Application of zizania sinensis ZlLG1 gene in regulation and control of rice panicle type

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of ZlLG1 gene of zizania sinensis in regulation and control of rice panicle types, the nucleotide sequence of the ZlLG1 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the ZlLG1 gene is shown as SEQ ID NO: 2. The invention aims to realize regulation and control of the rice panicle type by overexpressing the ZlLG1 gene. Under the condition that the growth environment of the ZlLG1 gene overexpression rice is consistent with the growth environment of a control plant, the ZlLG1 gene overexpression rice generates a tissue similar to a convex structure at the base part of a first-level branch, so that the included angle between a spike stalk and the first-level branch becomes larger, and the spike shape becomes scattered.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to an application of a Chinese wild rice Z1LG1 gene in regulating rice panicle type. Background Art

[0002] Rice panicle type refers to the morphological characteristics and spatial distribution of the rice panicle, mainly including panicle-grain structure, panicle length, panicle thickness, panicle weight and other indicators. High-quality panicle types usually have longer panicles, moderate panicle thickness and larger panicle weight, which are conducive to increasing the number of rice grains and yield. Rice panicle types can be divided according to different classification methods: according to the weight of a single panicle, they can be divided into light panicle type, medium panicle type and heavy panicle type; according to the panicle neck angle, they can be divided into upright panicle type, semi-upright panicle type and curved panicle type; according to the grain density, they can be divided into tight panicle type, semi-tight panicle type, semi-loose panicle type and loose panicle type; according to the branching pattern, they can be divided into dense type, intermediate type and loose type; according to the length of the panicle, they can be divided into long panicle type and short panicle type. The ideal panicle type is the best combination related to high yield and high quality under a specific ecological environment and variety type. The definition and design principle of the ideal rice panicle type aims to improve the yield and quality of rice by optimizing the panicle traits.

[0003] Inflorescence structural diversity plays an important role in the process of crop domestication. Adjustment of inflorescence structure is conducive to increasing the number of flowers and thus improving crop yield. Common wild rice presents a typical loose panicle phenotype, which is conducive to self-cross-pollination, seed dissemination after maturity, and reducing disease infection. This is an adaptability to the natural habitat. After common wild rice was domesticated, the panicle type of Asian cultivated rice became tighter. This phenotype is more conducive to high-density rice planting, and panicle branches are more likely to bear fruit and increase harvest efficiency. The OsLG1 gene encodes an SBP family transcription factor, which plays a key role in the process of rice domestication. By selecting the upstream cis-regulatory variation of this gene, the tight panicle phenotype of cultivated rice is caused. In addition, changes in the expression level of the OsLG1 gene will also affect the panicle type. For example, a 48bp deletion variation will lead to an increase in the expression level of the OsLG1 gene, resulting in a loose panicle type. It is worth noting that the rice grains of the tight-panicle type are relatively dense and have poor air permeability. In addition, they squeeze each other, which makes it easy for problems such as seedling lodging and heat damage to occur, which will have an adverse effect on the quality. The rice grains of the loose-panicle type are arranged more loosely, avoiding these problems of tight-panicle varieties, so loose-panicle varieties generally have better quality.

[0004] Chinese foxtail millet resources are very abundant in China. Studies have shown that Chinese foxtail millet and rice are very closely related. It is worth noting that as a species that has transitioned from wild to domesticated, Chinese foxtail millet is an ideal wild resource for expanding and enriching the rice gene pool. It retains a large number of natural excellent traits lost by domesticated crops, such as resistance to rice blast, loose panicle type, sturdy stems, strong tillering ability, low temperature resistance, flooding resistance, rapid filling and maturity, high biological yield, and higher content of grain protein, lysine, polyphenols, flavonoids and anthocyanins than ordinary rice, which can provide important excellent trait gene donor materials for overcoming the narrow bottleneck of rice breeding genetic resources. For example, distant hybridization of Chinese foxtail millet and rice has led to enhanced resistance to rice blast. There are very few Chinese foxtail millet species-specific DNA sequences in the distant hybridization materials of Chinese foxtail millet and rice, and it is speculated that the extensive cytosine methylation variation, transposon activation and the resulting sequence variation caused by the introduction of these exogenous DNA fragments into the rice genome may be the main reason for some trait variation in the introgression hybrid lines. Due to its many excellent traits, Chinese foxtail millet may become one of the ideal gene sources for rice molecular breeding in the future. OsLG1 regulates the panicle type of rice (dense panicle type or loose panicle type). Through the colinearity analysis of Chinese foxtail millet and rice genome, the homologous gene ZlLG1 of OsLG1 was cloned and identified in Chinese foxtail millet, which is expected to become a gene source for optimizing rice panicle type. Therefore, the discovery of important functional genes in Chinese foxtail millet has important practical significance and application prospects for the expansion of rice gene pool, trait optimization and variety improvement. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes an application of the Chinese wild rice ZlLG1 gene in regulating the panicle type of rice. The present invention is for the purpose of regulating the panicle type of rice, separates and applies a DNA fragment containing the ZlLG1 gene, and uses a constitutive promoter to drive the DNA fragment to over-express the ZlLG1 gene, resulting in a loose panicle type.

