Method and formulation for inducing abortion or deformation of plant seeds

Abstract

The present disclosure relates to the field of genetic breeding, in particular, to methods and formulations for inducing abortion or deformation of plant seeds by a VvDUF642 gene. The result of the present disclosure shows that the VvDUF642 gene is continuously expressed at a high level in seedless varieties, but there is no significant change in expression in nucleated varieties. After the VvDUF642 gene is transformed into an Arabidopsis, Arabidopsis seeds are deformed. After the VvDUF642 gene is transformed into a tomato, it causes abortion of tomato seeds. The gene or protein can be used for the construction or screening of seed abortion grape varieties.

Claims

1. A method for inducing abortion or deformation of a plant seed, comprising inducing the plant seed by a VvDUF642 protein or a nucleic acid molecule encoding the VvDUF642 protein, wherein the VvDUF642 protein is increased in activity in the plant seed, wherein the nucleic acid molecule encoding the VvDUF642 protein is overexpressed in the plant seed, wherein the amino acid sequence of the VvDUF642 protein is set forth in SEQ ID NO: 1, wherein a sequence of the nucleic acid molecule encoding the VvDUF642 protein is set forth in SEQ ID NO: 2, and wherein the plant seed is a grape seed, an Arabidopsis seed, or a tomato seed.

2. A method for causing abortion of a plant seed, comprising: enhancing a level and/or an activity of an endogenous VvDUF642 protein of claim 1 in a plant, or expressing the VvDUF642 protein in a plant that does not comprise a VvDUF642 gene, wherein the VvDUF642 protein is increased in activity in the plant seed, and wherein the plant seed is a grape seed, an Arabidopsis seed, or a tomato seed.

3. The method of claim 2, wherein a method of expressing the VvDUF642 protein in the plant that does not comprise the VvDUF642 gene comprises: constructing an expression vector comprising a nucleic acid encoding the VvDUF642 protein, transforming the expression vector into an Agrobacterium strain; and infecting the plant seed or an explant with the Agrobacterium strain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram showing the expression of the DUF642 gene transcription level; CS is Centennial Seedless, GR is Red Globe;

(2) FIG. 2 is a diagram showing the difference of the DUF642 protein expression between Red Globe and Centennial Seedless;

(3) FIG. 3 is a diagram showing the difference in expression of the DUF642 protein between nucleated and non-nucleated varieties;

(4) FIG. 4A is a diagram showing the size of Arabidopsis seeds; FIG. 4B shows that transgenosis of the DUF642 gene into Arabidopsis causes the shapes of Arabidopsis seeds to become smaller;

(5) FIG. 5 is a diagram showing that transgenosis of the DUF642 gene into Arabidopsis causes a reduction in thousand-seed weight of Arabidopsis seeds;

(6) FIG. 6 is a diagram showing expression of the DUF642 gene in wild-type Arabidopsis and DUF642 transgenic Arabidopsis;

(7) FIG. 7 is a diagram showing that the DUF642 gene causes tomato seeds abortion in tomatoes, forming a seedless fruit and reducing fruit volume;

(8) FIG. 8 is a diagram showing that transgenosis of the DUF642 gene into tomato causes a decrease in average fruit weight of tomatoes; and

(9) FIG. 9 is a diagram showing expression of the DUF642 gene in wild-type tomatoes and DUF642 transgenic tomatoes.

EMBODIMENTS

(10) The present disclosure provides a method for inducing abortion or deformation of a plant seed by a VvDUF642 gene. Those skilled in the art can learn from the content of this disclosure and appropriately improve process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present disclosure. The method and application of the present disclosure have been described through preferred embodiments. Relevant persons can obviously make changes or appropriate changes and combinations to methods and applications herein without departing from the content, spirit and scope of the present disclosure to achieve and apply the technology of the present disclosure.

(11) Reagents and consumables used in the present disclosure are all common commercial products, which can be purchased in the market.

(12) The nucleic acid molecule encoding a VvDUF642 protein includes genomic DNA, cDNA, recombinant DNA or mRNA, hnRNA encoding the VvDUF642 protein, or nucleic acid molecules that are reverse complementary to the above-mentioned DNA, cDNA, recombinant DNA or mRNA. In the present disclosure, VvDUF642 is the same as DUF642 and DUF.

