Gene mutant for promoting prematurity of rice and application of gene mutant

Abstract

The invention belongs to the technical field of biology, and particularly relates to a gene mutant for promoting prematurity of rice and application of the gene mutant. The invention provides a method for obtaining rice with advanced heading time, which comprises the step of increasing the content of EFL2m protein in rice, and the amino acid sequence of the EFL2m protein is shown as SEQ ID NO: 4. According to the method, the rice can flower early without reducing the yield, and the method has important application value in rice genetic breeding research and molecular breeding practice.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a gene mutant for promoting early maturity of rice and application thereof. BACKGROUND

[0002] The trend of global population growth puts a rigid demand for continuous improvement of grain yield, and the acceleration of urbanization process continuously encroaches on arable land, under this background, improving unit area grain yield has become one of the most effective ways to guarantee grain production. In the field of rice breeding, the contradiction between "high yield" and "early maturity" has existed for a long time, which seriously restricts the optimization process of rice varieties. From the biological mechanism, achieving high yield often requires rice to have a long enough growth cycle to accumulate biomass, while early maturity varieties are usually difficult to achieve the yield standard required by high yield due to the significant shortening of growth cycle and insufficient material accumulation. This contradiction makes it a major challenge to breed early-mature high-yield varieties in the field of rice breeding, which not only threatens the global food supply security, but also hinders the improvement and diversification of planting economic benefits and the satisfaction of diversified planting needs.

[0003] In view of this core difficulty, identifying and cloning new early-maturing genes and their alleles has become a key research direction. Ef-cd gene is a representative gene, which is a long non-coding RNA, and regulates the expression of flowering gene OsSOC1 / OsMADS50 by mediating the level of histone methylation, thereby promoting rice to achieve early maturity. Field test data in different latitudes across the country show that Ef-cd gene can make rice heading date advance significantly by 7-20 days, and show different degrees of yield increase in many ecological zones, which not only realizes the balance of early maturity and high yield, but also highlights the advantage of resource efficient utilization. JMJ720 gene is a gene that negatively regulates the growth period of rice through histone demethylation modification mechanism. JMJ720 gene not only negatively regulates the flowering time of rice, but also participates in the expression regulation of photoperiod related genes, and jmj720 mutant shows typical "early maturity without yield reduction" characteristics, which provides a new molecular target for solving the contradiction between "early maturity" and "stable yield" of rice in northeast cold region. The discovery of rice OsDREB1C gene also breaks the traditional breeding contradiction. As a transcription factor, the gene has a unique function of synergistically regulating photosynthetic efficiency, nitrogen utilization efficiency and heading date of rice: on the one hand, it improves photosynthetic efficiency and nitrogen utilization efficiency to achieve significant yield increase, on the other hand, it increases the content of florigen to promote rice to advance heading and shorten growth period.

[0004] Although relevant research has made some progress, but at present, excellent alleles that can synergistically regulate heading time and yield are still in short supply. SUMMARY

[0005] The inventors of the present application collected an early flowering mutant earlyflower2 (efl2) in Nipponbare background, which headed two weeks earlier than wild type. Field phenotyping of wild type Nipponbare and efl2 mutant showed that there was no significant difference in plant height, tiller number, panicle length and yield per plant between wild type and efl2 mutant. Backcrossing efl2 mutant with wild type Nipponbare showed that efl2 mutant was a single gene controlled semi-dominant mutant. Whole genome resequencing of efl2 mutant showed that a G to A base mutation occurred at the splicing site of the last exon of LOC_Os03g57940 gene (herein referred to as EFL2 gene) in efl2 mutant. The base mutation caused the mis-splicing of the EFL2 gene transcription product, leading to a frame shift and producing a wrong protein. The EFL2 gene mutant produced by the base mutation is named as EFL2m. The nucleotide sequence of EFL2 gene is shown as SEQ ID NO: 1, which encodes EFL2 protein, and the amino acid sequence of EFL2 protein is shown as SEQ ID NO: 2. The nucleotide sequence of EFL2m gene is shown as SEQ ID NO: 3, which encodes EFL2m protein, and the amino acid sequence of EFL2m protein is shown as SEQ ID NO: 4.

[0006] To verify whether the early flowering phenotype of efl2 mutant is caused by the new allelic mutation of EFL2 gene, overexpression of EFL2m gene in rice Nipponbare was performed, and the results showed that the heading time of EFL2m transgenic plants was significantly earlier than that of wild type Nipponbare Figure 4 C and Figure 4 D). Thus, it is shown that the early maturity feature of rice efl2 mutant is derived from the single base mutation at the splicing site of the last exon of EFL2 gene, which realizes the early maturity of rice without causing yield loss.

[0007] Based on the above research results, the present application provides a method for obtaining rice with advanced heading time, which comprises: increasing the content of EFL2m protein in rice, wherein the amino acid sequence of the EFL2m protein is shown as SEQ ID NO: 4.

[0008] In some embodiments, the content of EFL2m protein in rice is increased by overexpressing the coding gene of the EFL2m protein in rice.

