Application of AhPODS gene in increasing seed volume

Abstract

The present invention relates to the field of genetic engineering technology, and particularly to the application of AhPODS gene in increasing seed volume. The present invention creates an overexpressing plant of vector pCAMBIA1307 with the target gene through gene overexpression technology, thereby increasing the size of plant seeds. AhPODS ​

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of the 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 the growth rate and duration of vegetative growth of plants. The size of plant seeds is of great significance in the growth and development of plants. The size of seeds directly affects the nutrient reserves and growth potential of plants. Larger seeds contain relatively more nutrients and reserve substances, which is beneficial to the growth and development of plant seedlings, thereby enhancing the competitiveness of plants. The size of seeds is also closely related to the reproductive ability and adaptability of plants. Under adverse environmental conditions, the survival rate and growth rate of larger seeds are relatively higher, which is beneficial to the survival and reproduction of plants. Seed quality affects the size of other plant organs through a cascade effect during the ontogenetic process. For example, larger 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 a larger overall mature plant. There is a positive correlation between seed quality and seedling size at the intra- and inter-specific levels, as well as a proportional relationship between organ size and overall size. In addition, in a 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 the ontogenetic process and grow into larger mature plants. Therefore, increasing the size of seeds is very necessary for seed quality and plant size. In breeding production, it can effectively improve the yield traits of agriculture. Crop molecular breeding is a new breeding method different from traditional breeding methods. It is mainly based on the combination of biological genetics theory and related advanced scientific technologies. 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 the organism can be achieved, that is, cultivating new varieties that meet human requirements. Transgenic breeding is to introduce the desired genes into the recipient genes through some special means. Molecular design breeding is to find the varieties that best meet people's requirements through continuous practice, and finally determine the genes and propose reasonable breeding methods.

[0003] The gene overexpression technology (Overexpression) is to construct the target gene downstream of a constitutive expression promoter, a tissue-specific promoter, or the promoter of the target gene itself, and transfer it into plants to achieve the purpose of increasing the gene expression level. In plant research, it is of great significance for studying the functions of unknown genes, regulating the expression of known functional genes, and complementing mutant gene functions. So far, this technology has been widely used to overexpress genes in crops such as rice, corn, peanuts, and rapeseed, upregulating the gene expression higher than the original natural expression level. After overexpressing a certain gene, it may inhibit or activate a certain phenotype of biological characteristics, which is often the desired type. Of course, there may also be ineffective or other situations. Being able to observe the phenotype after the correct overexpression of the gene, obtaining beneficial agronomic traits, and increasing the yield of crops provide new ideas for crop variety improvement.

[0004] Gibberellin also plays a promoting role in the growth process of plants, especially in promoting root cell proliferation and cell expansion. In addition, the gibberellin signaling pathway also has important regulatory roles in the adaptation and resistance of plants to stress, and the defense response of plants to pathogenic bacteria, so as to ensure the survival of plants under stress. 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 important regulatory roles in pollen development, fruit setting, and fruit development. There are many enzyme genes in the plant genome involved in the control of seed size. For example, the process of converting the non-active components GA12 and GA53 of gibberellin into the active components GA1 and GA4 requires the catalytic activities of GA20ox and GA3ox to complete. AhPODS is the gibberellin 3-β-dioxygenase 1 gene in peanuts, and it is still unknown whether this gene plays a regulatory role in seed size. Summary of the Invention

[0005] To solve the above problems, the present invention provides the application of the AhPODS gene in increasing seed volume.

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

[0007] Preferably, the AhPODS gene is overexpressed to increase the volume of plant seeds.

[0008] Preferably, the reagent for overexpressing the AhPODS gene includes the pCAMBIA1307 plant binary expression vector with an MYC tag;

[0009]

[0010] Preferably, the reagent for overexpressing the AhPODS gene further comprises PrimeSTAR ® GXL DNA polymerase and restriction enzymes Eco R I, restriction enzymes Bam H I and T4 ligase.

[0011] 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 as SEQ ID NO.2 to SEQ ID NO.3.

[0012] Preferably, the method for overexpressing the AhPODS gene is to ligate the linearized pCAMBIA1307 plant binary expression vector with the MYC tag and the digested target gene using T4 ligase, transfer the ligation product into Escherichia coli competent cells, culture the transformed cells on a medium, and screen for positive clones with the overexpression vector carrying the AhPODS gene;

[0013] Extract the plasmid of the positive bacterial liquid with the overexpression vector carrying the AhPODS gene, and then transfer the plasmid into Agrobacterium tumefaciens for infecting to obtain transgenic plants and upregulate the expression level of the AhPODS gene.

