Application of Chinese bulrush ZlLG1 gene in regulating panicle type of rice

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a Potamogeton crispus ZlLG1 gene in regulation of panicle type of rice, wherein a nucleotide sequence of the ZlLG1 gene is shown as SEQ ID NO:1, and an amino acid sequence of the ZlLG1 gene is shown as SEQ ID NO:2. The application aims to realize regulation of panicle type of rice by overexpressing the ZlLG1 gene. In the case of being consistent with a growth environment of a control plant, the ZlLG1 gene overexpressed rice generates a tissue similar to a convex structure at a base of a primary branch, leading to a larger included angle between a panicle axis and the primary branch and a scattered panicle type.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a Chinese bulrush ZlLG1 gene in regulation of rice panicle type. BACKGROUND

[0002] Rice panicle type refers to the morphological characteristics and spatial distribution of the panicle of rice, mainly including panicle grain structure, panicle length, panicle thickness, panicle weight and the like. A high-quality panicle type usually has a longer rice panicle, moderate panicle thickness and larger panicle weight, and such a panicle type is conducive to increasing the number of rice grains and yield. The rice panicle type can be classified according to different classification methods: according to the weight of a single panicle, it can be classified into light panicle type, medium panicle type and heavy panicle type; according to the panicle neck angle, it can be classified into straight panicle type, semi-straight panicle type and curved panicle type; according to the grain density, it can be classified into tight panicle type, semi-tight panicle type, semi-loose panicle type and loose panicle type; according to the branching mode, it can be classified into dense type, intermediate type and loose type; and according to the length of the panicle, it can be classified into long panicle type and short panicle type. An ideal panicle type is the best combination related to high yield and high quality under 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] Panicle structure diversity plays an important role in the domestication process of crops, and the adjustment of panicle structure is conducive to increasing the number of flowers, thereby improving the yield of crops. The common wild rice presents a typical loose panicle phenotype, which is conducive to self-pollination, seed dissemination and reduction of disease infection, which is an adaptation to the natural habitat. After domestication, the panicle type of Asian cultivated rice becomes tight, which is more conducive to high-density planting of rice, and the panicle branching is more likely to increase, thereby improving the harvesting efficiency. The OsLG1 gene encodes an SBP family transcription factor, which plays a key role in the domestication process of rice, and the tight panicle phenotype of cultivated rice is caused by selecting a variation in the upstream cis-regulation of the gene. In addition, the expression level of the OsLG1 gene also affects the panicle type, for example, a 48bp deletion variation causes the expression level of the OsLG1 gene to increase, thereby causing the panicle type to become loose. It is worth noting that the grains of the tight panicle type are relatively dense, the ventilation is poor, and the grains are easily pressed against each other, which is prone to problems such as seedling lodging and heat damage, which will adversely affect the quality. The grains of the loose panicle type are arranged relatively loosely, avoiding the above problems of the tight panicle type, so the quality of the loose panicle type is generally better.

[0004] Zizania caduciflora is very rich in China. Research shows that Zizania caduciflora and rice have a very close relationship. It is worth noting that as a species from wild to domestication, Zizania caduciflora is an ideal wild resource for expanding and enriching the rice gene pool, and retains a large number of natural excellent traits lost by domesticated crops, such as resistance to rice blast, loose spike type, strong tillering, low temperature resistance, low irrigation resistance, fast grain filling, high biomass, and high grain protein, lysine, polyphenol, flavonoid and anthocyanin content than ordinary rice. It can provide important excellent trait gene donor materials to overcome the narrow bottleneck of rice breeding genetic resources. For example, Zizania caduciflora and rice distant hybridization leads to enhanced rice blast resistance. Zizania caduciflora and rice distant hybridization materials contain a small amount of Zizania caduciflora species specific DNA sequences, and it is speculated that the extensive cytosine methylation variation, transposon activation and sequence variation caused by the introduction of these foreign DNA fragments into the rice genome may be the main reason for some trait variation in the introgression hybrid line. Due to its many excellent traits, Zizania caduciflora has the potential to become one of the ideal gene sources for future rice molecular breeding. OsLG1 regulates the spike type of rice (dense spike type or loose spike type), and through genome collinearity analysis of Zizania caduciflora and rice, the homologous gene ZlLG1 of OsLG1 is cloned and identified in Zizania caduciflora, which is expected to become a gene source for optimizing the spike type of rice. Therefore, the exploration of important functional genes in Zizania caduciflora has important practical significance and application prospect for the expansion of rice gene pool, trait optimization and variety improvement. SUMMARY

[0005] The application aims at solving the above problems in the prior art, and provides application of Zizania caduciflora ZlLG1 gene in regulating rice spike type.

