CN119286925A - Application of AhPODS gene in increasing seed volume - Google Patents

Abstract

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

Description

Use of AhPODS gene in increasing seed volume

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of AhPODS genes in increasing seed volume.

Background

The size of organisms affects their ecological interactions and their effects on ecosystem processes, and most life-history features are related to body type. Seed size can affect the growth rate and nutrient growth duration of plants. The size of the plant seeds has important significance in the growth and development of plants. The size of the seeds directly affects the nutrition reserve and the growth vigor of plants, and the large seeds contain more nutrients and reserve substances, so that the growth and the development of plant seedlings are facilitated, and the competitiveness of plants is enhanced. The size of the seeds is also closely related to the reproductive capacity and fitness of the plant. Under unfavorable environmental conditions, the survival rate and the growth speed of the seeds with large volume are relatively higher, which is beneficial to the survival and the reproduction of plants. Seed quality affects the size of other plant organs through cascading effects during ontogenesis. For example, voluminous seeds usually germinate earlier in the season and grow into larger seedlings with larger organs. Regardless of the biomass per unit or resource availability per unit time, more biomass in the leaves and roots at the seedling stage has early advantages in stocking up available resources. This initial size advantage may lead to larger leaves, thicker stems and longer, heavier roots, resulting in an overall larger mature plant. There is a positive correlation between seed quality and seedling size at both intra-and inter-seed levels, as well as a direct proportional relationship between organ size and overall size. Furthermore, in global analysis of functional traits, plant size and seed quality vary together on the same axis of variation in plant traits. Thus, seeds that produce larger seedlings may amplify their effect and grow into larger mature plants during ontogeny. Increasing the size of the seeds is necessary for the quality of the seeds and the size of the plants. In breeding production, the yield traits of agriculture can be effectively improved. Crop molecular breeding is a novel breeding mode different from the traditional breeding mode. It is based mainly on a combination of biological genetic theory and related advanced science and technology. According to genetic theory, it is known that the biological phenotype is mainly determined by its gene, and by changing the gene of the organism, the purpose of changing the biological phenotype can be achieved, namely, a new variety meeting the requirements of human beings can be cultivated. Transgenic breeding is the introduction of a desired gene into a recipient gene by a number of specific means. Molecular design breeding is to find out the variety which best meets the requirements of people through continuous practice, finally establish genes and put forward a reasonable breeding method.

The gene over-expression technology (Overexpression) is to construct the target gene to the downstream of a constitutive expression promoter, a tissue specific promoter or a promoter of the target gene itself, and to realize the purpose of increasing the gene expression after transferring the target gene into plants. In plant research, the method has important significance for researching unknown gene functions, regulating and controlling the expression of known functional genes, complementing mutant gene functions and the like. Up to now, the technology has been widely used for over-expressing genes of crops such as rice, corn, peanut, rape and the like, and up-regulating the expression of the genes is higher than the original natural expression quantity. After overexpression of a gene, it will inhibit or activate a phenotype of the biological feature, which is often the desired type, but ineffective or otherwise may occur. The phenotype of the correctly over-expressed gene can be observed, the beneficial agronomic characters can be obtained, the yield of crops can be improved, and a new idea is provided for crop variety improvement.

Gibberellins also play a promoting role in the growth process of plants, particularly in promoting root cell proliferation and cell expansion. In addition, gibberellin signaling pathway also has important regulatory roles in adaptation and resistance of plants to stress and defense response of plants to pathogenic bacteria, and thus, survival of plants in stress is ensured. Meanwhile, the method can promote the transformation from vegetative growth to reproductive development and the initiation of flower development of plants, and has important regulation and control effects on pollen development, fruit setting and fruit development. There are many enzyme genes in the genome of plants involved in seed size control, such as the conversion of gibberellin inactive ingredients GA12 and GA53 to active ingredients GA1 and GA4, which require the catalytic activity of GA20ox and GA3 ox. AhPODS is gibberellin 3-beta-dioxygenase 1 gene in peanuts, and whether the gene plays a role in regulating the size of seeds is unknown.

