CN115725647A - Gene for regulating dormancy and germination of plant seeds and application thereof - Google Patents

Abstract

The invention provides a gene (GA 2ox 9) for regulating dormancy and germination of plant seeds and application thereof. The GA2ox9 influences the activity of alpha amylase and the content of soluble sugar by regulating the content of active Gibberellin (GA) in endosperm, changes the signal of abscisic acid (ABA) in embryos, and further regulates the dormancy of seeds and the germination of plant ears. GA2ox9 gene deletion causes seed dormancy to be reduced, and the phenomenon of sprouting of ears occurs; and the overexpression of the GA2ox9 gene improves the dormancy of seeds. The invention provides a new way for improving the dormancy of seeds of plants and reducing the sprouting of ears.

Description

Gene for regulating dormancy and germination of plant seeds and application thereof

Technical Field

The invention belongs to the field of botany and genetic engineering, and particularly relates to a gene for regulating dormancy and germination of plant seeds and application thereof.

Background

With the acceleration of industrialized information process and the improvement of human medical level, the world population is rapidly increased. Grain yield and excellent germplasm resources are important factors for people's livelihood. The theoretical method of modern molecular genetics is utilized to deeply research the molecular genetic mechanism formed by crop yield and combine with a molecular design breeding technology, so that people can be helped to better utilize high-quality germplasm resources.

Cereal crops are important crops and how to effectively grow such crops on limited fields of cultivation has been the focus of research among agricultural workers. The research on means for regulating the crop planting is very important work. Especially, the gramineous plant rice (Oryza sativa) is one of the most important food crops in the world, and provides staple food for more than half of the population in the world. With the continuous growth of the world population and the ever-decreasing amount of arable area, as well as the increasing amount of environmental pollution, food production faces more serious challenges. For some population countries, the high yield and high quality of rice directly influence the quality of life of people, have important significance for guaranteeing the national civilization, and are directly related to the global food safety.

In the breeding process, varieties with rapid and regular seed germination and fast seedling formation and capable of obtaining higher economic yield are often selected for production, but the selection mode also causes some undesirable phenomena, such as that the cultivated high-yield varieties lose seed dormancy, and show stronger sprouting of ears in the weather of high temperature and rainy days in the harvest season, thereby seriously affecting the yield and quality of rice (Yangdong, etc., 1991). Pre-harvest sprouting (PHS) refers to a phenomenon that seeds germinate on the ears when rice is subjected to high-temperature and high-humidity conditions in a harvest period, and is closely related to dormancy and germination of the seeds. The pre-harvest sprouting not only causes the yield reduction and the edible quality reduction of grain crops, but also influences the seed production quality of the crops. Statistics show that conventional rice has about 6% of sowing area, while hybrid rice has the emergence of sprouting in up to 20% of area, because southern rice harvest season often encounters high temperature and raininess.

The phenomenon of pre-harvest sprouting has been noticed as early as the eighties of the last century, and a plurality of QTLs related to pre-harvest sprouting are positioned, so that clues are provided for the subsequent excavation and cloning of key genes. The regulation and control of the ear sprouting is a complex physiological process, and a plurality of influencing factors exist. Besides external factors such as moisture, temperature, light and the like, the seed is also influenced by hormones, saccharides, active oxygen, NO, microRNA and the like in the seed.

Although the pre-harvest sprouting problem has been gradually noticed, and the research on the model plant Arabidopsis is more, the research foundation of the pre-harvest sprouting problem in crops is still weaker.

Disclosure of Invention

The invention aims to provide a gene for regulating dormancy and germination of plant seeds and application thereof.

In a first aspect of the invention, there is provided a method of regulating dormancy and germination of a seed of a graminaceous plant, comprising: modulating expression or activity of a GA2ox9 gene or a protein encoded thereby in a plant, thereby modulating seed dormancy and seed germination in a plant.

In one or more embodiments, the method is selected from: (a) Up-regulating the expression or activity of the GA2ox9 gene or the protein encoded thereby, thereby improving seed dormancy or reducing germination (e.g., ear germination); or, (b) down-regulating the expression or activity of a GA2ox9 gene or a protein encoded thereby, thereby reducing seed dormancy or promoting germination (e.g., ear germination) in a plant.

In one or more embodiments, the up-regulating the expression or activity of a GA2ox9 gene or a protein encoded thereby comprises: transferring the GA2ox9 gene or an expression construct or a vector containing the gene into a plant; or a mutation resulting in a function of the GA2ox9 gene in a plant having a low expression or activity of the GA2ox9 gene (including no expression or no activity).

