Rice photoperiod adaptive leaf angle regulation gene and application thereof
By regulating the leaf angle of rice through the light signal response pathway of miR172 and its downstream target genes IDS1, SNB1 and photoreceptor PhyB, the problem of insufficient plant architecture adaptability of rice under photoperiod changes was solved, and efficient photosynthesis and yield improvement were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INSTITUTE OF CROP SCIENCE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2024-12-23
- Publication Date
- 2026-06-23
AI Technical Summary
The lack of effective photoperiod regulation gene modules in existing technologies to regulate the leaf angle of rice leads to insufficient plant adaptability to changes in light environment, affecting yield and photosynthetic efficiency.
By utilizing miR172 and its downstream target genes IDS1, SNB1 and photoreceptor PhyB, a photoresponse pathway was constructed. By regulating the expression and activity of miR172, the leaf angle of rice was adjusted, thereby achieving adaptive regulation of photoperiod.
By regulating the expression and activity of miR172, the leaf angle of rice can be adjusted under different photoperiods, thereby improving photosynthetic efficiency and biomass yield, and providing options for compact and ornamental plant types.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant molecular biology, and in particular to rice photoperiod adaptive leaf angle regulation genes and their applications. Background Technology
[0002] Rice is the world's most important staple crop, and designing rice plant architecture to achieve high yields is of great significance for rice breeding and production. The leaf angle, the angle between the leaf and the stem, is a crucial component of rice plant architecture. An ideal rice plant should have corresponding leaf angles at different canopy levels. A compact plant architecture reduces canopy density, ensuring that both the upper and lower leaves receive sufficient sunlight simultaneously, facilitating efficient photosynthesis for nutrient accumulation and growth. Compact plant architecture allows for increased planting density, increasing the overall biomass of the rice population and ultimately boosting crop yield.
[0003] Current research has found that the leaf angle in rice is regulated by plant hormones. Brassinosteroid (BR) can inhibit proximal cell division within the leaf angle tissue and conversely promote distal cell elongation, thereby increasing the leaf angle (Gan L, Wu H, Wu D, Zhang Z, Guo Z, Yang N, Xia K, Zhou X, Oh K, Matsuoka Met al: Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice. Plant science: an international journal of experimental plant biology 2015, 241:238-245.). Numerous genetic evidence supports this conclusion. Mutants of key BR synthesis-related genes, dwarf4-1, d2, dwarf1, and the BR signaling gene mutation d61, all exhibit erect leaf morphology. Furthermore, BR regulates the development of the leaf angle in rice by manipulating GA synthesis.Reducing the expression of the GA negative regulator SPINDLY can increase the leaf angle in rice (Hong Z, Ueguchi-Tanaka M, Umemura K, Uozu S, Fujioka S, Takatsuto S, Yoshida S, Ashikari M, Kitano H, Matsuoka M: A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell 2003, 15(12):2900-2910; Sakamoto T, Morinaka Y, Ohnishi T, Sunohara H, Fujioka S, Ueguchi-Tanaka M, Mizutani M, Sakata K, Takatsuto S, Yoshida S et al: Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice. Nat Biotechnol 2006, 24(1):105-109;Strable J,Wallace JG,Unger-Wallace E, Briggs S, Bradbury PJ, Buckler ES, Vollbrecht E:Maize YABBY Genes drooping leaf1 and drooping leaf2 Regulate Plant Architecture. Plant Cell 2017, 29(7):1622-1641.). High concentrations of auxin also regulate the development of leaf angle in rice, acting in a BR synergistic manner. Inhibiting the expression of auxin regulators OsAFB2 and OsTIR1 can increase the level of leaf angle. ABA can also regulate leaf angle by interfering with BR homeostasis, for example, the rice gain-of-function mutant RELATED TO ABSCISIC ACID INSENSITIVE3 has a BR homeostasis imbalance defect and an increased leaf angle.Downstream of hormonal signaling, many transcription factors directly regulate cell division and elongation in the leaf angle, and also regulate the leaf angle in rice. For example, the vernalization insensitive class 3 protein LC2 negatively regulates distal cell division, thereby regulating the size of the leaf angle; HLH1 transcription factors ILI1, BU1, BUL1, and BC1 increase the leaf angle in rice by increasing the elongation of cells in the leaf angle (Zhang LY, Bai MY, Wu J, Zhu JY, Wang H, Zhang Z, Wang W, Sun Y, Zhao J, Sun X et al: Antagonistic HLH / bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis. Plant Cell 2009, 21(12):3767-3780.). IBH1 can interact with BC1, BUL1, and ILI1 to regulate cell elongation development. These transcription factors form a regulatory network pathway to integrate developmental signals and regulate the morphogenesis of the leaf angle.