[0006] The technical solution of the present invention is:

[0007] Application of ZlLG1 gene of Chinese wild rice in regulating rice panicle type, wherein the nucleotide sequence of the ZlLG1 gene is shown in SEQ ID NO: 1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the ZlLG1 gene is shown in SEQ ID NO:2.

[0009] Furthermore, the constructed ZlLG1 gene overexpression vector was transferred into rice to obtain a vector capable of overexpressing ZlLG1.

[0010] Furthermore, the overexpression vector is transformed into Agrobacterium by chemical transformation, and callus tissue is infected with Agrobacterium to obtain independent transformants, and transgenic rice with loose panicle shape is obtained by plant regeneration.

[0011] Beneficial effects of the present invention:

[0012] (1) The present invention adopts PCR technology to amplify a genomic DNA fragment containing the ZlLG1 gene coding sequence from a Chinese wild rice cDNA library, constructs this sequence into a PC1300S-GFP overexpression vector, and uses the vector to transform rice, thereby obtaining transgenic rice plants with loose panicle shape by increasing the expression level of the ZlLG1 gene.

[0013] (2) The present invention provides an application of ZlLG1 gene polymerization of Chinese wild rice in regulating the panicle shape. Under the condition of constant growth environment, compared with the control plant, the rice with overexpression of ZlLG1 gene produces a tissue similar to a convex structure at the base of the primary stalk, resulting in a larger angle between the panicle axis and the primary stalk and a loose panicle shape.

[0014] (3) Based on the sequencing results of the Chinese wild rice genome, the present invention cloned the gene ZlLG1 that regulates panicle type in Chinese wild rice by performing colinearity analysis with the rice genome. Biological function verification showed that overexpression of the ZlLG1 gene caused the panicle type of transgenic rice to become loose, and the present invention confirmed the application approach and method of the Chinese wild rice ZlLG1 gene in regulating the panicle type of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The nucleotide sequence of the ZlLG1 gene was compared with the nucleotide sequence of the homologous gene in rice using ClustalΩ software (publicly available software) in Example 1. Figure 1 It can be seen that the nucleotide sequences of ZlLG1 and OsLG1 have a high degree of similarity, and several sites are conserved; Explanation of the reference numerals: Figure 1 OsLG1 is a gene homologous to the present invention in rice, and ZlLG1 gene is a gene cloned by the present invention.

[0016] Figure 2 This is the physical map of the Z1LG1 overexpression vector in Example 2.

[0017] Figure 3 This is an agarose electrophoresis gel image of the transgenic rice positive plant identification in Example 2; M represents DL2000 marker, B represents blank control, P represents positive control, and 1-32 are transgenic rice plant numbers.

[0018] Figure 4The phenotypes of the control and Z1LG1 overexpressing rice panicles in Example 3. Explanation of the accompanying figures: (A) is the phenotype of the control rice panicle; (B) is the phenotype of the rice panicle obtained by culturing the control rice seeds after the Z1LG1 gene was transferred. The scale bar in the figure is 5 cm.

[0019] Figure 5 The paraffin sections of the tissue of the control and ZlLG1 gene overexpressing rice spike axis and primary stalk angle in Example 4. Explanation of the accompanying figures: (A) is the paraffin section of the tissue of the control rice; (B) is the paraffin section of the tissue of the rice obtained by culturing the control rice seeds after the ZlLG1 gene was overexpressed. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, and the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] The biological material used in the following examples, Zizyphus chinensis, was collected in Huai'an City, Jiangsu Province, China; the control rice was Nipponbare rice, the seeds of which were sourced from Wuhan City, Hubei Province, China.

[0023] Example 1 Obtaining Z1LG1 gene

[0024] 1.1 Extraction of total RNA and preparation of cDNA from Zizyphus chinensis

[0025] 1.1.1 Extraction of total RNA from Zizyphus chinensis

[0026] Polysaccharide and polyphenol plant RNA extraction kit ( Universal Plant Total RNA Isolation Kit, Vazyme) was used to extract RNA from Chinese wild rice leaves and reverse transcribed into cDNA. The RNA extraction from Chinese wild rice leaves was carried out according to the instructions of the plant RNA extraction kit. The specific experimental method steps are as follows:

[0027] (1) The leaf samples of Zizania latifolia were quickly ground into powder in liquid nitrogen. 50 mg of the ground sample was weighed and added with 500 μL of Bufer PRL preheated at 65 °C. The mixture was immediately vortexed for 60 s.

[0028] (2) Incubate the lysate in a 65°C water bath for 5 min, invert twice, and centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 0.5 times the volume of the supernatant with anhydrous ethanol, and mix immediately by pipetting.

