Rice ABD gene Os06g23650.1 and application of rice ABD gene Os06g23650.1 in influence on rice organ development and plant type establishment

Abstract

The invention relates to a rice ABD gene Os06g23650.1. The nucleotide sequence of the rice ABD gene Os06g23650.1 is as shown in SEQ ID No. 1. The amino acid sequence of the gene coding protein is as shown in SEQ ID No.2. The invention also discloses application of the gene in influencing organ development and plant type establishment of rice. According to the invention, a new function of the rice ABD gene Os06g23650.1 in the aspects of influencing organ development and plant type establishment is found; the gene regulates and controls the development of the leaf pillow of rice through the leaf pillow development gene OsLG1 so as to influence the size of a leaf included angle; the gene regulates tillering starting through a tillering starting gene MOC1, so that the tillering state is influenced; the gene also can activate seed shattering related genes qSH1, SHAT1 and OsYABBY2, so that the seed shattering is influenced; the content of the invention enriches the regulation and control mechanism of establishment of the rice organ boundary, and provides a new application thought for molecular design and breeding of rice.

Description

Technical Field

[0001] The present invention belongs to the field of plant gene functions, and relates to a rice ABD gene Os06g23650.1 and its application in affecting rice organ development and plant type establishment. Background Art

[0002] Between the plant meristem and organ primordia, there is a special region called the boundary region. The establishment of the boundary region not only affects the initiation of organ primordia, but also plays a role in the structural and functional stability of the shoot apical meristem. The research on the boundary between the meristem and organ primordia mainly focuses on dicotyledonous plants such as the model plant Arabidopsis thaliana. In recent years, with the improvement of technical means, relevant experimental results show that there may be a new type of boundary region in monocotyledonous plants, called the internal organ boundary. Taking the monocotyledonous plant rice as an example, the rice leaf can be divided into two parts: the leaf blade and the leaf sheath. The leaf blade mainly performs photosynthesis to accumulate organic matter for the plant individual, while the leaf sheath mainly plays the role of transporting water from bottom to top and mechanical support. Between the two, the region composed of the ligule, auricle and collar divides them into two parts, forming an obvious demarcation line, and this region is a typical internal organ boundary. At present, the research on the internal organ boundary of monocotyledonous plants is less, and the genetic regulation mechanism of the establishment of this boundary is still unknown.

[0003] The earliest liguleless mutant found in monocotyledonous plants is liguless1 of maize, lg1 and the mutant completely loses the collar. LG1 belongs to the SPL transcription factor family, and there is a conserved SBP domain in its protein structure, which can bind to downstream genes. The oslg1 mutant found in rice also shows a similar phenotype, indicating that the regulation of collar development by this gene is conserved in gramineous crops. Another type of mutant similar to the lg1 phenotype is lg2, whose collar development is abnormal and the ligule on the leaf margin is reduced. Genetic proof shows that LG1 and LG2 are located on the same regulatory pathway, and the expression of LG2 is earlier than that of LG1. In rice, there are two homologous genes of LG2, namely OsLG2 and OsLG2-like. Only when both are deleted will the mutant lose the collar, indicating that OsLG2 and OsLG2-like have functional redundancy and jointly regulate the collar development of rice.

[0004] Plant architecture is an important measurement index for plants, which is related to the agronomic traits of plants and has a direct relationship with agronomic traits. As a gramineous plant, rice is one of the food crops with the widest planting area in the world. The plant architecture of rice is closely related to its yield. The concept of "ideal plant architecture" refers to a rice model in which various rational traits are integrated together to achieve multiple functions such as increased yield, lodging resistance, disease resistance, and favorable for close planting. In the process of rice breeding, semi-dwarfing genes have also been introduced SD1 ( SEMI DWARF1 ), which has achieved the "Green Revolution" of rice. In addition to plant height, tiller number and leaf angle are also directly related to rice yield. Tillering is a branching mode during the growth and development of rice, and the number of effective tillers has a positive relationship with the yield per unit area of rice. Leaf angle is an important reference index affecting whether individual plants can achieve intensive planting and improve the light energy utilization efficiency. The regulation of rice tiller bud initiation is similar to that of axillary meristem initiation in dicotyledonous plants. Tiller buds originate from axillary meristems, and the GRAS transcription factor family is involved in the regulation, such as MOC1 moc1 The tiller number of the moc1 mutant decreases, and the number of branchlets on its panicle also decreases significantly and it cannot set seeds, indicating that MOC1 is involved in the regulation of rice tillering. In rice, MOC1 can be bound by the DELLA protein SLR1, a negative regulator of gibberellin GA, to prevent degradation, and when gibberellin degrades SLR1, MOC1 will be degraded by the ubiquitination system mediated by the E3 ligase. Another early identified tillering mutant in rice moc3 is similar to the moc1 mutant in phenotype, with a reduced tiller number and abnormal panicle development. By mapping, it was found that MOC3 is a homologous gene of the stem apical meristem stem cell population regulatory gene WUS and regulates the initiation of tillering together with cytokinin and auxin in rice moc3 It was found that the transcriptional expression of multiple cytokinin response regulatory factors OsRR was altered in the moc3 mutant, and knocking out the auxin signaling-related gene ASP1 in the moc3 mutant could complement the defect that the mutant could not form axillary buds

