Methods for improving plant growth, stress tolerance, productivity and seed quality

Abstract

The present invention provides a method for improving the growth, stress tolerance and productivity of a plant. The present invention also provides a method for improving the seed quality of a plant. In particular, the present invention provides a method for improving the growth, stress tolerance and productivity of a plant, comprising providing a transgenic plant having reduced expression of a MYBS2 gene compared to its wild-type counterpart; and a method for improving the seed quality of a plant, comprising providing a seed from a transgenic plant, the seed from the transgenic plant over-expressing a full-length or mutant MYBS2 gene compared to its wild-type counterpart.

Description

Technical Field

[0001] The present invention relates to a method for improving the growth, stress tolerance and productivity of plants. The present invention also relates to a method for improving the seed quality of plants. Background Art

[0002] As autotrophic organisms, plants constantly monitor and respond to sugar status to maintain sugar homeostasis, which is essential for growth regulation, tolerance to environmental stress, and productivity. Plants have evolved mechanisms to respond to fluctuations in sugar levels by regulating metabolism to balance physiology. From sugar production in source tissues to sugar utilization or storage in sink tissues, sugar homeostasis is tightly coordinated through a comprehensive signaling network involving crosstalk between sugars, hormones, and environmental cues to regulate developmental and stress adaptation processes. Sugars regulate nearly all essential processes throughout the plant life cycle. In general, sugar availability upregulates genes involved in biosynthesis, transport, storage of reserves, and cell growth, while downregulating genes involved in photosynthesis, reserve mobilization, and stress responses, but sugar starvation has the opposite effect.

[0003] After assimilation in photosynthetic source leaves, the newly fixed carbon is used for cellular respiration and metabolism, temporarily stored in vacuoles as sucrose, temporarily stored in plastids as starch, and transported as sucrose to sink tissues such as growing tissues (for energy production) or developing organs (for long-term storage). Although sugars are essential for plant growth, excess sugars can be harmful. For example, ectopic expression of yeast invertase can convert sucrose to glucose and fructose in the apoplast, resulting in reduced sucrose export and accumulation of carbohydrates in leaves, which in turn causes photosynthesis inhibition, stunted growth, impaired root formation, and tobacco necrosis. Rice and maize mutants defective in the tonoplast sucrose transporter SUT2 accumulate higher concentrations of sugar in their leaves but show slow growth and reduced biomass and grain yield, which may be due to reduced sucrose transport from the vacuole in the source leaves to the sink tissues / organs with high sugar demand.

[0004] Sugar starvation-induced nutrient recycling represents a fundamental strategy for survival or continued growth under adverse conditions. Plants experience sugar starvation at specific times in their life cycle and in certain non-green organs, such as roots, stems, and flowers, which do not photosynthesize. In addition, sugar starvation or depletion may occur under extreme environmental conditions, or under attack by pathogens or pests that significantly reduce the efficiency of photosynthesis, or after defoliation during the quiescent phase or when photosynthesis is shut down. During prolonged darkness, sugar starvation leads to profound metabolic readjustment and initiates the autophagic pathway, which partially degrades chloroplasts in the vacuole, thereby recycling amino acids and other molecules that can be used to feed young tissues and produce seeds.

[0005] Starch accounts for about 75% of the dry weight of cereal grains and provides the main carbon source for energy and metabolites during germination and seedling growth. αAmy is the most abundant hydrolase and plays a central role in starch mobilization and seedling growth rate. Prior art studies in rice have shown that sugar deficiency can upregulate αAmy expression by controlling transcription rate and mRNA stability. αAmy transcriptional regulation is mediated by the sugar response complex (SRC) in the αAmy promoter, of which the TA box is a key cis-acting element. MYBS1 is a single DNA binding repeat (R1) MYB transcription factor that interacts with the TA box and induces the αAmy promoter in response to sugar deficiency. Sugar deficiency activates MYBS1 expression and promotes its nuclear import, while the provision of sugar has the opposite effect.

[0006] GA activates the αAmy promoter via the GA response complex (GARC), in which the adjacent GA response element (GARE) and TA box are key elements for synergistic action. The MYBS1-TA box interaction is essential for GARC and SRC functions, indicating that MYBS1 is a key node in the cross-signaling between GA and glucose starvation. In rice and barley, MYBGA is a GA-inducible R2R3 MYB transcriptional factor that binds to GARE and activates αAmy and hydrolase promoters in aleurone cells surrounding the starchy endosperm. GA antagonizes sugar-mediated repression of αAmy expression by enhancing the co-nuclear transport of MYBGA and MYBS1 and forming a stable bipartite MYB-DNA complex to activate αAmy and hydrolase gene promoters.

[0007] The 14-3-3 protein family is a highly conserved group of dimeric proteins that dock on phosphorylated serine (Ser) and threonine (Thr) residues in target proteins. In plants, target proteins of 14-3-3 proteins are involved in signal transduction and gene regulation of various biological processes, and the binding of 14-3-3 proteins to client proteins may lead to conformational changes, alternations in activity and stability, or sequestration in subcellular compartments. It has been reported that 14-3-3 proteins are involved in sugar regulation in yeast cells. In Saccharomyces cerevisiae, interaction with HSP70 (Ssb) requires 14-3-3 proteins (Bmh1) to recruit phosphatases (Glc7) to dephosphorylate and inactivate protein kinase SNF1, a process required for glucose repression. In plants, the regulation of sugar homeostasis mediated by 14-3-3 proteins has not yet been explored.

[0008] In the past, we identified another R1 MYB transcription factor, MYBS2, which is upregulated by sugar and also binds to the TA box of the oAmy promoter. Here, by using αAmy as a biochemical marker, we show that sugar availability / starvation neutralizes each other by regulating the competition between MYBS1 and MYBS2 for binding to the TA box, which acts as a transcriptional activator and repressor of the αAmy promoter. In particular, phosphorylation regulates the sugar-dependent nucleocytoplasmic shuttling of MYBS2 and its cytoplasmic interaction with 14-3-3 proteins, which plays a key role in regulating the on / off switching of reversible gene expression to maintain sugar homeostasis. We also observed that manipulation of MYBS2 and αAmy can have beneficial effects on plant growth, stress tolerance and grain productivity, which provides a novel approach for crop improvement. Summary of the invention

[0009] The main object of the present invention is to provide a method for improving plant growth, stress tolerance and productivity, comprising: (a) providing a transgenic plant having reduced expression of a MYBS2 gene compared to its wild-type counterpart; and (b) growing the transgenic plant in a normal environment or an environment containing an abiotic stress factor.

[0010] According to one embodiment of the present invention, the transgenic plant has increased expression of an αAmy3 gene compared to the wild-type counterpart.

[0011] According to one embodiment of the present invention, the MYBS2 gene encodes a MYBS2 transcription factor, and the MYBS2 transcription factor binds to a TA box of the αAmy3 gene promoter.

[0012] According to an embodiment of the present invention, the plant is a monocotyledon or a dicotyledon.