[0004] Besides the developmental system regulating leaf angle, external environmental factors also regulate leaf uprightness in rice. For example, silicon deposition in rice leads to upright leaves, as does phosphorus (Pi) deficiency. Downstream transcription factors PHR1, Syg1, and SPX1 / 2 are involved in changes in leaf angle induced by Pi supply. The first gene discovered to regulate the response of leaf angle to light intensity is from maize. lac1 It is a naturally mutated base site; the upper leaves are upright, while the middle and lower leaves are relatively flat. lac1 The plant's shape allows it to capture more light and adapt to environmental changes under dense planting conditions. Photoreceptor A accumulates in darkness and interacts with RAVL1 to promote its degradation, thus inhibiting [the growth of light]. lac1 The activity of the gene was reduced, thus lowering the accumulation level of BR. This is currently the only reported application of photosensitive leaf angle regulation in crops, while such gene modules and related applications are lacking in rice. Summary of the Invention
[0005] This invention provides the application of miR172 and its downstream target genes and upstream photoreceptor genes in regulating plant leaf angle.
[0006] This invention provides for the first time a series of regulatory sites for the regulation of leaf angle in rice photoresponse, including one non-coding miRNA (miR172), two AP2-type transcription factors (IDS1 and SNB1), and one photoreceptor gene (PhyB). In this pathway, the photoreceptor gene affects the expression of miRNA by sensing changes in light signals such as light temperature. miR172, as a photoperiod-adaptive miRNA, regulates the leaf angle in rice by modulating the expression of downstream target transcription factors. Experiments demonstrate that miR172 has the function of regulating leaf angle; overexpression of miR172 decreases the leaf angle, while downregulation of miR172 increases it. Similarly, the AP2-type transcription factors IDS1 and SNB1, as well as the photoreceptor PhyB, also have the function of regulating leaf angle.
[0007] Specifically, the present invention provides the following technical solutions.
[0008] In a first aspect, the present invention provides the application of rice miR172 or its encoding gene in regulating plant leaf angle, wherein miR172 is miR172a and / or miR172b.
[0009] miR172 is a known miRNA in rice, including miR172a, miR172b, miR17c, and miR172d. In this invention, miR172 is preferably miR172a or miR172b, and those skilled in the art can obtain its sequence based on the above names. Preferably, the mature form of miR172a in this invention has the sequence shown in SEQ ID NO.4, and the mature form of miR172b has the sequence shown in SEQ ID NO.5.
[0010] This invention provides a novel non-coding RNA, miR172, for regulating the angle of the flag leaf in rice. The genomic DNA sequence of miR172a is shown in SEQ ID NO: 1, its precursor nucleotide sequence is shown in SEQ ID NO: 2, and its mature form (mature miR172a) sequence is shown in SEQ ID NO: 4; the precursor nucleotide sequence of miR172b is shown in SEQ ID NO: 3, and its mature form (mature miR172b) sequence is shown in SEQ ID NO: 5. Overexpression of miR172 in rice can reduce the angle of the flag leaf in transgenic plants, thereby increasing the photosynthetic efficiency and biomass yield of rice plants; downregulation of miR172 expression can increase the angle of the flag leaf in rice, producing a new drooping plant shape and providing a unique plant type for ornamental purposes.
[0011] In a second aspect, the present invention provides the application of rice miR172 or its encoding gene in regulating the leaf angle of plants in response to light signals, wherein miR172 is miR172a and / or miR172b.
[0012] Preferably, the light signal is photoperiod. miR172, as a photoperiod-adaptive miRNA, can respond to photoperiod regulation of the leaf angle.
[0013] Preferably, the applications described in the first and second aspects above include: reducing the leaf angle of the plant by upregulating the expression of miR172, or increasing the leaf angle of the plant by downregulating the expression of miR172.
