Application of AhPODS gene in increasing seed volume

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an AhPODS gene to increase of seed volume. According to the invention, an overexpression plant of a vector pCAMBIA1307 with a target gene AhPODS is created through a gene overexpression technology, so that the seed size of the plant is increased.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of AhPODS gene in increasing seed volume. Background Art

[0002] The size of an organism affects its ecological interactions and its impact on ecosystem processes, and most life history traits are related to body size. Seed size affects plant growth rate and duration of vegetative growth. Plant seed size is of great significance in plant growth and development. Seed size directly affects plant nutrient reserves and growth potential. Large seeds contain relatively more nutrients and reserve substances, which is beneficial to the growth and development of plant seedlings, thereby enhancing plant competitiveness. Seed size is also closely related to plant reproductive capacity and adaptability. Under adverse environmental conditions, the survival rate and growth rate of large seeds are relatively higher, which is beneficial to the survival and reproduction of plants. Seed mass affects the size of other plant organs through cascading effects during individual development. For example, large seeds usually germinate earlier in the season and grow into larger seedlings with larger organs. Regardless of the resource acquisition rate per unit biomass or per unit time, more biomass in leaves and roots at the seedling stage has an early advantage in hoarding available resources. This initial size advantage may lead to larger leaves, thicker stems, and longer and heavier roots, resulting in larger mature plants overall. There is a positive correlation between seed quality and seedling size at both intraspecific and interspecific levels, as well as a positive proportional relationship between organ size and overall size. In addition, in the global analysis of functional traits, plant size and seed quality co-vary on the same axis of plant trait variation. Therefore, seeds that produce larger seedlings may amplify their effects during individual development and grow into larger mature plants. Therefore, increasing seed size is very necessary for seed quality and plant size. In breeding production, agricultural yield traits can be effectively improved. Crop molecular breeding is a new breeding method that is different from traditional breeding methods. It is mainly based on the combination of biogenetic theory and related advanced science and technology. According to genetic theory, the phenotype of an organism is mainly determined by its genes. By changing the genes of an organism, the purpose of changing the phenotype of an organism can be achieved, that is, to cultivate new varieties that meet human requirements. Transgenic breeding is to introduce the desired gene into the receptor gene through some special means. Molecular design breeding is to find out the variety that best meets people's requirements through continuous practice, and finally establish the gene and propose a reasonable breeding method.

[0003] Gene overexpression technology is to construct the target gene to the downstream of the constitutive expression promoter, tissue-specific promoter or the promoter of the target gene itself, and then transfer it into the plant to achieve the purpose of increasing the gene expression. In plant research, it is of great significance to study the functions of unknown genes, regulate the expression of genes with known functions, and complement the functions of mutant genes. So far, this technology has been widely used to overexpress genes in crops such as rice, corn, peanuts, and rapeseed, and to increase the expression of genes to a level higher than the original natural expression. After overexpressing a gene, a phenotype of a biological feature will be inhibited or activated, which is often the type you want to get. Of course, there will be invalid situations or other situations. The phenotype after the gene is correctly overexpressed can be observed, beneficial agronomic traits can be obtained, and the yield of crops can be increased, providing new ideas for crop variety improvement.

[0004] Gibberellins also play a promoting role in the growth of plants, especially in promoting root cell proliferation and cell expansion. In addition, the gibberellin signaling pathway also plays an important regulatory role in the adaptation and resistance of plants to adverse stress and the defense response of plants to pathogens, thereby ensuring the survival of plants under adverse conditions. At the same time, it can also promote the transition of plants from vegetative growth to reproductive development and the initiation of flower development, and also has an important regulatory role in pollen development, fruit setting and fruit development. There are many enzyme genes in the genome of plants that are involved in the control of seed size. For example, the process of converting the inactive gibberellin components GA12 and GA53 into active components GA1 and GA4 requires the catalytic activity of GA20ox and GA3ox to complete. AhPODS is the gibberellin 3-β-dioxygenase 1 gene in peanuts. It is still unknown whether this gene plays a regulatory role in seed size. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of AhPODS gene in increasing seed volume.

[0006] The application of AhPODS gene in increasing seed volume, the amino acid sequence encoded by the AhPODS gene is shown in SEQ ID NO.1; the seeds are plant seeds.

[0007] Preferably, the seed volume of the plant is increased by overexpressing the AhPODS gene.

[0008] Preferably, the reagent for overexpressing the AhPODS gene includes a MYC-tagged pCAMBIA1307 plant binary expression vector;

[0009] Preferably, the reagent for overexpressing the AhPODS gene further comprises PrimeSTAR ® GXL DNA polymerase, restriction endonuclease Eco RI, restriction endonuclease Bam HI and T4 ligases.

