Application of Rice GS2 Gene in Regulating Rice Panicle Type

Abstract

The present invention discloses the application of the rice GS2 gene in regulating the panicle type of rice, belonging to the technical field of plant genetic engineering. The amino acid sequence of the protein encoded by the GS2 gene is as shown in SEQ ID No: 3; it can regulate rice panicle traits such as grain shape, grain number per panicle, and panicle length. The GS2 gene provided by the present invention can be used for breeding high-yield rice varieties and has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and specifically relates to the application of the rice GS2 gene in regulating the panicle type of rice. Background Art

[0002] Rice is one of the most important food crops in the world, and its yield and quality are affected by various genetic and environmental factors. The panicle type and grain type of rice are important traits determining rice yield and are also the main objectives of rice breeding. The yield of rice is directly determined by the number of panicles per plant, the number of grains per panicle, and the grain weight. The number of grains per panicle depends on the number of primary and secondary branches. Elucidating the genetic and molecular mechanisms controlling these traits helps to cultivate high-yield and high-quality rice varieties.

[0003] As an important determinant of rice yield, the panicle morphology is closely related to it. So far, many genes controlling the panicle type have been discovered, which encode proteins with different functions, such as heterotrimeric G proteins and transcription factors. DEP1 is one of the members of the G protein γ subunit, and its mutant dep1 is a gain-of-function mutation, resulting in shortened panicle length, increased number of branches and grains per panicle, thus increasing the yield. DEP1 is positively regulated by IPA1, which is a member of the SPL transcription factor family and is regulated by miRNA156. The point mutation of OsSPL14 disrupts the regulation of OsSPL14 by miRNA156, producing a large-panicle phenotype, with increased panicle length, number of branches and grains per panicle, and also increased rice yield. Therefore, DEP1 and IPA1 play key roles in regulating the panicle type and the number of grains per panicle in rice.

[0004] The pleiotropic gene GS2 isolated by QTL analysis has been proven to be one of the key regulatory factors determining grain size. GS2 encodes the GRF4 transcription factor, and the mutation of TC(GS2)→AA(GS2 TM ) affects the cleavage of miRNA396, resulting in a large-grain phenotype. The inventors of the present invention found that in addition to regulating the grain size of rice, GS2 is also crucial for the panicle structure of rice. Therefore, it is urgent to deeply study the regulatory mechanism of the GS2 gene and the protein encoded by it on the panicle type and grain type of rice, which has important production application value for increasing rice yield. Summary of the Invention

[0005] To solve the above technical problems, the specific technical solutions provided by the present invention are as follows:

[0006] The present invention provides the application of the rice GS2 gene in regulating the panicle type of rice,

[0007] The gDNA sequence of the GS2 gene is SEQ ID NO:1, the cDNA sequence is SEQ ID NO:2, and the amino acid sequence of the coding region is SEQ ID NO:3;

[0008] The GS2 gene positively regulates panicle length and negatively regulates the number of grains per panicle;

[0009] The protein GS2 encoded by the GS2 gene interacts with the transcription factor IPA1 to jointly regulate the expression of the DEP1 gene, thereby regulating the panicle type of rice.

[0010] In a specific embodiment, by silencing GS2, the expression level of GS2 was reduced, and a GS2 TM -Ri expression vector was constructed, and it was found that the transgenic plants of GS2 TM -Ri had reduced panicle length, increased branch number and the number of grains per panicle; while the homozygous transgenic plants of GS2 TM -OE had increased panicle length, reduced branch number and the number of grains per panicle.

[0011] In a specific embodiment, GS2 was pyramided in hybrid rice TM and IPA1 TM1 , which could significantly increase panicle length and the number of grains per panicle, thereby increasing the rice yield.

[0012] The present invention provides a plasmid containing the rice GS2 gene.

[0013] The present invention provides a plant expression vector containing the rice GS2 gene, and the vector can express the polypeptide or homologous analog encoded by the rice GS2 gene as claimed in claim 1.

[0014] The present invention provides a prokaryotic expression vector containing the rice GS2 gene.

[0015] The present invention provides a host cell containing the rice GS2 gene, and the host cell includes one of Escherichia coli cells, Agrobacterium cells, and plant cells.

[0016] The present invention provides a method for regulating the panicle type of rice, which comprises transforming the rice GS2 gene into rice cells and then cultivating the transformed rice cells into plants.

[0017] The present invention provides a long-panicle rice variety in which the GS2 gene is overexpressed.

[0018] The present invention provides the application of the rice GS2 gene in improving the panicle type of rice and molecular design-assisted breeding.

[0019] The specific technical steps for implementing the present invention are as follows:

[0020] I. GS2 positively regulates panicle length and negatively regulates the number of grains per panicle

[0021] On the basis of previous studies, the inventor of the present invention used ZH11 and GS2 TMLocate the GS2 gene. It is known that GS2 can positively regulate rice grain size. To further explore the effect of GS2 on rice panicle type, the inventor used gene editing technology to construct vectors of GS2-OE and GS2-KO, and transferred them into ZH11 respectively to obtain homozygous transgenic plants of GS2-OE-1, GS2-OE-2, GS2-OE-3, and GS2-KO-1 (deleting 1 bp G), GS2-KO-2 (deleting 2 bp AG), GS2-KO-3 (deleting 5 bp CAGAG), and statistically analyzed the differences in their panicle types ( Figure 1 A). The inventor found that there were no significant differences in panicle length, panicle branch number, grain number per panicle, etc. between the three lines of GS2-KO and the three lines of GS2-OE and ZH11 ( Figure 1 B). Analyzing the reasons, in GS2-KO-1, GS2-KO-2, and GS2-KO-3, due to the deletion of different bases in GS2, protein translation frameshift termination occurred, and the GS2 protein had no function; in the lines of GS2-OE, it may be because the GS2 mRNA was inhibited by miRNA396 cleavage, resulting in the degradation of the GS2 protein, so there was no phenotype.

