CN117844827A - Application of rice OsFLD gene in regulation of rice plant height formation - Google Patents

Abstract

The invention discloses a riceOsFLDApplication of gene in regulation of rice plant height formation and editing of rice by targetingOsFLDThe double target site sequences shown as SEQ ID NO.2 and SEQ ID NO.3 in the genes obtain rice mutants with nucleotide sequences shown as SEQ ID NO.10 or SEQ ID NO.11, and the rice plant height is effectively reduced.

Description

Application of rice OsFLD gene in regulation of rice plant height formation

Technical Field

The invention relates to application of a rice OsFLD gene in regulation and control of rice plant height formation, and belongs to the technical fields of biotechnology and plant genetic engineering.

Background

Rice is one of the important food crops in the world and is a staple food for more than half of the population worldwide. The plant type is an important factor influencing the rice yield, the ideal plant type can greatly improve the rice yield, and the cultivation of new rice varieties of the ideal plant type is always the focus of scientific researchers and breeders. Plant height is one of key characters for shaping ideal plant type of rice, and mainly consists of the number and length of elongation internodes. The high-stalk rice variety is easier to lodge during the growth period, especially in the later period of grouting, so that the yield is reduced in a large area. Although the utilization of dwarf genes significantly improves the yield and lodging resistance, the potential problem of single dwarf source and narrow genetic background of rice is not fundamentally solved.

Because the rice plant height is a complex quantitative trait controlled by multiple genes, the traditional manual selective breeding needs to be subjected to multi-year hybridization and backcross, so that the breeding period is long, the consumption cost is high, and the linkage encumbrance is difficult to avoid, so that a breeder is often accompanied with other unfavorable traits when introducing dwarf genes. The gene editing technology can realize the efficient and accurate variation of the target gene of the organism, thereby changing the genetic information and phenotype of the organism. The gene editing technology is used for directionally improving the rice plant height related genes, so that new dwarf gene resources can be quickly created, the cultivation process of new rice dwarf and lodging-resistant varieties is accelerated, and the method has important significance for high-yield rice breeding.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides an application of a rice OsFLD gene in regulating and controlling the formation of rice plant height, so that the rice plant height is effectively reduced.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides an application of a rice OsFLD gene in regulation of rice plant height formation, and a CRISPR/Cas9 technology is utilized to target and edit double-target site sequences shown as SEQ ID NO.2 and SEQ ID NO.3 in the rice OsFLD gene, so that a rice mutant with a nucleotide sequence shown as SEQ ID NO.10 or SEQ ID NO.11 is obtained.

Furthermore, the CRISPR/Cas9 technology is utilized to target and edit the double-target site sequences shown as SEQ ID NO.2 and SEQ ID NO. 3.

In a second aspect, the present invention provides a mutant characterised by being obtained according to the use as described above.

With reference to the first aspect, further, the application specifically includes:

designing two pairs of target primers according to a sequence shown by SEQ ID NO.1 in the OsFLD, and constructing an OsFLD gene editing vector;

transferring the OsFLD gene editing vector into agrobacterium EHA105, and transforming rice by using an agrobacterium-mediated method to obtain a transgenic rice plant;

and (3) taking DNA of the transgenic rice plant as a template, performing PCR amplification by using identification primers with sequences shown as SEQ ID NO.8 and SEQ ID NO.9, and screening out homozygous osfld mutants.

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

the invention provides application of a rice OsFLD gene in regulation of rice plant height formation, and the OsFLD gene and protein encoded by the gene can regulate the rice plant height.

The invention obtains the homozygous mutant material with obviously reduced plant height by a mode of editing the OsFLD gene in the rice variety Suyu glutinous rice by genes, and can be used for regulating and controlling the rice plant height.

Drawings

FIG. 1 is a schematic diagram showing the sequencing results of osfld-1 and osfld-2 mutant types and wild type Suyu Waxy (WT) target genes thereof under the Suyu waxy genetic background provided by the embodiment of the invention;

FIG. 2 is a plant morphology diagram of osfld-1 and osfld-2 mutants and their wild type Suyu Waxy (WT) in a Suyu waxy genetic background according to an embodiment of the present invention;

FIG. 3 is a histogram of statistical results of osfld-1 and osfld-2 mutants and wild type plant heights thereof in a Suyu waxy genetic background over 2022 provided by the example of the present invention;

FIG. 4 is a histogram of the statistical results of osfld-1 and osfld-2 mutants and their wild type strain heights in a Suyu waxy genetic background over 2023 provided by the examples of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and the following examples are only for more clearly illustrating the technical aspects of the present invention, and are not to be construed as limiting the scope of the present invention.