[0006] The technical scheme of the application is as follows:

[0007] The application of Zizania caduciflora ZlLG1 gene in regulating rice spike type, wherein the nucleotide sequence of the ZlLG1 gene is shown as SEQ ID NO: 1.

[0008] Further, the amino acid sequence of the ZlLG1 gene coding protein is shown as SEQ ID NO: 2.

[0009] Further, the constructed ZlLG1 gene overexpression vector is transformed into rice to obtain a vector capable of overexpressing ZlLG1.

[0010] Further, the overexpression vector is transformed into Agrobacterium by chemical transformation, and the independent transformants are obtained by Agrobacterium infection of callus, and the spike type of the transgenic rice is changed by plant regeneration.

[0011] The present application has the following advantages:

[0012] (1) The present application adopts PCR technology to amplify the genomic DNA fragment containing the coding sequence of ZlLG1 gene from the cDNA library of Zizania latifolia Turcz, and constructs the sequence into PC1300S-GFP overexpression vector, and then transforms the rice by using the vector, so that the transgenic rice plant with changed spike type is obtained by increasing the expression of ZlLG1 gene.

[0013] (2) The present application provides the application of Zizania latifolia Turcz ZlLG1 gene in regulating the spike type of rice. Under the same growth environment, compared with the control plants, the ZlLG1 gene overexpression rice produces the tissue similar to the protruding structure at the base of the primary branch, so that the angle between the panicle axis and the primary branch is increased and the spike type is changed.

[0014] (3) Based on the genome sequencing results of Zizania latifolia Turcz, the gene ZlLG1 regulating the spike type is cloned from Zizania latifolia Turcz by collinearity analysis with the rice genome. The biological function verification shows that the overexpression of ZlLG1 gene leads to the change of the spike type of the transgenic rice, and the present application proves the application approach and method of Zizania latifolia Turcz ZlLG1 gene in regulating the spike type of rice. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For example 1, the Clustal Omega software (publicly used software) is used to compare the nucleotide sequence of ZlLG1 gene with the homologous gene nucleotide sequence in rice. Figure 1 It can be seen that the ZlLG1 and the OsLG1 nucleotide sequence have high similarity, and some sites are conserved; the reference signs are as follows: in Figure 1 OsLG1 is the gene homologous to the present application in rice, and ZlLG1 gene is the gene cloned in the present application.

[0016] Figure 2 It is the physical map of the ZlLG1 overexpression vector in example 2.

[0017] Figure 3 It is the agarose electrophoresis gel map of the identification of the positive plant of the transgenic rice in example 2; M represents DL2000 marker, B represents blank control, P represents positive control, and 1-32 is the transgenic rice plant number.

[0018] Figure 4This is an observation of the panicle phenotypes of control and ZlLG1 overexpressing rice in Example 3. Figure labels: (A) shows the panicle phenotype of control rice; (B) shows the panicle phenotype of rice obtained from control rice seeds after being transfected with the ZlLG1 gene. The scale bar in the figure is 5 cm.

[0019] Figure 5 Paraffin sections of tissue from the control and ZlLG1 gene-overexpressing rice panicles and the angle between their primary branches are shown in Example 4. Figure labels: (A) Paraffin section of control rice; (B) Paraffin section of rice obtained by culturing control rice seeds after transfection with the ZlLG1 gene. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] The biological material used in the following examples, *Zizania latifolia*, was collected from Huai'an City, Jiangsu Province, China; the control rice was *Nipponbare* rice, with seeds sourced from Wuhan City, Hubei Province, China.