[0014] Thirdly, the present invention provides the application of rice AP2 transcription factor or its encoding gene in regulating plant leaf angle, wherein the AP2 transcription factor includes the IDS1 and / or SNB1 genes.
[0015] In this invention, the gene accession number of rice IDS1 is LOC_Os03g60430, and its amino acid sequence is the amino acid sequence encoded by the sequence shown in SEQ ID NO: 9; the gene accession number of rice SNB1 is LOC_Os07g13170, and its amino acid sequence is the amino acid sequence encoded by the sequence shown in SEQ ID NO: 10.
[0016] This invention provides two novel AP2 transcription factor functional genes, LOC_Os03g60430 (OsIDS1) and LOC_Os07g13170 (OsSNB1), which regulate the angle of the flag leaf in rice. The CDS sequences encoding the genes of OsIDS1 and OsSNB1 are shown in SEQ ID NO: 9 and 10, respectively. Overexpression of the IDS1 or SNB1 gene in rice can increase the angle of the flag leaf in transgenic plants; downregulation of the expression of the IDS1 or SNB1 gene can decrease the angle of the flag leaf in rice, thereby increasing the photosynthetic efficiency and biomass of rice plants.
[0017] Fourthly, the present invention provides the application of rice AP2 transcription factor or its encoding gene in regulating plant leaf angle in response to light signals, wherein the AP2 transcription factor includes the IDS1 and / or SNB1 genes.
[0018] Preferably, the optical signal is an optical period.
[0019] AP2 transcription factors IDS1 and SNB1, as downstream target genes of the photoperiod-adaptive miRNA miR172, can regulate leaf angle in response to changes in photoperiod.
[0020] Preferably, the applications described in the third and fourth aspects above include: increasing the leaf angle of the plant by upregulating the expression and / or activity of the AP2 transcription factor, or decreasing the leaf angle of the plant by downregulating the expression and / or activity of the AP2 transcription factor.
[0021] Fifthly, this invention provides the application of rice PhyB protein or its encoding gene in regulating plant leaf angle.
[0022] In this invention, the gene accession number of rice PhyB is LOC_Os03g19590, and its amino acid sequence is the amino acid sequence encoded by the sequence shown in SEQ ID NO: 11.
[0023] The photoreceptor gene LOC_Os03g19590 (PhyB) provided by this invention has the function of regulating the leaf angle in rice. The nucleotide sequence of the PhyB gene is shown in SEQ ID NO: 11. Downregulating the expression of the PhyB gene in rice can increase the leaf angle.
[0024] In a sixth aspect, the present invention provides the application of rice PhyB protein or its encoding gene in regulating the leaf angle of plants in response to light signals.
[0025] PhyB, as a photoreceptor, can respond to changes in light signals by regulating the expression of miRNAs, thereby altering the expression of downstream target genes of miRNAs and ultimately playing a role in regulating leaf angle.
[0026] Preferably, the optical signal is an optical period.
[0027] In the applications described in the fifth and sixth aspects above, the leaf angle of plants is increased by downregulating the expression and / or activity of PhyB.
[0028] In a seventh aspect, the present invention provides the application of a light signal response signaling pathway in regulating the leaf angle of plants, the signaling pathway including rice PhyB protein, miR172 and rice AP2 transcription factor; The AP2 transcription factor includes the IDS1 and / or SNB1 genes, and the miR172 is miR172a and / or miR172b.
[0029] In this invention, the plant is a monocotyledonous plant, preferably a grass, more preferably rice, and even more preferably japonica rice. For rice, the leaf angle regulation described in this invention is a regulation method specific to japonica rice.
[0030] Eighthly, the present invention provides the application of miR172 or rice AP2 transcription factor or rice PhyB protein or their encoding gene in constructing transgenic rice with reduced leaf angle or upright plant type; The AP2 transcription factor includes the IDS1 and / or SNB1 genes, and the miR172 is miR172a and / or miR172b.
[0031] In a ninth aspect, the present invention provides the application of miR172 or rice AP2 transcription factor or rice PhyB protein or their encoding gene in rice leaf angle breeding or plant type breeding; The AP2 transcription factor includes the IDS1 and / or SNB1 genes, and the miR172 is miR172a and / or miR172b.
