ZaSCL3 gene for regulating and controlling plant growth and development as well as encoding protein and application of ZaSCL3 gene

Abstract

The invention discloses a ZaSCL3 gene for regulating and controlling plant growth and development as well as an encoding protein and application thereof, and belongs to the technical field of gene engineering. According to the invention, an over-expression vector of the ZaSCL3 gene is constructed and expressed in a wild tomato, so that stamens of a ZaSCL3 over-expression plant are aborted, ovary is spontaneously expanded without pollination, and seedless fruits are generated, which indicates that the over-expression ZaSCL3 gene can realize the effect of inducing male sterility and apomixis of the plant; a key gene resource is provided for molecular breeding and yield character improvement, and the important application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a ZaSCL3 gene for regulating plant growth and development, its encoded protein and application. Background Art

[0002] Sichuan pepper is an important economic crop in my country, with a long history of cultivation and widespread application. Similar to male sterility, Sichuan pepper's unique apomixis gives it significant potential for application in crop breeding and production. Apomixis allows crops to produce seeds without fertilization, preserving the maternal genotype. This characteristic is crucial for the stable reproduction of high-quality hybrids, preventing the decline of heterosis and improving seed production efficiency. Male sterility can be used in hybrid seed production to increase crop yield and quality. Using male sterile lines can avoid artificial emasculation, reduce hybrid seed production costs, improve seed production efficiency, and enhance hybrid vigor. Together, these two approaches can significantly improve breeding efficiency and enhance crop adaptability and production stability.

[0003] The GRAS gene family is a unique class of transcription factors in plants. It can be divided into eight subfamilies: DELLA, HAM, LISCL, PAT1, LS, SHR, SCR, and SCL3. These subfamilies are widely involved in important physiological processes, including root development, meristem formation, light signal transduction, gibberellin signaling, and stress responses. The DELLA subfamily, a star subfamily within the GRAS family, is particularly noteworthy for its role in regulating floral development. In Arabidopsis, the DELLA proteins RGA, RGL1, and RGL2 act synergistically to inhibit stamen development. Studies have revealed that rga-28 / gai-td1 double mutant plants exhibit complete male sterility, characterized by abnormal meiosis in their pollen mother cells, resulting in an inability to form fertile pollen. In rice, the DELLA mutant slr1 exhibits reduced male fertility, while in tomato, a single DELLA gene mutant, pro, exhibits parthenocarpic phenotypes, further demonstrating the critical regulatory role of DELLA proteins in floral development. Furthermore, in Arabidopsis, SCL3 promotes GA signaling by antagonizing DELLA proteins, while in rice, RID1 interacts with DELLA proteins to activate SCL3 expression, thereby regulating endogenous GA levels and promoting floral transformation. Currently, there are no reports on GARS genes regulating flower development in Zanthoxylum bungeanum. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a ZaSCL3 gene for regulating plant growth and development, its encoded protein and application, so as to solve the technical problems of low efficiency and long cycle of traditional breeding methods.

[0005] To achieve the above objectives, the first aspect of the present invention is to provide a ZaSCL3 gene for regulating plant growth and development. The nucleotide sequence of the ZaSCL3 gene is shown in SEQ ID NO.1.

[0006] The second aspect of the present invention is to provide a protein encoded by the ZaSCL3 gene, the amino acid sequence of the protein being shown in SEQ ID NO.2.

[0007] The third aspect of the present invention is to provide a plasmid comprising the ZaSCL3 gene.

[0008] The fourth aspect of the present invention is to provide an overexpression vector comprising the ZaSCL3 gene.

[0009] The fifth aspect of the present invention is to provide an engineered bacterium comprising the above-mentioned ZaSCL3 gene.

[0010] A sixth aspect of the present invention is to provide a preparation for regulating plant growth and development, the preparation comprising one or more of the following:

[0011] a. ZaSCL3 gene disclosed in the first aspect of the present invention;

[0012] b. the plasmid disclosed in the third aspect of the present invention;

[0013] c. the overexpression vector disclosed in the fourth aspect of the present invention;

[0014] d. The engineered bacteria disclosed in the fifth aspect of the present invention.

[0015] The seventh aspect of the present invention is to provide an application of the ZaSCL3 gene in regulating plant growth and development.

[0016] On the basis of the above technical solution, the present invention can also be improved as follows:

[0017] Furthermore, the application is to achieve regulation of plant growth and development by overexpressing the ZaSCL3 gene.

[0018] Furthermore, the growth and development are specifically as follows: the angle between the leaves of the plant increases, the plant is male sterile, and there is no fusion reproduction.