[0023] Example 1: Obtaining the ZlLG1 gene

[0024] 1.1 Extraction of total RNA and preparation of cDNA from Zizania latifolia

[0025] 1.1.1 Extraction of total RNA from Chinese wild rice

[0026] Using a polysaccharide and polyphenol plant RNA extraction kit ( RNA was extracted from *Zizania latifolia* leaves using the Universal Plant Total RNA Isolation Kit (Vazyme) and then reverse transcribed into cDNA. RNA extraction from *Zizania latifolia* leaves was performed according to the instructions for the plant RNA extraction kit; the specific experimental steps are as follows:

[0027] (1) The leaf samples of Chinese wild rice were rapidly ground into powder in liquid nitrogen. 50 mg of the ground sample was weighed and 500 μL of Bufer PRL preheated at 65 °C was added. The sample was then immediately subjected to violent vortexing for 60 s.

[0028] (2) The lysate was placed in a 65°C water bath for 5 min, inverted twice, and centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new 1.5 mL RNase-free centrifuge tube, and 0.5 times the volume of absolute ethanol was added and mixed immediately.

[0029] (3) The mixture was transferred to a FastPure gDNA-Filter Column II, centrifuged at 12000 rpm for 2 min, and the filtrate was discarded.

[0030] (4) 500 μL of Buffer PRLPlus was added to the FastPure gDNA-Filter Column II, centrifuged at 12000 rpm for 30 s, and the filtrate was collected.

[0031] (5) 0.5 times the volume of absolute ethanol was added to the filtrate and mixed immediately. The mixture was transferred to a FastPure RNA Column IV, centrifuged at 12000 rpm for 2 min, and the filtrate was discarded.

[0032] (6) 700 μL of Buffer PRW1 was added to the FastPure RNA Column IV, and it was left at room temperature for 1 min, centrifuged at 12000 rpm for 30 s, and the filtrate was discarded.

[0033] (7) 500 μL of Buffer PRW2 was added to the FastPure RNA Column IV, centrifuged at 12000 rpm for 30 s, and the filtrate was discarded. This step was repeated once.

[0034] (8) The FastPure RNA Column IV was centrifuged at 12000 rpm for 2 min to remove the residual Buffer PRW2 in the FastPure RNA Column IV.

[0035] (9) The FastPure RNA Column IV was transferred to a new RNase-free 1.5 mL centrifuge tube, and 40 μL of RNase-free ddH2O was added to the center of the adsorption membrane, left at room temperature for 2 min, and centrifuged 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 used as the 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 later use.

[0038] Table 1 PCR system and procedure for reverse transcription

[0039]

[0040] 1.2 Amplification of ZlLG1 gene

[0041] The primers designed according to the sequence of ZlLG1 gene 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 cDNA of ZlLG1 prepared was used as a template, and the 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 sequence was 1071 bp, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

[0048] Example 2 Construction of ZlLG1 gene overexpression vector and genetic transformation

[0049] 2.1 Construction of ZlLG1 gene overexpression vector

[0050] The PC1300S-GFP vector was digested by restriction endonuclease Kpnl and BamHI, the enzyme digestion products were separated by agarose gel electrophoresis, and the linearized PC1300S-GFP large fragment was recovered by a gel recovery kit. Recombination was performed with the PCR amplification product ZlLG1, the target gene was connected to the vector, and the ZlLG1 overexpression vector was obtained by transformation into E. coli competent DH5a, and the physical map is shown in Figure 2 .

[0051] The ligation product was transformed into E. coli DH5a. The specific transformation steps were as follows: the DH5a stored in a-80°C refrigerator was placed on ice for about 10 min for ice bath freeze-thawing, 10 μL of the ligation product was added to 100 μL of the DH5a, and the mixture was mixed by blowing and then was placed in an ice bath for 30 min; then the mixture was heat-shocked in a 42°C water bath for 90 s, and was quickly transferred to an ice bath for 2 min; 1 mL of LB liquid medium without antibiotics was added in a clean bench, and the mixture was recovered and cultured at 37°C in a 220 rpm shaker for 45-60 min; the recovered bacterial solution was centrifuged at 6000 rpm for 5 min, 500 μL of supernatant was taken out with a pipette, 500 μL of the culture medium was used to resuspend the precipitated bacterial cells, and the resuspended mixture was plated and cultured overnight in a 37°C incubator, and a positive clone was picked and cultured overnight at 37°C in a 220 rpm shaker.

