CN113832163B - Method for effectively improving plant fruit biomass - Google Patents

Abstract

The invention relates to a method for effectively improving plant fruit biomass, belonging to the field of molecular biology. The invention discloses application of a gene AT5G02580 in effectively improving plant fruit biomass, which comprises the following steps: the nucleotide sequence of the gene AT5G02580 is shown in SEQ ID NO: 1. The gene AT5G02580 negatively regulates the number of the fruits and the weight of seeds of the Arabidopsis, and specifically comprises the following steps: overexpression of this gene reduces the number of fruits and seed weight of arabidopsis; the gene deletion mutation increases the number of horn fruits and seed weight of Arabidopsis thaliana.

Description

Method for effectively improving plant fruit biomass

Technical Field

The invention relates to a method for effectively improving plant fruit biomass, belonging to the field of molecular biology.

Background

The Horn fruit is a fruit formed by the development of pistil consisting of two synbiotic carpels, is special for cruciferous plants, and can be divided into long horn fruits and short horn fruits in morphology. The long horn fruits are slender and cylindrical, the short horn fruits are almost flat and elliptical in length and width. The fruits of crucifers have great application value. Rape is silique, its mature seed oil content can reach 35-45%, it is one of the important sources of edible vegetable oil, it can also be used for making lubricant, varnish, nylon, plastics, insect repellent, stabilizing agent and medicines, etc.. The crassostrea tenuissima is characterized in that the mature seeds of the crassostrea tenuissima contain 20-30% of oil, and the oil can be used for preparing soap or used as paint. The herba Thlaspis has the keri formed by the two carpels, has peculiar shape and 28-34% of oil content in seeds, and can be used for preparing soap or lubricating oil when being used for pressing oil. The Horn fruit of white mustard can be eaten by human beings as Chinese medicine, and can also be used for squeezing oil or flavoring. At present, research on cruciferous plants focuses on biomass characters such as the number of fruits, thousand seed weight and the like, has large contribution to yield, and is controlled by inheritance. The arabidopsis thaliana is dicotyledonous mode plant arabidopsis thaliana of cruciferae, has a horn shape, is about 10-14 mm long and less than 1mm wide, can grow 50-60 seeds per pod, is an ideal material for researching genetic regulation of horn biomass, and has important reference significance for crop breeding.

The invention patent (the gene for enhancing plant disease resistance and the function thereof) (the application number: CN 202110236767.4) discloses the sequence of the AT5G02580 gene, and the function of enhancing the disease resistance of Arabidopsis plants after knocking out the AT5G02580 gene.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a new application of AT5G02580 gene, namely, a method for regulating and controlling the biomass of the fruits, namely, a method for effectively improving the biomass of the fruits of plants.

In order to solve the technical problems, the invention provides application of a gene AT5G02580 in effectively improving plant fruit biomass: the nucleotide sequence of the gene AT5G02580 is shown in SEQ ID NO: 1.

As an improvement of the application of the invention: negative control of the number of horn fruits and seed weight of arabidopsis thaliana; overexpression of this gene reduces the number of fruits and seed weight of arabidopsis; the gene deletion mutation can increase the number and seed weight of the fruits of the arabidopsis thaliana, and the nucleotide sequence after the gene deletion mutation is any one of the following: SEQ ID NO: 2. SEQ ID NO. 3.

The technical scheme of the invention is as follows:

the invention constructs an AT5G02580 gene over-expression vector by utilizing a transgenic technology, obtains a positive transgenic plant by agrobacteria-mediated genetic transformation of wild arabidopsis thaliana, and identifies the transgenic plant over-expressing the AT5G02580 gene by gene expression analysis. Meanwhile, the invention also utilizes the gene editing technology to edit the AT5G02580 gene in wild type arabidopsis thaliana, so as to obtain two fragment deletion mutants of the gene. Then, the numbers of the fruits and the thousand kernel weights of the seeds on the main stems of the deletion mutant, the over-expressed plant and the wild type control are compared, and as a result, the numbers of fruits and the thousand kernel weights of the seeds of the deletion mutant are found to be significantly higher than those of the wild type control, and the phenotype of the plant over-expressed with the AT5G02580 gene is just opposite.

