Application of DF gene in regulating plant tiller number and yield

Abstract

This invention discloses a DF The application of genes in regulating plant tiller number and yield falls under the field of genetic engineering. This invention discovers a gene for regulating rice tillering. DF By building DF Mutants and overexpression lines of the gene were found to have overexpression DF The gene can increase the number of effective tillers in rice, thereby increasing the yield per plant; while knocking out the gene will reduce the number of effective tillers in rice, thus reducing the yield per plant. This proves that... DF Genes have a positive regulatory effect on the number of tillers and yield in rice. Therefore, this invention provides important genetic resources for the breeding of high-yielding rice varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a DF application of a gene in regulating plant tiller number and yield. BACKGROUND

[0002] Rice is one of the main food crops in the world. With the increase of population and the decrease of arable land, increasing rice yield has been the main goal of rice breeding. Rice yield is a complex agronomic trait, which is composed of three key factors: panicle number per unit area, grain number per panicle, and 1000-grain weight. Among them, tiller number, as a core parameter directly determining panicle number per unit area, plays a crucial role in regulating rice yield, and increasing tiller number is one of the effective strategies to improve rice yield. Tiller number is a complex quantitative trait regulated by multiple genes, and has been the focus of genetic research and rice breeding for a long time.

[0003] Rice tiller number is one of the key factors determining yield, and nitrogen plays a crucial role in regulating the tillering process. Nitrogen is an essential macronutrient for plant growth and development, and its adequate supply can significantly promote the occurrence and growth of rice tillers. When the soil nitrogen is sufficient, the photosynthetic capacity of rice plants is enhanced, and the synthesis of organic matter is increased, providing a rich nutrient basis for the germination and growth of tiller buds, thereby enabling more tiller buds to break dormancy and successfully develop into effective tillers. At the same time, nitrogen can also regulate the hormone balance in plants, such as promoting the synthesis and transport of hormones such as cytokinins, which play a key regulatory role in the differentiation, growth, and elongation of tiller buds, further promoting tiller formation and increasing tiller number. However, excessive nitrogen supply can also lead to excessive ineffective tillers, consuming excessive nutrients and resources, which is not conducive to the final yield formation. Therefore, reasonable regulation of nitrogen supply level is of great significance for optimizing rice tiller number and improving yield.

[0004] During plant development, purine catabolism is one of the core pathways of nitrogen remobilization. In particular, in legumes, fixed nitrogen elements in root nodules are transported to the aboveground part in the form of allantoin through purine catabolism, and are further metabolized into ammonia, providing a stable nitrogen source for plant growth and development. In the process of tissue senescence in non-legume plants such as rice, the nitrogen generated by the decomposition of DNA / RNA purine bases becomes an important endogenous nitrogen source through remobilization mechanisms. DF is a uric acid amide hydrolase in the purine metabolic pathway, and its function in regulating rice tillers has not been reported. SUMMARY

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a DFThe application of genes in regulating the number of tillers and yield in plants to increase the number of tillers and yield in rice.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method is provided. DF Application of genes in regulating plant tiller number and yield.

[0007] further, DF The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1.

[0008] further, DF The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Furthermore, plants include both dicotyledonous and monocotyledonous plants.

[0010] Furthermore, monocotyledons include plants of the Poaceae family.

[0011] Furthermore, grasses include rice.

[0012] Furthermore, regulating plant tiller number and yield is achieved through knockout. DF Genes can reduce the number of tillers in rice, leading to a decrease in yield.

[0013] Furthermore, the regulation of plant tiller number and yield is achieved through overexpression. DF Genes increase the number of rice tillers, leading to increased yield.

[0014] This invention provides a formulation for increasing the number of tillers and yield in rice, the formulation comprising the above-mentioned... DF Genes and the above DF At least one of the proteins encoded by the gene.

[0015] This invention also provides a method for preparing a high-yield rice variety, through overexpression of rice DF Genes are used to develop high-yield rice varieties.

[0016] This invention has the following beneficial effects: This invention discovers a gene for regulating rice tillering. DF By building DF Mutants and overexpression lines of the gene were found to have overexpression DF The gene can increase the number of effective tillers in rice, thereby increasing the yield per plant; while knocking out the gene will reduce the number of effective tillers in rice, thus reducing the yield per plant. This proves that... DF Genes have a positive regulatory effect on the number of tillers and yield in rice. Therefore, this invention provides important genetic resources for the breeding of high-yielding rice varieties. Attached Figure Description

[0017] Figure 1 The pYLCRISPR / Cas9-MH vector spectrum;

[0018] Figure 2 Map of the pYLsgRNA-OsU3 vector;

[0019] Figure 3 for DF The image shows the gene structure and the editing results of the two CRISPR editing targets;