[0052] The colony PCR primers PN272JCR1: 5'-GGTCTCGCACACCTTGTG-3' (SEQ ID NO: 5) and PEGFPN3: 5'-CGTCGCCGTCCAGCTCGACCAG-3' (SEQ ID NO: 6) were designed for detecting the positive ZlLG1 monoclonal by colony PCR. After electrophoresis of the colony PCR product, a positive single colony with a target band was screened, and sequencing verification was performed.

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

[0054] (1) The competent cells of Agrobacterium EHA105 stored at-80°C were taken out and placed at room temperature or in the palm of the hand for a while to partially melt, and were inserted into ice when they were in an ice-water mixed state.

[0055] (2) 5 μL of the extracted plasmid was added to each 100 μL of the competent cells of Agrobacterium EHA105, the bottom of the tube was mixed by hand, and the mixture was sequentially placed 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) 700 μL of LB liquid medium without antibiotics was added, and the mixture was cultured at 28°C for 2-3 h.

[0057] (4) The mixture was centrifuged at 6000 rpm for 1 min to collect the bacteria, about 100 μL of supernatant was taken out, the bacterial block was resuspended by gently blowing, and the resuspended mixture was plated on an LB plate containing kanamycin, and was cultured in a 28°C incubator for 2-3 d.

[0058] 2.3 Obtaining of the rice overexpressing ZlLG1 gene

[0059] (1) Preparation of callus

[0060] Step 1, Sterilization of mature rice seeds. With appropriate tools, the mature seeds are dehulled, and seeds with mildew and poor embryo development (shriveling, brown) are discarded to ensure the integrity and cleanliness of the seeds. After dehulling, the rice seeds are first washed with 75% ethanol for 1 min, then sterilized with 0.15% HgCl2 for 15-20 min, and finally washed with sterile ddH2O for 4-5 times. The last time is overnight soaking.

[0061] Step 2, Induction of rice callus. The sterilized seeds are washed with sterile water, and the embryos are peeled off along the aleurone layer with a scalpel. 8-12 sterilized rice seeds are inoculated into each induction medium, the temperature is set to 30°C, and dark culture is performed for 40-45 days to induce the generation of callus.

[0062] Step 3, Subculture of callus. The light yellow, granular, dry, and strong callus is selected from the induced callus and transferred to the subculture medium for dark culture for 20 days. During the first subculture, the other tissues attached to the callus (such as endosperm and buds) are removed completely. The callus subcultured once can be used for Agrobacterium tumefaciens infection, and the callus used for transformation can be subcultured at most twice. Multiple subcultures can cause somatic variation in callus and reduce the transformation efficiency.

[0063] Step 4, Pre-culture of callus. The light yellow, granular, dry, and strong callus is selected from the subcultured callus and transferred to the pre-culture medium. 60-80 callus particles the size of mung beans are inoculated into each dish, and the larger callus particles are crushed with tweezers. The pre-culture is performed at 28°C in the dark for 3-4 days. After the pre-culture, the small particles with good condition and vigorous division are collected with a small spoon into a 250 mL sterile Erlenmeyer flask for Agrobacterium infection.

[0064] (2) Preparation of Agrobacterium

[0065] Step 1, Activation of Agrobacterium. Two days before the experiment, the Agrobacterium tumefaciens strain containing the target gene is streaked on LA plates containing the corresponding antibiotic, and then incubated at 28°C for 2 days.

[0066] Step 2, Resuspension of Agrobacterium. The Agrobacterium tumefaciens streak plate is taken out, and about one ring of Agrobacterium is inoculated into 100 mL of suspension medium with a inoculating loop. 100 μL of acetosyringone stock solution and 2 mL of 50% glucose are added, and the culture is incubated at 28°C, 200 rpm for 30 min on a constant temperature shaker. The concentration of the Agrobacterium suspension is about OD 600 = 0.3.

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

[0068] Step 1, Agrobacterium infection. Pour the prepared Agrobacterium suspension into the flask containing callus until all the callus is immersed, and stand for 10 min. Pour out the bacterial solution. Take a sterilized petri dish containing water-absorbing paper and filter paper, open the petri dish, and invert the flask containing callus on the filter paper of the petri dish, and try to drain the bacterial solution as much as possible. Then spread the callus on the filter paper of a sterile large dish, cover it with a sterilized filter paper, and press the filter paper with a pair of tweezers to absorb the bacterial solution on the surface of the callus. Then remove the filter paper after absorbing the moisture, and repeat the above steps four times. Finally, cover the callus with a filter paper, cover the large dish, and dry naturally for 1-2 h.