Disclosure of Invention

In order to solve the problems, the invention provides application of AhPODS genes in increasing seed volume.

The application of AhPODS gene in increasing seed volume, the sequence of the AhPODS gene coded amino acid is shown as SEQ ID NO.1, and the seed is plant seed.

Preferably, the AhPODS gene is overexpressed to increase plant seed volume.

Preferably, the agent that overexpresses the AhPODS gene comprises a MYC-tagged pCAMBIA1307 plant binary expression vector;

The sequence of the pCAMBIA1307 plant binary expression vector of the MYC tag is ：CCATGACAGTTTAATCATTTCTTTTAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCATGGGCGAGCTCCGACGGTATCGATTTAAAGCTATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGAGCTTGGGCGACCTCACCATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAACTCGGTATCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCGGGTACTCCGCAAAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTTCTCCAGAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTGACCAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAAGGAAGGTGGCACCTACAAATGCCATCATTGGCGATAAAGGAAAGCTTATCGTTCAAGAATGCCTCTGCCGACAGGTGTCCAAGAGATGGAACCCCCACCCCACAAGAAGACATTCTGTGAGAAAT, and is recorded as SEQ ID NO.4.

Preferably, the agent for over-expressing the AhPODS gene further comprises PRIMESTAR ^® GXL DNA polymerase, restriction enzyme EcoRI, restriction enzyme BamHI and T4 ligase.

Preferably, the reagent for over-expressing AhPODS genes further comprises a specific primer for amplifying AhPODS genes, and the sequence of the specific primer is shown as SEQ ID NO. 2-SEQ ID NO. 3.

Preferably, the method for over-expressing AhPODS gene is to connect the linearized MYC tagged pCAMBIA1307 plant binary expression vector and the target gene after enzyme digestion with T4 ligase, transfer the connected product into competent cells of escherichia coli, culture the transformed cells on a culture medium, and screen out positive clones of the over-expression vector with AhPODS gene;

extracting positive bacterial liquid plasmid with AhPODS gene over-expression vector, transferring the plasmid into agrobacterium for infection to obtain transgenic plant and up-regulating AhPODS gene expression.

Preferably, the MYC tagged pCAMBIA1307 plant binary expression vector is digested with restriction enzymes bamhi and Hind III to yield the linearized MYC tagged pCAMBIA1307 plant binary expression vector.

Preferably, the plant is tobacco.

According to the sequence information of the AhPODS gene downloaded from the database PeanutBase, a specific primer with an enzyme cutting site is designed by using Snap gene software, and the cDNA of the peanut strain C18 is used as a template to amplify the coding sequence of AhPODS. The amplified fragment was constructed on the intermediate vector QVB to construct the recombinant vector QVB-AhPODS, and the target fragment was recovered by double digestion with BamH I and HindIII. Simultaneously, the pCAMBIA1307 plant binary expression vector with MYC tag is subjected to enzyme tangency by two restriction enzymes of BamH I and Hind III, and a vector fragment is recovered. Subsequently, agrobacterium GV3101 competent cells are used for transformation, and the target recombinant plasmid DNA is mixed with the agrobacterium GV3101 competent cells, and the bacterial solution is fully mixed. And (3) adding an LB culture solution without antibiotics into the mixture, and then recovering the culture on a shaking table at the temperature of 28 ℃ for 2-3 hours. Finally, the cultures were plated on solid media containing the corresponding antibiotics for selection. The technology of agrobacterium-mediated transformation is adopted, and the technology refers to the technology of inserting a target gene into a transformed T-DNA region, realizing the transfer and integration of an exogenous gene into plant cells by means of agrobacterium infection, and regenerating transgenic plants by cell and tissue culture technology. The method utilizes the over-expression vector technology and the agrobacterium infection transformation technology to obtain the mode crop with phase change and directly obtain the transgenic material of the target crop.

Compared with the prior art, the invention has the beneficial effects that:

The present invention finds AhPODS to play an important role in increasing plant seed size.