The application provides an application of a rice OsFLD gene in regulation of rice plant height formation, which comprises the following steps:

designing a target primer according to a sequence shown in SEQ ID NO.1 in the rice OsFLD gene, and constructing an OsFLD gene editing vector;

transferring the OsFLD gene editing vector into agrobacterium, transforming rice by using agrobacterium-mediated method, and screening to obtain transgenic rice plants;

and (3) taking DNA of the transgenic rice plant as a template, performing PCR amplification by using identification primers with sequences shown as SEQ ID NO.8 and SEQ ID NO.9, and screening out homozygous osfld mutants.

Embodiment one:

the embodiment constructs an OsFLD gene editing vector, which specifically comprises the following steps:

step 1, submitting MSU_Locus of an OsFLD gene to a CRISPR-GE online website, selecting a sequence with high efficiency and good specificity in a 5' UTR region and a1 st exon region respectively, and designing two pairs of target primers, wherein F1 and R1 are a pair of target primers, and the sequences are shown as SEQ ID NO.4 and SEQ ID NO.5 respectively; f2 and R2 are a pair of target primers, the sequences of which are shown as SEQ ID NO.6 and SEQ ID NO.7 respectively, and the specific sequences of which are as follows:

forward primer F1:5'-GGCAGCGCGCCTGCTCGATAAATC-3'

Reverse primer R1:5'-AAACGATTTATCGAGCAGGCGCGC-3'

Forward primer F2:5'-GGCATGGACGCGGGATCGGGGGTT-3'

Reverse primer R2:5'-AAACAACCCCCGATCCCGCGTCCA-3'

And 2, adding 20 mu l of each of the upstream and downstream primers with the concentration of 100 mu M into a centrifuge tube, uniformly mixing, denaturing at 100 ℃ for 5min, and naturally cooling at room temperature to finish annealing.

As shown in FIG. 1, the two pairs of annealed targeting linkers were ligated to AarI digested intermediate vector SKm-gRNA, respectively, to give ligation products SKm-gRNA-1, SKm-gRNA-2, PCR procedure: 30min at 22℃and 2min at 4 ℃.

Step 3, the connection product is transformed into escherichia coli, and the specific process is as follows:

(1) Adding all the connection products in the step 2 into DH5 alpha competence of the escherichia coli, flicking the bottom of the tube by using fingers, completely mixing the connection products with DH5 alpha competence of the escherichia coli, and standing on ice for 5min;

(2) Heat shock is carried out at 42 ℃ for 45s, and the mixture is quickly transferred to ice for 2min;

(3) Adding 500 μl of LB liquid medium, and incubating at 37deg.C and 220rpm for 20min;

(4) The above E.coli was plated on solid LB medium containing 50. Mu.g/ml of ampicillin resistance.

Step 4, obtaining positive clones, wherein the process is as follows:

the above-mentioned monoclonal on LB medium was picked up and incubated in about 5ml of LB liquid medium (50. Mu.g/ml of ampicillin) at 37℃for about 12 hours at 220rpm, and plasmid extraction was performed by referring to the plasmid extraction kit for Vazyme (DC 201). And sequencing by using a universal primer M13R or T7, and comparing the sequencing result with a correct plasmid for constructing a final vector.

Step 5, obtaining a final carrier:

and (3) comparing the sequencing results, performing enzyme digestion on the correctly positive SKm-gRNA-1 and SKm-gRNA-2 by KpnI/SalI, and simultaneously performing enzyme digestion on the final vector pC1300-Cas9 by KpnI/BamHI, and performing gel recovery on the fragments after enzyme digestion.

The gel recovery method refers to a gel recovery kit of Vazyme, fragments SKm-gRNA-1 and SKm-gRNA-2 are gradually connected to a final vector pC1300-Cas9, escherichia coli DH5 alpha competence is transformed, a monoclonal extraction plasmid is selected, a universal primer M13R and M13F are used for sequencing, and the plasmid with correct sequencing is named as OsFLD-Cas9 and is preserved at minus 20 ℃ for standby.

Embodiment two:

the agrobacterium EHA105 was transformed in this example as follows:

(1) The EHA105 competence was taken out of the-80℃ultra-low temperature refrigerator and placed on ice for thawing.