[0013] According to one embodiment of the present invention, the dicotyledonous plant is selected from the group consisting of: Cucumber (Cucumis sativus), Comb (Ricinus communis), Tomato (Solanum lycopersicum), Potato (Solanum tuberosum), Grape (Vitis vinifera), Populus trichocarpa, Arabidopsis thaliana, Arabidopsis lyrata, and Platycodon randifloras.

[0014] According to an embodiment of the present invention, the plant is a crop.

[0015] According to an embodiment of the present invention, the crop is rice, corn, wheat, barley, sugar cane, banana, cotton, soybean, pea, potato, tomato, brassica, orchid, balloon flower, yam, sweet potato, cassava, rose, petunia, chrysanthemum, lily, or carnation.

[0016] According to an embodiment of the present invention, the plant is an angiosperm.

[0017] According to one embodiment of the present invention, the MYBS2 gene encodes a MYBS2 transcription factor, and the MYBS2 transcription factor binds to a TA box of the αAmy3 gene promoter.

[0018] In one embodiment of the present invention, the abiotic stress factor is osmotic pressure, salt, dehydration or heat.

[0019] Another object of the present invention is to provide a method for improving the seed quality of a plant, comprising: (a) providing a seed from a transgenic plant, wherein the transgenic plant over-expresses a full-length or mutant MYBS2 gene compared to its wild-type counterpart; and (b) growing the seed from the transgenic plant in an environment comprising a normal or an abiotic stress factor.

[0020] In one embodiment of the present invention, the seeds from the transgenic plant have reduced expression of an αAmy3 gene compared to the wild-type counterpart.

[0021] According to one embodiment of the present invention, the mutant MYBS2 gene encodes a truncated MYBS2 transcription factor, which includes deletion of the first to fifty-third amino acid residues.

[0022] According to an embodiment of the present invention, the plant is a monocotyledon or a dicotyledon.

[0023] According to one embodiment of the present invention, the monocotyledonous plant is selected from the group consisting of: Zea mays, Sorghum bicolor, Setaria italica, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Triticum spp., and Saccharum spp.

[0024] According to one embodiment of the present invention, the dicotyledonous plant is selected from the group consisting of: Cucumber (Cucumis sativus), Comb (Ricinus communis), Tomato (Solanum lycopersicum), Potato (Solanum tuberosum), Grape (Vitis vinifera), Populus trichocarpa, Arabidopsis thaliana, Arabidopsis lyrata, and Platycodon randifloras.

[0025] According to an embodiment of the present invention, the plant is a crop.

[0026] According to an embodiment of the present invention, the crop is rice, corn, wheat, barley, sugar cane, banana, cotton, soybean, pea, potato, tomato, brassica, orchid, balloon flower, yam, sweet potato, cassava, rose, petunia, chrysanthemum, lily, or carnation.

[0027] According to an embodiment of the present invention, the plant is an angiosperm.

[0028] In one embodiment of the present invention, the abiotic stress factor is osmotic pressure, salt, dehydration or heat.

[0029] In summary, the present invention provides a method for improving plant growth, stress tolerance, productivity and seed quality, and a method for improving plant seed quality, which has the benefits of promoting plant growth and / or yield under normal or stress conditions to cope with global farmland water shortage and global warming; protecting plants from damage caused by water shortage; maintaining grain productivity under stress-free and stressful conditions; maintaining high-quality rice under global warming; enhancing public acceptance of new varieties and their products; reducing the transparency of milled rice and providing high-quality sake-brewing rice for the brewing industry; meeting the international market demand for high-quality rice and improving the transparency of milled rice; maintaining sugar homeostasis in plants; increasing sugar content and stress tolerance; and producing new drought varieties in various plant species. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings constitute part of this specification and are included here to further illustrate some aspects of the present invention, which aspects may be better understood by referring to one or more of these drawings in combination with the detailed description of the embodiments presented herein.

[0031] Figure 1A is a data graph showing that MYBS2 is a negative regulator of germination and plant growth and represses the expression of αAmy, wherein total mRNA was extracted from 7-day-old seedlings of over-expressing (Ox) or under-expressing (Ri) Ubi:MYBS2 transgenic strains and subjected to qRT-PCR analysis; the inset shows the comparison of MYBS2 mRNA levels between isolated wild type (sWT) and Ri strains.

[0032] Figure 1B is a data graph demonstrating that MYBS2 is a negative regulator of germination and plant growth and inhibits αAmy expression, wherein seeds were germinated in sugar-free 1 / 2 MS medium at 28° C. for 5 days before determining the germination rate; error bars represent standard deviation (SD).

[0033] Figure 1C is a data graph demonstrating that MYBS2 is a negative regulator of germination and plant growth and represses αAmy expression, wherein two-day-old seedlings of sWT and MYBS2 Ox and Ri strains with similar shoot length were grown in 1 / 2MS for up to 14 days; seedling growth was determined by measuring shoot length.

[0034] Figure 1Dis a data graph demonstrating that MYBS2 is a negative regulator of germination and plant growth and represses αAmy expression, wherein two-day-old seedlings of sWT and MYBS2 Ox and Ri strains with similar shoot length were grown in 1 / 2MS for up to 14 days; seedling growth was determined by measuring shoot length.

[0035] Figure 1E This is a photo showing that MYBS2 is a negative regulator of germination and plant growth and inhibits αAmy expression. Figure 1C and Figure 1D The plants were transferred to a greenhouse for continued growth, and the morphology of 90-day-old plants was evaluated.

[0036] Figure 1F This is a photo showing that MYBS2 is a negative regulator of germination and plant growth and inhibits αAmy expression. Figure 1C and Figure 1D The plants were transferred to a greenhouse for continued growth, and the morphology of 90-day-old plants was evaluated.

[0037] Figure 2A This is a data graph demonstrating that MYBS2 inhibits αAmy promoter activity through the TA box, wherein seedlings of sWT, MYBS2 (full-length or truncated cDNA) (XM_015757381.2) Ox and Ri strains were cultured in sugar-free 1 / 2 MS medium for 10 days; total RNA was extracted from leaves and used for qRT-PCR analysis using αAmy (e.g., αAmy3)-specific primers (including sequences of SEQ ID NO: 1 and SEQ ID NO: 2).

[0038] Figure 2BSchematic diagram demonstrating that MYBS2 inhibits αAmy promoter activity via TA box, wherein rice embryo callus was transfected with effector (XM_015757381.2, AK068565) and reporter plasmids (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963–1980) and cultured in –S medium for 24 hours before measuring luciferase activity. In the absence of effector, the luciferase activity value of the reporter construct was set to 1x, and all other values ​​were calculated relative to this value. The error bar represents the standard error (SE) of three replicates. In the case of effector constructs, effector constructs in rice embryo callus carrying reporter constructs αAmy3-35Smp:Luc and 6xTA-35Smp:Luc (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963-1980).