[0020] Figure 4 The pCAMBIA1392 vector spectrum;

[0021] Figure 5 for DF Figure showing the expression levels of the protein in the two overexpression lines;

[0022] Figure 6 for DF Phenotypic observation and statistical data of overexpression lines. Detailed Implementation

[0023] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Example 1: Preparation of rice DF mutant

[0025] This invention uses CRISPR / Cas9 technology to study rice. DF Gene editing successfully obtained DF Genetically modified rice with gene mutations. The specific operational steps are as follows:

[0026] (1) Target sequence selection:

[0027] Target T1: 5'-GGGAGGAGATGGGCTCTACA-3' (SEQ ID NO.3), located at nucleotides 105-124 of SEQ ID NO.1;

[0028] Target T2: 5'-TGAGCACCGTCTCATCCCTG-3' (SEQ ID NO.4), located at nucleotides 135-154 of SEQ ID NO.1.

[0029] (2) Construction of CRISPR / Cas9 vector:

[0030] The two target sequences mentioned above were inserted into the pYLCRISPR / Cas9-MH vector using the following method (vector map is shown below).Figure 1 ), the expression cassette for targeting T1 and T2 was constructed, and the specific method was as follows: the positive strand (F strand) and the reverse strand (R strand) of each target in Table 1 were mixed and subjected to denaturation and annealing treatment to prepare T1 target linker and T2 target linker, respectively.

[0031] Table 1. Target linker primer sequences

[0032]

[0033] The T1 target linker and the T2 target linker were mixed with Bsa I-HF enzyme, pYLsgRNA-OsU3 vector (vector map see Figure 2 ) and T4 ligase for mixed reaction, thereby constructing T1 target gRNA gene expression cassette (gRNA-U3-DF-T1) driven by OsU3 promoter and T2 target gRNA gene expression cassette (gRNA-U3-DF-T2) driven by OsU3 promoter. The gRNA-U3-DF-T1 and gRNA-U3-DF-T2 were subjected to two rounds of PCR amplification with amplification primers for connection to pYLCRISPR / Cas9-MH vector, and the amplification primer information was as follows:

[0034] First round of amplification primers:

[0035] U-F: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO. 9);

[0036] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO. 10).

[0037] Second round of amplification primers:

[0038] Uctcg-B1: 5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3' (SEQ ID NO. 11);

[0039] gRcggt-BL: 5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 12).

[0040] The amplified gRNA expression cassette was digested with Bsa I enzyme, and then ligated with the Bsa I-digested plasmid pYLCRISPR / Cas9-MH. The ligation product was transformed into DH5α competent cells, and recombinant plasmids were screened and sequenced for verification. The recombinant plasmids with correct sequencing results were named pYLCRISPR / Cas9-MH-DF-T1 and pYLCRISPR / Cas9-

[0041] MH-DF-T2.

[0042] (3) Obtaining genetically modified rice:

[0043] The pYLCRISPR / Cas9-MH-DF-T1 and YLCRISPR / Cas9-MH-DF-T2 vectors obtained in step (2) were transformed into Agrobacterium tumefaciens competent cells EHA105 via freeze-thaw transformation, respectively, to obtain recombinant Agrobacterium strains EHA105 / pYLCRISPR / Cas9-MH-DF-T1 and EHA105 / YLCRISPR / Cas9-MH-DF.

[0044] -T2. Then, using Agrobacterium-mediated genetic transformation, the above-mentioned recombinant Agrobacterium strain was used to transform wild-type Zhonghua 10 rice, ultimately obtaining... DF Homozygous mutant rice with gene mutation CR-df-1 and CR-df-2 The specific conversion steps are as follows:

[0045] ① Soak the seeds in 75% ethanol for 1 minute, then soak them in 2.5% sodium hypochlorite for 30 minutes, and finally rinse them 5 times with sterile water.

[0046] ② Place the sterilized seeds on callus induction medium N6D (4 g / L Chu (N6) Basal Salt Mixture Powder + 2 mg / L glycine + 0.5 mg / L niacin + 0.5 mg / L vitamin B6 + 1 mg / L vitamin B1 + 1 mg / L 2,4-D + 0.3 g / L hydrolyzed casein + 2.878 g / L proline + 0.1 g / L inositol + 30 g / L sucrose + 4 g / L plant gel, pH=5.8) and culture at 32℃ under 24-hour light for about 6 days. Cut off the grown callus tissue and transfer it to fresh callus induction medium N6D, and continue culturing at 32℃ for 3 days.