[0069] Step 2, co-culture. Transfer the dried callus particles to the co-culture medium with tweezers, and seal with sealing glue. Place in a dark environment at 19°C for 3 d of co-culture.

[0070] Step 3, water washing. Transfer the callus tissue after co-culture to a water washing cup, pour in sterilized distilled water until the callus is completely immersed, cover with a lid and shake for 20-30 s, pour out the sterilized distilled water, and repeat the water washing for 3-4 times. Observe if the distilled water in the water washing cup is clear, which indicates that the Agrobacterium has been basically cleaned, otherwise it needs to be washed continuously. Finally, pour out the sterilized distilled water, add sterilized distilled water containing 500 mg / L carbenicillin, and stand for 30 min. Pour out the sterilized distilled water containing 500 mg / L carbenicillin.

[0071] Step 4, callus screening. After the callus is dried, transfer the callus particles to the screening medium with tweezers, seal with sealing glue, and place in a dark culture room for 20 d of screening culture (first screening S1). Select the dried callus without Agrobacterium contamination from the S1 medium, and transfer it to the S2 medium. Dark culture for 20 d, and observe if fresh and tender yellow resistant callus grows. If there is no resistant callus, continue to transfer the dish for S3 screening culture. Generally, the japonica rice variety can grow resistant callus after two screenings, i.e. at the S2 stage.

[0072] Step 5, differentiation culture. Pick up small pieces of light yellow, dense, dry and vigorous growth resistant callus, and only one resistant callus is picked up for each piece. Pay attention not to pick up callus with Agrobacterium. Place 3-4 small pieces of resistant callus evenly in each piece of differentiation medium, because the callus cells will continue to grow on the differentiation medium, and too dense placement will easily lead to different callus pieces growing together and being indistinguishable. 28°C light culture for 30-40 d, with a light cycle of 16 h light / 8 h dark. After the seedlings are 3-5 cm high, they can be subjected to rooting culture. During the light culture, the contaminated materials should be cleaned up in time.

[0073] Step 6, plant rooting. The differentiated shoots were removed from the differentiation medium with a gun-shaped forceps and placed in a sterile empty Petri dish. Only one healthy seedling was taken from one callus. The seedling was cleaned with scissors and the dead or yellow leaves and roots growing on the differentiation medium were cut off. One seedling was placed in each tube. The seedlings were cultured in the light culture room for 15-20 days. After the new roots grew sufficiently, the seedlings were transplanted.

[0074] Step 7, plant transplantation. The sealing film of the rooting tube was removed and tap water was added. The seedlings were cultured in the light culture room for 3-4 days. During the culture, a small sample of leaves was taken for transgenic positive detection. The transformed seedlings were removed from the rooting tube, washed to remove the adhering medium on the roots, and transplanted into pots or barrels with prepared soil.

[0075] (4) Preparation of reagents and media used in the transformation:

[0076] 1) Abbreviations of reagents and solutions:

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

[0078] 2) Main solution formula:

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

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

[0081] N6 max stock (10x): 28.3 g KNO3, 4.63 g (NH4)2SO4, 4.0 g KH2PO4, 1.85 g MgSO4-7H2O, 1.25 g CaCl2or 1.66 g CaCl2-2H2O, dissolved one at a time, then bring to 1000 mL at room temperature.

[0082] N6 min stock (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, dissolved one at a time, then bring to 1000 mL at room temperature.

[0083] Fe 2+ - EDTA stock (100x): To one reagent bottle, add about 300 mL dH2O and 2.78 g FeSO4-7H2O; to another reagent bottle, add about 300 mL dH2O and heat to 70°C, then add 3.73 g Na2-EDTA-2H2O; after both are dissolved, allow the solutions to cool to room temperature, then mix the solutions from both bottles, then bring to 1000 mL with dH2O, store at 4°C in the dark.