In one or more embodiments, the downregulating the expression or activity of a GA2ox9 gene or a protein encoded thereby comprises: knocking out or silencing a GA2ox9 gene in a plant, or inhibiting the activity of a GA2ox9 protein; preferably, it comprises: performing gene editing by using a CRISPR system to knock out a GA2ox9 gene, silencing the GA2ox9 gene by using an interference molecule which specifically interferes with the expression of the GA2ox9 gene, and knocking out the GA2ox9 gene by using a homologous recombination method or performing loss-of-function mutation on the GA2ox9 gene; preferably, the method of down-regulation comprises: engineering the GA2ox9 gene to frame shift or pre-terminator (the GA2ox9 protein encoded by it provides termination); preferably, the modification targets 367 to 410 th (preferably, 384 to 403 th positions are gRNA target binding regions, and 381 to 383 th positions are PAM sequences) of the nucleotide sequence shown in SEQ ID NO 1; preferably, the nucleotide sequence of sgRNA used for gene editing is shown in SEQ ID NO. 3.

In one or more embodiments, the GA2ox9 gene or protein encoded thereby increases seed dormancy or reduces germination by decreasing the level of active gibberellin (preferably gibberellin in the endosperm of seeds), decreasing alpha-amylase activity, decreasing the level of soluble sugars, increasing abscisic acid (preferably abscisic acid in the embryo) sensitivity.

In one or more embodiments, down-regulation of the GA2ox9 gene or protein encoded thereby increases alpha amylase activity, increases soluble sugar content, decreases abscisic acid (preferably, abscisic acid in the embryo) sensitivity, decreases plant seed dormancy or promotes germination by increasing the content of active gibberellins, preferably, gibberellin in the embryo of a seed.

In another aspect of the present invention, there is provided the use of a GA2ox9 gene, its encoded protein, or a modulator thereof, for modulating seed dormancy and seed germination in gramineae.

In one or more embodiments, the modulator is a GA2ox9 gene or a protein encoded thereby is an expression construct or vector comprising a GA2ox9 gene for use in increasing seed dormancy or reducing germination (e.g., ear germination).

In one or more embodiments, the modulator is a down-regulator of the GA2ox9 gene or protein encoded thereby, for use in reducing seed dormancy or promoting germination (e.g., ear germination) in a plant.

In one or more embodiments, the down-regulating agent comprises: a gene-editing reagent that specifically edits the GA2ox9 gene, an interfering RNA molecule (e.g., siRNA, shRNA) or antisense nucleotide that specifically interferes with the expression of the GA2ox9 gene, a reagent that knocks out the GA2ox9 gene by homologous recombination, and the like.

In another aspect of the present invention, there is provided a method of screening for a regulator for regulating dormancy and germination of a graminaceous plant seed, the method comprising: (1) Adding the candidate substance to a system containing a GA2ox9 gene or a protein encoded by the gene; (2) Detecting said system and observing the expression or activity of the GA2ox9 gene or of the protein encoded by it; if the candidate substance is up-regulated (significantly up-regulated, e.g., up-regulated by 10%, 20%, 40%, 60%, 80%, 90% or more) the GA2ox9 gene or protein encoded thereby, then it is an indicator that the candidate substance is a modulator that increases seed dormancy or reduces germination; if the candidate substance down-regulates (significantly down-regulates, such as by 10%, 20%, 40%, 60%, 80%, 90% or more) the expression or activity of the GA2ox9 gene or the protein encoded thereby, it is indicative that the candidate substance is a modulator that reduces dormancy or promotes germination in plant seeds.

In one or more embodiments, the screening further comprises observing a change in the signaling pathway "GA2ox 9-gibberellin- α -amylase-soluble sugar-abscisic acid" in which the GA2ox9 gene or its encoded protein is involved; if the candidate substance acts on the GA2ox9 gene or the protein encoded thereby, thereby reducing the content of active gibberellin, reducing alpha amylase activity, reducing the content of soluble sugars or increasing the sensitivity of abscisic acid, preferably abscisic acid in the embryo, it is an indication that the candidate substance is a modulator for increasing seed dormancy or reducing germination.

In one or more embodiments, the screening further comprises observing a change in the signaling pathway "GA2ox 9-gibberellin- α -amylase-soluble sugar-abscisic acid" in which the GA2ox9 gene or its encoded protein is involved; if the candidate substance acts on GA2ox9 gene or protein coded by the gene, and further the content of active gibberellin is increased, the activity of alpha amylase is increased, the content of soluble sugar is increased or the sensitivity to abscisic acid is reduced, the candidate substance is a regulator for reducing dormancy of plant seeds or promoting germination.