[0047] ③ When the OD of the culture medium of recombinant bacteria EHA105 / pYLCRISPR / Cas9-MH-DF-T1 and EHA105 / YLCRISPR / Cas9-MH-DF-T2... 600nmWhen the value reaches about 0.5-0.8, the two recombinant bacteria are collected respectively. Then, using 30-40 mL AAM (including: 0.25 g / L MgSO4·7H2O, 0.15 g / L NaH2PO4·2H2O, 0.15 g / L CaCl2·2H2O, 3 g / L KCl, 10 mg / L MnSO4·4H2O, 2 mg / L MnSO4·4H2O, 0.025 mg / L CuSO4·5H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CoCl2·6H2O, 0.75 mg / L KI, 3 mg / L H3BO3, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2-EDTA, 0.5 g / L hydrolyzed casein, 7.5 mg / L glycine, 0.1767 g / L arginine, 0.9 g / L glutamine, 0.3 g / L aspartic acid, 0.1 g / L inositol, 1 mg / L nicotinic acid, 1 mg / L vitamin B6, 10 mg / L vitamin B1, 68.5 g / L sucrose, 36 g / L glucose and 10 mg / L acetoin, pH=5.2) solution, they are resuspended to prepare EHA105 / pYLCRISPR / Cas9-MH-DF-T1 infection solution and EHA105 / pYLCRISPR / Cas9-MH-DF-T2 infection solution respectively. Then the callus is immersed in the above two infection solutions for infection treatment, and the infection time is 5 minutes.

[0048] ④The infected callus is placed on the co-culture medium 2N6-AS (including: 4 g / L Chu (N6) Basal Salt Mixture powder, 2 mg / L glycine, 0.5 mg / L nicotinic acid, 0.5 mg / L vitamin B6, 1 mg / L vitamin B1, 1 mg / L 2,4-D, 0.3 g / L hydrolyzed casein, 0.1 g / L inositol, 30 g / L sucrose, 30 g / L glucose, 5 g / L plant gel and 10 mg / L acetoin, pH=5.2) covered with sterile filter paper, and cultured at 25°C in the dark for 3 days.

[0049] ⑤ Rinse the callus tissue repeatedly with sterile water until all residual Agrobacterium is completely removed. Then, wash the callus tissue in sterile water containing carbenicillin (500 mg / L) and blot dry on sterile filter paper. Next, transfer the callus tissue to selection medium S1 (containing: N6D medium, 25 mg / L hygromycin, and 400 mg / L carbenicillin, pH=5.8) and incubate for 3 days in the dark at 25°C. Then, transfer the callus tissue to selection medium S2 (containing: N6D medium, 50 mg / L hygromycin, and 400 mg / L carbenicillin, pH=5.8) and incubate for approximately 2 weeks in a continuous light incubator at 32°C. Finally, the callus tissue was transferred to differentiation medium RE-III (containing 4.4 g / L MS medium (Sigma), 2 g / L hydrolyzed casein, 0.0002 mg / L NAA, 2 mg / L KT, 30 g / L sucrose, 30 g / L sorbitol, 4 g / L plant gel, 50 mg / L hygromycin and 200 mg / L carbenicillin, pH=5.8).

[0050] ⑥ The callus tissue was cultured in differentiation medium RE-III for 6 days, and then transferred to a new differentiation medium for another 6 days. During the second culture, seedlings will emerge from the callus tissue. At this time, the seedlings can be transplanted from the differentiation medium into the soil until they mature. The mature seedlings are marked as T0 generation rice.

[0051] (4) Identification of genetically modified rice:

[0052] Genomic DNA was extracted from the T0 generation rice obtained in step (3), and then PCR amplification was performed using the following primer pairs.

[0053] DF -F:5'-CTACGCCCTCTTCCTCCTCCCAATC-3' (SEQ ID NO.13);

[0054] DF -R:5'-ACAACACCAAGCGGATAACGAAGG-3' (SEQ ID NO. 14).

[0055] The PCR amplification products were sequenced and analyzed to obtain... DF Gene mutation information. The T0 generation rice mutant... DF Genes and wild-type rice DF Genes were compared. The results are as follows: Figure 3 As shown, the T0 generation rice mutant CR-df-1 of DF The gene has an inserted T base at position 122 after the translation start site ATG; T0 generation rice mutant CR-df-2 ofDF The gene is missing a base G at the 139th position after the translation initiation site ATG.

[0056] Example 2: Preparation of DF overexpression strain

[0057] (1) Vector construction:

[0058] Using DF genomic DNA as a template, high-fidelity polymerase KOD and primer pair 1392-OEDF-F and 1392-OEDF-R were used to amplify the genomic DNA of the DF gene by PCR. After the pCAMBIA1392 vector (vector map see Figure 4 ) was treated with BamHI, the amplified product was ligated using homologous recombination enzyme, and then sequencing was performed after ligation. After the sequence was correct, E. coli DH5a was transformed, and the plasmid was extracted to obtain the recombinant expression vector pCAMBIA1392-DF. The nucleotide sequences of primer pair 1392-OEDF-F and 1392-OEDF-R are as follows:

[0059] 1392-OEDF-F: 5'-TGTTTGGTGTTACTTCTGCAGATGTACCCATACGACGTCCCAGACTACGCTATGGCTCTCCTCCTCTCC-3' (SEQ ID NO. 15);

[0060] 1392-OEDF-R: 5'-CTCCTCGCCCTTGCTCACCATGGATCCCGATTCCGCCGCCGTCGCCGCCGCCGCATCCACCGCGTTCT-3' (SEQ ID NO. 16).