[0039] Figure 2C Schematic diagram demonstrating that MYBS2 inhibits αAmy promoter activity via TA box, wherein rice embryo callus was transfected with effector (XM_015757381.2, AK068565) and reporter plasmids (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963–1980) and cultured in –S medium for 24 hours before measuring luciferase activity. In the absence of effector, the luciferase activity value of the reporter construct was set to 1x, and all other values ​​were calculated relative to this value. The error bar represents the standard error (SE) of three replicates. In the case of effector constructs, effector constructs in rice embryo callus carrying reporter constructs αAmy3-35Smp:Luc and 6xTA-35Smp:Luc (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963-1980).

[0040] Figure 3Ais a data graph demonstrating that reduced MYBS2 expression up-regulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein ten-day-old sWT rice seedlings were treated with the indicated abiotic stresses; total RNA was extracted using αAmy3- and MYBS2-specific primers for qRT-PCR analysis; the upper and lower figures show the expression of MYBS2 and αAmy3, respectively; CK: untreated control group.

[0041] Figure 3B is a data graph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2 MS medium without 400 mM sorbitol; germination rate was measured daily until day 5; germination and plant growth without sorbitol treatment (-sorbitol).

[0042] Figure 3C is a data graph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2 MS medium without 400 mM sorbitol; sprout length was measured on day 8; error bars represent SD (n=10); germination and plant growth without sorbitol treatment (-sorbitol).

[0043] Figure 3D is a photograph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2MS medium without 400 mM sorbitol; plant morphology was photographed on the 8th day; germination and plant growth without sorbitol treatment (-sorbitol).

[0044] Figure 3Eis a data graph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2 MS medium with 400 mM sorbitol; germination rate was measured daily until day 5; error bars represent SD (n=10); germination and plant growth after sorbitol treatment (+sorbitol).

[0045] Figure 3F is a data graph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2 MS medium with 400 mM sorbitol; sprout length was measured on day 8; error bars represent SD (n=10); asterisks indicate significant differences (Student's t-test, *P<0.05, **P<0.01, ***P<0.001); germination and plant growth after sorbitol treatment (+sorbitol).

[0046] Figure 3G is a photograph demonstrating that reduced MYBS2 expression upregulates αAmy3 under abiotic stress and that ectopic expression of αAmy3 enhances osmotic stress tolerance in rice, wherein transgenic rice seeds overexpressing Ubi:αAmy3 or αAmy3:αAmy3 were germinated in 1 / 2MS medium with 400 mM sorbitol; plant morphology was photographed on the 8th day; germination and plant growth treated with sorbitol (+sorbitol).

[0047] Figure 4A Schematic representation demonstrating that reduced MYBS2 expression enhances osmotic and drought stress tolerance and grain yield in rice, wherein 10-day-old seedlings were treated with 15% PEG for 7 days and 20% PEG for an additional 16 days before determining survival; n=10; isolated wild type (sWT), MYBS2-Ox and MYBS2(54-265)-Ox and MYBS2-Ri strains were used.

[0048] Figure 4B is a schematic diagram showing that reduced MYBS2 expression enhances drought stress tolerance in rice, wherein ten-day-old seedlings were transferred to soil with regular water for 3 weeks and then watering was withheld for the next 21 days, and then the survival rate was determined; n=10; sWT, MYBS2-Ox, MYBS2(54-265)-Ox and MYBS2-Ri strains.

[0049] Figure 4C is a data graph demonstrating that reduced MYBS2 expression enhances rice grain yield, wherein the grain yield of field-grown rice. n=12, significance level of t-test: *P<0.05, **P<0.01, ***P<0.001.

[0050] Figure 4D is a data graph demonstrating that decreased MYBS2 expression enhances rice grain yield, wherein the grain yield of field-grown rice. *P<0.05, **P<0.01, ***P<0.001.

[0051] Figure 4E is a data graph demonstrating that decreased MYBS2 expression enhances rice grain yield, wherein the grain yield of rice grown in a greenhouse. **P<0.01, ***P<0.001.

[0052] Figure 4F is a data graph demonstrating that decreased MYBS2 expression enhances rice grain yield, wherein the grain yield of rice grown in a greenhouse. *P<0.05, **P<0.01, ***P<0.001.

[0053] Figure 4G This is a data graph demonstrating that reduced MYBS2 expression enhances rice grain yield, wherein sWT, OX-1, OX-2, OX-3, OX-(tr)-1, OX-(tr)-2, RNAi-1, RNAi-2, and MYBS2-Cas9 (knockout) strains were analyzed for grain weight of rice grown in the field in 2020.

[0054] Figure 5 is a schematic diagram demonstrating that overexpression of MYBS2 reduces chalky grains (ie, improves rice quality), whereas downexpression of MYBS2 (or overexpression of αAmy3) increases chalky grains. DETAILED DESCRIPTION

[0055] In the following detailed description of the embodiments of the present invention, reference is made to the accompanying drawings, which are shown to illustrate specific embodiments in which the present disclosure may be implemented. These embodiments are provided to enable those skilled in the art to practice the present disclosure. It should be understood that other embodiments may be used and that the embodiments may be changed without departing from the scope of the present invention. Therefore, the following description should not be considered to limit the scope of the present invention.

[0056] Sugar homeostasis is a unique feature of continuous sugar production and utilization in autotrophic plants. Plants have developed mechanisms to cope with fluctuations in sugar levels by regulating metabolism to balance physiological responses. On / off switching of reversible gene expression by sugar deficiency treatment / supply is the main mechanism to maintain sugar homeostasis, but the details of the mechanism are still unclear. α-Amylase (αAmy) is a key enzyme that hydrolyzes starch to provide sugar for plant growth, so sugar homeostasis regulation is required: induction caused by sugar deficiency treatment and inhibition caused by sugar supply. αAmy is induced by various stresses, but its physiological significance is also unclear. The present invention found that the on / off switching of αAmy expression is regulated by two MYB transcription factors competing for the same promoter element: MYBS1 promotes but MYBS2 inhibits αAAmy expression. Sugar deficiency treatment promotes nuclear import of MYBS1, but promotes nuclear export of MYBS2, and vice versa with sugar supply. Phosphorylation of MBYS2 at different serine residues plays a key role in regulating its sugar-dependent nucleocytoplasmic shuttling and retention in the cytoplasm by 14-3-3 proteins. In addition, MYBS2 expression is inhibited by dehydration and heat, leading to the induction of αAmy3, whereas activation of αAmy3 and inhibition of MYBS2 lead to improved rice plant growth, stress tolerance and grain yield. The present invention demonstrates a unique role for a key regulatory mechanism for on / off switching of reversible gene expression in maintaining sugar homeostasis, tightly regulating plant growth and development, and also highlights MYBS2 and αAmy3 as potential targets for crop improvement.