[0029] (3) Transfer the above mixed solution to FastPure gDNA-Filter Column II, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0030] (4) Add 500 μL Buffer PRLPlus to FastPure gDNA-Filter Column II, centrifuge at 12,000 rpm for 30 s, and collect the filtrate.

[0031] (5) Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix immediately by pipetting; transfer the mixture to FastPure RNA Column IV, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0032] (6) Add 700 μL of Buffer PRW1 to FastPure RNA Column IV, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0033] (7) Add 500 μL of Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12,000 rpm for 30 s, discard the filtrate, and repeat this step once.

[0034] (8) Centrifuge the FastPure RNA Column IV adsorption column at 12,000 rpm for 2 min to remove the residual Buffer PRW2 in the FastPure RNA Column IV.

[0035] (9) Transfer FastPure RNA Column IV to a new RNase-free 1.5 mL centrifuge tube, add 40 μL of RNase-free ddH2O to the center of the adsorption column membrane, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

[0036] 1.1.2 Preparation of cDNA

[0037] After RNA extraction, the RNA concentration was determined, and 2.0 μg of each sample was taken as a substrate for reverse transcription. Reverse transcription was performed using a reverse transcription kit to obtain cDNA products, which were stored in a -20°C refrigerator for future use.

[0038] Table 1 Reverse transcription PCR system and procedure

[0039]

[0040] 1.2 Amplification of Z1LG1 gene

[0041] The primers designed according to the Z1LG1 gene sequence are as follows:

[0042] ZlLG1-F: 5'-ATGATGAACGTACCATCTGCCAACT-3' (SEQ ID NO: 3);

[0043] ZlLG1-R: 5'-CTAGTGATCGAAGTCGAGATCAAAC-3' (SEQ ID NO: 4).

[0044] The prepared ZlLG1 cDNA was used as a template and primers were used for PCR amplification to obtain the target fragment ZlLG1. The PCR amplification system and reaction procedure are as follows:

[0045] Table 2 PCR system and procedure

[0046]

[0047] The PCR product was sequenced, and the full length of the sequence was 1071 bp. The nucleotide sequence is shown in SED ID NO.1, and the amino acid sequence is shown in SED ID NO.2.

[0048] Example 2 Construction and genetic transformation of Z1LG1 gene overexpression vector

[0049] 2.1 Construction of ZlLG1 gene overexpression vector

[0050] The PC1300S-GFP vector was digested with restriction endonucleases KpnI and BamHI, and the digestion products were separated by agarose gel electrophoresis. The linearized PC1300S-GFP large fragment was recovered using a gel recovery kit. It was recombined with the PCR amplification product ZlLG1, and the target gene was connected to the vector and transformed into Escherichia coli competent DH5α to obtain the ZlLG1 overexpression vector. Its physical map is shown in Figure 2 .

[0051] The ligation product was transformed into Escherichia coli DH5α. The specific transformation steps were as follows: DH5α stored in a -80°C refrigerator was placed on ice for about 10 minutes for ice-thaw, 10 μL of the ligation product was added to 100 μL DH5α, the mixture was mixed by pipetting and then placed on ice for 30 minutes; then, it was heat-shocked in a 42°C water bath for 90 seconds, and the mixture was quickly transferred to an ice bath for 2 minutes; 1 mL of LB liquid culture medium without antibiotics was added to the clean bench, and the mixture was revived and cultured in a shaker at 220 rpm at 37°C for 45-60 minutes; the revived bacterial solution was centrifuged at 6000 rpm for 5 minutes, 500 μL of supernatant was removed with a pipette, 500 μL of culture medium was retained to resuspend the precipitated bacteria, the plate was coated after resuspension, and the mixture was inverted in a 37°C incubator for overnight culture, and the positive clones were picked and cultured in a shaker at 220 rpm at 37°C overnight.

[0052] Design bacterial PCR primers PN272JCR1: 5'-GGTCTCGCACACCTTGTG-3' (SEQ ID NO: 5) and PEGFPN3: 5'-CGTCGCCGTCCAGCTCGACCAG-3' (SEQ ID NO: 6) to detect ZlLG1 monoclonal positive colonies by colony PCR. After electrophoresis of the colony PCR products, positive single colonies with target bands were screened and sequenced for verification.

[0053] The extraction plasmid was transformed into Agrobacterium EHA105 competent cells, and the operation steps were as follows:

[0054] (1) Take the competent cells of Agrobacterium tumefaciens EHA105 stored at -80°C and place them in the room temperature or in the palm of your hand for a while until they are partially thawed. When they are in a state of ice-water mixing, place them in ice.

[0055] (2) Add 5 μL of the extracted plasmid to every 100 μL of Agrobacterium EHA105 competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0056] (3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2-3 h.

[0057] (4) Collect the bacteria by centrifugation at 6000 rpm for one minute. Take about 100 μL of the supernatant and gently blow to resuspend the bacteria. Apply the suspension to an LB plate containing kanamycin antibiotics and place it upside down in a 28°C incubator for 2-3 days.