[0014] Preferably, use restriction enzymes Bam H I and Hind III to digest the pCAMBIA1307 plant binary expression vector with the MYC tag to obtain the linearized pCAMBIA1307 plant binary expression vector with the MYC tag.

[0015] Preferably, the plant is tobacco.

[0016] Download the AhPODS sequence information of the gene according to the database PeanutBase, design specific primers with restriction sites using Snap gene software, and use the cDNA of peanut line C18 as a template to amplify AhPODS the coding sequence. Construct the amplified fragment into the intermediate vector QVB3 to construct the recombinant vector QVB3- AhPODS vector, and use Bam H I and Hind III to perform double digestion on it and run gel electrophoresis to recover the target fragment. At the same time, use Bam H I and HindIII was digested with two restriction endonucleases to linearize it and the vector fragment was recovered. Subsequently, Agrobacterium tumefaciens GV3101 competent cells were used for transformation. The target recombinant plasmid DNA was mixed with Agrobacterium tumefaciens GV3101 competent cells, and the bacterial solution was thoroughly mixed. An LB culture medium without antibiotics was added to the mixture, and then it was recovered and cultured on a shaker at 28 °C for 2 h to 3 h. Finally, the culture was spread on a solid medium containing the corresponding antibiotics for screening. Subsequently, the Agrobacterium-mediated transformation technology was adopted. This technology refers to inserting the target gene into the modified T-DNA region, and realizing the transfer and integration of foreign genes into plant cells by means of the infection of Agrobacterium tumefaciens, and then regenerating transgenic plants through cell and tissue culture techniques. Using the overexpression vector technology and Agrobacterium-mediated infection and transformation technology, transgenic materials of model crops were obtained indirectly and those of target crops were obtained directly.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention discovers AhPODS plays an important role in increasing the size of plant seeds.

[0019] The present invention creates an overexpression vector carrying the target gene through gene overexpression technology AhPODS and then cultivates plant varieties with large seeds. Experiments show that through gene overexpression technology, a pair of primers with high specificity and restriction enzyme sites are designed according to the AhPODS CDS sequence described in the present invention. Using the cDNA reverse transcribed from the RNA of the Changhua 18 variety as a template, the CDS sequence was specifically amplified, and the linearized vector was ligated with the target fragment using T4 ligase, so that AhPODS gene overexpression can increase the size of tobacco seeds. It shows that through gene overexpression technology, the size of tobacco seeds can be genetically improved using the AhPODS gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows AhPODS 1050 bases of the coding region sequence.

[0021] Figure 2 shows AhPODS a sequence encoding 349 amino acids.

[0022] Figure 3 is a schematic diagram of the gene overexpression vector pCAMBIA1307.

[0023] Figure 4 is the sequencing map of the gene overexpression AhPODS vector.

[0024] Figure 5Transgenic tobacco plants, where A is WT, B is OE-1, and C is OE-2.

[0025] Figure 6 Gel image for positive identification of T1 generation tobacco seedlings. From left to right, the first and second columns are 2 negative controls, the third to twelfth columns are the results of 10 tobacco seedlings of T1 generation, and the thirteenth column is Marker.

[0026] Figure 7 Tobacco seeds, where A is WT, B is OE-1, C is OE-2, and the scale bar is 1000 μm.

[0027] Figure 8 Phenotypic analysis of tobacco overexpressing the target gene. Hypocotyl elongation of tobacco seeds in the dark environment, where A is WT, B is OE-1, and C is OE-2. Detailed implementation mode

[0028] The following is a detailed description of the specific implementation mode of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation mode. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0029] Example 1

[0030] AhPODS Construction and genetic transformation of gene overexpression vector

[0031] Using AhPODS A pair of specific primers was 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.

[0032] AhPODS The base sequence of the coding region of Figure 1 is as shown AhPODS The amino acid sequence encoded is as shown Figure 2 is as shown AhPODSThe amino acid sequence encoded by the gene is: MATSESCAVETKTLPIQLPLDFSSIQSVPESHAWPESNEDQVENNGDGSLVLPIIDLNDPKAMELIGYACENWGAFQLKNHGISKSVIEELEVETKRLFDLPKEQKLKALRSRDNPTGYGTFWITPFFQQRMWQEGFTIIASAVQDAKKIWPNDYQRFCDAMKKFEDESRVLIEKLIHLSFKFLGISEEEEKNWVGPNNHAGAIQLNSYPICPKPENAMGIAPHTDTSIFTLLHQSQSSGLHIFKDGSGWFTVPLVPDTIVINTGDVLHMLSNARFKSALHKVSVNNVKHRYSMVYFYRPTMDQVVSPLVPSNNSDEEPRFRALTFKEFVGIKDKYLDKALSIVSVKED, denoted as SEQ ID NO.1.

