Application of rice OsFLD gene in regulating rice plant height formation

Abstract

The application discloses a rice OsFLD gene in regulating the formation of rice plant height, through targeted editing of double-target site sequences as shown in SEQ ID NO. 2 and SEQ ID NO. 3 in a rice OsFLD gene, a rice mutant with a nucleotide sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 11 is obtained, and the plant height of the rice is effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of rice OsFLD gene in regulating the formation of rice plant height, and belongs to the technical field of biotechnology and plant genetic engineering. BACKGROUND

[0002] Rice is one of the important food crops in the world, and is the staple food of more than half of the world's population. Plant type is an important factor affecting rice yield, and ideal plant type can greatly improve rice yield. The cultivation of new rice varieties with ideal plant type has always been the focus of researchers and breeders. Plant height is one of the key traits for shaping ideal plant type of rice, which is mainly composed of the number and length of elongated internodes. High-stalk rice varieties are more prone to lodging during the growth period, especially in the late filling stage, resulting in large-scale yield reduction. Although the use of dwarf genes has significantly improved yield and lodging resistance, the potential problems of single dwarf source and narrow genetic background of rice have not been fundamentally solved.

[0003] Because rice plant height is a complex quantitative trait controlled by multiple genes, traditional artificial selection breeding needs to go through years of hybridization and backcrossing, which not only has a long breeding cycle and high cost, but also is difficult to avoid linkage drag, resulting in the introduction of dwarf genes by breeders, which is often accompanied by other adverse traits. Gene editing technology can achieve efficient and precise mutation of target genes in organisms, thereby changing their genetic information and phenotype. Through targeted improvement of rice plant height-related genes by gene editing technology, new dwarf gene resources can be quickly created, and the breeding process of new rice varieties with dwarf and lodging resistance can be accelerated, which is of great significance for high-yield breeding of rice. SUMMARY

[0004] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide an application of rice OsFLD gene in regulating the formation of rice plant height, which effectively reduces the plant height of rice.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0006] In a first aspect, the present application provides an application of rice OsFLD gene in regulating the formation of rice plant height, which utilizes CRISPR / Cas9 technology to target edit double target site sequences as shown in SEQ ID NO. 2 and SEQ ID NO. 3 in rice OsFLD gene, and obtains a rice mutant with a nucleotide sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 11.

[0007] Further, the present application utilizes CRISPR / Cas9 technology to target edit double target site sequences as shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0008] In a second aspect, the application provides a mutant, characterized by being obtained according to the above-mentioned application.

[0009] In combination with the first aspect, further, the application specifically includes:

[0010] Two pairs of target site primers are designed according to the sequence shown in SEQ ID NO. 1 in OsFLD to construct an OsFLD gene editing vector;

[0011] The OsFLD gene editing vector is transformed into Agrobacterium EHA105, and the Agrobacterium-mediated method is used to transform rice to obtain transgenic rice plants;

[0012] The DNA of the transgenic rice plant is used as a template, and the identification primers with sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9 are used for PCR amplification to screen out homozygous osfld mutants.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The application provides an application of a rice OsFLD gene in regulating the formation of rice plant height, and the OsFLD gene and the protein coded thereby can regulate the plant height of rice. The application significantly reduces the plant height of rice by targeted editing of the gene sequence, provides excellent genetic germplasm resources for ideal plant type and rice lodging resistance and high-yield breeding, and has a wide application prospect.

[0015] The application obtains a homozygous mutant material with significantly reduced plant height by editing the OsFLD gene in the rice variety Sugunuo, and the homozygous mutant material can be used for regulating the plant height of rice. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The application provides a schematic diagram of sequencing results of osfld-1 and osfld-2 mutant types and a wild-type Sugunuo (WT) target gene in a Sugunuo genetic background for an embodiment of the application;

[0017] Figure 2 The application provides a plant morphology diagram of osfld-1 and osfld-2 mutants and a wild-type Sugunuo (WT) in a Sugunuo genetic background for an embodiment of the application;

[0018] Figure 3 The application provides a columnar diagram of plant height statistical results of osfld-1 and osfld-2 mutants and a wild-type in a Sugunuo genetic background in 2022 for an embodiment of the application;

[0019] Figure 4A columnar chart of plant height statistics of osfld-1 and osfld-2 mutants and wild type strains in the genetic background of Su Gulu in 2023 is provided for the embodiments of the present application. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with the drawings, and the following embodiments are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0021] The present application provides an application of a rice OsFLD gene in regulating the formation of rice plant height, comprising:

[0022] A target primer is designed according to the sequence shown in SEQ ID NO. 1 in the rice OsFLD gene to construct an OsFLD gene editing vector;

[0023] The OsFLD gene editing vector is transferred into Agrobacterium, and Agrobacterium-mediated transformation is used to transform rice, and transgenic rice plants are screened and obtained;

[0024] The DNA of the transgenic rice plant is used as a template, and the identification primers with sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9 are used for PCR amplification, and homozygous osfld mutants are screened.