[0084] Vitamin stock (100x): 0.1 g nicotinic acid, 0.1 g nicotinamide thiamine, 1 g pyridoxine hydrochloride, 10 g myo-inositol, 0.2 g glycine, bring to 1000 mL with dH2O, store at 4°C.

[0085] AA max stock (10x): 29.50 g KCl, 2.50 g MgSO4-7H2O, 1.50 g NaH2PO4, 1.50 g CaCl2-2H2O, bring to 1000 mL with dH2O, store at room temperature in the dark.

[0086] AA min stock (100x): 1.0 g MnSO4-H2O, 0.2 g ZnSO4-7H2O, 0.0025 g CuSO4-5H2O, 0.3 g H3BO3, 0.075 g KI, 0.0025 g CoCl2-6H2O, 0.025 g NaMoO4-2H2O, bring to 1000 mL with dH2O, store at room temperature in the dark.

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

[0088] KT stock solution (1 mg / mL): 100 mg KT, add 1.0 mL 1 N KOH and shake until KT is dissolved, then add dH2O 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 1 N KOH and shake for 5 min, then add 10 mL dH2O and shake until 2,4-D is dissolved, add dH2O to 100 mL and store at room temperature.

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

[0091] IAA stock solution (1 mg / mL): 100 mg IAA, add 1.0 mL 1 N KOH and shake until IAA is dissolved, then add dH2O to 100 mL and store at room temperature in the dark.

[0092] NAA stock solution (1 mg / mL): 100 mg NAA, add 1.0 mL 1 N KOH and shake until NAA is dissolved, then add dH2O to 100 mL and store at room temperature in the dark.

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

[0094] 0.15% HgCl2: 1.5 g HgCl2, partially or completely dissolve in 1 mL absolute ethanol and add dH2O to 1000 mL, stir for 4-8 h and store at room temperature.

[0095] 3) Medium formulations for genetic transformation of rice:

[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, Phytagel 3 g, adjust 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 (100x) 10 mL, 2,4-D stock (1 mg / mL) 2.0 mL, Hydrolyzed casein 0.6 g, Proline 0.5 g, Sucrose 30 g, Phytagel 3 g, adjust pH to 5.9, supplement with dH20 to 1000 mL.

[0098] Pre-culture medium: N6max stock (10x) 12.5 mL, N6min stock (100x) 1.25 mL, Vitamin stock (100x) 2.5 mL, Fe 2+ - EDTA stock (100x) 25 mL, 2,4-D stock (1 mg / mL) 0.75 mL, Acetylpyrocatechol stock 300 μL, 50% glucose solution 5 mL, Hydrolyzed casein 0.15 g, Sucrose 5 g, Agarose 1.75 g, adjust pH to 5.4, supplement with dH20 to 250 mL.

[0099] Co-culture medium: N6max stock (10x) 12.5 mL, N6min stock (100x) 1.25 mL, Vitamin stock (100x) 2.5 mL, Fe 2+ - EDTA stock (100x) 25 mL, 2,4-D stock (1 mg / mL) 0.75 mL, Acetylpyrocatechol stock 300 μL and 50% glucose solution 5 mL, Hydrolyzed casein 0.2 g, Sucrose 5 g, Agarose 1.75 g, adjust pH to 5.4, supplement with dH20 to 250 mL.

[0100] Suspension medium: N6max stock (10x) 5 mL, N6min stock (100x) 0.5 mL, Vitamin stock (100x) 1 mL, Fe 2+ - EDTA stock (100x) 0.5 mL, 2,4-D stock (1 mg / mL) 0.2 mL, Acetylpyrocatechol stock 100 μL and 50% glucose solution 2 mL, Hydrolyzed casein 0.08 g, Sucrose 2 g, adjust pH to 5.4, supplement with dH20 to 100 mL.

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

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

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

[0104] 2.4 ZlLG1 gene overexpression plant identification

[0105] The transformed plant leaves were cut and DNA was extracted using CTAB method and PCR detection was performed using specific primers of the selection marker gene. The operation method is as follows:

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

[0107] Cross-promoter design of overexpression vector transformation positive identification primers: GFP-420F: 5'-CCACAAGTTCAGCGTGTCC-3' (SEQ ID NO:7), GFP-420R: 5'-TTCACCTTGATGCCGTTCT-3' (SEQ ID NO:8). Agarose gel images are shown below. Figure 3 .