[0009] In some embodiments, the nucleotide sequence of the coding gene of the EFL2m protein is shown as SEQ ID NO: 3.

[0010] The coding gene of the EFL2m protein can be obtained by any suitable method. In some embodiments, the coding gene of the EFL2m protein is obtained by a DNA chemical synthesis method. In other embodiments, based on the difference between the 3' end nucleotide sequence of the EFL2m gene shown in SEQ ID NO: 3 and the 3' end nucleotide sequence of the EFL2 gene shown in SEQ ID NO: 1, a suitable upstream primer and a downstream primer containing the difference sequence are used to obtain the coding gene of the EFL2m protein by PCR method with cDNA of wild type rice as template.

[0011] In some embodiments, the method for obtaining rice with advanced heading time comprises: constructing a recombinant expression vector for overexpressing the coding gene of the EFL2m protein in rice; introducing the recombinant expression vector into rice callus, inducing positive callus differentiation and regeneration into plants, and screening positive plants carrying the coding gene of the EFL2m protein.

[0012] The recombinant expression vector for overexpressing the coding gene of the EFL2m protein in rice can be constructed using any suitable plant expression vector.

[0013] In some embodiments, the recombinant expression vector is introduced into rice callus by Agrobacterium-mediated genetic transformation method.

[0014] In some embodiments, the positive plants carrying the coding gene of the EFL2m protein are screened by polymerase chain reaction (PCR) and sequencing method.

[0015] In some embodiments, the rice is Oryza Sativa L. spp. Japonica, var. nipponbare.

[0016] The present application also provides an EFL2 gene mutant for promoting the heading time of rice, wherein the nucleotide sequence of the EFL2 gene mutant is shown in SEQ ID NO: 3; compared with the EFL2 gene shown in SEQ ID NO: 1, the nucleotide sequence at positions 2066-2121 of the EFL2 gene mutant is changed and 12 nucleotides are added at the 3' end.

[0017] The EFL2 gene mutant can be obtained by any suitable method. In some embodiments, the EFL2 gene mutant is obtained by a DNA chemical synthesis method. In other embodiments, based on the difference between the 3' end nucleotide sequence of the EFL2 gene mutant and the 3' end nucleotide sequence of the EFL2 gene, a suitable upstream primer and a downstream primer containing the difference sequence are used to obtain the EFL2 gene mutant by PCR method with cDNA of wild type rice as template.

[0018] The application also provides an expression cassette, a recombinant vector or a recombinant bacterium comprising the EFL2 gene mutant for promoting the heading time of rice.

[0019] The recombinant vector can be a cloning vector or an expression vector. The recombinant bacterium can be a cloning strain or an expression strain.

[0020] The application also provides a protein encoded by the EFL2 gene mutant for promoting the heading time of rice, and the amino acid sequence of the protein is shown as SEQ ID NO: 4.

[0021] The application also provides the use of the EFL2 gene mutant for promoting the heading time of rice or the protein in cultivating early-maturing rice.

[0022] Experiments prove that the EFL2 overexpression plant (Act:EFL2-GFP) presents a late-maturing phenotype, and the EFL2m overexpression plant (Act:EFL2m-GFP) presents an early-maturing phenotype (P<0.001). Figure 4 C and Figure 4 D). The method provided by the application can promote the heading of rice, and has important application value in genetic breeding research and molecular breeding practice of rice. The application further expands the gene resource library for early-maturing and high-yield breeding of rice, and provides a new technical path and theoretical support for coping with the global food security challenge. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Phenotype analysis of rice elf2 mutant. Figure 1 A shows the phenotype of wild type rice Nipponbare (WT(NP)) and rice elf2 mutant (elf2) at the heading stage, and the scale length is 20 cm. Figure 1 B is a statistical chart of the heading time (days after sowing) of the first ear of wild type rice Nipponbare (WT(NP)) and rice elf2 mutant (elf2), P<0.001 (***). WT is the abbreviation of wild type, indicating wild type; NP is the abbreviation of nipponbare, indicating Nipponbare; DAS is the abbreviation of days after sowing, indicating days after sowing.

[0024] Figure 2 Field phenotype analysis of rice elf2 mutant. Figure 2 A is the field phenotype of wild type rice Nipponbare (NP) and rice elf2 mutant (elf2) at the heading stage. Figure 2B is a graph showing the plant height of wild type rice Nipponbare (NP) and rice elf2 mutant (elf2), and the values are mean ± SD (wild type n = 21, mutant n = 11). Figure 2 C is a graph showing the tiller number of wild type rice Nipponbare (NP) and rice elf2 mutant (elf2), and the values are mean ± SD (wild type n = 21, mutant n = 11). Figure 2 D is a graph showing the panicle length of wild type rice Nipponbare (NP) and rice elf2 mutant (elf2), and the values are mean ± SD (wild type n = 13, mutant n = 11). Figure 2 E is a graph showing the yield per plant of wild type rice Nipponbare (NP) and rice elf2 mutant (elf2), and the values are mean ± SD (n = 10). NP is the abbreviation of nipponbare, which means Nipponbare. ns means not significant difference.