[0019] Furthermore, the plant is a dicotyledonous plant.

[0020] Furthermore, the plant is a tomato.

[0021] The beneficial effects of the present invention are as follows: by constructing an overexpression vector of the ZaSCL3 gene and expressing it in wild-type tomatoes, it was found that the stamens of the ZaSCL3 overexpressing plants were aborted, the ovaries spontaneously expanded without pollination, and seedless fruits were produced, indicating that overexpression of the ZaSCL3 gene can induce plant male sterility and apomixis, providing a theoretical basis for breeding new varieties of male sterile and apomictic plants, and having important significance for further understanding the development of floral organs. It can not only accelerate the breeding process, but also provide a new way to breed excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For the evolutionary analysis of ZaSCL3 gene;

[0023] Figure 2 This is the adventitious bud of ZaSCL3 transgenic tomato;

[0024] Figure 3 Rooting ZaSCL3 transgenic tomato seedlings;

[0025] Figure 4 This is the positive identification of ZaSCL3 transgenic tomato;

[0026] Figure 5 is the expression level of ZaSCL3 gene in transgenic tomatoes;

[0027] Figure 6 Comparison of plant types between wild type and ZaSCL3 overexpressing plants;

[0028] Figure 7 This is the histological analysis of leaf angles in wild-type and ZaSCL3-overexpressing plants;

[0029] Figure 8 Comparison of leaf angles between wild type and ZaSCL3 overexpressing plants;

[0030] Figure 9 To analyze the spatiotemporal dynamics of floral organ morphogenesis in wild-type and ZaSCL3-overexpressing plants;

[0031] Figure 10 Comparison of stamen morphology between wild type and ZaSCL3 overexpressing plants;

[0032] Figure 11 Histological analysis of stamens of wild-type and ZaSCL3-overexpressing plants;

[0033] Figure 12 Comparison of ovary morphology between wild type and ZaSCL3 overexpressing plants;

[0034] Figure 13 This is a spatiotemporal dynamic analysis of fruit development in wild-type and ZaSCL3-overexpressing plants;

[0035] Figure 14 Histological analysis of young fruits of wild-type and ZaSCL3-overexpressing plants;

[0036] Figure 15 Analysis of IAA content in wild-type and ZaSCL3-overexpressing plants;

[0037] Figure 16 Analysis of GA4 content in wild-type and ZaSCL3-overexpressing plants;

[0038] Figure 17 Analysis of cZT content in wild-type and ZaSCL3-overexpressing plants;

[0039] Figure 18 Analysis of iPT content in wild-type and ZaSCL3-overexpressing plants;

[0040] Figure 19 Analysis of tZR content in wild-type and ZaSCL3-overexpressing plants;

[0041] Figure 20 Analysis of tZT content in wild-type and ZaSCL3-overexpressing plants. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0043] The nucleotide sequence of the ZaSCL3 gene is as follows:

[0044]

[0045] The amino acid sequence of the protein encoded by the ZaSCL3 gene is as follows:

[0046] MFKDDGSSSSVTSSSPLQFFSMMPPSLGSPYPWLKELKSEERGLYLIHLLLGCANHVANGSLENANLALDQISQLASPEGDTMQRIAAYFTEALAHRILRAWPGLHKALNSTRICLVS DEILVRKLFFDMFPFLKVAFVLTNQAIIEAMEGEKMVHVIDLHAAEPAQWIALIQALSTRPEGPPHLRITGIHPQQEVLDQMARRLTEEAEKLDIPFQFNSVVSTLGSLDFEKLRVK TGEALAISSVLQLHSLLAIDDELLQLNRHSLALKNPNGVHLPRALGELLEKDLANGYSRSPDSASSSPLSPNASVKMDSFLNSLWSLSPKIMLVTEQDSNHNGSTLMERLLEALYSY AALFDCLESTVARTSIERLKVEKMLFGEEIRNIIACEGAERKERHEKIEKWIQRFDLACFGNVPLSYCGMLQARRLLQSYNCDGYRIAEDNRCVLICWQDRPLFSVSAWRCRK(SEQ ID NO.2).

[0047] The sequence information of the primers used in the following experiments is shown in Table 1.