(13) The above-mentioned nucleic acid molecules can be modified or optimized according to actual needs to make gene expression more efficient. For instance, (1) according to preferred codons of a recipient plant, codons of the VvDUF642 gene are changed to conform to preference of the recipient plant while maintaining the amino acid sequence of the VvDUF642 gene of the present disclosure. (2) Or the gene sequence adjacent to the initiating methionine is modified to enable efficient translation initiation. For example, a known effective sequence in plants is used for modification. (3) The above-mentioned nucleic acid molecules are linked to promoters expressed by various plants to facilitate their expression in plants. The promoter may include constitutive, inducible, timing adjustment, developmental regulation, chemical regulation, tissue preference, and tissue-specific promoters. The choice of a promoter will vary with needs of expression time and space, and also depends on the target species. (4) An enhancer sequence is introduced, such as an intron sequence (e.g. from Adhl and bronzel), and a viral leader sequence (e.g. from TMV, MCMV, and AMV).

(14) In the present disclosure, the vector can be a plasmid, a cosmid, a phage, or a virus vector. The host can be a fungus, a bacterium, algae, or a cell.

(15) For a plant that does not contain the VvDUF642 gene, a chemical method, a shotgun method, microinjection, electroporation and other methods can be used to introduce a VvDUF642 gene fragment into a plant cell.

(16) The present disclosure is further described below with reference to examples:

Example 1

(17) 1. The expression of the DUF642 gene in seedless varieties is significantly higher than that in nucleated varieties. Transcriptome sequencing was performed on five stages of fruit development [ovule stage (before flowering), fertilization stage (flowering), seed coat development stage (5-10 days after flowering), endosperm development stage (11-30 days after flowering), embryo development stage (31-40 days after flowering)] of a seedless variety (Centennial Seedless) and a nucleated variety (Red Globe). The result showed that the expression of this gene did not differ significantly between the ovule stage and the fertilization stage; from the seed coat development stage, the expression of the DUF642 gene in the seedless variety continuously increased, but the expression in the nucleated variety did not change significantly (FIG. 1).

(18) 2. During fruit development, the expression of the DUF642 protein in seedless varieties is significantly higher than that in nucleated varieties (FIG. 2). Detected by western blot, the expression of the DUF642 protein in Seedless and White was much higher than that in Rose Fragrant (FIG. 3).

(19) 3. Large sample verification: eight varieties and two de-seeded fruits at the developmental stage were selected as samples for western blot verification. During fruit development, the DUF642 protein was highly expressed in seedless varieties, while low expressed in nucleated varieties. The DUF642 protein expression increased after seedless treatment of Drunk Gold Fragrant and Sunshine Rose. These results confirmed the close linkage between the expression of the DUF642 gene and the seedless trait of grapes (FIG. 3).

(20) 4. RNA extraction

(21) Young leaves of a grape vine were selected to extract plant total RNA by a Bioteke plant RNA extraction kit. Thermo Scientific Nanodrop 1000 Micro UV-Vis Spectrophotometer was used to measure the concentration and to confirm the integrity of RNA by agarose gel electrophoresis. A RR047 reverse transcription kit of TakaRa was used to obtain the first strand of cDNA as a template for PCR cloning.

(22) 5. Obtaining of the DUF642 cDNA sequence:

(23) The Primer Find in Vector NTI 11 was used to design primers to clone the DUF642 gene sequence. The primers are shown in Table 1. NEB's Phusion ultra-fidelity enzyme was used for PCR amplification. The reaction system was: HF buffer 10 L, 2.5 mmol/L dNTPs 2.5 L, template 2 L, upstream and downstream primers (10 mmol/L) 2.5 L each, Phusion ultra-fidelity enzyme 0.5 L, and double distilled water to 50 L. The PCR reaction program was: pre-denaturation at 98 C. for 3 min; denaturation at 98 C. for 10 s, annealing at 58 C. for 10 s, extending at 72 C. for 30 s, a total of 31 cycles; the final extending at 72 C. for 10 min, and storage at 4 C. The PCR products were electrophoresed on 2% agarose gel, and the amplified products were recycled by an Omega Gel extract recovery kit. TaKaRa EXTaq polymerase plus A were used on the products purified, and the reaction system was: 10 buffer 2 L, 2.5 mmol Mg.sup.2+ 2 L, 2.5 mmol/L dNTPs 2 L, and template 14 L. Reaction program: 72 C. for 10 min. A DUF642-T vector was constructed according to the pGEMT-easy TM vector construction instruction, and transformed into DH5a Escherichia coli (E. coli) for blue-white spot screening. A single clone was selected for sequencing by Sangon Biotech (Shanghai) Co., Ltd. Sequencing primers were universal primers of T7 and T7 Terminal.

(24) TABLE-US-00001 TABLE1 cloningprimersoftheDUF642gene Primername Primersequence5-3 DUF642F atgagagctgtggcgtttcttttgcta (SEQIDNO:3) DUF642R ttagatgtgcctaggaggagtgtgcgga (SEQIDNO:4)

(25) 6. The cDNA sequence of the DUF642 gene is shown in SEQ ID NO: 2.