[0025] Figure 3 It is shown that rice elf2 mutant is a semi-dominant early flowering mutant. Figure 3 A is a graph showing the phenotype of the segregation of the hybrid F2 generation of wild type rice Nipponbare and rice elf2 mutant, when the wild type (EFL2 / EFL2) has not yet headed, the hybrid material (EFL2 / elf2) is in the flowering stage, and the homozygous material (elf2 / elf2) has basically completed the filling. Figure 3 B is Figure 3 A is the spike phenotype of the three plants.

[0026] Figure 4 It is the cloning and functional verification of EFL2 gene and EFL2m gene mutant. Figure 4 A is a schematic diagram of the mutation site of EFL2 gene, showing that a G to A base mutation occurs at the splicing site of the last exon of EFL2 gene in rice elf2 mutant. Figure 4 B is the expression level of EFL2 gene in wild type rice Nipponbare (WT) and rice elf2 mutant (elf2), and ns means not significant difference. Figure 4 C shows the phenotype of wild type rice Nipponbare (WT (NP)), EFL2 overexpression plant (Act: EFL2-GFP) and EFL2m overexpression plant (Act: EFL2m-GFP) at the heading stage, and the ruler length is 20 cm. Figure 4D is a histogram of the first panicle heading time (days after sowing) of wild type rice Nipponbare (WT(NP)), EFL2 overexpression plant (Act:EFL2-GFP) and EFL2m overexpression plant (Act:EFL2m-GFP), and the values in the figure are the mean ± SD (WT(NP) n = 12, Act:EFL2-GFP n = 12, Act:EFL2m-GFP n = 12), P < 0.01 (**) and P < 0.001 (***), and DAS is the abbreviation of days after sowing, indicating the days after sowing to heading. DETAILED DESCRIPTION

[0027] The present application is described in detail below with reference to examples, and the examples are given only to illustrate the present application, not to limit the scope of the present application.

[0028] The wild type rice Nipponbare used in the following examples is a known general rice material, which is preserved in the laboratory. The rice material can be purchased.

[0029] The pAHLG-GFP vector used in the following examples is a binary vector AHLG described in the method section "Plasmid construction and transformation" of the literature "Jiang, L., Liu, X., Xiong, G., Liu, H., Chen, F., Wang, L., Meng, X., Liu, G., Yu, H., Yuan, Y., et al. (2013). DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401-405.", which is a binary vector obtained by adding an Actin promoter, a multiple cloning site, a GFP coding sequence and a NOS terminator to a pCAMBIA1300 vector as a framework. The pAHLG-GFP vector can be obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0030] The Agrobacterium EHA105 strain used in the following examples is preserved in the laboratory. The strain can be purchased.

[0031] The experimental methods used in the following examples are conventional methods in the art, and the steps or conditions described in the literature or product instruction manual in the art can be referred to if not otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels if not otherwise specified.

[0032] The data in the following examples were processed using GraphPad Prism 8 statistical software, and the experimental results were expressed as mean ± standard deviation, using two-tailed Student's t-test, P<0.05 (*), P<0.01 (**), P<0.001 (***), ns (not significant).

[0033] Example 1 Phenotype and genetic analysis of rice elf2 mutant

[0034] 1. Phenotype analysis

[0035] The rice elf2 (earlyflower2) mutant is an early-maturing mutant in the background of Nipponbare (Oryza Sativa L. spp. Japonica, var. nipponbare) collected by the previous laboratory. The mutant is naturally mutated from wild-type Nipponbare. Field experiments of the rice elf2 mutant were carried out in a rice field in Beijing (40°13'54" N, 116°33'50" E). After the rice seeds were soaked in water for 2 days, they were moved to a 37°C incubation room for 3 days, and then the seeds that sprouted were sown in a seedbed for seedling raising. When the seedlings reached the 4-leaf stage, they were transplanted into the rice field. Wild-type rice Nipponbare and rice elf2 mutant were planted in the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing, with 24 plants per strain, with a plant spacing and row spacing of 17 cm. The growth period was from May to November each year. The field phenotype investigation of the rice materials was carried out from August 8 to October 8 each year, and the earliest time for each single plant to sprout the first ear was counted.

[0036] As shown in Figure 1 , compared with wild-type rice Nipponbare (WT (NP)), the flowering time of the rice elf2 mutant was about 2 weeks earlier, which was a typical early-maturing mutant. The field phenotype investigation results showed that there was no significant difference in plant height, tiller number, ear length and yield per plant between wild-type rice Nipponbare (NP) and rice elf2 mutant Figure 2 ).

[0037] 2. Genetic analysis

[0038] Backcross was carried out with wild-type rice Nipponbare as the female parent and rice elf2 mutant as the male parent, and F1 generation seeds were harvested. The F1 generation seeds were planted in the field of the experimental base, and the grown F1 generation plants showed an early-maturing phenotype earlier than wild-type rice Nipponbare, indicating that the early-maturing phenotype was controlled by a dominant gene.