[0048] Table 1 Primer sequence list

[0049]

[0050] Example 1 Construction of recombinant expression vector

[0051] 1. Use heterologous recombination technology to connect the ZaSCL3 gene. The coding frame of the ZaSCL3 gene amplified using primers ZaSCL3-F (SEQ ID NO. 3) and ZaSCL3-R (SEQ ID NO. 4) is ligated into the cloning vector. Then, the coding frame of the ZaSCL3 gene is ligated into the K303 final vector through homologous recombination. The specific steps include:

[0052] (1) The full-length sequence of ZaSCL3 was amplified using primers ZaSCL3-F and ZaSCL3-R. The growth and development tree of the ZaSCL3 gene is shown in Figure 2. Figure 1 After the amplified sequence was cut and recovered, TAKARA The amplified fragment was ligated with the intermediate vector 8GWN after double digestion with NotⅠ and Sbfi using the HD Cloning Kit. The transformed DH5α strain was plated on a plate containing 100 μg / mL spectinomycin for screening. Monoclonal strains were selected and colony PCR was performed. The strain that amplified the target fragment was sent for sequencing to obtain a positive strain (8GWN-ZaSCL3).

[0053] (2) The positive strain was shaken to extract the plasmid, which was used as a template for sequence amplification using primers ZaSCL3-F and ZaSCL3-R. The plasmid with the correct amplified band was homologously recombined with the plant expression vector CaMV35S-K303 using the Gateway LR Clonase II kit. The product was transformed into DH5α strain and spread on a plate containing 100 μg / mL kanamycin for screening. Monoclonal strains were selected and colony PCR was performed. The strains that amplified the target band and whose fragment size was consistent with the expected size were sequenced. After correct sequencing, they were identified as positive strains, and the recombinant vector K303-ZaSCL3 was constructed.

[0054] 2. Transformation of microorganisms: Prepare engineered bacteria (Agrobacterium tumefaciens GV3101).

[0055] The obtained positive strain was shaken, and the plasmid K303-ZaSCL3 plasmid, which is the recombinant expression vector, was transformed into Agrobacterium tumefaciens GV3101. Single clones were screened and selected on a plate containing 100 μg / mL kanamycin and 50 μg / mL rifampicin for colony PCR verification. The strain that passed the verification was the engineered bacterium containing the recombinant expression vector (K303-ZaSCL3).

[0056] Example 2 Obtaining transgenic plants

[0057] 1. Transform tomato explants with Agrobacterium containing K303-ZaSCL3 by the leaf disc method. The specific steps are as follows:

[0058] (1) Seed disinfection and culture: Place an appropriate amount of wild-type tomato seeds in a sterile tissue culture flask. Add 75% anhydrous ethanol for 30 seconds and rinse three times with sterile water. Then, add 1.5% sodium hypochlorite solution for 15 minutes and rinse five times with sterile water. Place the flask in a shaker and shake until the seeds germinate. Finally, spread the germinated seeds evenly on pre-prepared MS solid culture medium and culture in an artificial climate chamber.

[0059] (2) Explant cutting: After about 14-20 days of culture, when the first pair of true leaves grow to about 2-5 mm, cut the cotyledons and hypocotyls into segments and place them in the pre-culture medium in the dark for 1 day.

[0060] (3) Activation and infection of Agrobacterium: Add 50-60 μL of pre-stored Agrobacterium glycerol culture to 20 mL of LB liquid medium containing antibiotics (20 μL Kan and 20 μL Rif), and then place in a 28°C constant temperature shaker at 200 rpm for 1-2 days. Shake and culture until the OD 600 = around 1.0, secondary activation to OD 600 = 0.8, remove the bacterial solution and centrifuge at 6000 rpm for 10 min to collect the bacteria. Then resuspend the bacterial solution in KCMS liquid medium to OD 600 = 0.1. Finally, the explants pre-cultured for 1 day were soaked in the diluted Agrobacterium solution for 20 minutes, the solution was aspirated with filter paper, and then the explants were transferred to the co-culture medium and co-cultured with Agrobacterium for 2 days.

[0061] (4) Differentiation culture and rooting culture: Transfer the co-cultured explants to differentiation culture medium and place them in a light incubator for culture. Replace the differentiation culture medium every half a month. Figure 2 ), cut the seedlings and transfer them to the rooting medium. Figure 3 ) can be transferred to the smart greenhouse for hardening, the conditions are: the light intensity during the day is 250μmol·m -2 ·s -1 , the light duration is 16h, the night duration is 8h; the temperature is 22℃, the relative humidity is 80%, water on time and irrigate with nutrient solution regularly.

[0062] 2. PCR identification and screening were performed on the positive seedlings to obtain transgenic lines. Using K303-F (SEQ ID NO. 5) as the forward primer and ZaSCL3-R (SEQ ID NO. 4) as the rear primer, and then using T0 generation plants as materials, the transgenic positive plants (ZaSCL3-OX1, ZaSCL3-OX4 and ZaSCL3-OX6) were identified by direct PCR. The identification results were as follows: Figure 4 A band of approximately 1400 bp was amplified in all three strains, which is consistent with the length of the ZaSCL3 gene sequence.