[0022] To further study the function of GS2, the inventor considered that gene knockout directly disrupted the protein structure of GS2, resulting in the GS2 protein having no function. Therefore, a GS2 TM -Ri expression vector was constructed to reduce the expression level of GS2 by silencing GS2. And it was found that the transgenic plants of GS2 TM -Ri showed an upright and dense panicle type phenotype ( Figure 2 A). A detailed statistical analysis was carried out on the panicle phenotypes of GS2 TM -Ri-1, GS2 TM -Ri-2, and GS2 TM -Ri-3 plants, and it was found that the three lines of GS2 TM -Ri all significantly reduced the panicle length and increased the number of primary branches, secondary branches, and grains per panicle ( Figure 2 B). This indicates that the silencing of GS2 led to changes in panicle development, making the panicle more compact and plump. To compare the effects of GS2 TM -Ri and overexpression, a statistical analysis was also carried out on the panicle phenotypes of the homozygous transgenic plants of GS2 TM -OE, and it was found that GS2 TM -OE-1, GS2 TM -OE-2, and GS2 TM -OE-3 increased the panicle length and reduced the number of secondary branches and grains per panicle ( Figure 2 B). This indicates that GS2 TMOverexpression of GS2 promoted panicle elongation but inhibited panicle branching and grain number per panicle. These results suggest that GS2 TM expression levels have an important impact on panicle development, and GS2 TM can positively regulate panicle length and negatively regulate panicle branching number and grain number per panicle.

[0023] II. GS2 Physically Interacts with IPA1

[0024] To elucidate the regulatory mechanism of GS2 on rice plant architecture and panicle development, a yeast two-hybrid (Y2H) screening method was used. GS2 and GS2 TM were constructed into the pGBKT7 vector to search for interacting proteins of GS2 ( Figure 3 A). The results showed that GS2 and GS2 TM could interact with IPA1. To further explore the interaction region between GS2 and IPA1, according to the protein domains of GS2, it was truncated into 7 proteins of different lengths (amino acids 1-63, amino acids 1-99, amino acids 1-124, amino acids 1-169, amino acids 100-259, amino acids 125-259, and amino acids 170-259). The results showed that the GS2 protein containing the C-terminal amino acid region of 170-259 could interact with IPA1 ( Figure 3 A).

[0025] To verify the existence of the interaction between GS2, GS2 TM and IPA1 in plant cells, the present invention used bimolecular fluorescence complementation (BiFC) technology to express fusion proteins of GS2, GS2 TM and IPA1 in tobacco epidermal cells. The results showed that when co-infecting tobacco epidermal cells with GS2-YC and IPA1-YN or GS2 TM -YC and IPA1-YN, obvious green fluorescence signals could be observed, indicating that an interaction occurred between GS2, GS2 TM and IPA1, and this interaction mainly occurred in the nucleus ( Figure 3 D). In addition, the association between GS2 and IPA1 was further verified by in vivo co-immunoprecipitation experiments (Co-IP) and in vitro pull-down experiments (Pull-down) ( Figure 3 B, C). These results indicate that GS2 and IPA1 may form a complex in rice and jointly participate in the regulation of rice panicle type and grain type.

[0026] III. Identification of GS2 Downstream Target Genes

[0027] The present invention conducted a yeast one-hybrid (Y1H) experiment to verify the interaction between DEP1 and GS2. The promoter of DEP1 was cut into 5 different fragments, and then the 5 fragments were further subdivided to obtain 10 different fragments ( Figure 4 B). It was found that blue spots appeared in the yeast cells of DEP1-1, DEP1-2, DEP1-3, DEP1-6, DEP1-7, DEP1-8, DEP1-9, DEP1-10 and DEP1-11, indicating that GS2 can activate the LacZ reporter gene by binding to their promoters ( Figure 4 A). In addition, the present invention also uses ChIP-PCR and EMSA experiments to confirm that GS2 can bind to DEP1-6, DEP1-7, DEP1-8, DEP1-9, DEP1-10 and DEP1-11 ( Figure 4 C, E). The transient transcription activity of LUC was determined in rice protoplasts. The results showed that GS2 and GS2 TM Both of them can activate the transcription of proDEP1::LUC reporter gene, while the negative control does not, indicating that GS2 is a direct activator of DEP1 ( Figure 4 D).

[0028] 4. GS2, IPA1 and DEP1 affect panicle shape

[0029] In order to explore the role and relationship of GS2, IPA1 and DEP1 in panicles, the present invention constructed different genetic materials ( Figure 5 ). After counting the spike data, it was found that 9311-GS2 TM and WY-GS2 TM Compared with WT, the spike length increased, while 9311-IPA1 TM1 and WY-IPA1 TM2 Compared with WT, not only the ear length increased, but also the number of ear branches and the number of grains per ear increased. However, compared with WT, the ear length of 9311-dep1 and WY-dep1 decreased, but the number of ear branches and the number of grains per ear increased ( Figure 5 In addition, the double mutant 9311-GS2 TM -IPA1 TM1 and WY-GS2 TM -IPA1 TM2 Compared with WT, the ear length, number of ear branches and number of grains per ear increased ( Figure 5 ). This shows that GS2 TM and IPA1 TM1 The effects on ear development are additive rather than offsetting. This additive effect may be due to the TM and IPA1TM1 Simultaneously affect the expression or activity of DEP1, thereby enhancing the regulatory effect of DEP1 on panicle development. In addition, 9311-dep1-GS2 TM , WY-dep1-GS2 TM , 9311-dep1-IPA1 TM1 and WY-dep1-IPA1 TM2 Compared with WT, the number of primary and secondary branches and the number of grains per panicle also increased significantly, but there was no significant difference compared with dep1 ( Figure 5 ). Similar results were also observed in the triple mutants 9311-dep1-GS2 TM -IPA1 TM1 and WY-dep1-GS2 TM -IPA1 TM2 , indicating that DEP1 masks the effects of GS2 TM , IPA1 TM1 and IPA1 TM2 on panicle branch number and grain number per panicle. This masking effect may be due to the fact that DEP1 functions downstream of GS2 and IPA1 during panicle development.