[0039] After planting F1 generation plants, F2 generation seeds were harvested. 301 F2 generation plants were planted in the field and phenotypes were observed. 74 single plants showed very early heading phenotype, 152 single plants showed sub-early maturity phenotype, and 75 single plants showed late maturity phenotype (similar to wild type rice Nipponbare). Chi-square test (χ2=0.0825<χ20.05,1) showed that the early maturity and late maturity segregation ratio was consistent with 3:1, indicating that the early maturity phenotype was controlled by a single gene as a semi-dominant trait. Figure 3

[0040] 3. Gene mutation analysis

[0041] In order to analyze which gene mutation causes the rice efl2 mutant, whole genome sequencing was performed on the rice efl2 mutant. Genomic DNA was extracted from rice leaves using a modified CTAB method (Mou Z, He Y, Dai Y, et al. Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology. The Plant Cell. 2000, 12, 405-418.). 100 mg of rice leaves were frozen in liquid nitrogen and ground into powder in a 5 cm diameter mortar. The powder was transferred to a 1.5 mL centrifuge tube for DNA extraction, and finally the obtained DNA pellet was dissolved in 100 μL H2O. The extracted DNA was sent to Beijing Novogene for whole genome resequencing.

[0042] By comparing the differences in genomic sequences between the rice efl2 mutant and wild type rice Nipponbare, it was found that the splicing site of the last exon and intron of the LOC_Os03g57940 gene in the rice efl2 mutant had a G to A base mutation Figure 4 A). This base mutation caused incorrect splicing of the gene transcription product, leading to a frameshift and producing a wrong protein. There is no report on this allelic variation of the LOC_Os03g57940 gene. The LOC_Os03g57940 gene of wild type rice Nipponbare is named as EFL2 gene. The EFL2 gene mutant contained in the rice efl2 mutant is named as EFL2m gene. The coding sequence (CDS) of the EFL2 gene is shown in SEQ ID NO: 1, which encodes the EFL2 protein with the amino acid sequence shown in SEQ ID NO: 2. The coding sequence of the EFL2m gene is shown in SEQ ID NO: 3, which encodes the EFL2m protein with the amino acid sequence shown in SEQ ID NO: 4.​

[0043] CDS sequence of the EFL2 gene (2121 bp):

[0044]

[0045] Amino acid sequence of EFL2 protein (from N- to C-terminus, 707 aa):

[0046] MPELRGGVWRARLRSKKVYDVQDADPAASPVSPAPRGRTGRRGGAAAGRGNKTVAEGGGRKALKPRGKGCRAVDLCKDQPCKDLPEVIARKAVTGKAQEDLGLNKVADRAANLMMDGESGDKFAAAEDESTTTPVPERVQVGNSPEYITDRKLGKGGFGQVYVGRRVSGGGSRTGPDAQEVALKFEHRSSKGCNYGPPYEWQVYHTLNGCYGIPSVHYKGRLGDYYILVMDMLGPSLWDVWNSVGQAMSAHMVACIAVEAISILEKLHSKGFVHGDVKPENFLLGHPGSVDEKKLFLIDLGLASRWKEASSGQHVDYDQRPDVFRGTIRYASVHAHLGRTGSRRDDLESLAYTLIFLIRGRLPWQGYQGDNKSFLVCKKKMATSPELLCCFCPAPFKHFLEMVTNMKFDEEPNYPKLISLFDGLIEGPASRPIRIDGALKVGQKRGRMVVNLDDDEQPKKKVRLGSPATQWISVYNARRPMKQRYHYNVADSRLHQHIEKGNEDGLYISCVSSSANFWALIMDAGTGFCSQVYELSQVFLHKDWIMEQWEKNYYITAIAGATNGSSLVVMSKGTPYTQQSYKVSESFPYKWINKKWKEGFHVTSMATAGNRWGVVMSRNAGYSHQVVELDFLYPSEGIHRRWETGYRITSTAATPDQAAFILSIPKRKPMDETQETLRTSSFPSNHVKEKWSKNLYIASICYGRTVC (SEQ ID NO: 2)

[0047] CDS sequence of EFL2m gene (2133 bp):

[0048]

[0049] Amino acid sequence of EFL2m protein (from N-terminal to C-terminal, 711 aa):