[0057] As autotrophic organisms, plants constantly monitor sugar status and respond to it to maintain sugar homeostasis, which is crucial for growth regulation, tolerance to environmental stresses, and productivity. αAmy is a key enzyme that hydrolyzes starch to sugars and is therefore subject to sugar homeostasis regulation: induction by sugar deficiency but repression by sugar supply. Two MYBs compete for binding to the same promoter element and mediate this counteracting regulation, with MYBS1 promoting but MYBS2 repressing αAmy expression. Induction of αAmy expression by repressing MYBS2 improves stress tolerance and productivity. Phosphorylation of MYBS2 plays a key role in regulating its sugar-dependent nucleocytoplasmic shuttling and interaction with 14-3-3 proteins, constituting a novel regulatory mechanism for reversible gene expression in sugar homeostasis.

[0058] During plant growth and development, sugar supply and starvation (i.e., sugar deficiency treatment) regulate gene expression in opposite directions in time and space, which plays a key role in determining the productivity and quality of crops. The present invention identifies MYBS2, a sugar inducible transcription factor that negatively regulates plant growth and development by binding to the TA box and inhibiting αAmy promoter activity. The MYBS2 promoter has specific activity in sugar-rich rice tissues. The present invention demonstrates that MYBS2 regulates sugar homeostasis in rice and shows that reduced MYBS2 expression leads to increased accumulation of α-amylase gene (αAmy3) expression, which in turn enhances rice plant growth, abiotic stress tolerance and grain yield. The purpose of the present invention is to manipulate the expression of MYBS2 and αAmy3 to improve the growth, abiotic stress tolerance and grain yield of rice and other plant species.

[0059] definition

[0060] The numerical values ​​used herein are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and optimally within a range of 5%. Therefore, the term "approximately" means within 20%, preferably within 10%, and optimally within 5% of a given value or range.

[0061] Materials and Methods

[0062] Plant materials

[0063] Rice (Oryza sativa cv Tainung 67) and barley (Hordeum vulgaris cv Himalaya) were used in the present invention. Seedlings were grown in a growth chamber at 28°C under a 16-hour light and 8-hour dark cycle.

[0064] Prior art shows that sugar regulates the function of MYBS1 in barley aleurone, SnRK1A (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963-1980; Chung-An Lu et al., (2007), Plant Cell, 19(8): 2484-2499; Kuo-Wei Lee et al., (2009), Sci Signal, 2(91): ra61; Ya-Fang Hong et al., (2012), Plant Cell, 24(7): 2857-73; Alan H. Christensen and Peter H. Quail ,(1996), Transgenic Research, volume 5, pages 213–218) using rice embryo calli to modulate MYBS1 function, CIPK15 using rice suspension cells to modulate SnRK1A expression, and rice and barley aleurone to modulate MYBS1 and MYBGA interaction and MYBS1 nucleocytoplasmic shuttling. Regardless of the system used, the regulation of SnRK1A and MYBS1 activity and nucleocytoplasmic shuttling using rice embryo calli and barley aleurone was consistent. For transient expression assays of luciferase activity, rice embryo calli were chosen because they are easier to handle for large-scale sample preparation, particle bombardment, and protein extraction.

[0065] Introduction

[0066] Tables 1 and 2 list the primers used for plasmid construction and RT-PCR analysis.

[0067] Table 1. Primers used for RT-PCR and qRT-PCR analysis

[0068]

[0069] Table 2. Primers used for plasmid construction and DNA sequence mutation

[0070]

[0071] Plastid

[0072] Plasmid p6XTA containing six copies of a fragment comprising positions -134 to -82 upstream of the transcription start site of αAmy3 (SEQ ID NO: 21) was fused to the CaMV35S minimal promoter (SEQ ID NO: 18)-luciferase gene (Luc) (see Chung-An Lu et al., (2002), Plant Cell, 14(8): 1963–1980). Plasmid pUG containing the Ubiquitin (Ubi) promoter fused to the β-glucuronidase gene (GUS) was used as an internal control for transient expression assays. Plasmid p3Luc.18 contains αAmy3 SRC (-186 to -82 upstream of the transcription start site) fused to the CaMV35S minimal promoter-Adh1 intron-Luc cDNA fusion gene.

[0073] Plastid construction

[0074] In order to overexpress full-length MYBS2 (SEQ ID NO: 13) and N-terminal deleted MYBS2 (tr) in transgenic rice, a MYBS2 cDNA fragment (SEQ ID NO: 14) (AK121235) containing nucleotides 1-795 (full-length version) and nucleotides 160-795 (truncated version) was inserted downstream of the Ubi promoter and upstream of the Nos terminator (SEQ ID NO: 19) to generate pU-MYBS2 and pU-MYBS2 (tr) (see Manuel Cercós et al.,(2002),theplant journal, doi.org / 10.1046 / j.1365-313X.1999.00499.x ). The two plasmids were hydrolyzed with Hind III and the binary vector pCambia1301 (CAMBIA) was inserted at the same site to generate pAU-MYBS2 and pAU-MYBS2 (tr) (AK121235). The amino acid sequence of the full-length MYBS2 transcription factor is shown in SEQ ID NO: 22.

[0075] For knocking down and knocking out MYBS2 in transgenic rice, the present invention uses a 485 bp (SEQ ID No. 15) partial cDNA fragment of MYBS2 3'UTR to generate a MYBS2 RNA interference (RNAi) construct and a 24 bp MYBS2 leader RNA sequence of MYBS2 with a monogram. The MYBS2 RNA interference (RNAi) part fuses the MYBS2 cDNA fragment with the green fluorescent protein gene (GFP) and then inserts it into the pSMY1H binary vector (AK121235); while the MYBS2 gene knockout part inserts the 24 bp MYBS2 leader RNA sequence into the pRGEB31 binary vector.

[0076] To generate a universal destination vector for constructing plasmids used in the transient expression assay of the present invention, the present invention digested the plasmid pAHC18 with BamHI to remove the Luc cDNA insert before inserting Not 1, Bgl II and Spe 1 restriction site sequences between the Ubi or CaMV35S promoter and the Nos terminator, generating the destination vector pUbi-Not1-BglII-SpeI-Nos.

[0077] Rice transformation

[0078] Plasmids pUbi-MYBS2, pUbi-MYBS2 RNAi, MYBS2-CAS9 / pRGEB331 (SEQ ID NO: 16) and pUbi-MYBS2(tr) (see Alan H. Christensen and Peter H. Quail ,(1996), Transgenic Research , volume 5, pages 213–218; ROBERT KAY et al., (1987), Science, Vol. 236, Issue 4806, pp. 1299-1302) was introduced into Agrobacterium tumefaciens strain EHA105 (Lifeasible, Cat# ACC-103), and then rice (Tainong 67 (TN67)) was transformed.

[0079] Determination of transient performance of rice embryo callus and barley aleurone

[0080] Rice embryo callus tissue was used for sample preparation and particle bombardment transient expression assay. Error bars indicate standard error of three replicates. Plasmid pRS426, an unrelated yeast plasmid with a molecular weight similar to that of the effector plasmid, was used as a control plasmid.