[0033] The nucleotide sequences of a pair of specific primers are:

[0034] AhPODS- EcoRI-F: ccgGAATTCATGGCGACAAGTGAATCTTGT, denoted as SEQ ID NO.2.

[0035] AhPODS- BamHI-R: cgcGGATCCATCTTCTTTGACGCTAACAATTG, denoted as SEQ ID NO.3.

[0036] Table 1 PCR reaction system

[0037]

[0038] Table 2 PCR amplification program

[0039]

[0040] Clone the amplified target gene into pCAMBIA1307 to construct the gene overexpression vector pCAMBIA1307- AhPODS , specifically:

[0041] Construct the amplified target gene fragment into the intermediate vector QVB3 to construct the recombinant vector QVB3- AhPODS , using Bam H I and HindIII and two restriction enzymes were used to linearize the vector in a 50 μL digestion reaction system in a PCR instrument. The reaction conditions were carried out at 37 °C for 1.5 h, and the recombinant vector was digested with double enzymes and run on a gel to recover the target fragment. Using the same digestion reaction system, the pCAMBIA1307 plant binary expression vector with the MYC tag was simultaneously digested and linearized with Bam H I and Hin dIII two restriction endonucleases, and the linearized vector fragment was recovered. Using the T4 ligase from Takara (China) company, the linearized vector fragment was ligated with the target fragment to obtain the target recombinant plasmid, namely AhPODS the gene overexpression vector. Among them, the schematic diagram of the pCAMBIA1307 vector is as shown in Figure 3 , and the system for the ligation process is shown in Table 3:

[0042] Table 3 Ligation system

[0043]

[0044] Reaction conditions: 4 °C for 12 h.

[0045] The target recombinant plasmid was transferred into Escherichia coli, and positive clones with the overexpression vector of the AhPODS gene were screened out:

[0046] (1) Take 100 µL of competent cells melted on ice, add the target recombinant plasmid, mix gently, and let it stand on ice for 30 min.

[0047] (2) Heat shock in a 42 °C water bath for 50 s, quickly transfer to an ice bath, and let it stand for 2 min. Do not shake the sample during the standing on ice, otherwise the transformation efficiency will be reduced. It should be noted that heat shock in a 42 °C water bath for 45 s - 60 s can achieve similar effects.

[0048] (3) Add 700 µL of sterile liquid LB medium without antibiotics to the centrifuge tube, mix well, and incubate at 37 °C and 200 rpm for 60 min to obtain the liquid medium that has been transformed with the target recombinant plasmid after resuscitation.

[0049] (4) According to the experimental needs, pipette 50 µL of the liquid medium that has been transformed with the target recombinant plasmid after resuscitation in step (3) and spread it evenly on the LB solid medium containing kanamycin resistance. Invert the plate and place it in a 37 °C incubator overnight.

[0050] (5) The sequencing diagram of the gene overexpression AhPODS vector is as shown in Figure 4 , and positive clones with the overexpression vector of the AhPODS gene were screened out.

[0051] The plasmid of the positive bacterial liquid with the overexpression vector carrying the AhPODS gene was introduced into Agrobacterium tumefaciens GV3101.

[0052] The steps of introducing the plasmid of the positive bacterial liquid with the overexpression vector carrying the AhPODS gene into Agrobacterium tumefaciens GV3101 are as follows: Transformation was carried out using Agrobacterium tumefaciens GV3101 competent cells. First, 1 μL of the positive plasmid DNA of the overexpression vector carrying the AhPODS gene was mixed with 100 μL of Agrobacterium tumefaciens GV3101 competent cells, and the bacterial liquid was thoroughly mixed. Then, it was left standing on ice for 5 min, followed by treatment with liquid nitrogen for 5 min, immediately followed by treatment in a water bath at 37°C for 5 min, and then placed on ice again for 5 min to cool. After completing these steps, 700 μL of antibiotic-free LB culture medium was added to the mixture, and then it was recovered and cultured on a shaker at 28°C for 2 h. Finally, the culture was spread on a solid medium containing the corresponding antibiotic for screening.