Therefore, the gene is shown to participate in the growth and development of the horn fruits of the arabidopsis, is a key factor for negatively regulating the biomass of the horn fruits of the arabidopsis, and the functional discovery of the gene has certain reference significance for the horn fruit research of other crops of the cruciferae.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the analysis of the expression level of AT5G02580 gene in Arabidopsis wild type, transgenic plants, wherein over-expression-1 and over-expression-2 are two different transgenic lines (same applies below);

FIG. 2 is a sequencing analysis of AT5G02580 gene editing targets in Arabidopsis wild type, deletion mutant-1 and deletion mutant-2 plants, wherein deletion mutant-1 and deletion mutant-2 are two different fragment deletion mutants of AT5G02580 gene (hereinafter the same);

FIG. 3 is a main stem and pod count statistic of Arabidopsis wild type, deletion mutation-1, deletion mutation-2, over-expression-1, over-expression-2 plants;

FIG. 4 is a seed thousand kernel weight statistic for Arabidopsis wild type, deletion mutant-1, deletion mutant-2, over-expression-1, over-expression-2 plants;

* Shows that there was a very significant (P < 0.01) difference in t-test between the other group and the wild-type control group.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1 PCR cloning of AT5G02580 Gene

Extracting total RNA of Arabidopsis leaves by adopting a RNeasy Plant Mini Kit (QIAGEN) reagent, wherein the specific operation is according to the product specification; for the obtained RNA, goScript was used ^TM Reverse Transcription System kit (Promega) first strand cDNA was synthesized, methods were performed as described with reference to the product instructions.

PCR primers (5' end with recognition sequence of restriction endonuclease, underlined) for amplifying AT5G02580 gene were synthesized AT biotechnology company (e.g. TaKaRa):

primer 1:5' -AGAGCTCATGGGTGATCATAATAGCTCGCA-3’；

Primer 2:5' -CGGATCCTTAGATTGTAGTTCGTGAAAGCA-3’。

From the cDNA obtained above, AT5G02580 gene was PCR-amplified using PrimerSTAR HS DNA Polymerase kit (TaKaRa) by the following method: primerSTAR HS DNA Polyrase0.2. Mu.L, 5× PrimerSTAR Buffer 4. Mu. L, cDNA 0.8.8. Mu.L, and 0.5. Mu. L, dNTP Mixture1.6. Mu. L, ddH each of primer 1 and primer 2 (10. Mu.M) were added to the PCR tube ₂ O12.4. Mu.L. The following procedure was used for PCR amplification:

step (1) 95 ℃ for 5min;

step (2) is repeated for 30 times at 98 ℃ for 10 seconds, 60 ℃ for 15sec,72 ℃ for 60 sec;

step (3) at 72 ℃ for 10min.

The nucleotide sequence of the gene AT5G02580 is shown in SEQ ID NO: 1.

Example 2 construction of AT5G02580 Gene overexpression vector

For the PCR product obtained in example 1, using

AxyPrep ^TM PCR Clean-Up Kit (Axygen) was purified and operated according to the product instructions. Then, 15. Mu.L of the purified PCR product was taken, 1.5. Mu.L of Sac I restriction enzyme, 1.5. Mu.L of BamH I restriction enzyme, 3. Mu.L of the corresponding Buffer was added thereto, and ddH was used ₂ O was added to 30. Mu.L, and the mixture was reacted in a water bath at 37℃for 5 hours. Preparing 1% agarose gel, adding 30 mu L of enzyme-cut product into a sample adding hole, carrying out electrophoresis for 30min at 100v voltage, and dyeing in Ethidium Bromide (EB) solution for 5-10 min; the stained gel was subjected to ultraviolet irradiation at 365nm, and the band of the target gene was cut with a small blade, followed by AxyPrep ^TM DNA Gel Extraction Kit (Axygen) the DNA was purified and the purified digested fragment of the gene of interest (SEQ ID NO: 1) was obtained according to the product specifications.

Cutting the purified enzyme into target gene slicesThe fragment was inserted into the expression vector pCAMBIA 1300S: purified enzyme-digested fragment of target gene 3. Mu.L, linearized pCAMBIA1300S vector 1. Mu. L, T4 DNA Ligase (Takara) 0.5. Mu. L, T4 DNA Ligase Buffer 1. Mu. L, ddH ₂ O was made up to 10. Mu.L and run on a PCR instrument at 4℃for 12h. The obtained linked product was subjected to transformation of E.coli JM109 by reference to "molecular cloning operation guide (second edition) (Beijing: scientific Press, 1992), to obtain a monoclonal colony containing pCAMBIA1300-35S: AT5G02580 vector, and then a plasmid vector of the monoclonal colony was extracted by a plasmid miniextract kit (Tiangen), and the method was operated according to the product specification.

Example 3 genetic transformation of AT5G02580 Gene overexpression vector

Agrobacterium-mediated method (Plant Journal,1998,16 (6): 735-743), the pCAMBIA1300-35S: AT5G02580 vector was genetically transformed into wild type Arabidopsis Col-0, and the corresponding transgenic plants were obtained by screening.

