for 24h recovery culture; place the Arabidopsis under light, pour the culture solution, and then normally culture; the transformed plants normally bloom and grow, and the seeds can be harvested after 2-3 weeks; store the seeds in 1.5ml centrifuge tube, add desiccant for short-term storage at room temperature, or store at -20°C for long-term storage.

[0054] Transgenic plant screening: after sterilization, sow the transformed seeds (T0 generation) on MS medium containing 50mg / L Kan, 4°C in the dark for 2 days, and then normally culture under light; after 10 days, the Arabidopsis seedlings (T1 generation) into which the foreign gene has been transferred can be distinguished, because the binary vector has a Kan resistance site, the cotyledon of the resistant seedling is green, the hypocotyl is longer, and two normal true leaves grow; the seedling without the binary vector does not have the ability to grow in the resistant medium, the cotyledon is yellow, and there is no normal true leaf. Transplant the resistant plants, culture under light, and collect T2 generation seeds; sow the T2 generation seeds on medium containing 50mg / L Kan for further resistance screening, and according to the resistance segregation ratio of the offspring (T2 generation), it can be determined whether it is a homozygote and the number of copies of the inserted foreign gene. Select single gene insertion transgenic plants for transplantation, collect seeds (T3 generation) from a single plant, and further screen homozygotes on medium containing 50mg / L Kan, and those without segregation are homozygotes.

[0055] The subcellular localization of the fusion protein in the transgenic plant 35S::SP-CaM2-GFPLer and its control plant 35S::SP-GFP Ler was observed by laser confocal microscope. The cells were treated with 50% sucrose solution to cause plasmolysis, and then observed by laser confocal microscope. After plasmolysis of the root cells in the 35S::SP-AtCaM2-GFPLer plant, the high-brightness fluorescence was mainly distributed on the cell wall, and the fluorescence in the plasma membrane and cytoplasm was weaker than that on the wall. After plasmolysis of the root cells in the 35S::SP-GFP Ler plant, the high-brightness fluorescence was mainly distributed on the cell wall, and the fluorescence in the plasma membrane and cytoplasm was weaker than that on the wall. After plasmolysis of the root cells in the 35S::GFPCol plant, the high-brightness fluorescence only existed in the cytoplasm of the cells, and no high-brightness fluorescence was detected on the cell wall, as shown in FIG. 4. The results show that the signal peptide can indeed guide the proteins AtCaM2-GFP and GFP fused thereto to the outside of the plant cells. Figure 3

[0056] Three transgenic 35S::SP-CaM2-GFPLer lines (L1, L2 and L3) of the T3 generation were selected and compared with three control 35S::SP-GFP Ler lines (L1 # , L2 # and L3 # ) and wild-type lines in terms of siliqua shape and seed number in siliquas, as shown in FIG. 5. Figure 4-8 Figure 4 FIG. 5A shows the transgenic overexpression L1 line, Figure 5 FIG. 5B shows the transgenic overexpression L2 line, Figure 6 FIG. 5C shows the transgenic overexpression L3 line, Figure 7 FIG. 5D shows the transgenic overexpression L4 line, Figure 8 FIG. 5E shows the comparison between the transgenic overexpression L2 line and the wild-type line, and the measurement of siliqua length and seed number in siliquas of each line is shown in Table 2.

[0057] Table 2

[0058] Strain Pod length (mm) Number of seeds in pod Wild type 14.26±0.85 40±3 Control line L1 # ]] 13.65±1.8 42±2 Control line L2 # ]] 14.78±1.21 41±1 Control line L3 # ]] 14.94±2.11 39±3 Strain L1 9.23±2.12** 53±5** Strain L2 10.13±2.01** 58±6** Strain L3 9.34±2.05** 57±7**

[0059] ** indicates that the numerical value reaches a significant difference level (P<0.01) between the transgenic line and the wild-type control.

[0060] As shown in the above table, the seed number in siliquas of the transgenic Arabidopsis line is significantly higher than that of the wild-type line and the control line, which is because the overexpressed calmodulin is transported to the outside of the cells by the signal peptide, affecting the culture environment of Arabidopsis, and increasing the seed number in siliquas of Arabidopsis.

[0061] ​​The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0062] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

[0063]

[0064]

[0065]

[0066]

[0067]

Claims

1. Use of a calcium modulator in regulating the number of seeds in a silique of Arabidopsis thaliana, characterized in that, The application relates to a method for increasing the number of seeds in a silique of Arabidopsis thaliana by applying calmodulin to the culture environment outside cells of the Arabidopsis thaliana. The method for applying calmodulin to the culture environment outside cells of the Arabidopsis thaliana comprises the following steps: Arabidopsis thaliana seeds are cultured in liquid MS medium containing calmodulin; After the culture, the Arabidopsis thaliana seeds are transplanted, and the culture of the Arabidopsis thaliana seeds is continued during the transplanting process. The nucleotide sequence of the calmodulin is shown in SEQ ID NO.

1.

2. The use of a calcium modulator for regulating the number of seeds in a pod of Arabidopsis thaliana according to claim 1, characterized in that, The method for culturing the Arabidopsis thaliana seeds in the liquid MS medium containing the calmodulin comprises the following steps: The Arabidopsis thaliana seeds are sterilized and placed in the liquid MS medium containing the calmodulin, and then the Arabidopsis thaliana seeds are placed in dark at 4 DEG C for 2 days; The Arabidopsis thaliana seeds and the medium are transferred into a transparent tube, and then the Arabidopsis thaliana seeds are shaken and cultured in a light culture room at 22 DEG C.

3. The application of the calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana according to claim 2, wherein the Arabidopsis thaliana is genetically modified to overexpress the calmodulin, and the calmodulin is transported to the culture environment outside cells by a signal peptide. The calcineurin concentration of the liquid MS medium containing calcineurin is 1 x 10 -7 M.

4. The application of the calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana according to claim 2, wherein the Arabidopsis thaliana is genetically modified to overexpress the calmodulin, and the calmodulin is transported to the culture environment outside cells by a signal peptide. The light intensity in the said rock culture is 120-130 μM.m -2 . -1 .

5. Use of a calmodulin to regulate seed number in a Brassica napus silique, characterized in that, The Arabidopsis thaliana is genetically modified to overexpress the calmodulin, and the calmodulin is transported to the culture environment outside cells by a signal peptide, so as to increase the number of seeds in a silique of the Arabidopsis thaliana. The method for genetically modifying the Arabidopsis thaliana comprises the following steps: A vector containing the calmodulin gene is constructed; The vector containing the calmodulin gene is introduced into Arabidopsis thaliana plants to perform genetic transformation of the Arabidopsis thaliana; The vector used is pBI121- 35S::SP-AtCaM2-GFP ; The nucleotide sequence of the pBI121- 35S::SP-AtCaM2-GFP in the middle SP-AtCaM2-GFP is shown as SEQ ID NO.

2.

6. The application of the calmodulin in regulating the number of seeds in a silique of Arabidopsis thaliana according to claim 5, wherein the method for genetically modifying the Arabidopsis thaliana further comprises the following steps: The method for genetically modifying the Arabidopsis thaliana further comprises the following steps: cultivating the transformed Arabidopsis thaliana plants, harvesting the Arabidopsis thaliana seeds, performing multi-generation breeding, and screening T3 generation homozygous plants.

Images