(2) 0.5-2. Mu.g of the plasmid of interest was added to 20ul EHA105 competence and left on ice for 5min.

(3) Rapidly placing in liquid nitrogen for 5min.

(4) After being taken out of the liquid nitrogen, the mixture is quickly placed in a water pre-pot at 37 ℃ for water bath for 5min.

(5) Transfer to ice for 2min.

(6) Adding 500 mu lLB liquid culture medium, placing on a shaking table, and incubating at 28 ℃ and 220rpm for about 2.5-3 hours.

(7) Most of the supernatant was centrifuged, and the remaining bacterial liquid was spread on LB solid medium containing kanamycin (50. Mu.g/m l) and rifampicin, and cultured at 28℃for about 2 days.

(8) After the colony grows out, selecting a monoclonal, carrying out colony PCR identification, and screening out positive clones.

(9) Selecting positive clone into 5ml LB liquid culture medium with corresponding antibiotic and rifampicin, culturing at 28 deg.C and 220rpm for 16-18 hr, storing the bacterial liquid in 50% glycerin at-80 deg.C refrigerator in the volume ratio of 1:1, and activating at-80 deg.C.

Embodiment III:

the osfld mutant was obtained in this example as follows:

step 1, infecting rice callus by agrobacterium, which comprises the following specific steps:

(1) The previously stored Agrobacterium was removed from the-80℃refrigerator and added to 5ml of LB medium containing kanamycin (50. Mu.g/ml) and rifampicin (50. Mu.g/ml) at a ratio of 1:100, and incubated overnight at 28℃at 220 rpm.

(2) The bacterial liquid after overnight culture was subjected to expansion culture in 50ml sterilized centrifuge tubes to have an OD of about 0.8 to 1.0, and the bacterial liquid was taken out of the incubator.

(3) Selecting Suyu glutinous callus with good growth state, about 200-300 grains.

(4) The callus was added to the liquid medium after the expansion culture, and 50ul of a liquid containing 20. Mu.g/ml AS (acetosyringone) was added, and then gently mixed for 1 to 2 minutes to perform infection, about 45 minutes, and gently shaking the medium for 30 seconds every 15 minutes.

(5) Pouring out the liquid culture medium, transferring the infected callus to a culture dish paved with filter paper, performing aseptic operation, adsorbing the redundant liquid culture medium, and repeating the process for about 3-5 min. Spreading a layer of filter paper on the solid co-culture medium, soaking the filter paper, and transferring the infected callus onto the solid culture medium, and culturing in dark at 28 ℃ for 2-3 days.

Step 2, screening culture

After co-cultivation 2-3, the calli on the medium were transferred to a screening medium containing 200. Mu.g/ml penicillin and 50. Mu.g/ml hygromycin. Transferring the strain to a sterile artificial climate culture room for culturing for about 15 days.

Step 3, pre-differentiation culture

The resistant calli on the screening medium are transferred to a pre-differentiation medium and placed in a sterile culture chamber for about 7-10 days.

Step 4, differentiation culture

Transferring the pre-differentiation culture callus to a differentiation medium, and placing 7-10 callus grains per bottle. The transgenic seedlings can be differentiated by placing the transgenic seedlings in a sterile culture room at 28 ℃ for culturing for about 30 days.

And 5, identifying transgenic seedlings, wherein the specific steps are as follows:

(1) Proper amount of rice leaves are put into a 2ml centrifuge tube, steel balls subjected to high-pressure sterilization are added, quick freezing is carried out by liquid nitrogen, and then vigorous shaking is carried out until the leaves are in powder form.

(2) Add 500. Mu.l of DNA lysate and place in an oven at 65℃for 60min, during which they are thoroughly lysed by shaking every 20 min.

(3) Add 500. Mu.l chloroform, mix vigorously upside down, and stand at room temperature for 5min.

(4) 12000rpm, and centrifuging at room temperature for 8min.

(5) Taking the supernatant into a new 1.5ml centrifuge tube, adding the isopropyl alcohol precooled in equal volume, mixing uniformly upside down, and placing in a refrigerator at-20 ℃ for 1h.

(6) 12000rpm, and centrifuging at room temperature for 10min.

(7) The supernatant is discarded, white sediment is seen at the bottom of the tube, the sediment is a mixture of DNA and protein, and 70% -75% ethanol is added for cleaning the sediment.

(8) 12000rpm, and centrifuging at room temperature for 10min. The supernatant is discarded and placed in a baking oven at 37 ℃ for drying, after ethanol is volatilized completely, 150-300 mu l of deionized water is added for dissolving and precipitating, and the liquid at the moment is the crude DNA extract of rice and can be used for the next step of PCR identification.