[0081] Real-time quantitative RT-PCR analysis

[0082] Total RNA was extracted from rice seedling tissues using Trizol reagent (Invitrogen) and treated with RNase-free DNase I (Promega) at 37°C for 30 min. TM III reverse transcriptase (Invitrogen), 1-5 μg of RNA was used for cDNA synthesis, and the cDNA was diluted 10 times for storage. 2 μL of cDNA was mixed with 10 μM primers and 10 μL Power SYBR Green PCR Master Mix reagent (Applied Biosystems) and applied to ABI7500 Real-Time PCR System (Applied Biosystems). The rice UBQ5 gene was used as an internal control to standardize all data.

[0083] Field trials

[0084] To evaluate grain yield in the field, 25-day-old seedlings were transplanted into the soil with a spacing of 25 x 25 cm between each plant. Seeds were harvested after maturity, dried and their yield was determined. Grain yield was evaluated in irrigated and non-irrigated fields. A total of 26 plants were planted per row in each plot, and only 12 plants were used to determine grain yield.

[0085] Statistical analysis

[0086] In the present invention, for all experiments, n = 30. Asterisks indicate significant differences (Student's t-test, *P < 0.05, ** < 0.01, *** < 0.001).

[0087] Example 1

[0088] MYBS2 is a negative regulator of germination and plant growth and inhibits αAmy expression

[0089] In this example, the present invention first studied the physiological role of MYBS2 in plant growth by gain-of-function and loss-of-function analysis in transgenic rice overexpressing MYBS2 cDNA or MYBS2 RNA interference (Ri) constructs under the control of the Ubi promoter (SEQ ID NO: 17). Relative to the isolated wild type (sWT), the level of recombinant MYBS2 mRNA in the transgenic seedlings of the two overexpressing (Ox) strains was increased by 52 to 60 times, while the endogenous MYBS2 mRNA in the two silenced (Ri) strains was reduced by 50-70% ( Figure 1A The present invention compared the phenotypes of these transgenic strains with sWT and found that the germination rate of the two Ox strains was reduced by 34-59%, but there was no change in the Ri strain ( Figure 1B ). In addition, seedling growth was delayed until 14 days in the Ox strain, but remained unchanged in the Ri strain ( Figure 1C , 1D In the Ox strain, plant height at 90 days (i.e., just before heading) was also shorter, but similar to that of sWT in the Ri strain ( Figure 1E , 1F ).

[0090] Example 2

[0091] MYBS2 represses αAmy promoter activity via TA box

[0092] The expression of αAmy3 in rice seedlings was suppressed (by 40-60%) in MYBS2-Ox strains but activated (by 5- to 6-fold) in MYBS2-RNAi strains ( Figure 2A ).like Figure 2AAs shown, overexpression of MYBS2 (full-length or truncated cDNA) inhibited the expression of αAmy3, whereas knockdown of MYBS2 increased the expression of αAmy3. In this example, isolated (sWT), MYBS2-Ox, MYBS2(54-265)-Ox, and MYBS2-Ri strains were used.

[0093] To understand the mechanism of MYBS2-mediated sugar repression of αAmy expression, the present invention used a rice germ transient expression system to evaluate MYBS1 (SEQ ID NO: 20), MYBS2 and MYBS2(Ri) (expression driven by the Ubi promoter) ( Figure 2B ) on the promoter activity of six tandem repeats (6xTA) containing αAmy3 SRC and TA box fused to CaMV35S minimal promoter. The results showed that the activity of both promoters was enhanced by MYBS1 and even significantly enhanced by MYBS2 Ri construct, but was inhibited by MYBS2 ( Figure 2C These gain- and loss-of-function analyses revealed that MYBS2 is a transcriptional repressor that counteracts the transcriptional trans-activation activity of MYBS1 on the TA box of the αAmy3SRC promoter.

[0094] Through transient expression assays, the present invention found that αAmy3 and 6xTA box promoters were repressed by MYBS2 overexpression (up to 50%) and activated by MYBS2(Ri) (2.2 to 3.7 fold) ( Figure 2C This result is consistent with the results in the gene transfection stable expression assay, in which αAmy3 mRNA accumulation was inhibited by MYBS2 overexpression (by 40-60%) and activated by MYBS2(Ri) (5.4-5.7-fold) ( Figure 2A ).

[0095] Example 3

[0096] Downregulation of MYBS2 expression upregulates αAmy3 under abiotic stress, and ectopic expression of αAmy3 enhances osmotic stress tolerance in rice seedlings

[0097] Since αAmy is activated by various biotic and abiotic stresses (such as water stress, viral / bacterial infection, wounding, heat or ABA) in different plant species, the present invention investigated whether MYBS2 regulates αAmy3 expression in response to abiotic stress. The present invention found that the expression of MYBS2 in rice seedlings was inhibited by dehydration (air drying) (up to 70%) and heating (42°C) (up to 80%), while the expression of αAmy3 was accompanied by a dramatic activation (up to 95 times) under conditions of dehydration (up to 2478 times) and heat stress (up to 2478 times). Figure 3A ), where CK represents the untreated control group.

[0098] To determine whether the expression of αAmy3 is associated with plant stress response, we generated transgenic rice carrying Ubi:αAmy3 or αAmy3:αAmy3. We found that the germination rate and shoot length of seedlings of all αAmy3-Ox strains were similar to those of sWT without sorbitol treatment ( Figure 3B -D), but significantly higher than that of sWT treated with sorbitol (i.e., simulated osmotic stress) ( Figure 3E -G), indicating ectopic expression of αAmy3 (most genes in cells have Tissue specificity and induced phenotype (the artificial manipulation of the introduction and expression of exogenous genes is called ectopic expression) to confer osmotic stress tolerance to rice.

[0099] In this example, it was shown that dehydration and heating in rice seedlings caused the expression of MYBS2 to be suppressed, while the expression of αAmy3 was induced ( Figure 3A ). The results suggest that sugar production via αAmy-mediated starch degradation may be involved in stress tolerance in rice. The present analysis showed that ectopic overexpression of αAmy3 promoted germination rate and seedling growth of rice under osmotic stress ( Figure 3E -G), this view is supported by the analysis of the present invention.