[0010] Preferably, the reagent for overexpressing the AhPODS gene further comprises specific primers for amplifying the AhPODS gene, and the sequences of the specific primers are shown in SEQ ID NO.2 to SEQ ID NO.3.

[0011] Preferably, the method for overexpressing the AhPODS gene is to connect the linearized MYC-tagged pCAMBIA1307 plant binary expression vector and the target gene after restriction digestion with T4 ligase, and the ligated product is transferred into Escherichia coli competent cells, and the transformed cells are cultured in a culture medium to screen out positive clones of the overexpression vector carrying the AhPODS gene; The positive bacterial liquid plasmid of the overexpression vector carrying the AhPODS gene is extracted, and then the plasmid is transferred into Agrobacterium to infect and obtain transgenic plants, thereby upregulating the expression level of the AhPODS gene.

[0012] Preferably, restriction endonucleases are used. Bam HI and Hind III. digesting the MYC-tagged pCAMBIA1307 plant binary expression vector to obtain the linearized MYC-tagged pCAMBIA1307 plant binary expression vector.

[0013] Preferably, the plant is tobacco.

[0014] Download from PeanutBase database AhPODS The sequence information of the gene was obtained, and specific primers with restriction sites were designed using Snap gene software. The cDNA of peanut strain C18 was used as a template to amplify AhPODS The amplified fragment was constructed into the intermediate vector QVB3 to construct the recombinant vector QVB3- AhPODS vector, and use Bam HI and Hind III for double enzyme digestion and gel run to recover the target fragment. Bam HI and Hind III two restriction endonucleases to linearize it and recover the vector fragment. Subsequently, Agrobacterium GV3101 competent cells were used for transformation, and the target recombinant plasmid DNA was mixed with Agrobacterium GV3101 competent cells, and the bacterial solution was fully mixed. LB culture medium without antibiotics was added to the mixture, and then the culture was resumed on a shaker at 28°C for 2h~3h. Finally, the culture was spread on a solid culture medium containing the corresponding antibiotics for screening. Agrobacterium-mediated transformation technology was used later, which refers to the technology of inserting the target gene into the modified T-DNA region, using Agrobacterium infection to achieve the transfer and integration of exogenous genes into plant cells, and then regenerating transgenic plants through cell and tissue culture technology. Using overexpression vector technology and Agrobacterium infection transformation technology, model crops are obtained in disguise and transgenic materials of target crops are directly obtained.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention finds AhPODS Plays an important role in increasing plant seed size.

[0016] The present invention creates a target gene by gene overexpression technology. AhPODS The experiment showed that the gene overexpression technology according to the present invention AhPODS A pair of highly specific primers with restriction sites were designed based on the CDS sequence of the Changhua 18 variety. The cDNA reverse transcribed from the RNA was used as a template to specifically amplify the CDS sequence. The linearized vector was ligated with the target fragment using T4 ligase to make AhPODS Gene overexpression can increase tobacco seed size. AhPODS Genes can be used to genetically improve tobacco seed size. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 show AhPODS The coding region sequence is 1050 bases.

[0018] Figure 2 show AhPODS A sequence encoding 349 amino acids.

[0019] Figure 3 Schematic diagram of gene overexpression vector pCAMBIA1307.

[0020] Figure 4 This is the sequencing map of the gene overexpression AhPODS vector.

[0021] Figure 5are transgenic tobacco plants, among which A is WT, B is OE-1, and C is OE-2.

[0022] Figure 6 This is a gel image of the positive identification of the T1 generation of tobacco seedlings. From left to right, the first and second columns are 2 negative controls, the third to twelfth columns are the results of the T1 generation of 10 tobacco seedlings, and the thirteenth column is the Marker.

[0023] Figure 7 These are tobacco seeds, where A is WT, B is OE-1, and C is OE-2. The scale bar is 1000 μm.

[0024] Figure 8 Phenotypic analysis of tobacco overexpressing the target gene. The elongation of the hypocotyl of tobacco seeds in a dark environment. A is WT, B is OE-1, and C is OE-2. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0026] Embodiment 1 AhPODS Construction of gene overexpression vector and genetic transformation use AhPODS A pair of specific primers were designed based on the specific DNA sequence of the gene coding region to amplify the sequence of the target gene. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2.

[0027] AhPODS The coding sequence bases are as follows Figure 1 As shown, AhPODS The amino acid sequence is Figure 2 As shown, AhPODSThe sequence of the amino acids encoded by the gene is: MATSESCAVETKTLPIQLPLDFSSIQSVPESHAWPESNEDQVENNGDGSLVLPIIDLNDPKAMELIGYACENWGAFQLKNHGISKSVIEELEVETKRLFDLPKEQKLKALRSRDNPTGYGTFWITPFFQQRMWQEGFTIIASAVQDAKKIWPNDYQRFCDAMKKFEDESRV LIEKLIHLSFKFLGISEEEEKNWVGPNNHAGAIQLNSYPICPKPENAMGIAPHTDTSIFTLLHQSQSSGLHIFKDGSGWFTVPLVPDTIVINTGDVLHMLSNARFKSALHKVSVNNVKHRYSMVYFYRPTMDQVVSPLVPSNNSDEEPRFRALTFKEFVGIKDKYLDKALSIVSVKED, recorded as SEQ ID NO.1.