Example 4 Positive identification of transgenic plants

Respectively taking 0.1g of leaves of wild arabidopsis thaliana and transgenic individual plants, grinding with liquid nitrogen, adding 600 μl of extracting solution (15.76 g Tris-cl,29.22g Nacl,15.0g SDS powder, adding ultrapure water to fix volume to 1L, and keeping the temperature at 65 ℃ for 1h; 200 μl of 5MKAC was added and the ice bath was performed for 10min; adding 500 μl chloroform, mixing, and centrifuging at 10000rpm for 5min; taking the supernatant, adding 500 μl of isopropanol, mixing well, centrifuging at 12000rpm for 3min, and discarding the supernatant; washing the precipitate with 500. Mu.l of 75% ethanol, centrifuging at 12000rpm for 3min, and discarding the supernatant; after drying the DNA upside down for 15min, 50. Mu.l of pure water was added to dissolve the DNA.

PCR primers for identifying the AT5G02580 gene transgene were synthesized AT biotechnology company (e.g., taKaRa):

primer 3:5'-ATGGGTGATCATAATAGCTCGCA-3';

primer 4:5'-TTAGATTGTAGTTCGTGAAAGCA-3'.

The above-extracted genomic DNA was used as a template, and PCR amplification was performed using 2X Taq PCR Master Mix (TIANGEN Co.) in a PCR amplification system of 20. Mu.l containing 2X Taq PCR MasterMix. Mu.l of each of primer 3 and primer 4 (10. Mu.M) in an amount of 0.5. Mu.l, and 1. Mu.l of the template DNA was used<1μg)，ddH ₂ O was made up to 20. Mu.L; the PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 55℃for 30sec, elongation at 72℃for 30sec,35 cycles; extending at 72℃for 10min. The PCR product was electrophoresed in 1% agarose gel for 30min, stained with Ethidium Bromide (EB), compared with the band of DL2000 Marker, the transgenic plant capable of amplifying the band of 276bp was positive plant, and the band of 275bp could not be amplified in wild type Arabidopsis genome DNA.

Example 5 analysis of expression of AT5G02580 Gene in transgenic plants

RNA from leaves of positive transgenic plants obtained in example 4 was extracted and cDNA was synthesized according to the method of example 1. PCR primers for AT5G02580 gene expression analysis were synthesized AT biotechnology Co (e.g., taKaRa):

primer 5:5'-AGTATTCAACATGGGCAAAG-3'

Primer 6:5'-TTCTCGGTCGGTATTTCTT-3'

The gene expression level was measured by qPCR method using TB Green ^TM Premix Ex Taq ^TM Kit (TaKaRa). The specific operation is as follows: cDNA 2. Mu.L, primer 5 and primer 6 (10. Mu.M) each 0.5. Mu.L, TB Green Premix Ex Taq. Mu. L, ddH ₂ O6.6. Mu.L, ROX Reference Dye 0.4.0.4. Mu.L. In a StepOne Plus ^TM Real-Time PCR System (Applied Biosystems) runs qPCR with the following procedure: pre-denaturation at 95 ℃ for 30s; the data obtained for 95℃for 5s,60℃for 30s,40 cycles were analyzed for significant differences using the t-Test method.

Compared to the wild type, the results are shown in fig. 1, two different lines were identified in the transgenic plants that overexpressed the AT5G02580 gene for subsequent phenotypic analysis.

EXAMPLE 6 construction of Arabidopsis AT5G02580 Gene deletion mutant

Designing an AT5G02580 gene edited targeting sgRNA sequence: 5'-GGAATGTATAGTATTCAACA-3', and synthesizing the corresponding annealing primer: 5'-TGATTGGAATGTATAGTATTCAACA-3' and 5' -AAACTGTTGAATACTATACATTCCA, and an AT5G02580 gene editing vector was constructed using CRISPR/Cas9 kit (Biogle, china) according to the product instructions. The AT5G02580 gene editing vector was genetically transformed into Arabidopsis wild type variety Col-0 in the same manner as in example 3 to obtain a corresponding transgenic Arabidopsis plant.