[0005] Rice leaf type is an important agronomic trait, which is related to the photosynthetic utilization efficiency of rice leaves and the planting density. After the concept of ideal plant type was proposed, corresponding reference indexes for rice leaf type were also available. During the modern rice breeding process, the domestication of leaf type tends to approach the five leaf type characteristics of "long, straight, narrow, concave, and thick". Among them, "straight" is most closely related to the leaf angle. The leaf angle refers to the angle formed between the leaf blade and the stem, and the size of this angle has a direct impact on yield. The size of the leaf angle is directly related to the development status of the leaf sheath. The rice gain-of-function lc1 mutant (LEAFINCLINATION 1) shows a phenotype of increased leaf angle. Through section observation, it is found that the main reason for this phenotype is that the adaxial leaf sheath of the lc1 mutant develops faster than the abaxial leaf sheath, resulting in the leaf blade drooping and increasing the leaf angle. LC1 encodes an IAA-amidosynthetase in rice, and the content of various auxins increases in the lc1 mutant, indicating that auxin has a direct effect on the regulation of leaf angle. After the discovery of LC1, a series of rice mutants related to leaf angle involving auxin were discovered, including lc3 and lc4. LC3 is a repressor transcription factor. In the lc3 mutant, the leaf angle increases due to the excessive elongation of cells at the adaxial leaf sheath. Experiments have proved that LC3 can interact with its binding protein LIP1 to regulate the auxin synthase genes OsIAA12 and OsGH3.2. LC4 is regulated by OsmiR394 in plants. Inhibiting the expression of OsmiR394 or overexpressing LC4 will both lead to an increase in the leaf angle, and the expressions of Osmir394 and LC4 are both regulated by auxin in the body. The two antagonistically regulate the parenchyma cells at the adaxial leaf sheath and thus affect the size of the leaf angle.

[0006] Therefore, based on the abnormal boundary development (abd) mutant of rice, the present invention identified a rice organ boundary development gene ABD. The rice ABD gene not only regulates boundary formation between the conserved shoot apical meristem and organ primordia, but also plays a similar role in the internal boundary of organs. The rice ABD gene can activate MOC1 and LG1 respectively on these two different boundaries to regulate the tiller initiation and leaf sheath development of rice, and ultimately affect the establishment of rice plant type. In addition, as a transcription factor, ABD can be expressed in the abscission zone and activate the abscission-related genes qSH1, SHAT1, and OsYABBY2 that function in this region. The content of the present invention enriches the regulatory mechanism of rice plant organ boundary formation and provides possible application ideas for the molecular design breeding of rice.

[0007] This patent is funded by the CAAS-CSIAF-202303 project of the Chinese Academy of Agricultural Sciences. Summary of the Invention

[0008] The first object of the present invention is to provide the rice ABD gene Os06g23650.1, its nucleotide sequence, and the amino acid sequence of the encoded protein.

[0009] The second object of the present invention is to provide the application of the above-mentioned rice ABD gene Os06g23650.1 in affecting rice organ development and plant type establishment.

[0010] The present invention is achieved through the following technical solutions: 1. A rice ABD gene Os06g23650.1, whose nucleotide sequence is shown in SEQ ID No.1.

[0011] Furthermore, the amino acid sequence of the protein encoded by the above-mentioned rice ABD gene Os06g23650.1 is shown in SEQ ID No.2.

[0012] 2. The application of the above-mentioned rice ABD gene Os06g23650.1 in affecting rice organ development and plant type establishment.

[0013] Furthermore, the application of the rice ABD gene Os06g23650.1 in regulating the development of the rice leaf sheath and the formation of the leaf angle.

[0014] Furthermore, the rice ABD gene Os06g23650.1 regulates the development of the rice leaf sheath through the leaf sheath development gene OsLG1, thereby affecting the size of the leaf angle.