[0058] 2.3 Obtaining rice with overexpression of ZlLG1 gene

[0059] (1) Callus preparation

[0060] Step 1: Sterilize mature rice seeds. Use appropriate tools to hull mature seeds, discard seeds with mold spots and undeveloped embryos (shriveled, brown), and ensure the integrity and purity of the seeds. After hulling, wash the rice seeds with 75% ethanol for 1 minute, sterilize with 0.15% HgCl2 for 15-20 minutes, and finally wash with sterile ddH2O for 4-5 times. Soak overnight for the last time.

[0061] Step 2, induction of rice callus. Wash the seeds overnight with sterile water, peel off the embryo along the aleurone layer with a scalpel, and inoculate them on the induction medium. Inoculate 8-12 sterilized rice seeds into each bottle of induction medium, set the temperature to 30°C, and culture in the dark for 40-45 days to induce the production of callus.

[0062] Step 3, callus subculture. Select pale yellow, granular, dry, and vigorous callus from the induced callus and transfer it to the subculture medium for dark culture for 20 days; during the first subculture, be sure to remove other tissues attached to the callus (such as endosperm, buds, etc.). Callus subcultured once can be infected with Agrobacterium tumefaciens. Callus used for transformation can be subcultured up to twice. Multiple subcultures can easily cause somatic mutations in callus and reduce transformation efficiency.

[0063] Step 4: Pre-culture of callus tissue. From the subcultured callus tissue, select the light yellow, granular, dry, and vigorous callus tissue and transfer it to the pre-culture medium. Inoculate 60-80 mung bean-sized callus particles in each dish. Large callus particles can be crushed with tweezers. Pre-culture at 28°C in the dark for 3-4 days. After the pre-culture, collect the small particles in good condition and with vigorous division with a small spoon into a 250mL sterile triangular flask for Agrobacterium infection.

[0064] (2) Preparation of Agrobacterium

[0065] Step 1: Agrobacterium activation: 2 days before the experiment, streak the Agrobacterium tumefaciens strain containing the target gene on an LA plate containing the corresponding antibiotics, and then culture it at 28°C for 2 days.

[0066] Step 2: Resuspend Agrobacterium. Take out the streaked plate of Agrobacterium tumefaciens, inoculate about one ring of Agrobacterium into 100 mL of suspension medium with an inoculation loop, add 100 μL of acetosyringone stock solution and 2 mL of 50% glucose, and place in a constant temperature shaker at 28°C and 200 rpm for 30 minutes. The concentration of the Agrobacterium suspension is about OD 600 =0.3 is sufficient.

[0067] (3) Agrobacterium infection and liquid co-culture

[0068] Step 1, Agrobacterium infection. Pour the prepared Agrobacterium suspension into the triangular flask containing callus until all callus are submerged, and let it stand for 10 minutes. Pour off the bacterial solution. Take a sterilized small plate containing absorbent paper and filter paper, open the small plate and invert the triangular flask containing callus on the filter paper of the small plate, drain as much bacterial solution as possible, then spread the callus on the filter paper of the sterile large plate, cover it with a piece of sterilized filter paper, press the filter paper lightly with tweezers to absorb the bacterial solution on the surface of the callus, and then remove the filter paper after moisture absorption. Change the filter paper four times up and down, and finally cover the callus with a piece of filter paper, cover the large plate, and dry it naturally for 1-2 hours.

[0069] Step 2: Co-culture. Use tweezers to transfer the dried callus particles to the co-culture medium and seal with sealing glue. Co-culture at 19°C in the dark for 3 days.

[0070] Step 3, washing. Transfer the co-cultivated callus to a washing cup, pour in sterile distilled water until the callus is completely submerged, cover with lid and shake for 20-30 seconds, pour out the sterile distilled water, and wash 3-4 times. Observe. If the distilled water in the washing cup is clear, it means that the Agrobacterium has been basically cleaned, otherwise it needs to be washed again. Finally, pour out the sterile distilled water, add sterile distilled water containing 500 mg / L carbenicillin, and let it stand for 30 minutes. Pour out the sterile distilled water containing 500 mg / L carbenicillin.

[0071] Step 4, callus screening. After the callus is dry, use tweezers to transfer the callus particles to the screening medium, seal with sealing glue, and place in a dark culture room for screening and culture for 20 days (the first screening S1). Select dry calli without Agrobacterium contamination from the S1 medium and transfer them to the S2 medium. Culture in the dark for 20 days and observe whether fresh and tender yellow resistant calli grow. If there are no resistant calli, continue to transfer the dish for S3 screening and culture. Generally, japonica rice varieties can grow resistant calli after two screenings, that is, in the S2 stage.