Extraction of transgenic Arabidopsis plant DNA obtained from example 6: 0.1g of fresh leaf tissue is weighed, ground into powder in liquid nitrogen, transferred into a 2mL centrifuge tube, added with 300 mu L of extracting solution (1.25 g/L SDS,500mmol/L NaCl,0.1mol/L Tris-HCl, pH 8.0) and ice-bathed for 1h at 65 ℃; adding 100 mu L of 5mol/L KAC, and ice-bathing for 10min; then adding 250 mu L of chloroform, uniformly mixing, and standing for 5min; centrifuging at 12000r/min for 10min, collecting supernatant, adding equal volume of isopropanol into 1.5ml tube, centrifuging at 12000r/min for 5min, discarding supernatant, adding 1mL of 70% ethanol for washing precipitate, centrifuging at 12000r/min for 7min, discarding supernatant, inverting, air drying at room temperature, and adding 100 μL of ddH2O to dissolve DNA.

Synthesizing PCR amplified primers:

primer 7:5'-GTGACTATAGCATCTATCATCCTTCA-3'

Primer 8:5'-TTTCGCTAGCTCTTTCCACAC-3'

Using the transgenic Arabidopsis plant DNA obtained in example 6 as a template, a 2×Taq PCR reagent (Tiangen) was used, and the PCR system was 20. Mu.L, which included 1. Mu.L of the template DNA, 0.5. Mu.L of each of primer 7 and primer 8 (10. Mu.M), and 10. Mu.L of 2× Taq PCR MasterMix II and ddH ₂ O8 μl; the PCR procedure was: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30sec, annealing at 55℃for 30sec, elongation at 72℃for 30sec,35 cycles; extending at 72℃for 2min.

After sequencing analysis of the PCR product, 2 fragment deletion mutants of the AT5G02580 gene were successfully identified: deletion mutation-1 plant, deletion 10 bases A (figure 2), its nucleotide sequence is SEQ ID NO 2; deletion mutation-2 plants, 29 bases A (figure 2) were deleted, and the nucleotide sequence was SEQ ID NO:3. Both fragment deletion mutations also resulted in frame shift mutation of the AT5G02580 gene.

EXAMPLE 7 statistical analysis of the number of Kernel and seed thousand seed weight of Arabidopsis plants

Taking seeds of five plants of wild arabidopsis Col-0, deletion mutation-1, deletion mutation-2, over-expression-1 and over-expression-2, putting the seeds into a conical flask, adding 800 mu l of 10% (V/V) sodium hypochlorite for sterilization, removing liquid in the conical flask after shaking table for 20min at 200rpm, cleaning the seeds with sterile water for multiple times, then performing vernalization in a 4 ℃ incubator, spreading the seeds on filter paper for air drying after 3d, spreading the seeds on a 1/2MS flat plate culture medium, culturing the seeds for 7-10 d in a 22 ℃ illumination incubator (16 h day and 8h night), and then transplanting seedlings with good growth vigor into nutrient soil for culturing, wherein the seedlings continue to grow to a mature period. Randomly selecting 9 plants of the five types, and counting the number of the horn fruits on the main stems of each plant; then, the seeds of each plant were collected separately from the seeds, air-dried at 28℃for two weeks, and weighed with an electronic scale. The t-Test method was used to analyze the significant differences between deletion mutation-1, deletion mutation-2, over-expression-1, over-expression-2 and wild-type control, respectively.

As a result of the identification, the numbers of the fruits of the deletion mutation-1 and deletion mutation-2 plants are obviously higher than that of the wild type, the fruits are respectively increased by 24.3 percent and 27.3 percent, and the numbers of the fruits of the overexpression-1 and the overexpression-2 transgenic plants are obviously lower than that of the wild type, and the fruits are respectively reduced by 25.9 percent and 31.0 percent (shown in figure 3); meanwhile, the thousand seed weights of the deletion mutation-1 and deletion mutation-2 plants are obviously higher than that of the wild type, and respectively increased by 6.3 percent and 5.8 percent, while the thousand seed weights of the over-expression-1 and over-expression-2 plants are obviously lower than that of the wild type, and respectively reduced by 5.9 percent and 6.9 percent (figure 4). The results show that the AT5G02580 gene negatively regulates the synthesis of the arabidopsis thaliana horn biomass, and two deletion mutations of the gene can improve the horn number and the seed weight of the arabidopsis thaliana.

Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.

Sequence listing

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Claims (2)

1. GeneAT5G02580The application in regulating plant fruit biomass is characterized in that: geneAT5G02580The nucleotide sequence of (2) is shown as SEQ ID NO:1 is shown in the specification; overexpression of this gene reduces the number of fruits and seed weight of arabidopsis; the gene deletion mutation increases the number of horn fruits and seed weight of Arabidopsis thaliana.

2. The use according to claim 1, characterized in that: the nucleotide sequence after gene deletion mutation is shown as SEQ ID NO: 2. SEQ ID NO. 3.

Images