[0015] Furthermore, the application of the rice ABD gene Os06g23650.1 in regulating rice tillering.

[0016] Furthermore, the rice ABD gene Os06g23650.1 regulates tillering initiation through the tillering initiation gene MOC1, thereby affecting the tillering state.

[0017] Furthermore, the application of the rice ABD gene Os06g23650.1 in regulating rice shattering.

[0018] Furthermore, the rice ABD gene Os06g23650.1 affects rice shattering by activating the shattering-related genes qSH1, SHAT1, and OsYABBY2.

[0019] Positive effects of adopting the above technical solutions: The present invention discovers a new function of the rice ABD gene Os06g23650.1 in affecting organ development and plant architecture formation; this gene regulates the development of the rice leaf collar through the leaf collar development gene OsLG1, thereby affecting the size of the leaf angle; this gene regulates tiller initiation through the tiller initiation gene MOC1, thereby affecting the tiller state; this gene can also activate the genes related to shattering, such as qSH1, SHAT1, and OsYABBY2, thereby affecting shattering; the content of the present invention enriches the regulatory mechanism of rice organ boundary formation and provides possible application ideas for the molecular design breeding of rice. Brief Description of the Drawings

[0020] Figure 1 For abd There are defects in the development of the leaf collar of the mutant. (a, b) Phenotypes of the wild type and the abd mutant, scale bar = 15 cm; (c, d) Front views of the leaf collars of the wild type and the abd mutant, scale bar = 5 mm; (e, f) Side views of the leaf collars of the wild type and the abd mutant, scale bar = 3 mm; (g - j) Section observations of the leaves, leaf sheaths, leaf collars of the wild type and the abd abnormal leaves of the mutant, scale bar = 400 μm; (k) Statistics of the leaf angles of the wild type and the abd mutant, **** indicates that the data is significantly different at the p < 0.0001 level by t - test analysis; Figure 2 It is that the tillering of the abd mutant is abnormal. (a - d) Tillering phenotypes of the wild type and the abd mutant. The wild type shows normal tillering, while the abd mutant shows ectopic leaves and deformed tillers, and individual mutant plants are unable to tiller, scale bar = 1.5 cm; (e) Statistics of the tillering morphology of the wild type and the abd mutant; Figure 3 It is that the organs of the abd mutant are fused and the meristem formation is abnormal. (a) The leaves of the abd mutant show fusion, scale bar = 1 cm; (b, c) Cross - section observations of the wild type and the abd mutant, the yellow arrow represents the fusion of leaf primordia, scale bar = 100 μm; (d - f) Longitudinal - section observations of the wild type and the abd mutant. The abd mutant is unable to initiate axillary meristems and there are defects in the formation of the shoot apical meristem. Individual mutants completely lose the shoot apical meristem. The red arrow represents the newly initiated axillary meristem, and the blue arrow represents the axillary meristem that cannot be initiated, scale bar = 100 μm; Figure 4 It is the map - based cloning of ABD. (a) Schematic diagram of the map - based cloning of ABD; (b) There is a 51 - bp base deletion in the first exon of ABD in the mutant; Figure 5 Complementary experiments and gene knockout of the ABD gene. (a) Schematic diagram of the construction of the complementation vector; (b) Phenotypes of the abd mutant, wild type, and complementation line abd-com1. The phenotypes of the wild type and the complementation line abd-com1 are consistent. Scale bar = 20 cm; (c) Schematic diagram of the gene knockout of ABD; (d) Phenotypes of the wild type and gene knockout lines abd-cr1 and abd-cr2. Scale bar = 15 cm; Figure 6 For observing GFP nuclear localization signals in the leaves and roots of overexpression lines ZmUBI:ABD-GFP (a) Nuclear localization signals in leaves. Scale bar = 60 μm; (b) Nuclear localization signals in roots. Scale bar = 50 μm; Figure 7 For overexpression lines ZmUBI:ABD-GFP producing ectopic leaf sheaths. (a) Phenotypes of the wild type and overexpression lines ZmUBI: ABD-GFP . Scale bar = 15 cm; (b, c) Front and back sides of the leaf sheaths of the overexpression lines ZmUBI:ABD-GFP . Scale bar = 5 mm; (d) Ectopic leaf sheaths are produced at the leaf margins of the overexpression lines ZmUBI:ABD-GFP . Scale bar = 1 cm; (e) Magnified view of the ectopic leaf sheaths on the leaf margins of the overexpression lines ZmUBI:ABD-GFP . Scale bar = 1 mm; (f) Statistics of the leaf angle sizes of the wild type and overexpression lines ZmUBI:ABD-GFP . **** indicates that the data are significantly different at the p < 0.0001 level analyzed by t-test; Figure 8 For the overexpression line ZmUBI:ABD-GFP to have an increased number of tillers at the initial stage of development. (a, b) Tiller bud phenotypes of the wild type and the overexpression line ZmUBI:ABD-GFP at 10 days of the growth period. Scale bar = 3 mm; (c) Tiller phenotypes of the wild type and the overexpression line ZmUBI:ABD-GFP at 20 days of the growth period. Scale bar = 5 cm; (d) Statistics of the number of tillers of the wild type and the overexpression line ZmUBI:ABD-GFP at 20 days of the growth period. *** indicates that the data are significantly different at the p < 0.001 level analyzed by t-test; Figure 9ABD directly binds to the promoters of OsLG1 and MOC1. (a, b) There are enrichment peaks of ABD in the promoter regions of OsLG1 and MOC1; (c, d) ChIP-qPCR proves that ABD binds to the promoter regions of OsLG1 and MOC1, **** indicates that the data is significantly different at the p<0.0001 level by t-test analysis; (e, f) The EMSA experiment proves in vitro that ABD can bind to the TGCGTG binding motif in the promoter regions of OsLG1 and MOC1; Figure 10 ABD directly binds to the promoters of qSH1, SHAT1 and OsYABBY2. (a) There are enrichment peaks of ABD in the promoter regions of qSH1, SHAT1 and OsYABBY2; (b) ChIP-qPCR proves that ABD binds to the promoter regions of qSH1, SHAT1 and OsYABBY2, ** indicates that the data is significantly different at the p<0.01 level by t-test analysis, **** indicates that the data is significantly different at the p<0.0001 level by t-test analysis; (c) The EMSA experiment proves in vitro that ABD can bind to the GCGTG binding motif in the promoter regions of qSH1, SHAT1 and OsYABBY2. Detailed implementation manners