[0100] Example 4

[0101] Reduced MYBS2 expression enhances osmotic and drought stress tolerance and grain yield in rice

[0102] To further investigate whether downregulation of MYBS2 enhances plant stress tolerance, the present invention subjected MYBS2-Ox, MYBS2(54-265)-Ox, and MYBS2-Ri strains to abiotic stress treatment. Using polyethylene glycol (PEG) treatment to simulate osmotic stress, the present invention observed that the survival rate of MYBS2-Ri strain (100%) was twice that of MYBS2-Ox strain (40-50%), and higher than sWT (90%) ( Figure 4AAfter drought treatment in soil, the survival rate of MYBS2-Ri strain (100%) was significantly higher than that of sWT strain (70%), and was significantly higher than that of MYBS2-Ox strain (0-17%) and MYBS2(54-265)-Ox strain (0-10%) ( Figure 4B The deletion of N-terminal amino acid residues 1-53 only caused nuclear localization of MYBS2, which is consistent with the extremely low expression of αAmy3 in rice seedlings ( Figure 2A ) and reduced survival of seedlings under drought stress ( Figure 4B ) is consistent. Since the present invention found that MYBS2 damages the growth of seedlings and plants ( Figure 1C , 1E ), so the present invention also evaluated the effect of MYBS2 expression on grain yield in field trials. These plants were grown in spring (February to June) ( Figure 4E greenhouse) or in autumn (July to November) ( Figure 4F greenhouse) or irrigated fields ( Figure 4D The present invention observed that grain yield was reduced by 40-70% for MYBS2-Ox strains but increased by 26-54% ( Figure 4C ). In addition, the grain weight of MYBS2-Ri strain (see Figure 4G RNAi-1 and RNAi-2) and MYBS2-Cas9 (knockout) strains (see Figure 4G Cas9-1 and Cas9-2) increased by 106-135% ( Figure 4G Together, these findings suggest that MYBS2 is a negative regulator of abiotic stress tolerance and grain yield.

[0103] Although MYBS2 expression was reduced, it did not affect plant growth under normal growth conditions ( Figure 1D , 1F ), but it did promote osmotic and drought tolerance in transgenic rice plants, whereas overexpression of MYBS2 had the opposite effect ( Figure 4A , 4B ).

[0104] Example 5

[0105] Overexpression of MYBS2 reduces chalky grains, whereas knockdown of MYBS2 (or overexpression of αAmy3) increases chalky grains

[0106] The present invention also evaluated the effect of MYBS2 expression and its downstream target αAmy3 expression on grain quality (obtained from Example 4). Overexpression of MYBS2 can improve seed quality by producing less chalky seeds. Conversely, reducing MYBS2 expression or αAmy3 expression increases the production of chalky ( Figure 5 , Ox indicates MYBS2 overexpression strain; Ubi-Amy3D indicates the use of Ubi as promoter; Amy3p-amy3D indicates the use of αAmy3’s own promoter).

[0107] In summary, the present invention provides a method for improving plant growth, stress tolerance, and productivity and a method for improving seed quality, which have the benefits of: promoting plant growth and / or yield under normal or stress conditions to cope with global farmland water shortage and global warming; protecting plants from damage caused by water shortage; maintaining grain productivity under stress-free and stressful conditions; maintaining high-quality rice under global warming; enhancing public acceptance of new varieties and their products; reducing the transparency of milled rice and providing high-quality sake-brewing rice for the brewing industry; meeting the international market demand for high-quality rice and improving the transparency of milled rice; maintaining sugar homeostasis in plants; increasing sugar content and stress tolerance; and producing new drought varieties in various plant species.