[0028] The nucleotide sequences of a pair of specific primers are: AhPODS - EcoRI-F: ccgGAATTCATGGCGACAAGTGAATCTTGT, recorded as SEQ ID NO.2.

[0029] AhPODS - BamHI-R: cgcGGATCCATCTTCTTTGACGCTAACAATTG, recorded as SEQ ID NO.3.

[0030] Table 1 PCR reaction system

[0031] Table 2 PCR amplification program

[0032] The amplified target gene was cloned into pCAMBIA1307 to construct the gene overexpression vector pCAMBIA1307- AhPODS , specifically: The amplified target gene fragment was constructed into the intermediate vector QVB3 to construct the recombinant vector QVB3- AhPODS ,use Bam HI and Hind III two restriction enzymes, set up 50 μL of restriction digestion reaction system in the PCR instrument to linearize the vector, the reaction condition is 37℃ for 1.5 h, and the recombinant vector is double-digested and run on the gel to recover the target fragment. Using the same restriction digestion reaction system, the pCAMBIA1307 plant binary expression vector with MYC tag was also cloned into Bam HI and Hin The linearized vector fragment was then ligated with the target fragment using T4 ligase from Takara (China) to obtain the target recombinant plasmid, i.e. AhPODS Gene overexpression vector. The schematic diagram of pCAMBIA1307 vector is as follows Figure 3 , the system of the connection process is shown in Table 3: Table 3 Connection system

[0033] Reaction conditions: 4℃ for 12 h.

[0034] The target recombinant plasmid was transferred into E. coli, and positive clones of the overexpression vector carrying the AhPODS gene were screened out: (1) Take 100 µL of competent cells melted on ice, add the target recombinant plasmid, mix gently, and let stand on ice for 30 minutes.

[0035] (2) Heat shock in a 42℃ water bath for 50 s, quickly transfer to an ice bath, and let stand for 2 min. Do not shake the sample during the ice bath, otherwise the transformation efficiency will be reduced. It should be noted that heat shock at 42℃ water bath for 45 s to 60 s can achieve similar results.

[0036] (3) Add 700 µL of sterile liquid LB medium without antibiotics to the centrifuge tube, mix well, and resuscitate at 37°C, 200 rpm for 60 min to obtain the resuscitated liquid medium that has been transformed with the target recombinant plasmid.

[0037] (4) According to experimental needs, take 50 µL of the liquid culture medium that has been transformed with the target recombinant plasmid after resuscitation in step (3) and evenly spread it on the LB solid culture medium containing kanamycin resistance, and place the plate upside down in a 37°C incubator for overnight culture.

[0038] (5) Sequencing diagram of gene overexpression AhPODS vector Figure 4 As shown, positive clones carrying the overexpression vector of AhPODS gene were screened.

[0039] The positive bacterial liquid plasmid carrying the AhPODS gene overexpression vector was introduced into Agrobacterium tumefaciens GV3101.

[0040] The steps for introducing the positive bacterial liquid plasmid of the overexpression vector with the AhPODS gene into Agrobacterium tumefaciens GV3101 are as follows: Use Agrobacterium GV3101 competent cells for transformation. First, mix 1 μL of the positive plasmid DNA of the overexpression vector with the AhPODS gene with 100 μL of Agrobacterium GV3101 competent cells, and mix the bacterial liquid thoroughly. After that, let it stand on ice for 5 min, then treat it with liquid nitrogen for 5 min, then treat it in a 37℃ water bath for 5 min, and place it on ice again for 5 min to cool. After completing these steps, add 700 μL of LB culture medium without antibiotics to the mixture, and then resume culture on a shaker at 28℃ for 2 h. Finally, spread the culture on a solid medium containing the corresponding antibiotics for screening.

[0041] Embodiment 2 AhPODS Creation and identification of gene overexpression tobacco materials Healthy and tender tobacco leaves with a growth period of 30 days were selected as explants. Genetic transformation of tobacco was carried out using the leaf disc method. AhPODS Transgenic tobacco materials, Nicotiana benthamiana was selected as the transformation background material. The specific experimental steps are as follows: (1) Sterilize the seeds with a 2.5% sodium hypochlorite solution for 8 min, and then sterilize them with a 75% anhydrous ethanol solution for 1 min.

[0042] (2) On a clean bench, wash the seeds five times with sterile water and let them stand for 3 minutes after each wash.