In one or more embodiments, the system comprises a compound selected from the group consisting of: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system, or an animal system.

In one or more embodiments, the candidate substance includes (but is not limited to): regulatory molecules (such as, but not limited to, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, compounds from libraries of compounds, and the like designed against the "GA2ox 9-gibberellin- α -amylase-soluble sugar-abscisic acid" signaling pathway, or pathway proteins thereof, or upstream or downstream proteins, genes, or signaling pathways thereof.

In another aspect of the present invention, there is provided the use of the GA2ox9 gene of Gramineae or a protein encoded thereby as a molecular marker for identifying seed dormancy and seed germination in plants (e.g., a tracer marker for a superior trait in progeny).

In another aspect of the present invention, there is provided a method for the directed selection of a graminaceous plant or a seed thereof whose seed dormancy and seed germination are regulated, said method comprising: identifying the expression or activity of the GA2ox9 gene or the protein encoded thereby in the test plant or seed thereof: (ii) a plant with increased seed dormancy or reduced germination if the test plant or seed thereof has a higher expression or activity of the GA2ox9 gene or protein encoded thereby than the average expression/activity value of the plant or seed thereof; a plant or seed thereof having reduced dormancy or improved germination of its seed if the test plant or seed thereof has an expression or activity of the GA2ox9 gene or protein encoded thereby that is significantly lower than the average expression/activity value for that plant or seed.

In one or more embodiments, the method of directionally selecting a plant or seed thereof having modulated seed dormancy and seed germination further comprises: the change of "GA2ox 9-gibberellin-alpha amylase-soluble sugar-abscisic acid" as a signaling pathway in which GA2ox9 gene or its encoded protein participates was observed.

In one or more embodiments, the use includes (but is not limited to): the identification of nucleic acid sequences is carried out by sequencing, PCR amplification, restriction analysis, probe, hybridization, chip, allelic polymorphism analysis.

In one or more embodiments, the GA2ox9 is derived from or is a plant of the family poaceae including (but not limited to): rice (Oryza sativa), millet (Setaria italica), wheat (Triticum aestivum), maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), millet (Panicum miliceum), rye (Secale cereale), oats (Avena sativa l.), brachypodium distachyon (brachyphidium distachyum).

In one or more embodiments, the protein encoded by the GA2ox9 gene is selected from the group consisting of: (a) a protein having an amino acid sequence shown in SEQ ID NO. 2; (b) A protein derived from (a) and having (a) a protein function, which is formed by substituting, deleting or adding one or more (e.g., 1 to 30 or 1 to 20; preferably 1 to 10; more preferably 1 to 5, 1 to 3 or 1 to 2) amino acid residues to the amino acid sequence shown in SEQ ID NO: 2; (c) A protein having an amino acid sequence which is 80% or more (preferably 85% or more, 90% or more, or 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence defined in (a) and having the function of the protein (a); or (d) a fragment of SEQ ID NO 2 having the function of the protein of (a); or (d) a protein formed by adding a tag sequence to the N-or C-terminus of the protein having the amino acid sequence shown in SEQ ID NO. 2, or adding a signal peptide sequence to the N-terminus thereof.

In one or more embodiments, the GA2ox9 gene or the protein encoded thereby comprises a homologue thereof.

In one or more embodiments, the plant is a plant expressing GA2ox9 or a homologue thereof.

In one or more embodiments, the plant is a plant whose germination and/or hibernation of seeds is regulated by gibberellin and abscisic acid.

In one or more embodiments, the high expression (or high expression) or high activity (or high activity) refers to a statistically significant increase in expression or activity, such as 10%, 20%, 40%, 60%, 80%, 90% or more, as compared to the average expression or activity of the same species or plant species.

In one or more embodiments, the low expression (or low expression) or low activity (or low activity) refers to a statistically significant decrease in expression or activity, such as a 10%, 20%, 40%, 60%, 80%, 90% or less decrease, as compared to the average value of expression or activity in the same or like plant.

In one or more embodiments, the term "improving seed dormancy or reducing germination" refers to a statistically significant increase/promotion of seed dormancy (e.g., a 5%, 10%, 20%, 40%, 60%, 80%, 90% or greater proportion of seeds in the dormant state) or a statistically significant reduction in seed germination (e.g., a 10%, 20%, 40%, 60%, 80%, 90% or greater proportion of seeds in the germinated state) as compared to the amount of the same or like plant.