The invention creates the over-expression vector with the target gene AhPODS by the gene over-expression technology, thereby cultivating the plant variety with large seeds. Experiments show that a pair of primers with high specificity and enzyme cutting sites are designed according to the CDS sequence of AhPODS by a gene overexpression technology, cDNA of RNA reverse transcription of Changhua 18 varieties is used as a template, the CDS sequence is specifically amplified, and a T4 ligase is utilized to linearize a vector and connect with a target fragment, so that AhPODS genes are overexpressed, and the size of tobacco seeds can be improved. It is shown that the AhPODS gene can be used to genetically modify the size of tobacco seeds by gene overexpression technology.

Drawings

FIG. 1 shows the 1050 bases of the coding region sequence of AhPODS.

FIG. 2 shows AhPODS sequences encoding 349 amino acids.

FIG. 3 is a schematic representation of the gene overexpression vector pCAMBIA 1307.

FIG. 4 is a sequencing diagram of the gene over-expression AhPODS vector.

FIG. 5 is a transgenic tobacco plant, wherein A is WT, B is OE-1, and C is OE-2.

Fig. 6 is a T1 generation positive identification gel diagram of tobacco seedlings, from left to right, the first and second columns are 2 negative controls, the third to twelfth columns are 10T 1 generation results of tobacco seedlings, and the tenth column is a Marker.

FIG. 7 shows tobacco seeds, wherein A is WT, B is OE-1, C is OE-2, and the scale bar is 1000. Mu.m.

FIG. 8 is a phenotypic analysis of tobacco over-expressing a gene of interest, elongation of the hypocotyl of a tobacco seed in a dark environment, where A is WT, B is OE-1, and C is OE-2.

Detailed Description

The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified.

Example 1

Construction and genetic transformation of AhPODS Gene overexpression vectors

A pair of specific primers is designed by utilizing a AhPODS gene coding region specific DNA sequence, the sequence of a target gene is amplified, a PCR reaction system is shown in a table 1, and a PCR amplification program is shown in a table 2.

The nucleotide sequence of the coding region of AhPODS is shown in figure 1, the nucleotide sequence of AhPODS is shown in figure 2, and the nucleotide sequence of AhPODS gene is ：MATSESCAVETKTLPIQLPLDFSSIQSVPESHAWPESNEDQVENNGDGSLVLPIIDLNDPKAMELIGYACENWGAFQLKNHGISKSVIEELEVETKRLFDLPKEQKLKALRSRDNPTGYGTFWITPFFQQRMWQEGFTIIASAVQDAKKIWPNDYQRFCDAMKKFEDESRVLIEKLIHLSFKFLGISEEEEKNWVGPNNHAGAIQLNSYPICPKPENAMGIAPHTDTSIFTLLHQSQSSGLHIFKDGSGWFTVPLVPDTIVINTGDVLHMLSNARFKSALHKVSVNNVKHRYSMVYFYRPTMDQVVSPLVPSNNSDEEPRFRALTFKEFVGIKDKYLDKALSIVSVKED, and is shown as SEQ ID NO.1.

The nucleotide sequences of a pair of specific primers are:

AhPODS-EcoRI-F CCGGAATTCATGGCGACAAGTGAATCTTGT, designated SEQ ID NO.2.

AhPODS-BamHI-R CGCGGATCCATCTTCTTTGACGCTAACAATTG, designated SEQ ID NO.3.

TABLE 1PCR reaction System

TABLE 2PCR amplification procedure

Cloning the amplified target gene to pCAMBIA1307 to construct a gene overexpression vector pCAMBIA1307-AhPODS, specifically comprising the following steps:

The amplified target gene fragment is constructed on an intermediate vector QVB to construct a recombinant vector QVB-AhPODS, two restriction enzymes BamH I and Hind III are utilized, 50 mu L of enzyme digestion reaction system is arranged in a PCR instrument to linearize the vector, the reaction condition is that 1.5 h is carried out at 37 ℃, and double enzyme digestion gel running is carried out on the recombinant vector to recover the target fragment. And simultaneously carrying out enzyme tangential linearization on the pCAMBIA1307 plant binary expression vector with MYC tag by using BamH I and Hind III restriction enzymes by using the same enzyme cleavage reaction system, recovering a linearization vector fragment, and connecting the linearization vector fragment with a target fragment by using T4 ligase from Takara (China) company to obtain the target recombinant plasmid, namely AhPODS gene overexpression vector. Wherein, the schematic diagram of pCAMBIA1307 vector is shown in FIG. 3, and the system of the connection process is shown in Table 3:

Table 3 connection system

The reaction conditions were 4℃12 h.