Example 2 DUF642 Gene Causes Seed Deformation in Arabidopsis

(26) 1. Construction of the plant expression vector: the sequence-verified DUF642-T plasmid was used as a PCR amplification template. pCAMBIA1303 was chosen as a plant expression vector. Agrobacterium strain was LBA4404. NEB's NCOI, Bst EII endonucleases, and Axygen small amount plasmid extraction kit were purchased from Zhengzhou Bomei Company. MS, Hygromycin B, Kanamycin, etc. were purchased from Zhengzhou Baosai Biology Company. PEG4000, Cellulase R10, Mecerozym R10, mannitol, potassium chloride, MES, BSA, 0.45 m filter head, etc. were purchased from Zhengzhou Chaoyan Biology Company. Then the plant expression vector pDUF642 was constructed.

(27) 2. With reference to the method of Maas C (Maas et al, 1989), an Arabidopsis protoplast was prepared and transformed. A confocal laser microscope was used to observe the Arabidopsis protoplast which was transformed and cultured. Nuc Pre was used to analyze and predict a nuclear localization signal.

(28) 3. Preparation of competent Agrobacterium LBA4404: LB solid medium containing 50 mg/L rifampicin was streaked. A single cell was picked out and shaken in LB liquid medium containing 50 mg/L rifampicin for 48 hours until bacteria solution became turbid. The bacterial solution was inoculated to 50 ml fresh LB liquid medium containing 50 mg/L rifampicin at a ratio of 1:100, and the bacteria were shaken for 5 h. The bacteria solution was placed in an ice bath at 4 C. for 30 min, and centrifuged at 5000 g for 5 min. The supernatant was discarded, and 1 ml of 0.1% calcium chloride was added to suspend; ice bath was performed at 4 C. for 5 min. Then, the solution obtained was centrifuged at 5000 g for 5 min. The supernatant obtained was discarded, and then 800 L of 0.02% calcium chloride was added for resuspension. The solution obtained was divided into 8 tubes, and 100 L of solution per tube was placed in an ice bath for later use.

(29) 4. 2 g of plasmid to be transformed was added to the competent Agrobacterium prepared. They were mixed, and subjected to liquid nitrogen quick freezing for 1 min, and placed in a water bath at 37 C. for 5 min. 1 ml of LB liquid medium was added into the solution obtained, and then the mixed solution was shaken at 28 C., 200 rpm for 5 h. The solution shaken was concentrated by centrifugation and spread on an LB plate containing 50 mg/L rifampicin and 50 mg/L kanamycin, and cultured at 28 C. for 48 h. Then a single colony was picked for verification.

(30) 5. Planting conditions of Arabidopsis were as follows: 22 C., 16 h light, 8 h dark, and water enough water two days before transformation. The Agrobacterium transformed was shaken to an OD value of about 2.0, and then concentrated and centrifuged at 5000 g for 5 min at room temperature. The supernatant was discarded, and 10% sucrose solution was added for resuspension. The solution obtained was centrifuged at 5000 g for 5 min at room temperature, and the precipitate obtained was resuspended to an OD value of 1.0 by adding 10% sucrose solution. Sillwet L-77 was added to a final concentration of 0.02%. An Arabidopsis flower was selected for transformation when a white spot just appeared on the flower. The inflorescence was soaked in an Agrobacterium liquid for 1 min, and the filter paper was taken out to absorb the excess liquid on the stem. It was placed in a dark condition at 22 C. for two days, and moisturizing was paid attention to, and then transformation was performed again 7 days later. About three weeks after transformation, the seeds were harvested when the fruit clips were mature.

(31) 6. The dried seeds were placed in a 2 ml EP tube, and 900 L of 70% ethanol containing 0.2% Tween 20 was added into the EP tube. Then the EP tube was shaken for 9 min. The supernatant was discarded, and 90% ethanol was added to wash 3 times. Finally, it was suspended in 100% ethanol and poured on sterilized filter paper to dry. A MS medium was prepared, and hygromycin B was added to a final concentration of 25 mg/L before pouring on the plate. Sterilized Arabidopsis seeds were evenly sprinkled on the MS medium, and then sealed and placed in a refrigerator at 4 C. for two days. Then the seeds were transferred into an incubator at 22 C. for cultivation. One week later, the screening result was observed, and the plant that could grow normally was a transgenic Arabidopsis positive plant. After continuous screening for two generations, pure T2 generation seeds were obtained for morphological observation.

(32) 7. Phenotypic Observation:

(33) As shown in FIGS. 4A and 4B, compared with wild-type Arabidopsis seeds, transgenosis of the VvDUF642 gene into Arabidopsis causes Arabidopsis seeds to become smaller.