[0021] The present invention is further described below through examples. It should be understood that these examples are only for illustrative purposes and in no way limit the scope of the present invention.

[0022] Sources of biological materials in the present invention: 1. All gene information in the present invention is obtained from the Rice Genome Annotation Project (MSU-RGAP); rice transgenic plants are generated from wild-type (WT) Nipponbare (NIP).

[0023] 2. This patent is funded by the project of the Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-CSIAF-202303). Example

[0024] This example shows that the abnormal development of the leaf sheath in the abnormal boundary development (abd) mutant leads to a smaller leaf angle.

[0025] The rice abd mutant is a natural mutant with a Nipponbare background found in the field. Compared with wild-type Nipponbare, the abd mutant plants show phenotypes such as short stature and difficulty in heading ( Figure 1a, b). Through further observation, it was found that the leaf angle of the abd mutant became smaller, mainly due to abnormal development of the leaf sheath base. The normal leaf sheath base is located between the leaf blade and the leaf sheath, and at this position, there are also symmetrically positioned ligule and auricle structures, forming a region similar to a boundary, clearly demarcating the boundary between the leaf blade and the leaf sheath. However, the leaf sheath base of the abd mutant showed obvious defects. The leaf sheath base structure did not form a complete and continuous boundary between the leaf blade and the leaf sheath, and only one side of the ligule and auricle was formed at this position, resulting in the appearance of leaf blade-like structures on its leaf sheath ( Figure 1 c - f). To more precisely observe the differences in leaf structure between the abd mutant and the wild type, paraffin sections were used for cytological observation of both. In the wild type, the rice leaf blade is mainly composed of mesophyll cells as the main body, with only two relatively large air cavities at the midrib; the leaf sheath is mainly composed of multiple air cavities; and the leaf sheath base has both abundant mesophyll cells and a certain number of air cavities, morphologically being between the leaf blade and the leaf sheath. In the leaf sheath of the abd mutant, it can be observed that at the same position, there are both leaf sheath structures and leaf blade structures, which are fused together and there is no obvious boundary between them. This phenotype indicates that ABD may be related to the establishment of the boundary between the leaf blade and the leaf sheath and regulate the development of the leaf sheath base ( Figure 1 g - j). Previous studies have shown that the development of the leaf sheath base is closely related to the leaf angle of rice. Therefore, the leaf angle traits of the wild type and the abd mutant were statistically analyzed. According to the statistical data analysis of the leaf angles of both, the average leaf angle of the wild type was 61 degrees, while the average leaf angle of the abd mutant was 32.7 degrees, showing a highly significant difference between the two, indicating that the abnormal development of the leaf sheath base in the abd mutant led to a smaller leaf angle. This shows that the mutant gene corresponding to the abd mutant can regulate the development of the leaf sheath base and the formation of the leaf angle. Example

[0026] This example shows that tillering of the abnormal boundary development (abd) mutant is defective.