Transferring the target recombinant plasmid into escherichia coli, and screening positive clones of the over-expression vector with AhPODS genes:

(1) 100. Mu.L of melted competent cells on ice was added to the recombinant plasmid of interest, gently mixed, and allowed to stand on ice for 30 min.

(2) The 42 ℃ water bath heat shock 50 s is quickly transferred to an ice bath, and the sample is kept stand 2 min on ice without shaking during the standing process, otherwise, the conversion efficiency is reduced. It should be noted that similar effects can be achieved by heat shock in a 42 ℃ water bath for 45-60 s.

(3) Adding 700 mu L of sterile liquid LB culture medium without antibiotics into a centrifuge tube, uniformly mixing, and resuscitating at 37 ℃ with 200 rpm to 60min to obtain a resuscitated liquid culture medium which has been transformed into the target recombinant plasmid.

(4) According to the experimental requirement, 50 mu L of the liquid culture medium which is recovered in the step (3) and has been transformed into the target recombinant plasmid is uniformly coated on LB solid culture medium containing kanamycin resistance, and the plate is placed in a 37 ℃ incubator for overnight culture.

(5) The sequencing diagram of the gene over-expression AhPODS vector is shown in FIG. 4, and positive clones of the over-expression vector with AhPODS gene are screened.

The positive bacterial liquid plasmid of the over-expression vector with AhPODS genes is introduced into agrobacterium tumefaciens GV 3101.

The step of introducing a positive plasmid of a positive bacterial liquid carrying an over-expression vector of AhPODS gene into Agrobacterium tumefaciens GV3101 is carried out by transforming with competent cells of Agrobacterium tumefaciens GV3101 by first mixing 1. Mu.L of positive plasmid DNA carrying an over-expression vector of AhPODS gene with 100. Mu.L of competent cells of Agrobacterium tumefaciens GV3101 and thoroughly mixing the bacterial liquid. Thereafter, it was left to stand on ice for 5min, followed by liquid nitrogen treatment for 5min, followed by treatment in a 37 ℃ water bath for 5min, and again placed on ice for 5min cooling. After completion of these steps, 700. Mu.L of LB medium without antibiotics was added to the mixture, and then the culture was resumed on a shaker at 28℃for 2h. Finally, the cultures were plated on solid media containing the corresponding antibiotics for selection.

Example two

Creation and identification of AhPODS Gene over-expressed tobacco Material

Healthy and tender tobacco leaves with a growth cycle of 30 days are selected as explants. Genetic transformation of tobacco is carried out by a leaf disc method to obtain AhPODS transgenic tobacco material, and Benshi tobacco is selected as a transformation background material. The specific test steps are as follows:

(1) Seed 8 min was sterilized with a sodium hypochlorite solution at a mass fraction of 2.5% followed by sterilization 1 min with an absolute ethanol solution at a volume fraction of 75%.

(2) On an ultra-clean bench, the seeds were washed 5 times with sterile water, and left to stand 3 min after each wash.

(3) Dibbling into sterile culture tank to culture into seedling.

(4) Agrobacterium carrying target gene is infected into tobacco leaf blade cell through agrobacterium mediated transformation method.

(5) The transformed leaves are placed in a SIM culture medium for co-cultivation, and then transferred into a screening culture medium for cultivation under the white light condition of 26 ℃ and the illumination intensity of 60 mu E/m ²/s and the illumination period of 14h darkness/10 h. After culturing, the tobacco leaves can generate white anti-callus, and then the white anti-callus is transferred into an SEM culture medium for differentiation into seedlings.