[0032] In a tenth aspect, the present invention provides a method for regulating the leaf angle of rice, the method comprising: regulating the expression of miR172 in rice, wherein miR172 is miR172a and / or miR172b; Alternatively, the method may include: regulating the expression and / or activity of AP2 transcription factors in rice, wherein the AP2 transcription factors include the IDS1 and / or SNB1 genes; Alternatively, the method may include: regulating the expression and / or activity of PhyB protein in rice.
[0033] Eleventhly, the present invention provides a method for constructing transgenic rice with reduced leaf angle or upright plant type, the method comprising: upregulating the expression of miR172 in rice, wherein miR172 is miR172a and / or miR172b; Alternatively, the method may include: downregulating the expression and / or activity of rice AP2 transcription factors, said AP2 transcription factors including the IDS1 and / or SNB1 genes.
[0034] In this invention, the method of downregulating gene expression can be selected from one or more of the following: 1) mutating the nucleotide sequence of the gene; 2) regulating gene expression using weaker transcriptional and / or translational regulatory elements. Mutating the nucleotide sequence of the gene includes, but is not limited to, nucleotide sequence mutations caused by various physical and chemical methods, such as mutagenesis caused by mutagens, radiation-induced mutagenesis, mutations caused by RNAi gene silencing, or mutations caused by TALLEN and CRISPR / Cas9 technologies. For example, downregulating gene expression can be achieved by knocking out the gene.
[0035] In this invention, gene expression upregulation can be achieved through one or more of the following methods: 1) mutating the nucleotide sequence of the gene; 2) increasing the gene copy number; 3) using stronger transcriptional and / or translational regulatory elements to regulate gene expression. Increasing the gene copy number can be achieved by introducing an overexpression vector carrying the gene, or by increasing the gene copy number on the chromosome. For example, gene expression upregulation is achieved by introducing an overexpression vector carrying the gene.
[0036] The beneficial effects of this invention include at least the following: the novel functions of miR172 and AP2 transcription factors IDS1, SNB1, and photoreceptor PhyB in leaf angle regulation provided by this invention offer new gene resources and methods for leaf angle regulation and plant type breeding in rice and other plants. At the same time, it provides gene resources and basis for plant type breeding of rice that adapts to photoperiodic changes, which is of great significance for creating compact plant type rice and increasing rice yield. Attached Figure Description
[0037] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0038] Figure 1 These are the experimental results of miR172 controlling leaf angle and erect plant type in japonica rice in Example 1 of this invention; where A and B are the phenotypes of increased leaf angle in miR172 knockout lines of the japonica rice varieties Nipponbare NIP and Kitaake, respectively; C and D are the leaf angle phenotypes of miR172a-OE overexpressing plants, miR172a-KO knockout plants, and miR172 STTM silent plants in field-grown wild-type NIP in different years, respectively. miR172a-OE all showed a smaller leaf angle and an erect and compact plant type; E is the leaf angle phenotype of miR172a-OE overexpressing plants, miR172a-KO knockout plants, and miR172a-KO silent plants in field-grown wild-type Kitaake. The leaf angle phenotype of STTM-silenced plants shows that the leaf angles of miR172a-OE are smaller, and the plant shape is upright and compact; F is a schematic diagram of the design of miR172 expression vector, STTM-RNAi vector, and CRISPR / Cas9 knockout vector.
[0039] Figure 2These are the experimental results of miR172 controlling the sensitivity of japonica rice leaf angle to photoperiod in Examples 1 and 2 of the present invention. Among them, A is the leaf angle change phenotype of wild type and miR172 overexpression, knockout and silence lines of japonica rice varieties Nipponbare NIP and Longjing 46 (LJ46) under two cultivation conditions of long day (14L) and short day (11L); B is the leaf angle change phenotype of wild type and miR172 knockout line of indica rice variety Kasalath; C is the leaf angle change phenotype of wild type and miR172 overexpression and silence lines of indica rice variety Huanghuazhan HZZ under two cultivation conditions of long day (14L) and short day (11L); D is the leaf angle change phenotype of wild type and miR172 overexpression, silence and miR172 knockout lines of indica rice varieties Kasalath and Huanghuazhan after grain filling at the mature stage.