[0025] Example 1

[0026] In this embodiment, an OsFLD gene editing vector is constructed, which specifically comprises the following steps:

[0027] Step 1, the MSU_Locus of the OsFLD gene is submitted to the CRISPR-GE online website, and a sequence with high efficiency and good specificity is selected in each of the 5'UTR region and the first exon region, and two pairs of target primers are designed, wherein F1 and R1 are a pair of target primers, and the sequences are shown in SEQ ID NO. 4 and SEQ ID NO. 5; F2 and R2 are a pair of target primers, and the sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7, and the specific sequences are as follows:

[0028] Forward primer F1: 5'-GGCAGCGCGCCTGCTCGATAAATC-3'

[0029] Reverse primer R1: 5'-AAACGATTTATCGAGCAGGCGCGC-3'

[0030] Forward primer F2: 5'-GGCATGGACGCGGGATCGGGGGTT-3'

[0031] Reverse primer R2: 5'-AAACAACCCCCGATCCCGCGTCCA-3'

[0032] Step 2, 20 μl of each of the upstream and downstream primers with a concentration of 100 μM were added to a centrifuge tube and mixed uniformly, denatured at 100°C for 5 min, and naturally cooled at room temperature to complete annealing.

[0033] As shown in Figure 1 , the above two pairs of annealed targeting adapters were respectively connected to the AarI enzyme-digested intermediate vector SKm-gRNA to obtain the ligation products SKm-gRNA-1 and SKm-gRNA-2. The PCR program was 22°C for 30 min and 4°C for 2 min.

[0034] Step 3, transformation of the ligation product into E. coli, the specific process is as follows:

[0035] (1) All the ligation products of step 2 were added to E. coli DH5a competent cells, the ligation products were mixed with the E. coli DH5a competent cells by flicking the bottom of the tube with a finger, and were static on ice for 5 min;

[0036] (2) 42°C, heat shock for 45 s, quickly transfer to ice for 2 min;

[0037] (3) Add 500 μl of LB liquid medium, incubate at 37°C, 220 rpm for 20 min;

[0038] (4) Spread the above E. coli on solid LB medium containing 50 μg / ml of ampicillin resistance.

[0039] Step 4, obtain positive clones, the process is as follows:

[0040] Pick a single colony on the above LB medium into about 5 ml of LB liquid medium (50 μg / ml of ampicillin), incubate at 37°C, 220 rpm for about 12 h, extract the plasmid, and the plasmid extraction method refers to the plasmid extraction kit of Vazyme (DC201). And use universal primer M13R or T7 for sequencing, and the sequencing results of the plasmid which is correct after comparison are used for the construction of the final vector.

[0041] Step 5, obtain the final vector:

[0042] The above positive SKm-gRNA-1 and SKm-gRNA-2 which are correct after comparison are digested with KpnI / SalI, and the final vector pC1300-Cas9 is digested with KpnI / BamHI, and the digested fragments are recovered by gel.

[0043] The gel recovery method is referred to the gel recovery kit of Vazyme. The fragments SKm-gRNA-1 and SKm-gRNA-2 are gradually connected to the final vector pC1300-Cas9. The E. coli DH5a competent cells are transformed. The single colony is picked and the plasmid is extracted. The universal primers M13R and M13F are used for sequencing. The plasmid with correct sequencing is named as OsFLD-Cas9 and is stored at -20℃ for standby.

[0044] Example two:

[0045] In this embodiment, the agrobacterium EHA105 is transformed. The specific steps are as follows:

[0046] (1) The EHA105 competent cells are taken out from the ultra-low temperature freezer at -80℃ and placed on ice to melt.

[0047] (2) 0.5-2 μg of the target plasmid is added to 20 ul of EHA105 competent cells and placed on ice for 5 min.