[0027] In addition to the leaf angle, the abd mutant also has defects in tillering. Normal rice tillering starts from the axillary meristem and develops into a stem with independent functions. In the wild type, seedlings that have grown for 6 weeks after germination can normally form several tillers. Larger tillers will form leaf blade and leaf sheath structures similar to the main stem, while smaller tillers have incompletely unfolded leaf blades. At the base of the main stem, the presence of the tiller base can be clearly observed. In the abd mutant, the tiller morphology is not completely consistent with that of the wild type. Through the observation of tillers, there are 4 types of tiller morphologies in the abd mutant, including: 1) normal tillers; 2) the formation of ectopic leaves at the tillering initiation site; 3) tillers can initiate normally, but there are abnormalities in later development; 4) tillers cannot initiate normally at all ( Figure 2a - d). Statistical results of tiller morphology of wild - type and abd mutants showed that all 36 wild - type plants could tiller normally; among the 35 abd mutants counted, 5 could tiller normally, 21 formed ectopic leaves, and in addition, 7 could not initiate tillering and 2 formed abnormal tillers. These results indicate that ABD may regulate the initiation of tillering in rice ( Figure 2 e). It shows that the mutant gene corresponding to the abd mutant can regulate the initiation of tillering. Example

[0028] This example shows that the abnormal boundary development (abd) mutant shows organ fusion.

[0029] The abd mutant also has a phenotype of leaf fusion. In wild - type plants, leaf development starts at the base of the stem and forms an alternate phyllotaxy on both sides of the stem. However, in a few abd mutants, adjacent leaves show a phenotype of fusion. The part where the leaves fuse has the leaf margins disappearing to form a whole, while the unfused leaf tips still have independent leaf margins ( Figure 3 a). Through section observation, it was found that two leaves cross - fuse in a cross - shape. This indicates that ABD has a function related to the establishment of organ boundaries. Organ boundary mutants often have defects in the establishment of the shoot apical meristem. Therefore, the base of the rice seedling stem was sectioned for observation. In transverse sections, it can be clearly observed that the edges of the wild - type leaf primordia P2 and P3 overlap with each other, while the edge of the leaf primordium P3 of the abd mutant hardly overlaps and has an obvious phenotype of organ fusion ( Figure 3 b, c). In longitudinal sections, the shoot apical meristem of wild - type plants is surrounded by symmetrically distributed leaf primordia such as P1 and P2 on both sides; while in some abd mutants, the shoot apical meristem shows abnormal development, and symmetrically distributed leaf primordia cannot be normally formed on both sides, and even in some extreme plants, the shoot apical meristem is completely lost. In addition, compared with wild - type plants, the formation of new axillary meristems was not observed at the base of most abd mutants ( Figure 3 d - f). The above results show that there are serious defects in the establishment of the shoot apical meristem and the initiation of axillary meristems in the abd mutant, indicating that ABD plays a role in the maintenance and regulation of meristems. It shows that the mutant gene corresponding to the abd mutant can regulate the formation of meristems. Example

[0030] This example describes the map - based cloning and identification of the mutant gene corresponding to the abd mutant We used map-based cloning to locate the mutant gene. The japonica rice Nipponbare background mutant abd was crossed with the indica rice variety TN1. After obtaining the F1 generation, it was self-crossed to obtain the F2 generation population. The abd mutants in the F2 generation population were used as the mapping population. The segregation ratio of the abd mutants was approximately 1:3, and a total of about 9000 mapping individuals were obtained. Through bulked segregant analysis (BSA) of 50 mapping individuals, the mutant gene was located within an approximately 8 Mb interval on chromosome 6 of rice by the mapping primers RM3183 and RM7193. Subsequently, the population was expanded for fine mapping. The number of recombinant individuals for RM3183 was 235, and the number of recombinant individuals for RM7193 was 623. Polymorphic primers were designed within this interval, and finally, the mapping interval was narrowed down to a 5 Mb interval (Chr06:13033959..17933612) by polymorphic primers such as X25, X21, X26, and X19. The polymorphic primers on both sides were X26 and X19, respectively. The number of recombinant individuals for X26 was 23, and the number of recombinant individuals for X19 was 17 ( Figure 4 a). According to the genomic data from the National Rice Data Center (www.ricedata.com), this interval contains 319 open reading frames (ORFs). Based on the abd mutant phenotype identified above, it was shown that ABD is a gene related to the regulation of organ boundary formation. Based on this, screening was carried out among the 319 genes in the mapping interval. Further, it was found that LOC_Os06g23650.1 was the target gene. Therefore, sequencing primers were designed based on this gene for amplification and sequencing. After sequence alignment with the wild-type Nipponbare, it was found that the abd mutant had a 51-base deletion from position 283 to position 333 starting from the start codon ATG in the first exon of LOC_Os06g23650.1, indicating that it was likely a mutation in the ABD gene ( Figure 4 b).