[0072] Step 5, differentiation culture. Pick small pieces of light yellow, dense, dry, and vigorously growing resistant callus. Pick only one resistant callus from each group, and be careful not to pick callus with Agrobacterium. Evenly place 3-4 small pieces of resistant callus in each bottle of differentiation medium, because callus cells will continue to grow on the differentiation medium. Placing them too densely will easily cause different callus pieces to grow together and be indistinguishable. Culture under light at 28℃ for 30-40 days, with a light cycle of 16h light / 8h dark. After the differentiated seedlings are 3-5cm high, rooting culture can be carried out. During the light culture, the contaminated materials with bacteria should be cleaned up in time.

[0073] Step 6: Plant rooting. Use gun-shaped forceps to take out the differentiated seedlings from the differentiation medium and place them in a sterilized empty plate. Take only one healthy seedling from each callus. Use scissors to clean the seedlings, cut off dead or yellow leaves and roots growing on the differentiation medium, and insert them into the rooting tubes, with one seedling in each tube. Rooting culture in the light culture room for 15-20 days, and transplant after the new roots grow fully.

[0074] Step 7, plant transplanting. Remove the sealing film of the rooting tube, add some tap water, and continue to harden the seedlings in the light culture room for 3-4 days. During the hardening period, small leaf samples can be taken for transgenic positive detection. Take the transformed seedlings out of the rooting tube, wash the attachment culture medium on the roots, and transplant them into a pot or bucket with prepared soil.

[0075] (4) Preparation of reagents and culture medium used in transformation:

[0076] 1) Abbreviations of reagents and solutions:

[0077] The abbreviations of the plant hormones used in the culture medium of the present invention are as follows: 6-BenzylaminoPurine (6-BA), 6-benzyladenine; Indole-3-acetic acid (IAA), indole-3-acetic acid; Napthalene acetic acid (NAA), naphthylacetic acid; 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenoxyacetic acid; Kinetin (KT), 6-glycosylaminopurine.

[0078] 2) Main solution formula:

[0079] MSmax stock solution (10x): 16.5 g NH4NO3, 1.7 g KH2PO4, 19.0 g KNO3, 3.7 g MgSO4·7H2O, 3.32 g CaCl2 or 4.4 g CaCl2·2H2O, dissolve them one by one, and then adjust the volume to 1000 mL at room temperature.

[0080] MSmin stock solution (100x): 2.23g MnSO4·4H2O, 0.86g ZnSO4·7H2O, 0.083g KI, 0.62g H3BO3, 0.025g Na2MoO4·2H2O, 0.0025g CoCl2·6H2O, 0.0025g CuSO4·5H2O, dissolve them one by one, and then make up to 1000mL at room temperature.

[0081] N6max stock solution (10x): 28.3g KNO3, 4.63g (NH4)SO4, 4.0g KH2PO4, 1.85g MgSO4·7H2O, 1.25g CaCl2 or 1.66g CaCl2·2H2O, dissolve them one by one, and then adjust the volume to 1000mL at room temperature.

[0082] N6min stock solution (100x): 0.08 g KI, 0.16 g H3BO3, 0.15 g ZnSO4·7H2O, 0.44 g MnSO4·4H2O or 0.3335 g MnSO4·H2O, dissolve them one by one, and then adjust the volume to 1000 mL at room temperature.

[0083] Fe 2+ -EDTA stock solution (100x): Add about 300mL dH2O and 2.78g FeSO4·7H2O to one reagent bottle; add about 300mL dH2O to another reagent bottle, heat to 70℃, and then add 3.73g Na2·EDTA·2H2O; after everything is dissolved, wait for the solution to cool to room temperature, mix the solutions in the two bottles, and then add dH2O to make up to 1000mL, and store at 4℃ away from light.

[0084] Vitamin stock solution (100x): 0.1g niacin, 0.1g nicotinic thiamine, 1g pyridoxine hydrochloride, 10g inositol, 0.2g glycine, add dH2O to make up to 1000mL, store at 4℃.

[0085] AAmax stock solution (10x): 29.50g KCl, 2.50g MgSO4·7H2O, 1.50g NaH2PO4, 1.50g CaCl2·2H2O, add dH2O to make up to 1000mL, store at room temperature away from light.

[0086] AAmin stock solution (100x): 1.0g MnSO4·H2O, 0.2g ZnSO4·7H2O, 0.0025g CuSO4·5H2O, 0.3g H3BO3, 0.075g KI, 0.0025g CoCl2·6H2O, 0.025g NaMoO4·2H2O, add dH2O to make up to 1000mL, and store at room temperature away from light.

[0087] 6-BA stock solution (1 mg / mL): 100 mg 6-BA, add 1.0 mL 1N KOH and shake until 6-BA is dissolved, then add dH2O to make up to 100 mL and store at room temperature.

[0088] KT stock solution (1 mg / mL): 100 mg KT, add 1.0 mL 1N KOH and shake until KT is dissolved, then add dH2O to make up to 100 mL and store at room temperature.

[0089] 2,4-D stock solution (1 mg / mL): 100 mg 2,4-D, add 1.0 mL 1N KOH and shake for 5 min, then add 10 mL dH2O and shake until 2,4-D is dissolved, make up to 100 mL with dH2O, and store at room temperature.