[0050] MPELRGGVWRARLRSKKVYDVQDADPAASPVSPAPRGRTGRRGGAAAGRGNKTVAEGGGRKALKPRGKGCRAVDLCKDQPCKDLPEVIARKAVTGKAQEDLGLNKVADRAANLMMDGESGDKFAAAEDESTTTPVPERVQVGNSPEYITDRKLGKGGFGQVYVGRRVSGGGSRTGPDAQEVALKFEHRSSKGCNYGPPYEWQVYHTLNGCYGIPSVHYKGRLGDYYILVMDMLGPSLWDVWNSVGQAMSAHMVACIAVEAISILEKLHSKGFVHGDVKPENFLLGHPGSVDEKKLFLIDLGLASRWKEASSGQHVDYDQRPDVFRGTIRYASVHAHLGRTGSRRDDLESLAYTLIFLIRGRLPWQGYQGDNKSFLVCKKKMATSPELLCCFCPAPFKHFLEMVTNMKFDEEPNYPKLISLFDGLIEGPASRPIRIDGALKVGQKRGRMVVNLDDDEQPKKKVRLGSPATQWISVYNARRPMKQRYHYNVADSRLHQHIEKGNEDGLYISCVSSSANFWALIMDAGTGFCSQVYELSQVFLHKDWIMEQWEKNYYITAIAGATNGSSLVVMSKGTPYTQQSYKVSESFPYKWINKKWKEGFHVTSMATAGNRWGVVMSRNAGYSHQVVELDFLYPSEGIHRRWETGYRITSTAATPDQAAFILSIPKRKPMDETQETLRTSSFPSNHVKVHTSSALPTSQCHTKSYDASNFF (SEQ ID NO: 4)

[0051] 4. Detection of EFL2 transcription level

[0052] Considering that exon and intron mis-splicing might affect the transcript of EFL2 gene, the EFL2 transcript of wild type rice Nipponbare and rice efl2 mutant was further detected. The experimental method is as follows:

[0053] Rice seedlings were grown in rice incubator (MLR-351H, SANYO, Japan) under 28°C with 16h light and 8h dark per day, light intensity (150-200μM m -2 s -1 ), and humidity 70%. After 2 weeks of culture in Kimura B nutrient solution, 1cm stem base tissue samples were collected from seedlings of different materials.

[0054] RNA was extracted from rice seedlings using Trizol method (Life Technologies: 15596-026), and the reagents and consumables used in the RNA extraction process were free of RNase. The tissue samples of rice seedlings were ground in liquid nitrogen, and the sample was kept frozen during the entire grinding process. 100-150mg of powder sample was loaded into a 1.7mL centrifuge tube, 1mL of Trizol was added, and the mixture was vortexed and mixed well. After standing at room temperature for 5min, 200μL of chloroform was added and immediately inverted for 15s, and then stood at room temperature for 5min. After centrifugation at 12,000g for 15min at 4°C, 500μL of supernatant was taken into a 1.7mL centrifuge tube, and an equal volume of isopropanol was added. After mixing well, the mixture was stood at room temperature for 10min, and then centrifuged at 12,000g for 10min at 4°C. The supernatant was discarded, and 1mL of 70%(v / v) ethanol was added to the precipitated RNA, which was inverted for 30 times and centrifuged at 7,500g for 5min. The supernatant was discarded, and after the precipitate was dried, 50μL of RNase-free water was added and placed on ice for more than 3h to allow the RNA to dissolve completely. The OD values at 230nm, 260nm and 280nm were measured using a NanoDrop 2000 ultraviolet-visible spectrophotometer to evaluate the overall quality of the RNA. The RNA sample was directly subjected to RNA reverse transcription experiment or stored at -20°C for later use.

[0055] The residual DNA in total RNA was removed using TURBO DNA-free TM Kit according to the operation steps recorded in the product manual, with a reaction system of 20μL, total RNA of 12.5μg, incubation at 37°C for 30min, addition of 2μL DNase inactivation reagent, thorough mixing at room temperature for 5min, centrifugation at 12,000g for 5min at room temperature, and taking 4μL of supernatant for reverse transcription experiment. The cDNA first strand was synthesized using III First-Strand Synthesis System reverse transcription kit according to the operation steps recorded in the product manual, and finally 180μL of nuclease-free double distilled water was added to dissolve the reverse transcription product, which was thoroughly mixed and used.

[0056] The transcription level of the EFL2 gene in the base of rice seedling stems was detected using quantitative real-time PCR (qPCR), with the Ubiquitin gene (NCBI accession number: NM_001056014) as an internal reference. Each 10 μL reaction volume in the qPCR consisted of 5 μL of SoFast EvaGreen supermix, 0.5 μL of forward primer (5 μM), 0.5 μL of reverse primer (5 μM), 2.0 μL of diluted cDNA, and 2 μL of ddH2O. The instrument used for qPCR was a BIO-RAD CFX96 real-time PCR instrument. The PCR program was: 98℃, 30 s; (98℃, 5 s → 60℃, 5 s → data acquisition) 40 cycles; 60-95℃, 0.5℃ / 5 s, data acquisition / 5 s. After the program was completed, the data was processed using BIO-RAD CFX Manager software.

[0057] Primers for quantitative real-time PCR of the EFL2 gene:

[0058] qEFL2-F:5'-AGGAACTCCATACACACAGCAG-3'(SEQ ID NO:5)

[0059] qEFL2-R:5'-CATGACAACTCCCCAACGGT-3'(SEQ ID NO:6)

[0060] Primers for real-time PCR of the internal reference gene:

[0061] qUbiquitin-F:5'-AACCAGCTGAGGCCCAAGA-3'(SEQ ID NO:7)

[0062] qUbiquitin-R:5'-ACGATTGATTTAACCAGTCCATGA-3'(SEQ ID NO:8)

[0063] The results are as follows Figure 4 As shown in Figure B, there was no significant difference in the EFL2 gene transcript between wild-type rice Nipponbare (WT) and the rice efl2 mutant, indicating that the mutation at the splice site of the EFL2 gene in the rice efl2 mutant did not affect the mRNA level of the EFL2 gene itself.