[0031] To further confirm whether LOC_Os06g23650.1 was the mutant gene, a complementation vector was constructed. The complementary region included 4.2 kb of the upstream gene sequence, the full-length gDNA sequence, and 2.3 kb of the downstream gene sequence ( Figure 5 a). Through genetic transformation, the complementation plasmid was transformed into the abd mutant background, and transgenic complementation lines were successfully obtained. By observing the phenotypes of the complementation lines, it was found that the ABD:ABD-gDNA in the complementation lines was similar to the wild type in plant height and the number of heading dates, and the phenotype of organ fusion on the leaves completely disappeared, indicating that LOC_Os06g23650.1 was the target gene ABD ( Figure 5b). The gene LOC_Os06g23650.1 was knocked out using the CRISPR / Cas9 vector vk005, and a forward gRNA target was set at the 10th base starting from the start codon ATG. Through genetic transformation and amplification sequencing of transgenic plants, two different knockout lines were finally identified. In the knockout line abd-cr1, a T was inserted at the 27th base starting from the start codon ATG, and in the knockout line abd-cr2, a 5-bp deletion occurred at the 30th to 34th bases starting from the start codon ATG, both causing frameshift mutations. Through phenotypic observation, the knockout lines abd-cr1 and abd-cr2 were consistent with the abd mutant in phenotypes such as plant height, leaf development, and tiller morphology, further proving that LOC_Os06g23650.1 is the target gene ABD ( Figure 5 c, d). Example

[0032] This example demonstrates that overexpression of the functional verification of the ABD gene promotes the formation of the leaf collar and affects the leaf angle.

[0033] To better analyze the function of ABD in rice, a fusion protein of ABD and GFP was driven by the maize UBI (Ubiquitin) promoter to construct overexpressing plants. Through genetic transformation, fluorescence observation of the leaves and roots of transgenic plants was performed using a confocal microscope. Clear green fluorescence signals with nuclear localization characteristics of transcription factors were observed on the leaves and roots of the positive overexpressing plants ZmUBI:ABD-GFP ( Figure 6 a, b).

[0034] Phenotypic identification of the overexpressing line ZmUBI:ABD-GFP found that its plant type was mainly short-stemmed. Compared with the abd mutant, the overexpressing line ZmUBI:ABD-GFP could normally heading and harvest seeds, but the overall seed setting rate was lower than that of Nipponbare ( Figure 7 a). The leaf collar of the abd mutant showed a phenotypic defect in development, so the leaf collar of the overexpressing line ZmUBI:ABD-GFP was observed. Most of the leaf collars of the overexpressing line ZmUBI:ABD-GFP showed a tendency to expand in area to different degrees towards the leaf direction. On the ventral side, the leaf collar area directly covered the leaf base, and on the dorsal side, the leaf collar extended irregularly along the main vein towards the leaf direction ( Figure 7 b, c). In addition, some leaf collars of the overexpressing line ZmUBI:ABD-GFP also showed a phenotype of extending towards the leaf direction along the leaf margin, and heterotopic and newly formed ligules appeared at the expanded position of the leaf margin. A few overexpressing lines ZmUBI:ABD-GFP had heterotopic leaf collars and ligules on the leaf margin, and there were tumor-like protrusions on the leaf surface ( Figure 7d). The above results all show that when ABD is overexpressed in rice, it will activate the formation of ectopic leaf sheaths on the leaves, including the following forms: (1) the expansion of the leaf sheath area; (2) the extension of the leaf sheath along the leaf margin and the generation of ectopic ligules; (3) the generation of ectopic leaf sheaths and ligules at the leaf margin. Combining with the leaf sheath defect phenotype of the abd mutant, it indicates that ABD has the function of regulating the leaf sheath development pathway. The abd mutant not only shows abnormal development of the leaf sheath, but also has a significantly reduced leaf angle. In order to explore whether the overexpressed ABD in plants will also affect the leaf angle of rice, the leaf angle of the overexpression line ZmUBI:ABD-GFP was measured. The measurement results show that the average leaf angle of the wild type is maintained at about 60 degrees, while the average leaf angle of the overexpression line ZmUBI:ABD-GFP is as high as about 130 degrees. There is a significant difference between the two, and the excessive leaf angle leads to the appearance of the spreading leaf phenotype in the overexpression line ZmUBI:ABD-GFP ( Figure 7 f). By synthesizing the changes in the relationship of the leaf angles among the abd mutant, the overexpression line ZmUBI:ABD-GFP and the wild type, it is proved that ABD is a negative regulator of the leaf angle of rice. Example

[0035] This example shows that overexpressing the ABD gene with verified function accelerates the tillering process.