[0040] Figure 3 The results of the differentiation analysis of the miR172 promoter sequence in indica and japonica rice in Example 3 of this invention are shown. By comparing the sequence resources of 3000 rice sequencing materials, the miR172 promoter sequences of 413 japonica rice materials (orange) and 817 indica rice materials (blue) with different geographical distributions were extracted and compared. A total of 57 variant sites were found (see the top of the table).
[0041] Figure 4 This is the experimental result of the photoreceptor phyB regulating the leaf angle of japonica rice through miR172 in Example 4 of the present invention; among them, the leaf angle of the phyB knockout line was significantly increased, and the leaf angle of phyBKO / miR172aOE was also increased to a certain extent relative to the wild type, but was significantly restored relative to the phyB knockout line.
[0042] Figure 5 This is the experimental result of the regulation of leaf angle in japonica rice by the AP2 transcription factor, a target gene of miR172, in Example 5 of the present invention. Among them, A shows that the leaf angle of the mutants of AP2 transcription factors SNB1 and IDS1 is significantly smaller and is not affected by changes in photoperiod; B and C show that the leaf angle of IDS1mut with IDS1-OE overexpression and the miR172 regulatory site mutation is significantly increased; D and E show that the leaf angle of the snb1 and ids1 mutants is significantly smaller than that of the wild type under field cultivation conditions in different years, and the plant type is upright and compact; F is a schematic diagram of the 21 nucleotides of the CDS coding region of the mRNA of miR172 binding target genes IDS1 and SNB1.
[0043] Figure 6The results of miR172 expression level detection in japonica and indica rice under long and short day conditions in Example 6 of the present invention are shown; where TC65, DJ, KOS, and 9311sd1 represent wild-type rice varieties Taichung 65, DongJing, Yueguang, and 9311, respectively.
[0044] In the above results graph, 172a represents miR172a, 172b represents miR172b, KO represents knockout, OE represents overexpression, and STTM represents silencing. Detailed Implementation
[0045] In specific embodiments of this invention, the functions of miR172 and AP2 transcription factors IDS1, SNB1, and the photoreceptor PhyB in regulating leaf angle were verified. Various vectors for overexpressing, knocking out, and silencing related genes were used in the functional verification experiments. The overexpression vector included an expression cassette element for overexpressing the gene regulating rice leaf angle. The promoter used in the expression cassette could be a natural or artificial promoter to drive gene expression in rice. The vector also contained selectable marker genes for selecting transformed cells or tissues, such as antibiotic resistance or herbicide resistance-related genes; selectable marker genes included, but were not limited to, resistance genes such as hygromycin, kanamycin, and streptomycin. The vector may also contain tag protein genes, such as green fluorescent protein genes or flag tag protein genes.
[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0047] The gene editing techniques involved in the following examples, such as overexpression, knockout, and silencing, can all be achieved using common plant genetic engineering methods. Specific methods will not be described in detail in the examples.
[0048] Example 1: Phenotypic analysis of miR172 regulating leaf angle in different japonica and indica rice backgrounds To analyze the function of miR172 in regulating the leaf angle of different rice varieties, this invention first analyzed the genome sequence, 2K promoter sequence, precursor sequence, and mature sequence of rice miR172. An expression vector was constructed using the miR172 precursor sequence capable of expressing small RNAs. An STTM-RNAi vector was designed using the mature miR172 sequence, and a CRISPR / Cas9 knockout vector was designed using the miR172 genome sequence. Figure 1 (F). Overexpression, silence, and knockout lines of miR172 were created in the japonica rice varieties Nipponbare NIP and Kitaake, the main cultivated variety in Northeast China Longjing 46, and the indica rice varieties Kasalath and Huanghuazhan, respectively. The phenotypes of the constructed rice lines were analyzed.