[0108] Example 3: Observation of ZlLG1 gene overexpression and control rice panicle phenotype

[0109] ZlLG1 gene overexpressing rice was cultured under the same greenhouse conditions as the control rice, with the same culture medium and temperature. All rice materials were grown to the heading stage under normal water and fertilizer conditions, and photographs were taken using a digital camera for data recording. The control rice exhibited a dense panicle phenotype, while the ZlLG1 gene overexpressing rice exhibited a loose panicle phenotype. Figure 4 As shown, overexpression of the ZlLG1 gene leads to a divergent panicle shape in rice.

[0110] Example 4: Paraffin sections of tissue from rice panicle axis and primary branch angle in ZlLG1 gene overexpression and control rice.

[0111] Rice panicles at the heading stage were harvested and subjected to the following steps: sampling, fixation, washing and dehydration, clearing, paraffin embedding, sectioning and spreading, dewaxing, staining, dehydration, clearing, and mounting. The angle between the panicle axis and the primary branch of ZlLG1 gene overexpression and control rice was observed under a microscope. The procedure is as follows:

[0112] Step 1, material collection and fixation. The primary branch of ZlLG1 gene overexpression and control rice panicle axis was cut with a blade and added to a 5 mL centrifuge tube that had been added with 5 mL of pre-cooled 50% FAA fixing solution (50 mL of absolute ethanol, 10 mL of 37% formaldehyde solution, 5 mL of glacial acetic acid, and make up to 100 mL with double distilled water). The fixing solution was placed on ice, vacuumed for 15 min, slowly released, and repeated twice until the material sank to the bottom. Fixation was performed for 2 days.

[0113] Step 2, washing and dehydration. The fixing solution was replaced with 50% ethanol solution, and the formaldehyde and acetic acid in the fixing solution were replaced 3 times, each time for 30 min. Then, the material was dehydrated with gradient ethanol, 70% ethanol for 2 h, 85% ethanol for 1 h, and 95% ethanol (containing eosin) for 2 h.

[0114] Step 3, continued dehydration and transparency. The following operations were performed respectively, soaking in absolute ethanol for 1 h, soaking in absolute ethanol for 1 h, soaking in 3 / 4 volume of absolute ethanol + 1 / 4 volume of xylene for 1 h, soaking in 1 / 2 volume of absolute ethanol + 1 / 2 volume of xylene for 1.5 h, soaking in 1 / 4 volume of absolute ethanol + 3 / 4 volume of xylene for 1.5 h, soaking in xylene for 1 h, and soaking in xylene for 1 h. Then, chloroform transparency was performed, soaking in absolute ethanol for 1 h, soaking in absolute ethanol for 1 h, soaking in 4 / 5 volume of absolute ethanol + 1 / 5 volume of chloroform for 3 h, soaking in 3 / 5 volume of absolute ethanol + 2 / 5 volume of chloroform for 3 h, soaking in 2 / 5 volume of absolute ethanol + 3 / 5 volume of chloroform for 3 h, soaking in 4 / 5 volume of absolute ethanol + 1 / 5 volume of chloroform for 1.5 h, soaking in pure chloroform for 1 h, and soaking in pure chloroform for 1 h.

[0115] Step 4, wax immersion. The sample was placed in xylene + broken wax (as much as possible) at 42°C overnight, and every 3-5 h in the next 48 h, the broken wax was added after the previous wax was dissolved. Then, the sample was placed in 1 / 2 volume of xylene + 1 / 2 volume of paraffin at 48°C for 2 h, continued to be placed in 1 / 4 volume of xylene + 3 / 4 volume of paraffin at 50°C for 2 h, and then placed in pure wax at 60°C for 1 h, and repeated 3 times by placing in new pure wax.

[0116] Step 5, embedding. The paper box was folded in advance, and new paraffin was melted on an electromagnetic stove and placed in a 60°C incubator for standby. A layer of embedding wax was first laid, then the material in the glass bottle was poured into the paper box, with a thickness of 1-2 cm. The material was evenly arranged by slightly heating the tweezers, and the material was allowed to cool naturally. The next day, the material was collected.