[0030] V. GS2 TM and IPA1 TM Promote the yield of hybrid rice

[0031] To explore the application value of GS2 and IPA1 in rice hybrid breeding, the present invention used the two-line sterile line PA64s as the female parent and crossed it with 9311, 9311-GS2 TM and 9311-IPA1 TM1 materials to cultivate a series of hybrid rice combinations ( Figure 6 ). The inventor of the present invention investigated the yield per plant and other panicle agronomic traits of these hybrid rice combinations and found that the panicle lengths were in ascending order as PA64s / 9311, PA64s / 9311-IPA1 TM1 , PA64s / 9311-GS2 TM and PA64s / 9311-GS2 TM -IPA1 TM . Compared with PA64s / 9311, the panicle lengths of PA64s / 9311-IPA1 TM1 , PA64s / 9311-GS2 TM and PA64s / 9311-GS2 TM -IPA1 TM1 increased successively ( Figure 6 A, B, D). PA64s / 9311 and PA64s / 9311-GS2 TMThe number of primary and secondary branch panicles and the number of grains per panicle were significantly lower than those of PA64s / 9311IPA1 TM1 and PA64s / 9311-GS2 TM -IPA1 TM1 ( Figure 6 D). In addition, the 1000-grain weight of PA64s / 9311-GS2 TM and PA64s / 9311-GS2 TM -IPA1 TM1 was significantly higher than that of PA64s / 9311 and PA64s / 9311-IPA1 TM1 ( Figure 6 D). Under the action of increasing the 1000-grain weight and the number of grains per panicle, the yield per plant of PA64s / 9311-GS2 TM -IPA1 TM1 increased by 21.32%, while the yield per plant of PA64s / 9311-IPA1 TM1 and PA64s / 9311-GS2 TM increased by 12.14% and 13.95% respectively( Figure 6 D). The research results showed that the polymerization of GS2 TM and IPA1 TM1 could significantly increase the panicle length, the number of grains per panicle and the yield per plant, and the polymerization of the two had great potential in super-high-yield rice.

[0032] Advantages of the present invention:

[0033] The present invention first discovered the application of the rice GS2 gene in regulating the rice panicle type, and this gene can regulate rice panicle traits such as grain shape, the number of grains per panicle, and panicle length. The GS2 gene provided by the present invention provides new gene resources and theoretical guidance for the improvement of rice panicle type and molecular design breeding, helps to shape the ideal rice panicle type, improves the yield of crops, and has important application value. Brief Description of the Drawings

[0034] Figure 1 Effects of GS2 on the panicle types of ZH11, GS2 TM , GS2-OE and GS2-KO plants

[0035] A Panicle types of ZH11, GS2 TM , GS2-OE and GS2-KO plants;

[0036] B Comparison of the panicle types of ZH11, GS2 TM , GS2-OE and GS2-KO;

[0037] C Mutation forms of GS2-KO-1, GS2-KO-2 and GS2-KO-3 transgenic plants.

[0038] Figure 2 GS2 on ZH11, GS2 TM , GS2 TM -Ri, GS2 TM -OE and GS2 TM -KO plant panicle type affects AZH11, GS2 TM , GS2 TM -Ri, GS2 TM -OE and GS2 TM -KO plant panicle type;

[0039] B ZH11, GS2 TM , GS2 TM -Ri, GS2 TM -OE and GS2 TM -KO panicle type comparison.

[0040] Figure 3 GS2 interacts with IPA1 protein

[0041] A GS2 and IPA1 protein interact in yeast;

[0042] B Co-immunoprecipitation experiment proves that GS2 and IPA1 protein can interact in vivo;

[0043] C Pull-down experiment proves that GS2 and IPA1 protein directly interact in vitro;

[0044] D BIFC proves the interaction between GS2 and IPA1 protein between tobacco leaves.

[0045] Figure 4 GS2 regulates the expression of DEP1

[0046] A Yeast one-hybrid interaction results of GS2 with different fragments of DEP1;

[0047] B Different fragment lengths of the DEP1 promoter;

[0048] C ChIP-PCR experiment detects the fragment of DEP1 directly bound by GS2;

[0049] D Relative activity of luciferase in rice protoplasts;

[0050] E EMSA detects different probes of GS2 binding to DEP1.

[0051] Panicle type traits of GS2, IPA1 and DEP1 genetic materials under the backgrounds of 59311 and WY

[0052] A Panicle types of GS2, IPA1 and DEP1 genetic materials under the 9311 background;

[0053] Panicle types of GS2, IPA1 and DEP1 genetic materials under the WY background;

[0054] C Comparison of panicle types of GS2, IPA1 and DEP1 genetic materials under the 9311 background;

[0055] D Comparison of panicle types of GS2, IPA1 and DEP1 genetic materials under the WY background.

[0056] Figure 6 Hybrid phenotypes of GS2 and IPA1 with PA64s

[0057] A Panicle types of the hybrids of GS2 and IPA1 with PA64s;

[0058] B Panicle types of the hybrids of GS2 and IPA1 with PA64s;

[0059] C Comparison of panicle types of the hybrids of GS2 and IPA1 with PA64s. Specific implementation manners

[0060] The specific implementation manners of the present invention are described below to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0061] Example 1. Construction of expression vectors

[0062] The nucleotide sequence of rice gene GS2 is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO: 3; a terminator * is provided at the end of SEQ ID NO: 3.