[0108] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made thereto without departing from the spirit and scope of the present invention shall be included in the appended claims. Sequence Listing <110> Liu Fudong <120> Methods for improving plant growth, stress tolerance, productivity and seed quality <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 1 gtaggcaggc tctctagcct ctagg 25 <210> 2 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 2 aacctgacat tatatattgc acc 23 <210> 3 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 3 ctcagggttc ctgccggtag aaagca 26 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 4 cgaaacgaac agtagctag 19 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 5 cagaccaacc ctggcaaaaa 20 <210> 6 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 6 gaggacttgg aagctgatca tca 23 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 7 accacttcga ccgccactac t 21 <210> 8 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 8 acgcctaagc ctgctggtt 19 <210> 9 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 9 gcgcggccgc accatggagc agcatgagga ggc 33 <210> 10 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 10 gcagatcttc acagcacctt gatggccgcc 30 <210> 11 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 11 gcgcggccgc atgcccaacc tcacctcca 29 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 12 gcagatctca gcaccttgat ggccg 25 <210> 13 <211> 1144 <212> DNA <213> Rice <400> 13 agtctcctcc aaaccgtcga gagcccaaca ataataatcc ccccttttca tcgcggcgac 60 gatacccgcc attttcatct cattctcacc accaaaagcg accacgcagc gaccggccac 120 cgatcgagat ggagcagcat gaggaggcag cggagaggaa gccttcgccg ccggtgatat 180 tccggctgtt cggcgtcgag gtccgcggcg gcggcggcgg agttgacgag gaggagtacg 240 aggaggagga ggtggagggt ggattgttca tcaagaagag ctccagtatg cccaacctca 300 cctccatcga cccgctgccg gtgccggccg acggcggcaa acggcgcgcc tccgacgact 360 ccgagctcgc ctccggccag cagaagcgcc gccgccgcaa ggtgcaggag aggaagaaag 420 gggtaccatg gactgaggag gagcacaaga aattcctgga agggctgagg cagctgggga 480 aaggggactg gagaggcatc tccaagaact ttgtgaccag caggacggcg actcaggtgg 540 ccagccacgc ccagaagtac ttcctccggc agaccaaccc tggcaaaaag aagcgccggg 600 ccagcctctt tgatgttgtt gctgagtgca gtgatgatca gcttccaagt cctcagagtg 660 ttggaactaa gcctcctacc caggatataa ttcatacaga tcgcggcgat gtcccgatac 720 taagctatcc agttgctaga ggctttagag gcgatagcgt gcaggttgat gaactaactg 780 aatatgtgaa gagattaaag gccgccgagg acatgtcgct ctccatgatc tctggactgg 840 aaatggcatc atcatccatc agcagtctag agctcagtat cgcgcctcct cattgcgcga 900 tcgaggcggc catcaaggtg ctgtgatcca gacgcaattc tccaccgaag gaatctggat 960 cggcttcagc tactgttctt cgtcgccgct gttgttgttt gttgttgttg tttttttttt 1020 ttttttgcgg gggttgtttg ttgttgttgt tgttgtagtt gtcatgctaa ctttgtattt 1080 gggtcatgtg gcgtttcttt cagcagtctt atataataca gtgagaatgt cagtaccttc 1140 cgag 1144 <210> 14 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> cDNA <400> 14 atgcccaacc tcacctccat cgacccgctg ccggtgccgg ccgacggcgg caaacggcgc 60 gcctccgacg actccgagct cgcctccggc cagcagaagc gccgccgccg caaggtgcag 120 gagaggaaga aaggggtacc atggactgag gaggagcaca agaaattcct ggaagggctg 180 aggcagctgg ggaaagggga ctggagaggc atctccaaga actttgtgac cagcaggacg 240 gcgactcagg tggccagcca cgcccagaag tacttcctcc ggcagaccaa ccctggcaaa 300 aagaagcgcc gggccagcct ctttgatgtt gttgctgagt gcagtgatga tcagcttcca 360 agtcctcaga gtgttggaac taagcctcct acccaggata taattcatac agatcgcggc 420 gatgtcccga tactaagcta tccagttgct agaggcttta gaggcgatag cgtgcaggtt 480 gatgaactaa ctgaatatgt gaagagatta aaggccgccg aggacatgtc gctctccatg 540 atctctggac tggaaatggc atcatcatcc atcagcagtc tagagctcag tatcgcgcct 600 cctcattgcg cgatcgaggc ggccatcaag gtgctgtga 639 <210> 15 <211> 485 <212> DNA <213> Artificial Sequence <220> <223> MYBS2RNAI ---- 3`UTR 485bp <400> 15 tccagacgca attctccacc gaaggaatct ggatcggctt cagctactgt tcttcgtcgc 60 cgctgttgtt gtttgttgtt gttgtttttt tttttttttt gcgggggttg tttgttgttg 120 ttgttgttgt agttgtcatg ctaactttgt atttgggtca tgtggcgttt ctttcagcag 180 tcttatataa tacagtgaga atgtcagtac cttccgagac atgtttaatt aagccttggt 240 tggttggtat aagttggtga aaagaaaacc agatagtagc ctgtctgtat tgtacggttg 300 caaaattgga gtatattgat gcagagatca tgaagggtct ccatcttgtt cataagtcta 360 gtttgtttcc tgttcctgtc acaggaggca gctgggacat cttcttgtgt ggctctgagc 420 ttcttccagt gcttcaataa ttggaccctt ccccttcatg tccttgtgac cattggtccc 480 agcca 485 <210> 16 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Guide RNA sequence of MYBS2 CRISPR / Cas9 strain <400> 16 ggcataatct gtgaaggggt accagttt 28 <210> 17 <211> 1986 <212> DNA <213> Oryza sativa <400> 17 gtgcagcgtg acccggtcgt gccccctct agagataatg agcattgcat gtctaagtta 60 taaaaaatta ccacatattt tttgtcac acttgtttga atgcagtttt atctatcttt 120 atacatatat ttaaacttta ctctacgaat atataatct atagtactac ataatatca 180 gtgttttaga gatcatata atgacagt tagacatggt ctaaaggaca attgagtatt 240 ttgacacag gactctacag ttttactt ttagtgtgca tgtgttctcc tttttttg 300 caatagctt cacctatata atactcatc cattttatta gtacatccat ttaggttta 360 gggttaatgg ttttataga ctaattttt tagtacatct attttattct attttagcct 420 ctaaattaag aaactaaaa ctctatttta gttttttat ttaataattt agataaaa 480 Tagaaaaa taaagtgact aaaaaaaaaccct Ttaagaatt aaaaaaacta 540 aggaaacatt tttctgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt 600 ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 660 cggcatctct gtcgctgcct ctggaccct ctcgagagtt ccgctccacc gttggacttg 720 ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 780 gcggcctcct cctctctca cggcacggca gctacgggg attcctttcc caccgctcct 840 tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc 900 aacctcgtgt tgttcggagc cacacacac acaaccagat ctcccccaaa tccacccgtc 960 ggcacctccg cttcaaggta cgccgctcgt cctcccccccc cccccctctc taccttctct 1020 agatcggcgt tccggtccat ggtagggcc cggtagttct acttctgttc atgtttgtgt 1080 tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac 1140 gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc 1200 tctagccgtt ccgcagacgg gatcgatttc atgattttt ttgtttcgtt catagaggtt 1260 tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt 1320 tcatgctttt tttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc 1380 ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg 1440 tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata 1500 ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg 1560 gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac 1620 tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct 1680 tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat 1740 gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1800 cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1860 acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1920 ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1980 tctgca 1986 <210> 18 <211> 588 <212> DNA <213> Rice <400> 18 ccgcggtgtc gtttcccgcc ttcagtttag cttcatggag tcaaagattc aaatagagga 60 cctaacagaa ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa 120 tgacaagaag aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa 180 tatcaaagat acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat 240 atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt 300 ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga 360 agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga 420 aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga 480 cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag 540 ttcatttcat ttggagagaa cacgggggac tcttgacgcg gccgcacc 588 <210> 19 <211> 256 <212> DNA <213> Oryza sativa <400> 19 ggatcccccg ggctgcagat cgttcaaaca tttggcaata aagtttctta agattgaatc 60 ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 120 taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc 180 aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 240 cgcgcgcggt gtcatc 256 <210> 20 <211> 1314 <212> DNA <213> Rice <400> 20 gtgcgagatc caccacccga tgacctccca ggcggcgacg acgacgacca cggcggcggc 60 ggcggcggcg tggaccaggg aggacgacaa ggcgttcgag aacgcgctcg cggcgtgcgc 120 ggcgccgccg cccgcggacg gaggcgcgcc cgacgacgac tggttcgccg cgctcgccgc 180 gagcgtgccc ggggcgaggt cggcggagga ggtgcggagg cactacgagg cgctggtgga 240 ggacgtcgcg gccatcgacg cgggccgcgt cccgctcccg cgctacgccg gggaggagtc 300 cgcggcgccg cccgacggag ccggagccgc cgccgccgcg tccaaggacg gcggacaccg 360 gcgcgacgag cgcaagggcg gcggcggcgg gtacgacggc ggcaagagct gctccaaggc 420 ggagcaggag aggcgcaagg gcatcccatg gacggaggaa gagcacaggc tgttcttgct 480 gggggctggac aagttcggca aggggactg gcggagcatc tcgcgcaact tcgtcatctc 540 gcggacgcca acgcaggtgg cgagccacgc gcagagtac ttcatccgcc tcaactccat 600 gaaccgcgac cgccgccgct ccagcatcca cgacatcacc agcgtcaccg ccggcgatca 660 ggtcgccgcg cagcagggcg ccccgatcac cggccaccag gccacgggca accccgcggc 720 ggcggcgctg ggcccgccgg gcatgaagca ccaccaccac caccacccgg gcggcgcgcc 780 gccgcccatg cccatgtaca gcgccgcgcc catgggccac cccgtcgccg gccacatggt 840 gcccgccgcc gtcggcacgc cggtggtgtt cccgccgggc cacgcgccgt acgtcgtgcc 900 cgtcggctac ccggcgccac cggccaagat gcacaatga cgcgccatgg agggacatga 960 gcagcatttc ttcctctcc tttcttgatg tgaatcttga tttgttcttt gtgtagtcgc 1020 cggctcatcg tccctgatca tcttgttctt ctcacaatct cactaatgta aacatacata 1080 gatcagatgc caagagtgca gggattgggg attaaaggcg aataagtaaa gtatttgct 1140 gactgtttgc aagtgatcat cacgtacacc cggtgaaagc ttagctccaa atgtggatgt 1200 aattagcagc ggccttccgt acgtggtggc gccgatcgat gatcttgcag gggttgcaat 1260 tagggattga tttccatttt gctgatgtaa atttgcctac tgtctcattg gacc 1314 <210> 21 <211> 1773 <212> DNA <213> Oryza sativa <400> 21 attcccctgc cacggtgacc gtgcccccgg tgttctatat atgccccccg acgtcgaggt 60 cattcgccac gaacacatcg atcatccatc atctacaaga gatcgatcag tagtggttag 120 cagcaactca ctatcgaaca cggtttcagc ttacacagat atgaagaaca ccagcagctt 180 gtgtttgctg ctcctcgtgg tgctctgcag cttgacctgt aactcgggtc aagcacaggt 240 cctcttccag ggtttcaact gggagtcgtg gaagcagcag ggtggctggt acaacatgtt 300 gaaaggccaa gtcgacgaca tcgccaaggc cggggtcacc cacgtctggc tgccgccgcc 360 gtcgcactcc gtggcgccgc aggggtacat gccggggcgt ctctacgacc tggacgcgtc 420 caagtacggc acggcggcgg agctcaagtc gctgatcgcg gcgttccacg ggaagggcgt 480 ccagtgcgtc gccgacgtcg tgatcaacca ccggtgcgcc gagaagaagg acgcccgcgg 540 cgtgtactgc gtgttcgagg gcgggacgcc cgacgaccgc ctcgactggg gccccggcat 600 gatctgcagc gacgacacgc agtactccga cggcacgggc caccgcgaca ccggcgaggg 660 gttcggcgcg gcgcccgaca tcgaccacct caacccgcgc gtccagcggg agctcaccga 720 ctggctcaac tggctcaagt ccgacgtcgg cttcgacggc tggcgcctcg acttcgccaa 780 gggatactcc acggacatcg ctaagatgta cgtcgagagc tgcaagccgg gcttcgtcgt 840 cgccgagata tggaactcgc tgagctacaa cggcgacggc aagccggcgg ccaaccagga 900 ccagggccgg caggagctgg tgaactgggt gaacgccgtc ggcgggccgg cgatgacgtt 960 cgacttcacc accaagggcc tcctgcaggc gggcgtccag ggcgagctgt ggcggctgcg 1020 cgacggcaac ggcaaggccg ccggcatgat cgggtggctg ccagagaagg ccgtcacgtt 1080 cgtcgacaac cacgacaccg gctcgacgca gaagctttgg ccgttcccct ccgacaaggt 1140 catgcagggc tacgcctaca tcctcaccca ccccggagtc ccctgcatct tctacgacca 1200 catgttcgac tggaacctga agcaggagat aaccgcgctg gcggcgatca gggagaggaa 1260 cggcatcaac gccgggagca agctccggat cgtcgtcgcc gacgccgacg catacgtcgc 1320 cgtcgtcgac gagaaggtca tggtgaagat cgggacgagg tacgacgtgg gcaacgcggt 1380 gccgtcggat ttccatcaga cggtgcacgg caaggactac agcgtctggg agaaggggtc 1440 cctccgcgtc ccggcggggc ggcacctata gcgggctcaa gccctaaact gaacgggata 1500 gtcatgctca aaccagtttc tacacggcaa gaatttactg attcttatac ttttgcagtc 1560 aattaaatta tggtttttat atatgtaatt ttgtatccga ttgtagcgtt cgaataagta 1620 ggcaggctct ctagcctcta ggttaattgc ggggcatatg tagcttgcca gttaattgtg 1680 tttgtatcac gcagtttgta accgttggtg caatatataa tgtcaggttc aggatgcagt 1740 aaaaaatcat actgcaccga tcagtgagtt ttt 1773 <210> 22 <211> 265 <212> PRT <213> Oryza sativa <400> 22 Met Glu Gln His Glu Glu Ala Ala Glu Arg Lys Pro Ser Pro Pro Val 1 5 10 15 Ile Phe Arg Leu Phe Gly Val Glu Val Arg Gly Gly Gly Gly Gly Val 20 25 30 Asp Glu Glu Glu Tyr Glu Glu Glu Glu Val Glu Gly Gly Leu Phe Ile 35 40 45 Lys Lys Ser Ser Ser Met Pro Asn Leu Thr Ser Ile Asp Pro Leu Pro 50 55 60 Val Pro Ala Asp Gly Gly Lys Arg Arg Ala Ser Asp Asp Ser Glu Leu 65 70 75 80 Ala Ser Gly Gln Gln Lys Arg Arg Arg Arg Lys Val Gln Glu Arg Lys 85 90 95 Lys Gly Val Pro Trp Thr Glu Glu Glu His Lys Lys Phe Leu Glu Gly 100 105 110 Leu Arg Gln Leu Gly Lys Gly Asp Trp Arg Gly Ile Ser Lys Asn Phe 115 120 125 Val Thr Ser Arg Thr Ala Thr Gln Val Ala Ser His Ala Gln Lys Tyr 130 135 140 Phe Leu Arg Gln Thr Asn Pro Gly Lys Lys Lys Arg Arg Ala Ser Leu 145 150 155 160 Phe Asp Val Val Ala Glu Cys Ser Asp Asp Gln Leu Pro Ser Pro Gln 165 170 175 Ser Val Gly Thr Lys Pro Pro Thr Gln Asp Ile Ile His Thr Asp Arg 180 185 190 Gly Asp Val Pro Ile Leu Ser Tyr Pro Val Ala Arg Gly Phe Arg Gly 195 200 205 Asp Ser Val Gln Val Asp Glu Leu Thr Glu Tyr Val Lys Arg Leu Lys 210 215 220 Ala Ala Glu Asp Met Ser Leu Ser Met Ile Ser Gly Leu Glu Met Ala 225 230 235 240 Ser Ser Ser Ile Ser Ser Leu Glu Leu Ser Ile Ala Pro Pro His Cys 245 250 255 Ala Ile Glu Ala Ala Ile Lys Val Leu 260 265