[0036] The above results show that the abd mutant has an abnormal tillering phenotype. Therefore, the tillering phenotype of the overexpression line ZmUBI:ABD-GFP was observed. The tillering morphology of the overexpression line ZmUBI:ABD-GFP is normal, and the number of tillers increases at the initial stage of development. By observing the tillering process of the wild type and the overexpression line ZmUBI:ABD-GFP at 10 days and 20 days after germination, it was found that at 10 days after germination, no new tillering buds could be observed at the base of most wild-type stems, and smaller tillering buds could be observed at the base of a few wild-type stems. However, tillering buds had grown at the base of most of the overexpression line ZmUBI:ABD-GFP, and the development degree was higher than that of the wild type ( Figure 8 a, b); at 20 days after germination, most of the seedlings of the overexpression line ZmUBI:ABD-GFP had produced 1-2 visible normal tillers, while most of the wild-type tillers were still in the initial stage of development, and a small number could produce at most 1 visible normal tiller ( Figure 8 c). By counting the number of tillers of the two, it was found that the number of tillers of the overexpression line ZmUBI:ABD-GFP was significantly higher than that of the wild type at the initial stage of development ( Figure 8d), indicating that the overexpression of ABD promotes the tiller number, which also shows that ABD is a positive regulator of rice tiller number. However, when the plants develop into the reproductive growth stage, the tiller number of the overexpression line ZmUBI:ABD-GFP does not increase significantly compared with that of the wild type in phenotypic observation, indicating that the downstream genes highly activated after ABD overexpression have a negative impact on plant development. Example

[0037] This example shows that ABD directly activates the leaf sheath development gene OsLG1 and the tiller initiation gene MOC1.

[0038] Single-gene analysis of OsLG1 and MOC1 was performed on the ChIP-seq data of ABD. After visualization analysis by IGV, it was found that there were binding peak phenomena in the promoter regions of both OsLG1 and MOC1. Among them, the binding peak of OsLG1 appeared at about -50 bp in its 5' UTR region, while the binding peak of MOC1 appeared at about -2100 bp in its promoter region. Through sequence alignment of the binding peak range and combined with the analysis results of MEME-ChIP above, it was found that there was a TGCGT binding motif at the upstream binding peaks of both OsLG1 and MOC1, indicating that ABD may perform its transcriptional activation function by recognizing and binding to this motif in the promoter regions of OsLG1 and MOC1 ( Figure 9 a, b). To verify this conjecture, ChIP-qPCR verification of the binding peaks of OsLG1 and MOC1 was carried out, using the transgenic line ZmUBI:GFP-NLS as the control group and the promoter segment of OsGAPDH (CYTOSOLIC GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE) as the control binding site. The ChIP-qPCR results showed that compared with the control group, ABD was enriched by about 2-fold at the upstream binding peak of OsLG1, while ABD was enriched by about 5-fold at the upstream binding peak of MOC1. Both showed significant differences from the control group, and there was no enrichment of ABD at the OsGAPDH control binding site. The above results indicate that ABD can bind to the DNA at the upstream binding peaks of OsLG1 and MOC1, further confirming that ABD can directly bind to the promoters of OsLG1 and MOC1 ( Figure 9c, d). To determine whether ABD binds to the promoters of OsLG1 and MOC1 through the binding motif TGCGTG, a gel electrophoresis mobility shift assay was performed to verify the binding ability of ABD. A 36-bp labeled probe was designed centered on the binding motif TGCGT upstream of OsLG1 and MOC1. The results of the gel electrophoresis mobility shift assay showed that the probes of OsLG1 and MOC1 could migrate normally in the absence of ABD protein; while when ABD protein was present, the migration speed of the probes of OsLG1 and MOC1 slowed down significantly. In addition, in the presence of ABD protein, after adding 20-fold excess of the competing probe, the slowed migration state of the labeled probe was restored; while after adding 20-fold excess of the mutant probe, the slowed migration state of the labeled probe could not be relieved (Figure 9e, f). These results indicate that ABD can bind to the promoters of OsLG1 and MOC1 through the TGCGTG motif. Example

[0039] This example demonstrates that ABD directly activates the genes qSH1, SHAT1, and OsYABBY2 related to seed shattering.