[0048] (3) Quickly place in liquid nitrogen for 5 min.

[0049] (4) After taking out from the liquid nitrogen, quickly place in a 37℃ water pre-pot for water bath for 5 min.

[0050] (5) Transfer to ice for 2 min.

[0051] (6) Add 500 ul of LB liquid medium, place on a shaker, incubate at 28℃, 220 rpm for about 2.5-3 h.

[0052] (7) Centrifuge, discard most of the supernatant, and spread the remaining bacterial solution on LB solid medium containing kanamycin (50 μg / ml) and rifampicin, and incubate at 28℃ for about 2 days.

[0053] (8) After the colonies grow, single colonies are picked and colony PCR identification is performed to screen out positive clones.

[0054] (9) The positive clones are picked into 5 ml of LB liquid medium containing the corresponding antibiotics and rifampicin, and incubated at 28℃, 220 rpm for about 16-18 h. At this time, the bacterial solution can be stored in a -80℃ freezer with 50% glycerol at a volume ratio of 1:1. When infecting callus, it can be activated and used after being taken out from the -80℃ freezer.

[0055] Example three:

[0056] In this embodiment, the osfld mutant is obtained. The process steps are as follows:

[0057] Step 1, agrobacterium infects rice callus. The specific steps are as follows:

[0058] (1) From the -80℃ refrigerator, take out the previously stored Agrobacterium, add it to liquid 5ml LB medium containing kanamycin (50μg / ml) and rifampicin (50μg / ml) at a ratio of 1:100, and incubate overnight at 220rpm and 28℃.

[0059] (2) After overnight incubation, the bacterial solution is expanded in a 50ml sterilized centrifuge tube to an OD value of about 0.8-1.0, and then taken out of the incubator.

[0060] (3) Select about 200-300 pieces of well-grown Suigulu callus.

[0061] (4) Add the callus to the expanded liquid medium and add 50ul of liquid containing 20μg / ml AS (acetyl-syringone), then mix gently for 1-2min to perform infection, and continue to gently shake the medium for 30s every 15min.

[0062] (5) Discard the liquid medium, then transfer the infected callus to a culture dish lined with filter paper, perform sterile operation, and adsorb the excess liquid medium repeatedly for about 3-5min. Place a layer of filter paper on the solid co-culture medium, soak the filter paper, then transfer the above-mentioned infected callus to the solid medium, and incubate at 28℃ in the dark for 2-3 days.

[0063] Step 2, screening culture

[0064] After 2-3 days of co-culture, transfer the callus on the medium to a screening medium containing 200μg / ml penicillin and 50μg / ml hygromycin. Transfer it to a sterile artificial climate incubator and culture for about 15 days.

[0065] Step 3, pre-differentiation culture

[0066] Transfer the resistant callus on the screening medium to a pre-differentiation medium, and incubate it in a sterile culture room for about 7-10 days.

[0067] Step 4, differentiation culture

[0068] Transfer the pre-differentiation callus to a differentiation medium, and place 7-10 calli per bottle in a sterile culture room at 28℃ for about 30 days to differentiate transgenic seedlings.

[0069] Step 5, identify the transgenic seedlings, the specific steps are as follows:

[0070] (1) Put the appropriate amount of rice leaves into a 2ml centrifuge tube, add sterilized steel balls, freeze with liquid nitrogen, then shake vigorously until the leaves are powdered.

[0071] (2) Add 500 μl of DNA lysis buffer and place in a 65°C oven for 60 min, shaking once every 20 minutes during the process to ensure complete lysis.

[0072] (3) Add 500 μl of chloroform, mix vigorously by inverting the container, and let stand at room temperature for 5 min.

[0073] (4) Centrifuge at 12000 rpm for 8 min at room temperature.

[0074] (5) Take the supernatant into a new 1.5ml centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inverting, and place in a -20℃ refrigerator for 1 hour.

[0075] (6) Centrifuge at 12000 rpm at room temperature for 10 min.

[0076] (7) Discard the supernatant, and you will see a white precipitate at the bottom of the tube. This precipitate is a mixture of DNA and protein. Add 70%-75% ethanol to wash the precipitate.

[0077] (8) Centrifuge at 12000 rpm for 10 min at room temperature. Discard the supernatant and place it in a 37℃ oven to dry. After the ethanol has evaporated completely, add 150-300 μl of deionized water to dissolve the precipitate. The liquid at this time is the crude DNA extract of rice, which can be used for the next step of PCR identification.