(34) 8. Data measurement:

(35) The thousand-seed weight of wild-type Arabidopsis seeds and the thousand-seed weight of Arabidopsis seeds into which the VvDUF642 gene was transferred was measured and counted. The result is shown in FIG. 5. The result shows that transgenosis of the DUF642 gene into Arabidopsis causes Arabidopsis seeds to become smaller. The statistical result of thousand-seed weight shows that high expression of the DUF gene causes Arabidopsis seeds to become smaller, from 0.023 g of wild type to 0.015 g.

(36) 9. DUF642 gene expression data of transgenic Arabidopsis:

(37) The expression of the DUF642 gene in wild-type Arabidopsis seeds and Arabidopsis seeds into which the VvDUF642 gene was transferred was detected and counted. The result is shown in FIG. 6. The result shows that the VvDUF642 gene is highly expressed in transgenic Arabidopsis.

Example 3 DUF642 Gene Causes Seed Abortion in Tomatoes

(38) 1. Tomato transgenic: the plant expression vector pDUF642 was constructed for tomato transgenic.

(39) Seed sterilization: a few grams of tomato seeds were placed in a sterile Erlenmeyer flask, and first washed with sterile water for 2 minutes, and then washed with 75% alcohol for 1 minute. The seeds were soaked in 5% hypochlorous acid solution for 5-8 minutes, and washed with sterile water for 20 minutes twice, and then placed in a sterile filter board to dry.

(40) 2. sowing: sterilized seeds were sown in a culture bottle with a plant spacing of 0.8-1.0 cm. The remaining sterilized seeds were sealed and placed in a dry place for next use. Frequent ultraviolet light irradiation in the ultra-clean table will reduce the germination rate of seeds. The culture bottle sown was placed in the dark for 2 to 3 days. After the seeds appeared white spots and germinated, they were placed in a lighted tissue culture box to grow for 4 to 5 days. Culture conditions: 232 C., 16 h/d light, and 8 h/d dark.

(41) 3. Preparation of the explants: after tomato seeds grew for 7-8 days, cotyledons were fully expanded. A cotyledon was fetched with a scalpel, and the cotyledon petiole and the cotyledon tip were cut off, leaving the middle part to be cut into 2 to 3 segments as explants.

(42) 4. Preculture of the explants: tomato explants were pre-cultured for 1-2 d to expand edges of the explants, which was good for infecting and transforming tomatoes.

(43) 5. Agrobacterium infection solution: Agrobacterium OD600 was 0.1-0.2, and infection time was 10-15 min; pH of MS suspension solution was 5.4. The explants were dried in a sterile filter paperboard, and the filter paperboard can be replaced multiple times to absorb excess Agrobacterium.

(44) 6. Co-culture: the dried explants were placed in a co-cultivation medium and cultured in the dark for 2 days. Temperature: 232 C. The co-cultivation medium was the same as the pre-cultivation medium.

(45) 7. Delayed screening: after co-cultivation, the explants were washed twice with 1 g/L cephalosporin aqueous solution for 15 minutes (or washed once with 1 g/L cephalosporin aqueous solution, and washed once with sterile water, each for 15 min). After the explants were dried in the filter paperboard, they were placed in a delayed screening medium, and cultivated under light for 3 to 5 days.

(46) 8. Induction and screening of calluses: 30-40 days. Callus differentiation and bud elongation (late seedling differentiation medium): 30-40 days. Rooting: the seedlings to be differentiated grew to about 2-3 cm, and they were excised from the calluses and transferred to a rooting medium; time: 10-15 days.

(47) 9. Phenotypic observation:

(48) The phenotypes of wild-type tomatoes and tomatoes into which the VvDUF642 gene was transferred were observed. The results showed that transgenosis of the VvDUF642 gene into tomatoes caused abortion of tomato seeds and formation of seedless fruits (FIG. 7). 10. Data measurement:

(49) The average fruit weight of wild-type tomato fruits and tomato seeds into which the VvDUF642 gene was transferred was measured and counted. The result is shown in FIG. 8. The result shows that transgenosis of the DUF642 gene into tomatoes causes tomato fruits to become smaller.

(50) 11. DUF642 gene expression data of transgenic tomatoes:

(51) The expression of the DUF642 gene in wild-type tomato seeds and tomato seeds into which the VvDUF642 gene was transferred was detected and counted. The result is shown in FIG. 9. The result shows that the VvDUF642 gene was highly expressed in transgenic tomatoes.

(52) The examples described above are just preferred examples of the present disclosure, it should be noted that improvements and modifications within the scope of the principle of the disclosure should be regarded as within the scope of protection of the present disclosure to those skilled in the art.