[0090] 100 mM acetosyringone stock solution: 0.196 g acetosyringone, 10 mL dimethyl sulfoxide, divided into 1.5 mL centrifuge tubes and stored at 4°C.

[0091] IAA stock solution (1 mg / mL): 100 mg IAA, add 1.0 mL 1N KOH and shake until IAA dissolves, then dilute to 100 mL with dH2O and store at room temperature away from light.

[0092] NAA stock solution (1 mg / mL): Add 1.0 mL 1N KOH to 100 mg NAA and shake until NAA is dissolved. Then add dH2O to make up to 100 mL and store at room temperature away from light.

[0093] 1N KOH stock solution: Dissolve 5.6 g KOH in 100 mL dH2O and store at room temperature.

[0094] 0.15% HgCl2: 1.5g HgCl2, first partially or completely dissolve it with 1mL of anhydrous ethanol and then make up to 1000mL with dH2O, stir for 4-8h, and store properly at room temperature.

[0095] 3) Culture medium formulation for rice genetic transformation:

[0096] Induction medium: N6max stock solution (10x) 100 mL, N6min stock solution (100x) 10 mL, Vitamin stock solution (100x) 10 mL, Fe 2+ -EDTA stock solution (100x) 10 mL, 2,4-D stock solution (1 mg / mL) 2.5 mL, hydrolyzed casein 0.6 g, proline 0.3 g, sucrose 30 g, plant gel (Phytagel) 3 g, adjust the pH to 5.9, and add dH2O to 1000 mL.

[0097] Subculture medium: N6max stock solution (10x) 100 mL, N6min stock solution (100x) 10 mL, Vitamin stock solution (100x) 10 mL, Fe 2+-EDTA stock solution (100x) 10 mL, 2,4-D stock solution (1 mg / mL) 2.0 mL, hydrolyzed casein 0.6 g, proline 0.5 g, sucrose 30 g, plant gel (Phytagel) 3 g, adjust the pH to 5.9, and add dH2O to 1000 mL.

[0098] Pre-culture medium: N6max stock solution (10x) 12.5 mL, N6min stock solution (100x) 1.25 mL, Vitamin stock solution (100x) 2.5 mL, Fe 2+ -EDTA stock solution (100x) 25 mL, 2,4-D stock solution (1 mg / mL) 0.75 mL, acetosyringone stock solution 300 μL, 50% glucose solution 5 mL, hydrolyzed casein 0.15 g, sucrose 5 g, agarose 1.75 g, adjust the pH to 5.4, and add dH2O to 250 mL.

[0099] Co-culture medium: N6max stock solution (10x) 12.5 mL, N6min stock solution (100x) 1.25 mL, Vitamin stock solution (100x) 2.5 mL, Fe 2+ -EDTA stock solution (100x) 25 mL, 2,4-D stock solution (1 mg / mL) 0.75 mL, acetosyringone stock solution 300 μL and 50% glucose solution 5 mL, hydrolyzed casein 0.2 g, sucrose 5 g, agarose 1.75 g, adjust the pH to 5.4, and add dH2O to 250 mL.

[0100] Suspension medium: N6max stock solution (10x) 5 mL, N6min stock solution (100x) 0.5 mL, Vitamin stock solution (100x) 1 mL, Fe 2+ -EDTA stock solution (100x) 0.5 mL, 2,4-D stock solution (1 mg / mL) 0.2 mL, acetosyringone stock solution 100 μL and 50% glucose solution 2 mL, hydrolyzed casein 0.08 g, sucrose 2 g, adjust the pH to 5.4, and add dH2O to 100 mL.

[0101] Screening medium: N6max stock solution (10x) 25mL, N6min stock solution (100x) 2.5mL, Vitamin stock solution (100x) 2.5mL, Fe 2+-EDTA stock solution (100x) 2.5 mL, 2,4-D stock solution (1 mg / mL) 0.625 mL, 400 μL of 400 mg / mL carbenicillin, 250 μL of 50 mg / mL hygromycin B, 5 mL of 50% glucose solution, 0.15 g of hydrolyzed casein, 7.5 g of sucrose, and 1.75 g of agarose, adjust the pH to 6.0, and add dH2O to 250 mL.

[0102] Differentiation medium: MSmax stock solution (10x) 100 mL, MSmin stock solution (100x) 10 mL, Vitamin stock solution (100x) 10 mL, Fe 2+ -EDTA stock solution (100x) 10 mL, 6-BA 2.0 mL, KT 2.0 mL, IAA 0.2 mL, NAA 0.2 mL, sucrose 30 g, hydrolyzed casein 1 g, plant gel (Phytagel) 3 g, adjust the pH to 6.0, and add dH2O to 1000 mL.

[0103] Rooting medium: MSmax stock solution (10x) 50 mL, MSmin stock solution (100x) 5 mL, Vitamin stock solution (100x) 10 mL, Fe 2+ -EDTA stock solution (100x) 10 mL, sucrose 20 g, plant gel (Phytagel) 3 g, adjust the pH to 5.8, and add dH2O to 1000 mL.