[0078] (9) Using the crude DNA extracted above as a template, amplification was performed using identification primers F3 and R3 according to the instructions of Vazyme's 2X Taq Master Mix (P112-03-AA). The PCR products were sent to Qingke Biotechnology for sequencing until homozygous osfld mutants with site editing were screened out. The corresponding sequences of primers F3 and R3 are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. The specific sequences are as follows:

[0079] F3:5'-CGGAAACCAGTACAGCGAAA-3'

[0080] R3:5'-CGGTTGATCACGATGATGTC-3'

[0081] like Figure 1 As shown, the editing types and sequencing results of two mutants, osfld-1 and osfld-2, obtained under the genetic background of Su Yunuo are shown. The nucleotide sequences are as shown in SEQ ID NO.10 or SEQ ID NO.11. The osfld mutants used in this embodiment are all homozygous gene editing materials.

[0082] Example 4:

[0083] The present embodiment carries out field experiment identification of rice agronomic traits, and the steps are as follows:

[0084] The mutants of rice osfld-1 and osfld-2 and wild type material SuGuliunuo (WT) were planted in the transgenic test field of Yangzhou University in Jiangsu Province in May 2022 and May 2023 for two consecutive years, and the plant height and other agronomic traits were counted in October of the same year.

[0085] Figure 2 The overall morphological diagram of the osfld mutant and its wild type in the genetic background of SuGuliunuo is shown in Figure 2 It can be seen that after targeted editing of the OsFLD gene, the rice plants become shorter compared with the wild type. Figure 3 The plant height statistics of osfld-1 and osfld-2 mutants and their wild type in the genetic background of SuGuliunuo in October 2022 are shown in Figure 4 The plant height statistics of osfld-1 and osfld-2 mutants and their wild type in the genetic background of SuGuliunuo in October 2023 are shown in The statistical results show that compared with the wild type, the plant height of the mutants osfld-1 and osfld-2 is significantly reduced.

[0086] The present application regulates rice plant height by editing the rice OsFLD gene, and after targeted editing of the OsFLD gene in the rice variety SuGuliunuo, the homozygous mutants osfld-1 and osfld-2 are screened out. After continuous field planting for two years, the agronomic traits of the two years are counted. The results show that the mutant pure line obtained has a significantly reduced plant height, indicating that the present application regulates the formation of rice plant height by editing the rice OsFLD gene, providing an excellent genetic germplasm resource for enriching ideal plant type and rice lodging resistance and high yield breeding.

[0087] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A type of rice OsFLD The application of gene mutants in reducing rice plant height formation is characterized by, OsFLD The gene mutant consists of osfld-1 and osfld-2, which are used to target and edit rice. OsFLD The rice mutant osfld-1, with a nucleotide sequence as shown in SEQ ID NO.2 and SEQ ID NO.3, was obtained by using the dual target site sequences in the gene. The rice mutant osfld-1, with a nucleotide sequence as shown in SEQ ID NO.10, and the rice mutant osfld-2, with a nucleotide sequence as shown in SEQ ID NO.11, is a Suyu Nuo variety.

2. The application according to claim 1, characterized in that, The dual-target site sequences shown in SEQ ID NO.2 and SEQ ID NO.3 were edited using CRISPR / Cas9 technology.

3. The application according to claim 1 or 2, characterized in that, include: According to rice OsFLD Primers targeting the gene sequence shown in SEQ ID NO.1 were designed and constructed. OsFLD Gene editing vectors; Will OsFLD Gene editing vectors were transferred into Agrobacterium, and rice was transformed using the Agrobacterium-mediated transformation method. Transgenic rice plants were then screened to obtain transgenic rice plants. Using DNA from transgenic rice plants as a template, PCR amplification was performed using identification primers with sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9 to screen for homozygous DNA. osfld Mutant.

4. The application according to claim 3, characterized in that, According to rice OsFLD Construction of dual target site sequences for genes OsFLD Gene editing vectors, including: Sticky ends were generated by digesting the intermediate vector SK-gRNA with the restriction endonuclease Aar I. A connector sequence is added to the adhesive end to form a leading sequence as shown in SEQ ID NO.4 and SEQ ID NO.6 and a trailing sequence as shown in SEQ ID NO.5 and SEQ ID NO.7.

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