[0063] (1) Knockout vector; To study the function of the gene, a GS2 knockout vector was constructed. The target site was designed according to the website http: / / skl.scau.edu.cn / home / developed by the team of Liu Yaoguang, and the GS2 knockout vector was constructed according to the operation method of the improved version of the multi-target pCRISPER vector (monocotyledonous plants).

[0064] (2) Interference vector: To study the function of the gene, a GS2 interference vector was constructed. The full-length antisense cDNA of GS2 was amplified in ZH11 and ligated into the pUbi::NOS vector.

[0065] (3) Overexpression vector: To study the function of the gene, a GS2 overexpression vector was constructed. ZH11 and GS2 were amplified respectivelyTM Full-length cDNA was ligated into the pUbi::GFP vector. The constructed GS2 overexpression vector was transformed into ZH11 plants to obtain GS2 overexpression transgenic lines.

[0066] (4) Yeast two-hybrid and yeast one-hybrid vectors: To search for GS2 interacting proteins, the full-length cDNA sequence of GS2 was amplified from ZH11 and constructed into the pGBK-T7 vector, and the full-length cDNA sequences of other proteins were constructed into the pGAD-T7 vector. The two constructed plasmids were co-transformed into yeast Y2H Gold competent cells to verify protein-protein interactions. To study the downstream target genes regulated by GS2, the full-length cDNA of GS2 and the promoter sequences of downstream target genes were amplified from ZH11 and constructed into the pB42-AD and pLac-ZU vectors, respectively.

[0067] (5) Bimolecular fluorescence complementation (BiFC) vectors: The full-length cDNAs of GS2 and its interacting protein were amplified from ZH11 and constructed into the pSPYCE and pSPYNE vectors, respectively.

[0068] (6) Co-immunoprecipitation (Co-IP) vectors: The full-length cDNA of GS2 was amplified from ZH11 and constructed into the pUbi-GFP-Flag vector; the full-length cDNA of other interacting proteins was amplified and ligated into the pUbi-RFP-HA vector.

[0069] (7) Prokaryotic expression vectors: To purify the GS2 protein and its interacting proteins, the full-length cDNA sequences of the relevant proteins were amplified from ZH11 and constructed into the pGEX-4T-1 and PET28a prokaryotic expression vectors, respectively.

[0070] Example 2. Genetic transformation of rice

[0071] I. Inducing callus

[0072] Prepare in advance: Sterile ddH 2 O, glass petri dishes with filter paper, 250 mL conical flasks; Prepare in advance: 70% alcohol, 80% 84 Blue Moon disinfectant.

[0073] (1) Select transparent rice seeds with intact embryos and place them in a 250 mL conical flask. Soak the seeds in 70% alcohol for 1 min, and wash the seeds 3 - 5 times with sterile ddH 2 O.

[0074] (2) Add 2 - 3 drops of Tween 20 to 80% 84 Blue Moon disinfectant, shake well and pour it into the 250 mL conical flask containing the seeds. Place it on a shaker at 200 rpm and shake for 20 min. Wash the seeds 5 times with sterile ddH 2 O.

[0075] (3) Add 80% Blue Moon disinfectant solution, place it in a shaker at 200 rpm, shake for 20 min, and then wash the seeds 5 times with sterilized ddH 2 O;

[0076] (4) Spread the seeds on a glass petri dish with filter paper, place it in a laminar flow hood to dry, and change to a clean glass petri dish with filter paper halfway to dry the seeds faster;

[0077] (5) Spread the dried seeds on N 6 D solid medium and culture them under continuous light at 32°C for about 15 days.

[0078] II. Agrobacterium tumefaciens transformation

[0079] Prepare in advance: Escherichia coli plasmid for transgenic plants, electroporation cuvette, liquid LB medium, solid rifampicin (Rif, 50 mM) and kanamycin (Kan, 50 mM) resistant LB medium.

[0080] (1) Wash the electroporation cuvette with sterilized ddH 2 O and 70% alcohol respectively, and place the electroporation cuvette in a laminar flow hood to dry;

[0081] (2) Add 1 μg of plasmid to 50 μL of Agrobacterium tumefaciens EHA105 competent cells, mix well and add it to the bottom of the electroporation cuvette, and electroporate the cuvette at 1800 V;

[0082] (3) Add 500 μL of liquid LB medium to the electroporation cuvette, place it in a shaker at 28°C at 200 rpm, shake for 2 - 3 hours, spread it on solid Rif + Kan resistant LB medium, and culture it at 28°C for 2 days.

[0083] III. Agrobacterium tumefaciens infection of callus

[0084] (1) Small shake of Agrobacterium tumefaciens: Pick a single colony on solid Rif + Kan resistant LB medium, shake the bacteria with liquid Rif + Kan LB medium in a shaker at 28°C at 200 rpm overnight;

[0085] (2) Large shake of Agrobacterium tumefaciens: When the Agrobacterium tumefaciens is shaken to orange, inoculate 300 - 500 μL of the bacterial solution into AAM liquid medium, add 15 mM acetosyringone (AS) to AAM liquid medium at a ratio of 1:1000, and shake it in a shaker at 28°C at 200 rpm overnight;

[0086] (3) Select the rice callus on N 6 D medium and transfer it to a sterilized 250 mL conical flask;

[0087] (4) Shake the large - scale bacterial solution until OD 600When the value is about 0.1, infect the selected rice callus for 1 minute;

[0088] (5) Spread the infected rice callus on a glass petri dish with filter paper, blow it in a laminar flow hood for 20 minutes, and place the petri dish in an incubator at 25 °C for 3 days of dark culture;

[0089] (6) Wash the callus cultured in the dark 3 times with sterile water containing 400 mg / L carbenicillin antibiotic;

[0090] (7) Shake the callus with carbenicillin water at 200 rpm for 25 minutes, and wash it 5 times with carbenicillin water;

[0091] (8) Repeat the operation in (7), spread the callus on a glass petri dish with filter paper, and place it in a laminar flow hood to dry;

[0092] (9) Spread the dried callus on N 6 DS solid medium, and place it in an incubator at 32 °C for continuous light for about 15 - 20 days.