[0043] (3) Sow seeds in sterile culture tanks and grow into seedlings.

[0044] (4) Through the Agrobacterium-mediated transformation method, Agrobacterium carrying the target gene is infecting tobacco leaf cells.

[0045] (5) The transformed leaves were placed in SIM medium for co-cultivation and then transferred to screening medium at 26°C and a light intensity of 60 μE / m 2 / s white light conditions, 14 h dark / 10 h light cycle. After cultivation, tobacco leaves will produce white anti-callus tissue, which will then be transferred to SEM medium for differentiation into seedlings.

[0046] Each liter of SIM medium contained: 4.3 g of M524, 30.0 g of sucrose, 20 μmol of 6-BA, 2.0 mg of 2.4-D, 5.0 μL of 0.5 mol / L NaOH, and double distilled water to 1 L; Each liter of SEM medium contains: 4.3 g of M524, 30.0 g of sucrose, 2 μmol of 6-BA, 2.0 mg of 2.4-D, 5.0 μL of 0.5 mol / L NaOH, and double distilled water to 1 L; M524 is Murashige & Skoog Basal Salt Mixture purchased from phytotech, Lot: HEW0524259A, packaging size: 100L.

[0047] The tobacco obtained by tissue culture was transferred to soil mixed with nutrient soil and vermiculite in a ratio of 1:1. After the tobacco survived, DNA was extracted from tobacco tissues, and PCR amplification was performed using AhPODS-specific primers to identify positive transformation strains. The identified transgenic strains were continued to be cultured in a greenhouse until the plants bloomed and bore fruit. The transgenic strains were recorded as OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, OE-8, OE-9, and OE-10, and two better transgenic plants were selected, namely OE-1 and OE-2. Figure 5 shown.

[0048] The collected AhPODS transgenic tobacco T1 seeds were dried and placed in a 4°C refrigerator for vernalization. After vernalization, the seeds were disinfected, cleaned, and sown on MS medium containing 25 μg / mL hygromycin and cultured at 25°C, 16 h light / 8 h dark. When the tobacco grew to maturity, it was moved to nutrient soil for culture to ensure appropriate water and nutrient conditions, and mature T2 seeds were harvested.

[0049] Results: The positive identification of tobacco seedlings T1 generation is shown in the figure Figure 6 As shown, among the 10 positive transgenic tobacco lines obtained, two lines with better growth were selected for observation. AhPODS Compared with wild-type tobacco seeds of the same background, the T1 generation seeds of gene overexpression transgenic tobacco have larger size, such as Figure 7 It was also observed that the hypocotyls of transgenic tobacco seeds grown under dark conditions were longer than those of wild-type seeds grown under the same period and conditions, as shown in Figure 2. Figure 8 shown.

[0050] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0051] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0052] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of AhPODS gene in increasing seed volume, characterized in that: The amino acid sequence encoded by the AhPODS gene is shown in SEQ ID NO.1; the seeds are plant seeds.

2. The use according to claim 1, characterized in that: By overexpressing the AhPODS gene, the seed volume of plants is increased.

3. The use according to claim 2, characterized in that: The reagent for overexpressing the AhPODS gene includes a MYC-tagged pCAMBIA1307 plant binary expression vector; The sequence of the MYC-tagged pCAMBIA1307 plant binary expression vector is shown in SEQ ID NO.

4.

4. The use according to claim 3, characterized in that: The reagent for overexpressing the AhPODS gene also includes PrimeSTAR ® GXL DNA polymerase, restriction endonuclease Eco RI, restriction endonuclease Bam HI and T4 ligases.

5. The use according to claim 4, characterized in that: The reagent for overexpressing the AhPODS gene also includes specific primers for amplifying the AhPODS gene, and the sequences of the specific primers are shown in SEQ ID NO.2 to SEQ ID NO.

3.

6. The use according to claim 5, characterized in that: The method for overexpressing the AhPODS gene is to connect the linearized MYC-tagged pCAMBIA1307 plant binary expression vector and the target gene after enzyme digestion with T4 ligase, and the ligated product is transferred into Escherichia coli competent cells, and the transformed cells are cultured in a culture medium to screen out positive clones of the overexpression vector carrying the AhPODS gene; The positive bacterial liquid plasmid of the overexpression vector carrying the AhPODS gene is extracted, and then the plasmid is transferred into Agrobacterium to infect and obtain transgenic plants, thereby upregulating the expression level of the AhPODS gene.

7. The use according to claim 6, characterized in that: Use restriction enzymes Bam HI and Hin The MYC-tagged pCAMBIA1307 plant binary expression vector was digested by enzyme d III to obtain the linearized MYC-tagged pCAMBIA1307 plant binary expression vector.

8. The use according to claim 1, characterized in that: The plant is tobacco.

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