Wherein each liter of SIM culture medium contains 4.3g of M524, 30.0g of sucrose, 20 mu mol of 6-BA, 2.0mg of 2.4-D, 5.0 mu L of 0.5mol/L NaOH and double distilled water to 1L;

Every liter of SEM culture medium contains 4.3g of M524, 30.0g of sucrose, 2 mu mol of 6-BA, 2.0mg of 2.4-D, 5.0 mu L of 0.5mol/L NaOH and double distilled water to 1L;

M524 is Murashige & Skoog Basal Salt Mixture, lot: HEW0524259A, package size: 100L from phytotech.

Transferring the tobacco obtained by tissue culture into soil mixed according to the proportion of nutrient soil and vermiculite 1:1, extracting DNA from tobacco tissue after the tobacco survives, carrying out PCR amplification by using AhPODS specific primers to identify positive transformant lines, continuously culturing the identified transgenic lines in a greenhouse until the plants bloom and fruit, respectively marking the transgenic lines as OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, OE-8, OE-9 and OE-10, and selecting 2 transgenic plants with better strains, namely OE-1 and OE-2, as shown in figure 5.

The harvested AhPODS transgenic tobacco T1 seeds are dried and placed in a 4 ℃ refrigerator for vernalization, and after vernalization, the seeds are sterilized, washed and broadcast on MS culture medium containing 25 mug/mL hygromycin, and are cultivated in an environment with 25 ℃ and 16 h illumination/8 h dark period. After the tobacco grows and matures, the tobacco is transferred to nutrient soil for culture, proper moisture and nutrient conditions are ensured, and mature T2 generation seeds are harvested.

As a result, the T1 generation positive identification gel diagram of the tobacco seedlings is shown in FIG. 6, and 2 strains with better growth vigor are selected from the 10 obtained positive transgenic tobacco strains for observation. The T1 generation seed of AhPODS gene over-expressed transgenic tobacco had a larger seed volume compared to wild type tobacco seed of the same background, as shown in fig. 7. And it was observed that the hypocotyl of the transgenic tobacco seed grown under dark conditions was longer than that of the wild-type seed during the same period and conditions, as shown in fig. 8.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (8)

1. The application of the AhPOSS gene in increasing the seed volume is characterized in that the sequence of amino acid coded by the AhPODS gene is shown as SEQ ID NO.1, and the seed is plant seed.
2. 2. The use according to claim 1, wherein the AhPODS gene is overexpressed to increase plant seed volume.
3. 3. The use of claim 2, wherein the agent that overexpresses the AhPODS gene comprises a MYC-tagged pCAMBIA1307 plant binary expression vector;

   the sequence of the pCAMBIA1307 plant binary expression vector of the MYC tag is shown as SEQ ID NO. 4.
4. 4. The use according to claim 3, wherein the agent overexpressing the AhPODS gene further comprises PRIMESTAR ^® GXL DNA polymerase, restriction enzyme EcoR I, restriction enzyme BamH I and T4 ligase.
5. 5. The use according to claim 4, wherein the agent for overexpressing AhPODS gene further comprises a specific primer for amplifying AhPODS gene, the sequence of the specific primer is shown as SEQ ID NO.2 to SEQ ID NO. 3.
6. 6. The use according to claim 5, wherein the method of over-expressing AhPODS gene is to connect the linearized MYC-tagged pCAMBIA1307 plant binary expression vector and the digested target gene with T4 ligase, transfer the linked product into competent cells of escherichia coli, culture the transformed cells on a culture medium, and screen out positive clones of the over-expression vector with AhPODS gene;

   extracting positive bacterial liquid plasmid with AhPODS gene over-expression vector, transferring the plasmid into agrobacterium for infection to obtain transgenic plant and up-regulating AhPODS gene expression.
7. 7. The use according to claim 6, wherein said MYC tagged pCAMBIA1307 plant binary expression vector is digested with restriction enzymes BamH I and Hind III to obtain said linearized MYC tagged pCAMBIA1307 plant binary expression vector.
8. 8. The use according to claim 1, wherein the plant is tobacco.