[0104] 2.4 Identification of plants overexpressing the ZlLG1 gene

[0105] Cut the leaves of the transformed plants, extract DNA using the CTAB method, and perform PCR detection using the specific primers for the screening marker gene. The operation method is as follows:

[0106] (1) Take 1-2 g of freshly transformed rice leaves, put them into a mortar precooled with liquid nitrogen, add liquid nitrogen to grind them into powder, and transfer them to a 2 mL centrifuge tube; (2) Add 600 μL of CTAB separation buffer, invert the centrifuge tube, mix well, and place it in a 65°C water bath for 30 min, shaking it gently every 3-4 min to mix well; (3) Add an equal volume of chloroform:isoamyl alcohol solution with a volume ratio of 24:1, invert the centrifuge tube, mix well, centrifuge at 12000 rpm for 15 min, and transfer the supernatant to a new 1.5 mL centrifuge tube; (4) Add 0.6 times the volume of isopropanol, mix gently, place it at -20°C to precipitate DNA for 1 h, centrifuge it at 12000 rpm for 15 min, and discard the supernatant; (5) Add 700 μL of DNA to the DNA precipitate. Wash with 70% ethanol, invert the centrifuge tube to mix well, centrifuge at 12000 rpm for 5 min, pour off the supernatant, and place the DNA precipitate in a clean bench to dry naturally; (6) Dissolve the DNA in ddH2O and store at -20°C for later use.

[0107] The positive identification primers for overexpression vector transformation were designed across the promoter, GFP-420F: 5'-CCACAAGTTCAGCGTGTCC-3' (SEQ ID NO: 7), GFP-420R: 5'-TTCACCTTGATGCCGTTCT-3' (SEQ ID NO: 8), agarose electrophoresis gel image is shown in Figure 3 .

[0108] Example 3 Observation of rice panicle phenotypes in Z1LG1 gene overexpression and control

[0109] The ZlLG1 gene overexpression and control rice were cultured under the same greenhouse conditions, and the same culture medium and culture temperature were used for growth conditions. All rice materials were grown under normal water and fertilizer conditions until the heading stage, and photos were taken with a digital camera. The control rice had a dense panicle phenotype, while the ZlLG1 gene overexpression rice had a loose panicle phenotype. Figure 4 As shown, overexpression of the ZlLG1 gene leads to loose panicle shape in rice.

[0110] Example 4 Paraffin sections of tissues showing the angle between the rice cob and the primary stalk in Z1LG1 gene overexpression and control

[0111] Cut the rice ears at the heading stage, and go through the steps of sampling, fixation, washing and dehydration, transparency, wax dipping, embedding, slicing and spreading, dewaxing, staining, dehydration, transparency, and sealing. Under a microscope, observe the angle between the ZlLG1 gene overexpression rice ear axis and the primary branch stalk. The operation method is as follows:

[0112] Step 1, sampling and fixation. Cut off the primary branches of the ZlLG1 gene overexpression and control rice panicle axis with a blade, add them to a 5 mL centrifuge tube that has been added with 5 mL of pre-cooled 50% FAA fixative (50 mL of anhydrous ethanol, 10 mL of 37% formaldehyde solution, 5 mL of glacial acetic acid, and double distilled water to 100 mL), put the fixative on ice, vacuum for 15 minutes, slowly deflate, repeat twice, until the material sinks to the bottom, and fix for 2 days.

[0113] Step 2, washing and dehydration. Replace the fixative with 50% ethanol solution, replace three times, 30 minutes each time for formaldehyde and acetic acid in the fixative. Then dehydrate with gradient ethanol, 70% ethanol for 2 hours, 85% ethanol for 1 hour, and 95% ethanol (containing eosin) for 2 hours.

[0114] Step 3, continue dehydration and transparency. Perform the following operations respectively: soak in anhydrous ethanol for 1 hour, soak in anhydrous ethanol for 1 hour, soak in 3 / 4 volume anhydrous ethanol + 1 / 4 volume xylene for 1 hour, soak in 1 / 2 volume anhydrous ethanol + 1 / 2 volume xylene for 1.5 hours, soak in 1 / 4 volume anhydrous ethanol + 3 / 4 volume xylene for 1.5 hours, soak in xylene for 1 hour, and soak in xylene for 1 hour. Then make it transparent with chloroform, soak in anhydrous ethanol for 1 hour, soak in anhydrous ethanol for 1 hour, soak in 4 / 5 volume anhydrous ethanol + 1 / 5 volume chloroform for 3 hours, soak in 3 / 5 volume anhydrous ethanol + 2 / 5 volume chloroform for 3 hours, soak in 2 / 5 volume anhydrous ethanol + 3 / 5 volume chloroform for 3 hours, soak in 4 / 5 volume anhydrous ethanol + 1 / 5 volume chloroform for 1.5 hours, soak in pure chloroform for 1 hour, and soak in pure chloroform for 1 hour.