[0093] IV. Screening of transgenic seedlings

[0094] (1) Select new callus with strong growth, transfer it to RE-III solid medium, and place it in an incubator at 32 °C for continuous light for about 15 days for induction and differentiation;

[0095] (2) Transfer the differentiated seedlings to HF solid medium, continue to induce root growth. When it grows for about 15 days, the rice seedlings can be transferred to water for acclimatization, and then transferred to the field two days later, and samples are taken to detect the transgenic positive situation.

[0096] Example 3. Preparation and transformation of protoplasts

[0097] I. Preparation of protoplasts

[0098] (1) Select plump seeds of rice 93-11. After soaking and germination, sow them in a basket for seedling cultivation. After 7 - 10 days of hydroponics, they are used for protoplast extraction;

[0099] (2) Cut the young stems of rice into slices about 0.5 mm thick with a blade, place them in a 0.6 M mannitol solution, and place them in the dark for 10 minutes to maintain the osmotic pressure of the cells;

[0100] (3) Filter out the 0.6 M mannitol solution with a 300-mesh cell sieve, transfer the cut rice slices to the enzyme solution, wrap the enzyme solution with tin foil, and place it in a shaker at 28 °C at 80 rpm for 4 hours of dark incubation;

[0101] (4) Add an equal volume of W5 solution, place it in a shaker at 28°C at 80 rpm, and continue culturing for 30 min;

[0102] (5) Filter the enzymolysis solution through a 300-mesh cell sieve, add 30 mL of W5 solution, place it in a shaker at 28°C at 80 rpm, and continue shaking for 10 min to elute the cells in the young stems;

[0103] (6) Filter the W5 solution through a 300-mesh cell sieve, collect the filtered liquid in a 50-mL round-bottom centrifuge tube, and repeat the above operation twice;

[0104] (7) Centrifuge the collected liquid at 80 rpm for 3 min, discard the supernatant, and the rice protoplasts are extracted at the bottom of the centrifuge tube;

[0105] (8) Add 5 mL of MMG solution, suspend the protoplasts at the bottom of the centrifuge tube, centrifuge at 80 rpm for 3 min, and discard the supernatant;

[0106] (9) According to the number of transformations, add MMG solution to 100 μL of protoplasts for each sample.

[0107] II. Transformation of Protoplasts

[0108] (1) Prepare 40% PEG 4000 solution in advance. For each sample, prepare 10 μg of plasmid, make up to 10 μL with water, add it to a 2.0-mL round-bottom centrifuge tube, add 100 μL of protoplasts, and 110 μL of 40% PEG4000 solution. Immediately mix gently and place it in an incubator at 28°C for dark incubation for 20 - 25 min;

[0109] (2) Add 440 μL of W5 solution, mix gently to terminate the transformation, centrifuge at 80 rpm for 3 min, and discard the supernatant;

[0110] (3) Add 1 mL of W5 solution, mix gently to wash the protoplasts, centrifuge at 80 rpm for 3 min, and discard the supernatant;

[0111] (4) Add 1 mL of W5 solution, mix gently, wrap the 2.0-mL centrifuge tube with tin foil, place it flat in an incubator at 28°C, and incubate in the dark for 14 - 16 h;

[0112] (5) For the cultured rice protoplasts, centrifuge at 80 rpm for 3 min, discard the supernatant, retain 100 μL of W5 solution, resuspend the protoplasts, and take pictures under a confocal microscope (Zeiss, LSM700).

[0113] Example 4, Yeast Two-Hybrid and Yeast One-Hybrid

[0114] I. Preparation of Yeast Competent Cells

[0115] (1) Streaking: Prepare YPDA solid medium, streak the Y2H Gold strain for yeast two-hybrid and the Y1H Gold strain for yeast one-hybrid on the YPDA solid medium respectively, and place them in an incubator at 28 °C for dark culture for 2 days;

[0116] (2) Small-scale shaking: Pick single colonies into 10 mL of YPDA culture medium respectively, shake at 200 rpm on a shaker at 28 °C overnight;

[0117] (3) Large-scale shaking: Add 1 mL of the small-scale shaken bacterial solution to 100 mL of YPDA culture medium according to a ratio of 1:100, and stop shaking when the OD 600 reaches 0.4 - 0.6;

[0118] (4) Collect the bacterial solution with a 50 mL centrifuge tube, centrifuge at 5000 rpm for 3 min, discard the supernatant, and perform subsequent operations according to the instructions (Coolaber, SK2401 - 200T).

[0119] II. Yeast transformation

[0120] (1) Yeast two-hybrid: Take 1 μg of the two plasmids for yeast two-hybrid respectively, mix them evenly, inject them into the prepared Y2H Gold yeast competent cells, add yeast transformation solution, incubate in a 30 °C water bath or metal bath for 1 h, and mix the yeast competent cells and the transformation solution every 15 min; centrifuge at 5000 rpm for 2 min, discard the supernatant, plate on SD - Leu - Trp solid medium, and grow in an incubator at 28 °C for 2 - 3 days; pick 3 - 5 single colonies into 50 μL of sterilized ddH 2 O, and dilute them at concentrations of 10, 100, and 1000 times; in different concentrations, respectively pipette 5 μL of the sample and spot it on SD - Leu - Trp and SD - Leu - Trp - His - Ade solid media, then place it in an incubator at 28 °C for 2 - 3 days, and take a photo with a camera.