[0053] Example 2

[0054] AhPODS Creation and identification of tobacco materials with gene overexpression

[0055] Select tobacco leaves with a growth period of 30 days, healthy and young as explants. Genetic transformation of tobacco was obtained by the leaf disc method AhPODS transgenic tobacco materials, and Nicotiana benthamiana was selected as the transformation background material. The specific experimental steps are as follows:

[0056] (1) Disinfect the seeds with a 2.5% mass fraction sodium hypochlorite solution for 8 min, and then disinfect with a 75% volume fraction absolute ethanol solution for 1 min.

[0057] (2) On the ultra-clean workbench, wash the seeds with sterile water 5 times, and let them stand for 3 min after each wash.

[0058] (3) Sow them in a sterile culture tank and culture them into seedlings.

[0059] (4) Through the Agrobacterium-mediated transformation method, Agrobacterium carrying the target gene was used to infect tobacco leaf cells.

[0060] (5) The transformed leaves were placed in SIM medium for co-culture, and then transferred to the screening medium. Under the white light conditions of 26°C and a light intensity of 60 μE / m 2 / s, cultured under a 14 h dark / 10 h light cycle condition. After culture, white anti-callus would be produced on the tobacco leaves, and then transferred to SEM medium for differentiation into seedlings.

[0061] Among them, each liter of SIM medium contains: 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 is added to make up to 1 L;

[0062] 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 is added to make up to 1 L;

[0063] M524 is Murashige & Skoog Basal Salt Mixture purchased from phytotech, Lot: HEW0524259A, packaging size: 100 L.

[0064] Transfer the tobacco obtained by tissue culture to the soil mixed with nutrient soil and vermiculite in a ratio of 1:1. After it survives, take tobacco tissue to extract DNA, and use AhPODS specific primers for PCR amplification to identify positive transformed lines. Continue to culture the identified transgenic lines in the greenhouse until the plants flower and bear fruit. The transgenic lines are respectively denoted as OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, OE-8, OE-9, OE-10. Select 2 better transgenic plants, namely OE-1 and OE-2 as Figure 5 shown.

[0065] Dry the harvested AhPODS transgenic tobacco T1 seeds and vernalize them in a 4°C refrigerator. After vernalization, disinfect, wash the seeds and sow them on the MS medium containing 25 μg / mL hygromycin, and culture them in an environment of 25°C, 16 h light / 8 h dark period. After the tobacco grows mature, transfer it to the nutrient soil for cultivation to ensure appropriate moisture and nutrient conditions, and harvest the mature T2 generation seeds.

[0066] Result: The gel picture of the positive identification of tobacco seedlings in the T1 generation is as Figure 6 shown. Among the 10 obtained positive transgenic tobacco lines, 2 lines with better growth vigor were selected for observation. AhPODS Compared with the wild-type tobacco seeds of the same background, the T1 generation seeds of the overexpressed transgenic tobacco of the gene have larger seed volume, as Figure 7 shown. And it was observed that the hypocotyls of the transgenic tobacco seeds growing under dark conditions are longer than those of the wild-type seeds at the same time and under the same conditions, as Figure 8 shown.

[0067] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0068] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0069] 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 equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Application 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; the plant is tobacco; The increase in seed volume refers to the increase in both the width and thickness of the seeds.

2. The application according to claim 1, characterized in that By overexpressing the AhPODS gene, the volume of tobacco seeds is increased.

3. The application according to claim 2, characterized in that, The reagent for overexpressing the AhPODS gene includes the pCAMBIA1307 plant binary expression vector with an MYC tag; The sequence of the pCAMBIA1307 plant binary expression vector with an MYC tag is shown in SEQ ID NO.

4.

4. The application according to claim 3, wherein The reagent for overexpressing the AhPODS gene further includes PrimeSTAR ® GXL DNA Polymerase, restriction endonucleases Eco R I, restriction endonucleases Bam H I and ligase T4 ligase.

5. The application 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~SEQ ID NO.

3.

6. The application according to claim 5, wherein The method for overexpressing the AhPODS gene is to ligate the linearized pCAMBIA1307 plant binary expression vector with an MYC tag and the digested target gene using T4 ligase, transfer the ligation product into Escherichia coli competent cells, culture the transformed cells on a medium, and screen for positive clones with the overexpression vector carrying the AhPODS gene; Extract the plasmid of the positive bacterial liquid with the overexpression vector carrying the AhPODS gene, and then transfer the plasmid into Agrobacterium for infecting to obtain transgenic plants and up-regulate the expression level of the AhPODS gene.

7. The application according to claim 6, characterized in that, Use restriction endonucleases Bam H I and Hin Digest the pCAMBIA1307 binary plant expression vector with the MYC tag using d III to obtain the linearized pCAMBIA1307 binary plant expression vector with the MYC tag.

Images