[0040] In addition to the fact that ABD can regulate the leaf angle and tillering of rice by activating the pulvinus development gene OsLG1 and the boundary-specific gene MOC1 as described above, we also attempted to find out whether ABD, as a transcription factor, is involved in other novel boundary formations. Among the results of candidate target genes in the Chip-seq analysis, there is binding enrichment of ABD in the promoter regions of some genes involved in the regulation of rice shattering, including qSH1, SHAT1, and OsYABBY2. Previous studies have shown that when these shattering-related genes are mutated or their expression is blocked, a normal abscission zone often cannot form between the mature seeds and the rachillae, resulting in the phenotype of difficult shattering; while when these genes are overexpressed in plants, an abscission zone can form between the mature seeds and the rachillae, leading to the phenotype of easy shattering. To further prove whether ABD can bind to the promoter regions of qSH1, SHAT1, and OsYABBY2, we performed Chip-qPCR for verification. The result analysis showed that compared with the negative control gene OsGAPDH, there was obvious enrichment of the ABD transcription factor in qSH1, SHAT1, and OsYABBY2 of the overexpression line ZmUBI:ABD-GFP, indicating that ABD can bind to and regulate the promoter regions of qSH1, SHAT1, and OsYABBY2 in vivo. In addition, through sequence alignment, we found that the GCGTG binding motif mentioned above exists in the ABD binding peak regions of qSH1, SHAT1, and OsYABBY2 in the Chip-seq peak map, meaning that ABD may bind to the promoter regions of qSH1, SHAT1, and OsYABBY2 through this element ( Figure 10 a, b). To verify this speculation, we used the DNA fragment containing this binding motif as a probe for gel electrophoresis mobility shift assay. The results of the mobility shift assay showed that when the ABD protein was present, there was obvious migration of the labeled probes of qSH1, SHAT1, and OsYABBY2; when the unlabeled cold probes of qSH1, SHAT1, and OsYABBY2 were added again, the originally migrated labeled probes significantly decreased; when only the labeled probes and unlabeled mutant probes of qSH1, SHAT1, and OsYABBY2 were added, the migrated probes were not greatly affected ( Figure 10 c). The above results indicate that ABD recognizes and binds to the GCGTG binding motif in the promoter regions of qSH1, SHAT1, and OsYABBY2, further suggesting that ABD, as a transcription factor, may directly activate the expression of qSH1, SHAT1, and OsYABBY2 in plants.

[0041] The present invention discovers a new function of the rice ABD gene Os06g23650.1 in affecting organ development and plant architecture formation; this gene regulates the development of the rice leaf sheath through the leaf sheath development gene OsLG1, thereby affecting the size of the leaf angle; this gene regulates tiller initiation through the tiller initiation gene MOC1, thereby affecting the tiller state; this gene can also activate the genes related to shattering, such as qSH1, SHAT1, and OsYABBY2, thereby affecting shattering; the content of the present invention enriches the regulatory mechanism of rice organ boundary formation and provides possible application ideas for the molecular design breeding of rice.

Claims

1. A rice ABD gene Os06g23650.1, whose nucleotide sequence is shown in SEQ ID No.

1.

2. The amino acid sequence of the protein encoded by the above rice ABD gene Os06g23650.1 is shown in SEQ ID No.

2.

3. Application of the above rice ABD gene Os06g23650.1 in affecting rice organ development and plant type establishment.

4. The application according to claim 3, characterized in that: The application of the rice ABD gene Os06g23650.1 in regulating the development of rice leaf axils and the formation of leaf angles.

5. The application according to claim 4, characterized in that: The above rice ABD gene Os06g23650.1 regulates the development of rice leaf axils through the leaf axil development gene OsLG1, thereby affecting the size of leaf angles.

6. The application according to claim 3, characterized in that: The application of the rice ABD gene Os06g23650.1 in regulating rice tillering.

7. The application according to claim 6, wherein: The above rice ABD gene Os06g23650.1 regulates tillering initiation through the tillering initiation gene MOC1, thereby affecting the tillering state.

8. The application according to claim 3, wherein: The application of the rice ABD gene Os06g23650.1 in regulating rice shattering.

9. The application according to claim 8, wherein: The above rice ABD gene Os06g23650.1 affects rice shattering by activating shattering-related genes qSH1, SHAT1 and OsYABBY2.

Images