[0117] Step 6, wax block trimming. The cooled wax block was taken out of the paper box and turned over. The material was trimmed into a trapezoidal platform with a narrow top and a wide bottom using a single-sided knife.

[0118] Step 7, Adhesion. A little piece of wax is heated on an alcohol lamp, and is applied to the wood block. The wax block is quickly adhered to the wood block, and is cooled.

[0119] Step 8, Sectioning. The small wood block is clamped in a microtome for sectioning.

[0120] Step 9, Unfolding. The sectioned sample is placed in a constant temperature water bath at 40°C, and is unfolded. An adhesive glass slide is used to pick up the sample section, and the sample section is adhered to the glass slide. The glass slide is moved to a 40°C unfolding table, and is placed in a sectioning box. The sectioning box is baked at 38-42°C for 3 days.

[0121] Step 10, De-waxing and rehydration. The following steps are performed in the following solutions, respectively: 1) immersion in xylene for 20 min, 2) immersion in xylene for 20 min, 3) immersion in 50% xylene + 50% anhydrous ethanol for 2 min, 4) immersion in anhydrous ethanol for 2 min, 5) immersion in anhydrous ethanol for 2 min, 6) immersion in 95% ethanol for 1 min, 7) immersion in 85% ethanol for 1 min, 8) immersion in 70% ethanol for 1 min, 9) immersion in 50% ethanol for 1 min, 10) immersion in 30% ethanol for 1 min, 11) immersion in 15% ethanol for 1 min, 12) immersion in double distilled water for 1 min, and 13) immersion in double distilled water for 1 min.

[0122] Step 11, Staining. The sample is stained with a 0.5% toluidine blue solution (0.5 g of toluidine blue is dissolved in 100 mL of double distilled water) for 30 min.

[0123] Step 12, Dehydration and transparency. The following steps are performed in the reverse order of step 10, 13) to 4) each for 1 min, and 2) to 1) each for 5 min.

[0124] Step 13, Mounting. A reagent bottle is first filled with a Canadian balsam solution, and then is diluted with xylene (1:2 by volume). The solution is stirred with a glass rod until it is uniform. One or two drops of mounting agent are added to the glass slide. A clean cover glass is picked up with a pair of tweezers. The left edge of the cover glass is contacted with xylene. The cover glass is slowly lowered so as to avoid air bubbles. The glass slide is blown in a fume hood for 15 min. The glass slide is placed in a sectioning box, and is baked in a constant temperature oven at 40°C overnight.

[0125] Step 14, Sectioning is observed under a microscope and is photographed.

[0126] The observation results are shown in Figure 5 The angle between the panicle axis and the primary branch of the control rice is small. The ZlLG1 gene overexpressed rice produces a protruding structure at the base of the primary branch, which results in a larger angle between the panicle axis and the primary branch of the ZlLG1 gene overexpressed rice.

[0127] Compared with the control rice, the ZlLG1 gene overexpressed rice produces tissue similar to the protruding structure at the base of the primary branch, resulting in the angle between the panicle axis and the primary branch becoming larger and the panicle type becoming scattered. Therefore, the ZlLG1 gene of Zizania caduciflora has important application value in regulating the panicle type of rice.

[0128] The above description is only the preferred embodiments of the present application, and is not a limitation on the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions recorded in the foregoing embodiments, or equivalently replace some technical features, without departing from the spirit and principle of the present application. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of overexpression of the ZlLG1 gene from *Zizania latifolia* in regulating panicle type in rice, characterized by: The nucleotide sequence of the ZlLG1 gene is shown in SEQ ID NO:1, and the regulation of rice panicle type is to cause rice panicle type dispersion.

2. The application 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 application according to claim 1, characterized in that, The constructed ZlLG1 gene overexpression vector was transferred into rice to obtain transgenic rice that could overexpress ZlLG1.

4. The application according to claim 3, characterized in that, The ZlLG1 gene sequence was constructed into an overexpression vector, and the overexpression vector was transferred into rice. By increasing the expression level of ZlLG1 gene mRNA, panicle type became more dispersed.

5. The application according to claim 4, characterized in that, The overexpression vector was chemically transformed into Agrobacterium, and independent transformants were obtained by infecting callus tissue with Agrobacterium. The transgenic rice was obtained through plant regeneration.

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