[0061] (2) Preparation of overexpression strain:

[0062] The pCAMBIA1392-DF vector obtained in step (1) was transformed into Agrobacterium tumefaciens competent cells EHA105 by freeze-thaw method, and the recombinant Agrobacterium strain EHA105 pCAMBIA1392-DF was obtained. Then, the above-mentioned recombinant Agrobacterium strain was used for transformation of wild-type Zhonghua 10 rice by Agrobacterium-mediated genetic transformation method, and finally the DF overexpression rice of the gene DF-OE1 and DF-OE2 were obtained. The specific transformation steps were the same as in Example 1.

[0063] (3) Western blot detection of the expression amount of DF in the overexpression strain:

[0064] Leaf protein was extracted from overexpression lines, and after adding SDS, it was boiled for 5 minutes, and then an equal amount of protein sample was loaded for loading. After completing electrophoresis, membrane transfer was performed, and the PVDF membrane after membrane transfer was immersed in 5% BSA blocking buffer, and incubated at room temperature for 1 hour. Then, the membrane was cut at 53 kDa, the upper part was added with GFP primary antibody, and the lower part was added with internal reference ACTIN primary antibody, and incubated at 4 DEG C overnight. After washing with TBST for three times, secondary antibody was added, and hybridization incubation was carried out at room temperature for 1 hour. After washing with TBST for three times again, luminescence detection was performed. The detection results are as shown in Figure 5 , DF The expression amount in two overexpression lines was significantly up-regulated compared with the wild type. This indicates that the expression amount of DF is significantly increased in overexpression lines.

[0065] (4) Phenotype observation of overexpression lines

[0066] The wild type rice flower 10 (WT), mutant CR-df-1 and CR-df-2 , overexpression DF-OE1 and DF-OE2 plants were planted in Beijing in summer, and the plants are as shown in Figure 6 A. After the plants grew up, the tiller number and yield per plant were counted. The tiller number results are as shown in Figure 6 B, CR-df-1 and CR-df-2 The tiller number of the mutant was reduced by 31.88% and 24.64% compared with the wild type. At the same time, the tiller number of DF-OE1 and DF-OE2 was increased by 8.70% and 10.14% compared with the wild type. The yield per plant results are as shown in Figure 6 C, CR-df-1 and CR-df-2 were reduced by 22.40% and 21.50% compared with the wild type, and the yield per plant of DF-OE1 and DF-OE2 was increased by 9.57% and 13.28% compared with the wild type.

[0067] The CDS region nucleotide sequence of DF in the application and the amino acid sequence of the encoded protein are as follows:

[0068]

[0069] (2) Protein coding: MALLLSYPRRHPSIHLLILSAYALFLLPILDGLELGGDGLYREILRDETVLRLKELGKISDGEGYLERTFLSPASIRASAVIISWMKDAGLTTWIDQMGNIHGRFEPTNSTKEALLIGSHMDTVIDAGMYDGALGIISAISALKVLKVTGRLQRLTRPVEVIAFSDEEGVRFQTTFLGSAAVAGTLPESILQVSDKSGTTVQDVLKLNSLEGTANALGEVRYSPESVGSYVEVHIEQGPVLEALRYPLGVVKGIAGQTRLKVIINGSQGHAGTVPMKLRRDPMVAAAELVLTLETLCKEPNKFLTYDEECGCFTEESLAGLVCTVGELLTWPSASNVIPGQVNFTVDIRAMDDKVRETIVTSFSRLVLQRCDDRLVDCAVEQKHAAAATPCDAELTSRLERATRSTISSMAAGVRRAGGETPVLMSGAGHDAMAMARLTKVGMLFVRCRGGVSHSPEESVMDDDVWAAGLALVNFIDQNAVDAAAATAAES (SEQ ID NO. 2).

[0070] The above description is merely the preferred embodiment of the present application, but not to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. DF Use of a gene in modulating tiller number and yield in plants, characterized in that, The regulation of plant tillering number and yield is overexpression DF The gene, the number of rice tillers is increased, and the yield is increased; wherein, the DF The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The DF The amino acid sequence of the encoded protein of the gene is shown as SEQ ID NO. 2.

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