[0121] (2) Yeast one-hybrid: Take 1 μg of the two plasmids for yeast one-hybrid respectively, mix them evenly, inject them into the prepared Y1H Gold yeast competent cells, and the transformation method is the same as the yeast two-hybrid transformation method in (1), plate on SD - Ura solid medium, and grow in an incubator at 28 °C for 2 - 3 days; the spotting method is the same as the yeast two-hybrid spotting method in (1), respectively pipette 5 μL of the sample and spot it on SD - Ura and SD - Ura / Raf / X - β - gal solid media, then place it in an incubator at 28 °C for 2 - 3 days, and take a photo with a camera.

[0122] Example 5. Prokaryotic induction expression and purification

[0123] I. Prokaryotic induction expression

[0124] (1) Extract the Escherichia coli plasmid from the successfully constructed vector, transform the BL21 competent cells, and place them in an incubator at 37°C overnight.

[0125] (2) Pick a single colony and inoculate it into the LB culture medium with the corresponding resistance. Incubate it on a shaker at 37°C at 200 rpm overnight.

[0126] (3) Add the overnight culture to the LB culture medium with the corresponding resistance at a ratio of 1:100, and stop shaking when the OD 600 reaches 0.4 - 0.6. Add 0.2 mM IPTG and incubate on a shaker at 18°C at 100 rpm for 14 - 16 h.

[0127] (4) Take samples of the bacterial solution before adding IPTG and after IPTG induction. Centrifuge the remaining bacterial solution at 5000 rpm for 2 min, discard the supernatant, add pre-cooled 1x PBS buffer (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, 140 mM NaCl, adjust the pH to 7.4 and autoclave) to suspend the bacterial solution, and add 1 mM protease inhibitor (PMSF) at a ratio of 1:100.

[0128] (5) Use an ultrasonic crusher (ultrasonic power 300 W, ultrasonic time 3 s, interval time 3 s, crush for 15 - 20 min) to lyse the cells. After the bacterial solution becomes clear, centrifuge at 5000 rpm for 2 min, and take samples of the supernatant and precipitate respectively.

[0129] (6) Centrifuge the bacterial solution before and after adding IPTG, suspend the bacterial solution and the precipitate after lysis with sterilized 1x PBS buffer. Add 5x protein loading buffer to the supernatant after lysis and the above samples, and denature at 100°C for 5 min.

[0130] (7) Perform SDS-PAGE electrophoresis on the samples before IPTG, after IPTG, supernatant after lysis, and precipitate after lysis at 160 V for 45 min.

[0131] (8) After electrophoresis, place the protein gel in Coomassie Brilliant Blue staining solution for 3 h, boil it in tap water for decolorization, and check whether there is a band with the size of the target protein in the bacterial solution after adding IPTG and the supernatant after lysis. If so, it proves that the target protein can be induced and expressed in the supernatant. If not, change the induction conditions.

[0132] II. Protein Purification

[0133] (1) Select different purification media (magnetic beads or agarose gel) according to different protein tags. For IPA1 protein, use the GST purification kit (Beyotime, P2262);

[0134] (2) Equilibrate the media with pre-cooled 1x PBS buffer so that the target protein and the media are in the same buffer system;

[0135] (2) Add the supernatant after cell disruption to the equilibrated media, incubate on a low-temperature shaker at 4°C for 2 h to allow the target protein to bind to the media, and collect the media using a magnetic stand or by centrifugation at 100 rpm;

[0136] (3) Wash the media with 10 - 15 volumes of pre-cooled 1x PBS buffer, and collect the media using a magnetic stand or by centrifugation at 100 rpm. Repeat this step twice;

[0137] (4) Add 1 mL of elution Buffer (weigh 0.154 g of glutathione powder, dissolve it in 50 mL of 50 mM Tris-Hcl, and adjust the pH to 8.0), incubate on a low-temperature shaker at 4°C for 1 h, and collect the supernatant using a magnetic stand or by centrifugation at 100 rpm. The supernatant is the purified protein;

[0138] (5) Take an appropriate amount of the sample for protein denaturation, perform SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining and decolorization, and observe whether the target band is purified.

[0139] Example 6. In vitro protein binding experiment (Pull-down)

[0140] (1) Pipette 50 μL of GST-beads (Beyotime, P2138), wash the GST-beads twice with Pull-down Buffer (50 mM Tris-Hcl, pH = 7.5, 150 mM KCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.01% Nonidet P-40), and aspirate the supernatant using a magnetic stand;

[0141] (2) Add an appropriate amount of IPA1-GST protein and His-GS2 protein (500 μg each) to 50 μL of GST-beads, add 600 μL of Pull-down Buffer, and incubate on a low-temperature shaker at 4°C for 4 h or overnight;

[0142] (3) Aspirate the supernatant of the above reaction solution using a magnetic stand, resuspend and mix it with 1 mL of Pull-down Buffer, aspirate the supernatant using a magnetic stand, and repeat this operation twice;

[0143] (4) Add 50 μL of 1x protein loading buffer, denature at 100 °C for 5 min, and subsequently detect the results by Western-blot.

[0144] Example 7: Co-Immunoprecipitation (Co-IP)

[0145] (1) Extract rice protoplasts according to the operation in Example 3 and transform the plasmid.

[0146] (2) Add 300 μL of IP Buffer (50 mM Tris-Hcl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1% glycerol, 1% Trito-x-100), vortex (vortex for 30 s, prevent on ice for 1 min, repeat 3 times), centrifuge at 12000 rpm for 2 min, and aspirate the supernatant.

[0147] (3) Aspirate 50 μL of Anti-DDDDK-tag mAb-Magnetic Beads (MBL, M185-11), wash the beads twice with IP Buffer, and aspirate the supernatant with a magnetic stand.

[0148] (4) Combine and mix the supernatant with the beads, and incubate on a low-temperature shaker at 4 °C for 4 h.

[0149] (5) Aspirate the supernatant of the above reaction solution with a magnetic stand, resuspend and mix with 1 mL of Washing Buffer (50 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Trito-x-100), aspirate the supernatant with a magnetic stand, and repeat this operation 2 times.