[0049] The results showed that ( Figure 1 and Figure 2 Both in the seedling and mature stages, miR172 participates in regulating the leaf angle of japonica rice, but not in the regulation of leaf angle in indica rice. The leaf angle of the miR172 knockout line (172aKO) of the japonica rice varieties Nipponbare NIP and Kitaake is increased. Figure 1 (A and B). Leaf angle phenotype results of miR172a overexpressing plants (172a-OE), miR172a knockout plants (172a-KO), miR172b knockout plants (172b-KO), and miR172a STTM-silenced plants (STTM) in field-grown wild-type NIP from different years showed that the overexpressing plant 172a-OE exhibited smaller leaf angles and a more upright and compact plant shape. Figure 1 Similarly, the leaf angle phenotype results of miR172a overexpressing plants (172a-OE), miR172a knockout plants (172a-KO), and miR172a STTM silent plants (STTM or 172-STTM) in wild-type japonica rice Kitaake showed that the leaf angles of the overexpressing plant 172a-OE were smaller at all leaf positions, and the plant shape was upright and compact. The silent or knockout lines showed larger leaf angles and looser plant shape. Figure 1 (E). Under short-day conditions, the leaf angle of japonica rice was smaller and the plant shape was more upright. However, the plant angle of the miR172a knockout line, miR172b knockout line, and miR172a silent line did not decrease, and the plant shape did not shrink. Figure 2 The A) indicates that the absence of miR172a or miR172b results in the loss of the adaptability of the leaf angle of japonica rice to photoperiod changes. In other words, the adaptability of the leaf angle of japonica rice to photoperiod depends on the presence of miR172.
[0050] However, the leaf angle phenotype results of miR172a overexpressing plants (172aOE), miR172a knockout plants (172aKO), and miR172 STTM silent plants (172aSTTM) against wild-type indica rice Kasalath and Huang Huazhan (HHZ) showed no significant changes in either leaf angle or plant architecture. Figure 2 (B, C, and D). The above results indicate that miR172 specifically regulates the leaf angle of japonica rice, but does not regulate the leaf angle of indica rice.
[0051] Example 2: Analysis of the differences in photoperiodic response phenotypes between indica and japonica rice Using artificial incubators of the same model purchased from the same batch, photoperiodic conditions of long day (14 light / 10 dark) and short day (11 light / 13 dark) were set to cultivate the japonica and indica rice materials in Example 1. The results showed that, compared with long day, the leaf angle of japonica rice seedlings was significantly smaller under short day conditions than under long day conditions, while the leaf angle of indica rice remained unchanged or slightly increased under short day conditions. Figure 2 (A, B, and C). Therefore, indica and japonica rice exhibit phenotypic differences in their responses to leaf angle, with miR172a occupying a core regulatory position in determining japonica rice's response to long and short days.
[0052] Example 3 miRNA sequence analysis To confirm the differentiation of miR172 in japonica and indica rice, the miR172 genome sequence was analyzed using 3K rice resource materials (http: / / snp-seek.irri.org / ). The promoter sequence 2K upstream of the miR172a transcript is SEQ ID NO: 6 in Nipponbare NIP. The results showed no significant differences or differentiation between indica and japonica rice in the precursor and mature sequence regions of miR172a; however, the promoter sequence 2K upstream of the transcript showed significant differentiation in 413 japonica and 816 indica rice accessions. The japonica rice group exhibited one haplotype, with its shared sequence shown in SEQ ID NO: 8, while the indica rice group exhibited another haplotype, with its shared sequence shown in SEQ ID NO: 7. Conserved shared sequences were extracted within the indica and japonica rice populations based on sequence characteristics, indicating that (…). Figure 3 Approximately 57 nucleotides between the core sequences of indica and japonica rice show clear inter- and inter-variety variation. These nucleotides are conserved across different japonica rice varieties and different indica rice varieties. The differentiation sequences of these promoters in indica and japonica rice varieties may determine the changes in miR172a expression levels with variations in light and temperature, thus leading to differences in the light and temperature sensitivity and responses of japonica and indica rice under different environmental conditions. Based on the above analysis, consensus sequences of the promoters of indica and japonica rice were extracted.
[0053] Example 4: Plant phenotypes with photoreceptor gene knockout A knockout line of the photoreceptor PhyB (its nucleotide sequence is shown in SEQ ID NO: 11) named phybKO was constructed using CRSIPR / Cas9 technology. Furthermore, miR172 was overexpressed in phybKO (phybKO / 172aOE), and plant phenotypes under short-day conditions were examined. The results showed that phybKO had a leaf angle greater than 90 degrees, exhibiting a drooping leaf shape and a relatively loose plant type. While the leaf angle of phybKO / 172aOE was also somewhat larger than that of the wild type, it was significantly restored compared to the phyB knockout line. Figure 4 ).