[0063] 3. Quantitative analysis of ZaSCL3 overexpression lines (ZaSCL3-OX1, ZaSCL3-OX4 and ZaSCL3-OX6) revealed that ( Figure 5 ), no background expression of ZaSCL3 was detected in the wild type, while the ZaSCL3 gene was overexpressed to varying degrees in the transgenic plants, indicating that the exogenous gene had been successfully integrated and achieved efficient and specific expression in the transgenic plants.

[0064] Example 3 Phenotypic Identification of Transgenic Plants Overexpressing the ZaSCL3 Gene

[0065] 1. Compare the plant types of WT and transgenic plants.

[0066] like Figure 6-Figure 8 As shown in the figure, in wild-type (WT) tomato plants, the angle between the leaves and the main stem is mainly distributed between 45° and 60°, showing a typical natural growth morphology. In contrast, the leaf angle of ZaSCL3-overexpressing plants is significantly increased, with most leaves being nearly perpendicular to the main stem (approximately 90°) or even slightly drooping (>90°), giving the overall plant shape a more stretched shape. The outward expansion of the main branches can reduce the overlap between leaves, improve the light absorption capacity of the group, and help improve photosynthetic efficiency.

[0067] 2. Compare the morphological characteristics of floral organs between WT and transgenic plants.

[0068] like Figure 9 As shown in Figure 2, compared with wild-type (WT) tomato plants, sepals of ZaSCL3 transgenic plants became longer; stamen development was significantly delayed and continued to be wrapped by sepals until the ovary expanded and was exposed through the cracking of the sepal apex. Figure 10 As shown in Figure 2, after the petals unfolded, the stamens of the WT plants turned bright yellow, while the stamens of the transgenic plants remained green. Figure 11 ) analysis further revealed that the anthers of WT plants contained a large number of normal, fertile pollen grains, while the anther locules of transgenic plants were completely devoid of mature pollen grains. These results suggest that overexpression of ZaSCL3 may cause male sterility by affecting anther development and pollen formation.

[0069] 3. Compare the morphological characteristics of ovaries and fruits between WT and transgenic plants.

[0070] Through stereomicroscope observation, it was found that the transgenic plants showed obvious parthenocarpy characteristics, and their ovaries could spontaneously swell without pollination ( Figure 12 ), fruit shape changes, loss of gelatin, seedless ( Figure 13 ). Through tissue section ( Figure 14 ) observed abnormal megaspore mother cells in transgenic fruits, with no mature embryo sac formed and ovules aborted, suggesting that ZaSCL3 is involved in fruit development and triggers apomixis.

[0071] Example 4 Determination of plant hormone content in transgenic plants after overexpression of ZaSCL3 gene

[0072] The phytohormone content of wild-type and transgenic plants was analyzed by LC-MS / MS. Figures 15-20As shown in the results, compared with the wild type, overexpression of ZaSCL3 significantly increased the levels of cytokinins (cZT, iPT, and tZR) in both lines, while significantly decreased the levels of auxin IAA, gibberellin GA4, and tZT in both lines. This suggests that ZaSCL3 induces male sterility and apomixis by regulating the levels of auxin, gibberellin, and cytokinin.

Claims

1. A ZaSCL3 gene for regulating plant growth and development, characterized in that: The nucleotide sequence of the ZaSCL3 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the ZaSCL3 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A plasmid comprising the ZaSCL3 gene according to claim 1. An overexpression vector comprising the ZaSCL3 gene according to claim 1 .

5. An engineered bacterium comprising the ZaSCL3 gene according to claim 1.

6. A preparation for regulating plant growth and development, characterized in that: The preparation contains one or more of the following: a. The ZaSCL3 gene according to claim 1; b. The plasmid according to claim 3; c. The overexpression vector according to claim 4; d. The engineered bacteria according to claim 5.

7. Use of the ZaSCL3 gene according to claim 1 in regulating plant growth and development.

8. The use according to claim 7, characterized in that The application is to achieve regulation of plant growth and development by overexpressing the ZaSCL3 gene.

9. The use according to claim 8, characterized in that The growth and development specifically includes: an increase in the angle of plant leaves, male sterility of the plant, and apomixis.

10. The use according to any one of claims 7 to 9, characterized in that: The plant is a dicotyledonous plant.

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