[0150] (6) Add 20 μL of 1x protein loading buffer, denature at 100 °C for 5 min, and subsequently detect the results by Western-blot.

[0151] Example 8: Bimolecular Fluorescence Complementation (BiFC)

[0152] (1) Plant Nicotiana benthamiana tobacco, with 14 h of light / 10 h of darkness, in a 25 °C light incubator, and grow for 4 - 6 weeks.

[0153] (2) Transform the plasmid containing the target gene into Agrobacterium.

[0154] (3) Pick a single clone into the resistant LB solution, shake at 28 °C and 200 rpm / min on a shaker for 14 - 16 h.

[0155] (4) Transfer 1 mL of the bacterial solution to 20 mL of resistant LB solution (containing 15 μM AS) for enlarged culture. Under the condition of a shaker at 28 °C and 200 rpm, shake the Agrobacterium until OD 600 = 0.4 - 0.6;

[0156] (5) Centrifuge at 5000 rpm for 3 min, collect the bacterial cells, and suspend the bacterial cells with the infection solution (10 mM MgCl 2 , 10 mM MES, 150 μM AS, pH = 5.6) until OD 600 = 0.8 - 1.0. Mix the bacterial cells of the two different plasmids in equal volume and culture in the dark at 28 °C for 2 - 3 h;

[0157] (6) Aspirate the bacterial solution with a 1 mL syringe and inject the bacterial solution into the epidermal cells on the back of the tobacco leaves. Mark the infection site with a marker. Transfer the injected tobacco to a light incubator at 21 °C for 2 - 3 d, and observe the fluorescence signal through a confocal microscope (Zeiss, LSM700).

[0158] Example 9: Electrophoretic Mobility Shift Assay (EMSA)

[0159] In this experiment, an EMSA probe biotinylation kit (Beyotime, GS008) was used to label the probe, and a chemiluminescent EMSA kit (Beyotime, GS009) was used to detect the biotinylation;

[0160] (1) Labeling of DNA probe: For the synthesized biotin single-stranded DNA probe, mix the forward and reverse primers in equal volume and anneal according to the following procedure: 95 °C for 2 min, decrease by 0.1 °C every 8 s until 25 °C, and store at 4 °C for a long time. After the reaction, the labeled biotin probe can be stored at -20 °C for a long time;

[0161] (2) EMSA binding reaction: Add various reagents in the following order: 5 μL of Nuclease-Free Water, 2 μL of 5x EMSA / Gel-Shift binding buffer, 2 μL of purified protein, mix well and place at room temperature (20 °C - 25 °C) for 10 min; then add 1 μL of the labeled probe, mix well and place at room temperature (20 °C - 25 °C) for 20 min;

[0162] (3) Preparation of EMSA gel: Use a mold for the conventional preparation of protein electrophoresis gel and prepare a 4% polyacrylamide gel according to the following formula: 2.0 mL of TBE buffer (5x), ddH 216.7 mL of O, 3 mL of 29:1 acrylamide / bisacrylamide, 625 μL of 80% glycerol, 500 μL of 10% ammonium persulfate (APS) were mixed evenly. 30 μL of TEMED was added and immediately mixed evenly. Then it was poured into the mold and the comb was inserted.

[0163] (4) Electrophoresis: Electrophoresis was carried out using 0.5x TBE. The reaction solution in (2) was loaded and electrophoresed at 100 V for about 1 h.

[0164] (5) Membrane transfer: The wet transfer method was used for membrane transfer. A nylon membrane similar in size to the protein gel was taken and placed in the transfer buffer (0.5x TBE) in the order of clamp - filter paper - protein gel - nylon membrane - filter paper - clamp, and transferred at 350 mA for 1 h.

[0165] (6) Cross - linking: After the membrane transfer was completed, the nylon membrane was placed with the sample side up, and the UV - light cross - linker was used to select a UV wavelength of 254 nm, 120 Mj / cm2, and cross - linked for 45 - 60 s.

[0166] (7) Detection by chemiluminescence method: The nylon membrane was placed in 15 mL of blocking solution and slowly shaken on a horizontal shaker for 15 min; the blocking solution was removed, and the nylon membrane was placed in a new 15 mL of blocking solution containing 7.5 μL of Streptavidin - HRP Conjugate, mixed evenly, and slowly shaken on a horizontal shaker for 15 min; the nylon membrane was rinsed with 1x washing solution for 1 min; the washing solution was removed, 15 mL of new 1x washing solution was added, and slowly shaken on a horizontal shaker for 5 min, and repeated 3 times; the nylon membrane was transferred to 15 mL of detection equilibration solution and slowly shaken on a horizontal shaker for 5 min; 5 mL of BeyoECL Moon A solution and 5 mL of BeyoECL Moon B solution were mixed evenly, the equilibration solution was poured out, 10 mL of BeyoECL Moon working solution was added, and left at room temperature for 2 - 3 min, and photographed using a cold light source (Biorad, ChemiDoc TM Touch).

[0167] Example 10: Chromatin Immunoprecipitation PCR Experiment (ChIP - PCR)

[0168] This experiment used the Chip Assay Kit (Beyotime, P2078) to detect the precipitated chromatin fragments.

[0169] (1) Weighed 2 g of wild - type and transgenic seedlings with Flag tags, wrapped them with gauze and immediately put them into 1% formaldehyde solution (36 mL of Extraction Buffer Ⅰ, 1 mL of 37% formaldehyde), and carried out cross - linking by vacuum pumping on ice for 30 min.