[0064] Example 2: Creation and Phenotypic Analysis of Transgenic Rice Materials

[0065] To further confirm that EFL2 gene is the gene controlling the dominant early flowering phenotype, we performed transgenic experiment on EFL2 gene. The coding sequence (CDS) of EFL2 gene and EFL2m gene were amplified using the transcript cDNA of wild type rice Nipponbare and rice efl2 mutant as templates, respectively. The gene CDS was cloned into pAHLG-GFP vector, and the obtained recombinant expression vector was used to transform wild type rice Nipponbare. The pAHLG-GFP vector is a binary vector described in the section of “Plasmid construction and transformation” in the article of Jiang, L., Liu, X., Xiong, G., Liu, H., Chen, F., Wang, L., Meng, X., Liu, G., Yu, H., Yuan, Y., et al.

[0066] (2013). DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401-405.

[0067] 1. Gene amplification

[0068] The total RNA of wild type rice Nipponbare and rice efl2 mutant was extracted, respectively, and the total RNA was reversely transcribed into cDNA by the same method as in Example 1. The coding sequence of EFL2 gene (SEQ ID NO: 1) was amplified by PCR using the cDNA of wild type rice Nipponbare as template and primers AHLG-EFL2-F and AHLG-EFL2-R. The coding sequence of EFL2m gene (SEQ ID NO: 3) was amplified by PCR using the cDNA of rice efl2 mutant as template and primers AHLG-EFL2m-F and AHLG-EFL2m-R. The nucleotide sequences of the primers are as follows:

[0069] AHLG-EFL2-F:

[0070]

[0071] AHLG-EFL2-R:

[0072] AHLG-EFL2m-F:

[0073] AHLG-EFL2m-R:

[0074] ​In the above primers, the black bold sequence is used to bind the cDNA of the target gene, and the underlined sequence is used for homologous recombination with the pAHLG-GFP vector.

[0075] The PCR reaction system (50 μL) was as follows: 5 μL of 10x PCR Buffer (KOD-Plus-neo enzyme buffer), 1 μL of KOD-Plus-neo enzyme (TOYOBO Co., Ltd., product number KOD-401), 5 μL of 2 mM dNTP, 3 μL of 25 mM magnesium sulfate, 1.5 μL of upstream primer (10 μM), 1.5 μL of downstream primer (10 μM), 1 μL of cDNA template, and 32 μL of sterile water. The PCR reaction program was as follows: 94°C pre-denaturation for 2 min, 1 cycle; (98°C denaturation for 10 sec, 55°C annealing for 30 sec, and 68°C extension for 2 min) 35 cycles; 68°C extension for 5 min, 1 cycle; and 12°C incubation. After the reaction, the PCR product was detected by agarose gel electrophoresis, and the EFL2 gene fragment and the EFL2m gene fragment were recovered by cutting the gel.

[0076] 2. Vector construction

[0077] The pAHLG-GFP vector was linearized by using ApaI and XbaI restriction enzymes. The recovered EFL2 gene fragment and the EFL2m gene fragment were subjected to homologous recombination with the linearized pAHLG-GFP vector, respectively, to obtain an EFL2 overexpression vector (AHLG-Act:EFL2-GFP) and an EFL2m overexpression vector (AHLG-Act:EFL2m-GFP). The homologous recombination reaction system (10 μL) was as follows: 1 μL of linearized pAHLG-GFP vector (30 ng / μL), 1 μL of gene fragment (200 ng / μL), 2 μL of recombinase (Clontech Co., Ltd., product number 639649), and 6 μL of double distilled water. The reaction conditions were as follows: 50 degrees Celsius for 15 min.

[0078] The EFL2 overexpression vector (AHLG-Act:EFL2-GFP) is a recombinant expression vector obtained by replacing the small fragment between the ApaI and XbaI enzyme recognition sites of the pAHLG-GFP vector with SEQ ID NO: 1, while maintaining the other nucleotide sequences of the pAHLG-GFP vector. The recombinant expression vector expresses a fusion protein of EFL2 protein and green fluorescent protein (GFP) (EFL2-GFP).

[0079] EFL2m overexpression vector (AHLG-Act:EFL2m-GFP) is a recombinant expression vector obtained by replacing the small fragment between the Apal and Xbal enzyme recognition sites of the pAHLG-GFP vector with SEQ ID NO: 3, and keeping the other nucleotide sequences of the pAHLG-GFP vector unchanged, which expresses the fusion protein (EFL2m-GFP) of EFL2m protein and GFP.