[0115] Step 4, wax immersion. At 42°C, place the sample in xylene + broken wax (as much as possible) overnight. In the next 48 hours, every 3-5 hours, wait for the last wax to dissolve and replenish the broken wax. Then, at 48°C, place the sample in 1 / 2 volume of xylene + 1 / 2 volume of paraffin for 2 hours. Continue to place the sample in 1 / 4 volume of xylene + 3 / 4 volume of paraffin for 2 hours at 50°C. Then, at 60°C, place the sample in pure wax for 1 hour. Repeat placing it in new pure wax 3 times.

[0116] Step 5: Embedding. Fold the paper box in advance, melt new paraffin wax on the induction cooker, and place it in a 60℃ incubator for later use. Spread a layer of embedding wax first, then pour the material in the glass bottle into the paper box with a thickness of 1-2cm. Heat the material slightly with tweezers, arrange the material evenly, let it cool naturally, and collect it the next day.

[0117] Step 6: Trim the wax block. Take the cooled wax block out of the paper box, turn it over, and use a single-edged knife to trim the material into a trapezoidal platform that is narrow at the top and wide at the bottom.

[0118] Step 7: Glue. Put a little wax on the blade and heat it on an alcohol lamp. Apply it on the wood block and quickly glue the wax block to the wood block and let it cool.

[0119] Step 8: Slice. Clamp the small wood block in the slicer and slice it.

[0120] Step 9, spread the slices. Put the cut sample slices into 40℃ constant temperature water to spread them out, pick them up with an adhesive slide so that the sample slices adhere to the slide, move them to a 40℃ spread table, put them in a slice box, and bake them at 38-42℃ for 3 days.

[0121] Step 10, dewaxing and rehydration. The dewaxing and rehydration were carried out in the following solutions respectively: 1) soaking in xylene for 20 min, 2) soaking in xylene for 20 min, 3) soaking in 50% xylene + 50% anhydrous ethanol for 2 min, 4) soaking in anhydrous ethanol for 2 min, 5) soaking in anhydrous ethanol for 2 min, 6) soaking in 95% ethanol for 1 min, 7) soaking in 85% ethanol for 1 min, 8) soaking in 70% ethanol for 1 min, 9) soaking in 50% ethanol for 1 min, 10) soaking in 30% ethanol for 1 min, 11) soaking in 15% ethanol for 1 min, 12) soaking in double distilled water for 1 min, 13) soaking in double distilled water for 1 min.

[0122] Step 11: Staining: Stain with 0.5% toluidine blue solution (0.5 g toluidine blue dissolved in 100 mL double distilled water) for 30 min.

[0123] Step 12, dehydration and transparency. Use the dyeing vat except 3) in step 10 in reverse order, soaking 13)-4) for 1 minute each, and 2)-1) for 5 minutes each.

[0124] Step 13, sealing. Pour Canadian gum stock solution into the reagent bottle first, then add xylene to dilute (volume ratio 1:2), stir evenly with a glass rod, add one or two drops of sealing agent on the slide, pick up a clean cover glass with tweezers, touch the left edge of the cover glass with xylene, slowly put down the cover glass, try to avoid bubbles, blow in the fume hood for 15 minutes, then spread it flat on the slide box, and dry it in a 40℃ constant temperature box overnight.

[0125] Step 14: Observe and photograph the slices under a microscope.

[0126] Observation results such as Figure 5 As shown, the angle between the panicle axis and the primary stalk of the control rice is smaller, while the rice with overexpression of the ZlLG1 gene produces a tissue similar to a bulge structure at the base of the primary stalk, resulting in a larger angle between the panicle axis and the primary stalk of the rice with overexpression of the ZlLG1 gene.

[0127] Compared with the control rice, the rice with overexpression of ZlLG1 gene in the present invention produces a tissue similar to a convex structure at the base of the primary stalk, which leads to a larger angle between the panicle axis and the primary stalk and a loose panicle shape. Therefore, the ZlLG1 gene of Chinese wild rice has important application value in regulating the panicle shape of rice.

[0128] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of Z1LG1 gene of Chinese wild rice in regulating rice panicle type, characterized in that: The nucleotide sequence of the ZlLG1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the ZlLG1 gene is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that: The constructed ZlLG1 gene overexpression vector was transferred into rice to obtain transgenic rice capable of overexpressing ZlLG1.

4. The use according to claim 3, characterized in that: The ZlLG1 gene sequence is constructed into an overexpression vector, and the overexpression vector is transferred into rice, so as to increase the expression amount of ZlLG1 gene mRNA, thereby causing the panicle shape to become loose.

5. The use according to claim 4, characterized in that: The overexpression vector is transformed into Agrobacterium by chemical transformation, and callus tissue is infected by Agrobacterium to obtain independent transformants, and the transgenic rice is obtained by plant regeneration.

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