[0170] (2) Add 2.5 mL of 2 M glycine solution, evacuate under vacuum on ice for 5 min, and stop cross-linking; wash the sample with ddH 2 O to remove formaldehyde, and grind the sample thoroughly in liquid nitrogen;

[0171] (3) Add 20 mL of CIB1 solution (10 mM Tris-HCl, pH = 8.0, 0.4 M sucrose, 10 mM MgCl 2 , 0.15% Triton X-100, 1 mM PMSF), mix well, filter through four layers of gauze and two layers of filter paper, and collect the filtrate;

[0172] (3) Centrifuge at 10,000 rpm for 10 min, wash the pellet with 10 mL of CIB2 solution (10 mM Tris-HCl, pH = 8.0, 0.25 M sucrose, 10 mM MgCl 2 , 0.15% Triton X-100, 1 mM PMSF);

[0173] (4) Centrifuge at 10,000 rpm for 10 min, resuspend the pellet with 10 mL of CIB3 solution (10 mM Tris-HCl, pH = 8.0, 1.7 M sucrose, 10 mM MgCl 2 , 0.15% Triton X-100, 1 mM PMSF), and slowly add an equal volume of CIB3 solution;

[0174] (5) Centrifuge at 12,000 rpm for 30 min, discard the supernatant;

[0175] (6) Resuspend the pellet in 1 mL of NLB solution (1% SDS, 50 mM Tris-HCl, pH = 8.0, 10 mM EDTA, pH = 8.0) per tube, and use an ultrasonic disruptor to disrupt (ultrasonic power 300 W, ultrasonic time 3 s, intermittent time 5 s, 30 cycles), centrifuge at 12,000 rpm for 15 min, aspirate the supernatant into a new centrifuge tube, and add 5.4 mL of ChIP Dilution Buffer;

[0176] (7) Pipette 600 μL as the Input sample and store it at -20 °C. Add 100 μL of Protein A+G Agarose / Salmon Sperm DNA to the remaining sample, and incubate on a low-temperature shaker at 4 °C for 1 h;

[0177] (8) Centrifuge at 100 g at 4 °C for 1 min, aspirate the supernatant, divide it equally into two parts, add Flag antibody to one part and HA antibody to the other part as a control, and incubate on a low-temperature shaker at 4 °C overnight;

[0178] (9) Add 100 μL of Protein A + G Agarose / Salmon Sperm DNA and incubate at 4°C on a low-temperature shaker for 1 h;

[0179] (10) Centrifuge at 100 g for 1 min at 4°C, discard the supernatant, and wash the pellet sequentially with 1 mL of the following solutions. After adding each solution, gently rotate on a low-temperature shaker at 4°C for 5 min, then centrifuge at 100 g for 1 min at 4°C and discard the supernatant; Low Salt Immune Complex Wash Buffer (once), High Salt Immune Complex Wash Buffer (once), LiCl Immune Complex Wash Buffer (once), TE Buffer (twice);

[0180] (11) Add 250 μL of Elution Buffer (1% SDS, 0.1 M NaHCO 3 ), elute on a horizontal shaker for 5 min, centrifuge at 100 g for 1 min, and transfer the supernatant to a new 1.5 mL centrifuge tube; Repeat the above operation and combine with the previous supernatant to obtain 500 μL of supernatant solution;

[0181] (12) Add 20 μL of 5 M NaCl, add 30 μL of 5 M NaCl to the Input sample, mix well and incubate in a metal bath at 65°C for 4 h; Add 20 μL of 1 M Tris-HCl pH = 6.5, 10 μL of 0.5 M EDTA, 1 μL of 20 mg / ml Proteinase K, mix well and incubate in a metal bath at 65°C for 1 h; Then purify and recover the sample using a DNA purification kit; The purified and recovered sample is used for qRT-PCR reaction.

[0182] Example 11. Dual-Luciferase Reporter Assay (LUC)

[0183] This experiment uses the Dual- Reporter Assay System kit (Promega, E1910) to detect the activity of luciferase.

[0184] (1) Transform the plasmid containing the target vector into rice protoplasts and culture in the dark at 28°C in an incubator for 14 - 16 h;

[0185] (2) Centrifuge at 100 rpm for 3 min, aspirate the supernatant, retain 50 μL of the solution, add 150 μL of 1x Passive Lysis Buffer, vortex for 30 s, place on ice for 1 min, and repeat 3 times;

[0186] (3) Centrifuge at 12,000 rpm for 5 min, aspirate 20 μL of the supernatant into a new 1.5 mL centrifuge tube, add 50 μL of Luciferase Assay Buffer, use a dual-luciferase detection instrument (Promega, GLO MAX) to record the RLU1 value (firefly luciferase activity), add 50 μL of Stop& Buffer to terminate the reaction, and at the same time record the RLU2 value (Renilla luciferase activity). The Ratio value, i.e., RLU 1 / RLU2, is the actual fluorescence intensity value, which can reflect the transfection efficiency of the cells.

[0187] The present invention has been described in detail above. The engineered bacteria or host cells used in the above transgenic process can be understood as the engineered bacteria or host cells used by those skilled in the art in the transgenic process. However, with the development of technology, the selection of the said engineered bacteria and host cells may change, or in the application fields other than the transgenic purpose, the utilization of vectors and engineered bacteria is also involved. But as long as they contain the gene or the vector described in the present invention, they are within the protection scope of the present invention. The preparation of transgenic rice is a conventional technical means in the art, and the present invention does not make further limitations. The technical solutions for rice transgenic using the gene described in the present invention are within the protection scope of the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art.

Claims

1. Application of rice GS2 gene in regulating rice panicle shape, characterized in that: The gDNA sequence of the GS2 gene is SEQ ID NO: 1, or the cDNA sequence is SEQ ID NO: 2, or the amino acid sequence of the coding region is SEQ ID NO: 3; the rice panicle type refers to the number of grains per panicle.

2. The use according to claim 1, wherein the GS2 gene negatively regulates the number of grains per ear.

3. A method for regulating rice panicle shape, comprising transforming the GS2 gene described in claim 1 into rice cells, and then cultivating the transformed rice cells into plants, wherein the GS2 gene is silenced in the rice plants.

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