[0080] 3. Induction and subculture of rice callus

[0081] Using wild type Nipponbare as the rice recipient, the seeds of Nipponbare to be transformed were shelled, the surface of the seeds was sterilized with 70% (v / v) ethanol for 1 min, then washed with 2.5% (w / v) sodium hypochlorite solution with a rotation speed for 45 min, and washed with sterile water for three times. The seeds were then sowed on NB solid medium (medium for inducing callus from rice seeds) and cultured at 28°C in the dark for two weeks. After the callus grew from the mature embryo shield, the callus was cut and subcultured on new NB solid medium. The callus was transferred to new NB solid medium every 7 days, and after 3-4 times of transfer, the callus was used for Agrobacterium-mediated genetic transformation. The formula of the medium used is as follows:

[0082] 1 liter of NB basic medium: potassium nitrate 2830 mg, ammonium sulfate 463 mg, potassium dihydrogen phosphate 400 mg, magnesium sulfate heptahydrate 185 mg, calcium chloride dihydrate 166 mg, ferrous sulfate heptahydrate 27.8 mg, disodium ethylenediaminetetraacetate 37.5 mg, manganese sulfate tetrahydrate 10 mg, boric acid 3 mg, zinc sulfate heptahydrate 2 mg, sodium molybdate dihydrate 0.25 mg, copper sulfate pentahydrate 0.025 mg, cobalt chloride hexahydrate 0.025 mg, potassium iodide 0.75 mg, vitamin B1 10 mg, vitamin B6 1 mg, nicotinic acid 1 mg, inositol 100 mg, hydrolyzed casein 300 mg, glutamine 500 mg, glycine 2 mg, proline 1000 mg, 2,4-dichlorophenoxyacetic acid 2 mg.

[0083] NB solid medium: 15 g / L agar was added to the NB basic medium.

[0084] 4. Transformation of rice callus

[0085] When the rice callus grows well, the prepared EFL2 overexpression vector (AHLG-Act:EFL2-GFP) and EFL2m overexpression vector (AHLG-Act:EFL2m-GFP) are respectively transformed into Agrobacterium EHA105 strain by high-voltage electric excitation, and are coated on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and are cultured at 28°C for 2-3 days; 4-5 single colonies of each transformation are inoculated in 7 mL LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and then are cultured at 28°C and 250 rpm overnight; the overnight cultured bacterial liquid is centrifuged at room temperature at 3,000 rpm for 5 min, the supernatant is discarded, and the bacterial pellet is resuspended with rice transformation liquid to obtain Agrobacterium infection liquid for rice callus transformation. The formulations of the used medium and transformation liquid are as follows:

[0086] The composition of LB liquid medium: 10 g / L proteose peptone, 5 g / L yeast extract, 10 g / L NaCl. The LB solid medium is based on the LB liquid medium and adds 15 g / L agar.

[0087] The composition of rice transformation liquid: 2 g of myo-inositol, 2 g of glutamine, 500 mg of hydrolyzed casein, 10 mL of 10% octylphenoxypolyethoxyethane, and 100 μmol of acetyl-syringone are added to 1 liter of NB basic medium.

[0088] The callus of wild-type rice Nipponbare in good condition is collected, an appropriate amount of Agrobacterium infection liquid is added until the callus is completely submerged, and it is placed at room temperature for 20 min with occasional shaking; then the transformed callus is taken out, the excess Agrobacterium infection liquid is absorbed with sterile filter paper, and the transgenic callus of EFL2 overexpression vector (Act:EFL2-GFP / NP rice callus) and the transgenic callus of EFL2m overexpression vector (Act:EFL2m-GFP / NP rice callus) are obtained by culturing in a 23°C incubator in the dark for 2-3 days.

[0089] 5. Planting of transgenic rice seedlings

[0090] The obtained Act:EFL2-GFP / NP rice callus and Act:EFL2m-GFP / NP rice callus were transferred to NB solid screening medium containing hygromycin (50 micrograms / milliliter), and after 7-10 days of culture at 28°C in the dark, they were subcultured to new NB solid screening medium containing hygromycin (50 micrograms / milliliter) for 3-4 rounds of hygromycin screening. The well-grown callus was expanded to differentiation medium, and after about a month of culture at 28°C under light, the grown seedlings were moved to rooting medium, and after about 4 weeks of culture at 28°C under light, the rooted seedlings were transplanted to soil, and after about a month of growth in a greenhouse, they were moved to a field. The formula of the used medium is as follows:

[0091] Differentiation medium: 100 milligrams of inositol, 2 grams of hydrolyzed casein, 0.2 milligrams of naphthalene acetic acid (NAA), 0.2 milligrams of kinetin, 2 milligrams of 6-benzylaminopurine (6-BA), 30 grams of sorbitol, 30 grams of sucrose, 3 grams of hygromycin, and 50 milligrams of plant gel were added to 1 liter of NB basic medium.

[0092] Rooting medium: 1.0-5.0 milligrams of methionine and 0.5 milligrams of IBA were added to 1 liter of NB basic medium.