[0054] Example 5: Leaf angle phenotype of target gene overexpression and mutants Based on bioinformatics predictions, miR172 can bind to the CDS coding region of the mRNA of target genes IDS1 and SNB1 in vivo (21 nucleotides). Figure 5 The F). Therefore, the overexpression lines (OE) and mutants of the target genes IDS1 and SNB1 were analyzed. snb1 , ids1-1, IDS1 MUT The phenotype of ) in which the mutant snb1 , ids1-1 These are loss-of-function mutants of this gene in the Tc65 and DJ cultivar backgrounds, respectively. IDS1 MUT The mutant plant was obtained by mutating the target site of miR172a in the IDS1 gene.
[0055] The results showed that, regardless of whether it was long-day or short-day, snb1 and ids1-1 The angles of the mutants were all significantly reduced. Figure 5 A). After IDS1 overexpression and miR172a target site mutation, the leaf angle of the plant was significantly increased ( ). Figure 5 (B and C); under field cultivation conditions, the ripening and grain-filling period snb1 and ids1-1 The mutant strain is significantly more compact than the wild type. Figure 5 The D and E components can increase the photosynthetic efficiency of the population under high-density planting conditions.
[0056] Example 6: Expression analysis of genes related to rice leaf angle Six to ten japonica and indica rice varieties were selected and cultured under long-day and short-day conditions, respectively. At the 4-5 leaf stage, leaf angles at the same leaf position were collected, RNA was extracted, reverse transcribed, and then analyzed for expression levels. The results showed that in japonica rice (… Figure 6(Left figure) Compared with long-day conditions, the expression level of miR172a increased significantly under short-day conditions; in indica rice, compared with long-day conditions, the expression level of miR172a remained unchanged or decreased under short-day conditions. Figure 6 (The image on the right).
[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. The application of rice miR172 or its encoding gene in regulating plant leaf angle, wherein miR172 is miR172a and / or miR172b.
2. Application of rice miR172 or its encoding gene in regulating plant leaf angle in response to light signals, wherein miR172 is miR172a and / or miR172b; Preferably, the optical signal is an optical period.
3. Use according to claim 1 or 2, characterized in that, The applications include: reducing the leaf angle of the plant by upregulating the expression of miR172, or increasing the leaf angle of the plant by downregulating the expression of miR172.
4. The application of rice AP2 transcription factor or its encoding gene in regulating plant leaf angle, wherein the AP2 transcription factor includes the IDS1 and / or SNB1 genes; Preferably, the application comprises: The leaf angle of the plant is increased by upregulating the expression and / or activity of the AP2 transcription factor, or the leaf angle of the plant is decreased by downregulating the expression and / or activity of the AP2 transcription factor.
5. Application of rice AP2 transcription factor or its encoding gene in regulating plant leaf angle in response to light signals, wherein the AP2 transcription factor includes the IDS1 and / or SNB1 genes; Preferably, the optical signal is an optical period.
6. Application of rice PhyB protein or its encoding gene in regulating plant leaf angle.
7. Application of rice PhyB protein or its encoding gene in regulating leaf angle in response to light signals; Preferably, the optical signal is an optical period.
8. The use according to any one of claims 1 to 7, characterized in that, The plant is a monocotyledonous plant, preferably a grass, more preferably rice, and even more preferably japonica rice.
9. A method for regulating leaf angle in rice, the method comprising, The method includes: regulating the expression of miR172 in rice, wherein miR172 is miR172a and / or miR172b; Alternatively, the expression and / or activity of AP2 transcription factors in rice can be regulated, including the IDS1 and / or SNB1 genes. Alternatively, it can regulate the expression and / or activity of PhyB protein in rice.
10. A method for constructing transgenic rice with reduced leaf angle or an upright plant type, characterized in that, The method includes: upregulating the expression of miR172 in rice, wherein miR172 is miR172a and / or miR172b; Alternatively, downregulate the expression and / or activity of rice AP2 transcription factors, including the IDS1 and / or SNB1 genes.