Claims

1. A method for improving stress tolerance and productivity of rice, comprising: (a) providing a transgenic rice having a MYBS2 reduced expression of genes; and (b) growing the transgenic rice in a normal environment or in an environment with abiotic stress factors; Compared to the wild-type counterpart, MYBS2 The expression of the gene is reduced, making the gene-transfected rice Amy3 Increased gene expression; The abiotic stress factor is osmotic pressure; Among them, MYBS2 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

22. Amy3 The nucleotide sequence of the gene is shown in SEQ ID NO:

21.

2. The method of claim 1, wherein the MYBS2 The gene encodes a MYBS2 transcription factor, which is associated with the Amy3 A TA box binds to the gene promoter.

3. The method of claim 1 or 2, wherein the abiotic stress factor is dehydration or heat.

4. A method for reducing the chalkiness of rice seeds, comprising: (a) providing a seed from a transgenic rice plant, which overexpresses a full-length encoding amino acid sequence as shown in SEQ ID NO: 22 MYBS2 Gene or mutation MYBS2 gene, compared to its wild-type counterpart; wherein the mutant MYBS2 The gene encodes a truncated MYBS2 transcription factor, which is a deletion of amino acid residues 1 to 53 based on SEQ ID NO: 22; and (b) growing the seed in a normal environment or in an environment with an abiotic stress factor; The abiotic stress factor is osmotic pressure.

5. The method of claim 4, wherein the mutant MYBS2 The nucleotide sequence of the gene is shown in SEQ ID NO:

14.

6. The method of claim 4 or 5, wherein the abiotic stress factor is dehydration or heat.

Images