[0093] 6. Identification of positive plants

[0094] The rice plants were respectively taken leaves by single plant, and the CTAB method was used to extract rice genomic DNA. The extracted DNA was used as a template for PCR amplification with identification primers EFL2-F and AHLG-R. The nucleotide sequences of the primers are as follows:

[0095] EFL2-F: 5'-AGGAACTCCATACACACAGCAG-3' (SEQ ID NO: 13)

[0096] AHLG-R: 5'-CTCGCCCTCGCCGGACACGC-3' (SEQ ID NO: 14)

[0097] The 50 microL PCR reaction system: 1 microL of DNA template, 2 microL of 10 microM primer EFL2-F, 2 microL of 10 microM primer AHLG-R, and 45 microL of gold medal Mix (Beijing Chengke Biological Technology Co., Ltd.). The PCR reaction program: 98°C pre-denaturation for 3 min; (98°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 15 s) for 35 cycles; 72°C extension for 5 min.

[0098] The PCR products were sent to Beijing Ruibo Biotechnology Co., Ltd. for Sanger sequencing. After obtaining the sequencing results, the sequences were aligned. The sequences consistent with the expected sequences were T0 generation positive plants. A total of 16 T0 generation positive single plants of the transgenic AHLG-Act:EFL2-GFP vector (Act:EFL2-GFP plants) and 20 T0 generation positive single plants of the transgenic AHLG-Act:EFL2m-GFP vector (Act:EFL2m-GFP plants) were obtained.

[0099] Eight Act:EFL2-GFP plants and eight Act:EFL2m-GFP plants were randomly selected for breeding, and eight T1 generation lines were planted. Six single plants were randomly collected from each T1 generation line for screening of homozygous positive single plants. The harvested T1 generation seeds were germinated, and 40 germinated seeds were placed on 0.5% agar solid medium containing 50 μg / mL hygromycin (Hyg). After 2-3 days, the root growth of the plants was observed. Normal root growth indicated a positive plant, and vice versa. If all the seeds of each plant could normally grow roots, it was a homozygous positive line. A total of six Act:EFL2-GFP homozygous positive single plants and five Act:EFL2m-GFP homozygous positive single plants were obtained.

[0100] 7. Phenotype statistics of homozygous positive plants

[0101] The experimental method is as follows: wild-type rice Nipponbare, Act:EFL2-GFP homozygous positive single plants (EFL2 overexpression plants), and Act:EFL2m-GFP homozygous positive single plants (EFL2m overexpression plants) were planted in the field of the experimental base of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing. Each line was planted with 24 plants at a plant spacing and row spacing of 17 cm. The growth period was from May to November each year. The field phenotype investigation of the rice material was from August 8 to October 8 each year, and the earliest time for each single plant to sprout the first ear was counted.

[0102] The experimental results are shown in Figure 4 C and Figure 4 D. Compared with the wild-type Nipponbare (WT(NP)), the Act:EFL2-GFP homozygous positive single plants (EFL2 overexpression plants) showed a late growth period phenotype. Compared with the wild-type Nipponbare (WT(NP)), the Act:EFL2m-GFP homozygous positive single plants (EFL2m overexpression plants) showed an early growth period phenotype.

[0103] The above results show that the rice elf2 mutant is a dominant mutant caused by a new allele mutation (EFL2m) of the EFL2 gene, which can make the rice present early flowering without yield reduction. Overexpression of the EFL2m gene in rice can promote early heading of the rice.

Claims

1. A method of obtaining heading time advancement in rice, characterized by, The method comprises: increasing the content of EFL2m protein in rice, wherein the amino acid sequence of the EFL2m protein is shown as SEQ ID NO:

4.

2. The method of claim 1, wherein, The content of EFL2m protein in rice is increased by overexpressing the gene encoding the EFL2m protein in rice.

3. The method of claim 2, wherein, The nucleotide sequence of the gene encoding the EFL2m protein is shown as SEQ ID NO:

3.

4. The method according to any of claims 1 to 3, characterized in that, The method comprises: constructing a recombinant expression vector for overexpressing the gene encoding the EFL2m protein in rice; introducing the recombinant expression vector into rice callus, inducing differentiation and regeneration of positive callus into plants; and screening positive plants carrying the gene encoding the EFL2m protein.

5. The method of claim 4, wherein, The recombinant expression vector is introduced into rice callus by an Agrobacterium-mediated genetic transformation method.

6. The method of claim 4, wherein, The positive plants carrying the gene encoding the EFL2m protein are screened by a polymerase chain reaction and sequencing method.

7. A EFL2 gene mutant for promoting the heading time of rice, characterized in that, The nucleotide sequence of the EFL2 gene mutant is shown as SEQ ID NO: 3; compared with the EFL2 gene with the nucleotide sequence shown as SEQ ID NO: 1, the nucleotide sequence at positions 2066-2121 of the EFL2 gene mutant is changed and 12 nucleotides are added at the 3' end.

8. An expression cassette, a recombinant vector or a recombinant bacteria comprising the EFL2 gene mutant for promoting early heading time of rice according to claim 7.

9. A protein encoded by the EFL2 gene mutant for promoting the heading date of rice according to claim 7, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO:

4.

10. Use of the EFL2 gene mutant for promoting early heading time of rice according to claim 7 or the protein according to claim 9 in cultivating early-maturing rice.

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