(9) The above crude DNA was used as a template, amplified with the identification primers F3 and R3 according to the above Vazyme company 2X Taq Master Mix (P112-03-AA) specification, and the PCR products were sent to the Optimum Trimeresurm Corp for sequencing until the site editing homozygous osfld mutant was selected, and the corresponding sequences of the primers F3 and R3 were shown as SEQ ID NO.8 and SEQ ID NO.9, respectively, with the following specific sequences:

F3:5'-CGGAAACCAGTACAGCGAAA-3'

R3:5'-CGGTTGATCACGATGATGTC-3'

as shown in FIG. 1, the results of editing type and sequencing of 2 mutants osfld-1 and osfld-2 obtained under the Suyu waxy genetic background show that the nucleotide sequences are shown as SEQ ID NO.10 or SEQ ID NO.11, and the osfld mutants adopted in the embodiment are all homozygous gene editing materials.

Embodiment four:

in this example, the rice agronomic traits were identified by field experiments, which were as follows:

mutant and wild material Suyu glutinous (WT) of rice osfld-1 and osfld-2 are planted in a transgenic test field of university of Yangzhou in Jiangsu province for 2 years continuously in 2022, 5 months and 10 months in the current year for statistics of agronomic characters such as plant height.

FIG. 2 is a schematic diagram showing the overall morphology of OsFLD mutant and its wild type in the Suyu waxy genetic background, and it can be seen from FIG. 2 that rice plants become shorter than wild type after targeted editing of the OsFLD gene. FIG. 3 shows statistics of osfld-1 and osfld-2 mutants and wild type strain heights thereof in a10 month Suyu waxy genetic background of 2022. FIG. 4 shows statistics of osfld-1 and osfld-2 mutants and wild type strain heights thereof in a Suyu genetic background of 10 months of 2023. Statistical results showed that mutant osfld-1 and osfld-2 strains were significantly reduced in height compared to the wild type.

According to the invention, the plant height of the rice is regulated and controlled by editing the OsFLD gene of the rice, and homozygous mutants OsFLD-1 and OsFLD-2 are screened after the OsFLD gene is edited in the rice variety Suyu glutinous rice in a targeted manner. Planting in continuous fields for two years, and respectively counting agronomic characters for two years. The results show that the obtained mutant pure line has obviously reduced plant height number, which indicates that the method participates in regulating and controlling the formation of the plant height of the rice by editing the rice OsFLD gene, and provides excellent genetic germplasm resources for enriching ideal plant types and rice lodging resistance and high-yield breeding.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Claims (6)

1. The application of the rice OsFLD gene in regulating and controlling the formation of rice plant height is characterized in that a double-target site sequence shown as SEQ ID NO.2 and SEQ ID NO.3 in the rice OsFLD gene is edited in a targeting manner, and a rice mutant with a nucleotide sequence shown as SEQ ID NO.10 or SEQ ID NO.11 is obtained.

2. The use according to claim 1, characterized in that the double target site sequences as shown in SEQ ID No.2 and SEQ ID No.3 are targeted-edited using CRISPR/Cas9 technology.

3. A rice mutant, characterized in that it is obtainable by the use according to claim 1 or 2.

4. Use according to claim 1 or 2, characterized in that it comprises:

designing a target primer according to a sequence shown in SEQ ID NO.1 in the rice OsFLD gene, and constructing an OsFLD gene editing vector;

transferring the OsFLD gene editing vector into agrobacterium, transforming rice by using agrobacterium-mediated method, and screening to obtain transgenic rice plants;

and (3) taking DNA of the transgenic rice plant as a template, performing PCR amplification by using identification primers with sequences shown as SEQ ID NO.8 and SEQ ID NO.9, and screening out homozygous osfld mutants.

5. The use according to claim 4, wherein constructing an OsFLD gene editing vector from a double-target site sequence of an OsFLD gene of rice comprises:

cleavage of intermediate vector SK-gRNA using restriction endonuclease Aar I to generate a cohesive end;

and adding a linker sequence at the sticky end to form a front primer sequence shown as SEQ ID NO.4 and SEQ ID NO.6 and a rear primer sequence shown as SEQ ID NO.5 and SEQ ID NO. 7.

6. The use according to claim 4, wherein said rice is transformed into a Suyu waxy variety by agrobacterium-mediated transformation.