In one or more embodiments, the phrase "reducing the dormancy or promoting germination of a seed of a plant" refers to a statistically significant decrease in the dormancy of the seed (e.g., a 10%, 20%, 40%, 60%, 80%, 90% or greater decrease in the proportion of seeds in the state of dormancy), or a statistically significant increase in the germination of the seed (e.g., a 10%, 20%, 40%, 60%, 80%, 90% or greater increase in the proportion of seeds in the state of germination) as compared to the amount of the same or like plant.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 sequencing analysis of OsGA2ox9 loss-of-function homozygous mutants edited differently. The green font indicates a PAM (Protospacer adjacent motif) sequence, the blue font indicates a gRNA sequence, and the red dots indicate regions of base deletion.

FIG. 2OsGA2ox9 functional deletion and statistics of seed germination rate of overexpressed transgenic material (upper panel). Taking seeds 27-29 days after pollination, and counting the germination number of the seeds at the same time point for 5 consecutive days under the conditions of germination temperature of 28 ℃,12 hours of illumination/12 hours of darkness. The materials used included Zhonghua 11, osGA2ox9-Cas9 transgenic lines (L2, L4), osGA2ox9-OE transgenic lines (L1, L2, L3). The phenomenon of sprouting of mature seeds of OsGA2ox9 function-deficient transgenic material (lower graph) appears.

FIG. 3 alpha amylase activity in mature seeds of OsGA2ox9-Cas9 transgenic material is increased.

After OsGA2ox9-Cas9 transgenic material and wild type mature seed are crosscut, endosperm parts without embryo are respectively placed on different GAs ₃ Alpha-amylase activity was measured at concentrations (0, 0.01mM, 0.1 mM).

FIG. 4 soluble sugar content in mature seeds of OsGA2ox9-Cas9 transgenic material is increased.

The trehalose, glucose, fructose and sucrose contents in mature seeds of OsGA2ox9-Cas9 transgenic material were determined by HPLC.

FIG. 5 seeds of OsGA2ox9-Cas9 transgenic material have reduced sensitivity to ABA compared to wild type (left panel). The qPCR results showed down-regulated expression of OsABI3 and OsABI5 in seeds 18 and 28 days after pollination of the OsGA2ox9-Cas9 transgenic material.

FIG. 6 homology analysis of OsGA2ox9 gene.

Detailed Description

The inventor discloses a novel gene GA2ox9 for regulating and controlling the dormancy and the germination of plant seeds through extensive research. The GA2ox9 influences the activity of alpha amylase and the content of soluble sugar by regulating the activity of Gibberellin (GA) in the endosperm, changes the signal of abscisic acid (ABA) in the embryo, and further regulates the dormancy of seeds and the germination of plant ears. The invention further discloses that GA2ox9 gene deletion causes seed dormancy to be reduced, and the phenomenon of sprouting of ears occurs; and the overexpression of the GA2ox9 gene can improve the seed dormancy. The invention provides a new way for improving the dormancy of seeds of plants and reducing the sprouting of ears.

Term(s) for

As used herein, the term "plant" includes plants that express GA2ox9 (including homologs thereof) or contain GA2ox9 and the signaling pathway in which it is involved. According to the knowledge in the art, a plant expressing GA2ox9, in which the mechanism of action as claimed in the present invention is present, can achieve the technical effects as claimed in the present invention. In some embodiments, the plant is a crop, preferably a cereal crop, the cereal crop being a crop with grain (ear). The "cereal crop" may be a gramineous plant. In some preferred forms, the gramineae comprises: rice, barley, wheat, oat, rye, corn, sorghum, brachypodium distachyon, and the like. The GA2ox9 includes its homologues (homologous genes and proteins encoded by them).

In some embodiments, the "plant" is a plant having an embryo, endosperm structure. It is well known to those skilled in the art that the composition of plant embryos or endosperm is similar, and that plants having an embryo or endosperm structure share common characteristics, with genes or regulatory elements present in the genome that are conserved to regulate transcription, expression of genes, e.g., a series of elements that regulate plant formation into embryo or endosperm. In the present invention, the "plant" expresses GA2ox9 or comprises GA2ox9, and the signal pathway in which it participates ("GA 2ox 9-gibberellin-. Alpha.amylase-soluble sugar-abscisic acid") is also disclosed. According to the knowledge in the art, a plant expressing GA2ox9, in which the action mechanism as claimed in the present invention is present, can achieve the technical effects as claimed in the present invention.

As used herein, the terms "increase", "improvement", "promotion" or "enhancement" are interchangeable and shall mean in the sense of application a modulation of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30% higher compared to a control plant as defined herein.

With regard to "control plants", the selection of suitable control plants is a routine part of the experimental design and may include corresponding wild-type plants or corresponding transgenic plants without the gene of interest. The control plant is typically the same plant species or even a variety that is the same as or belongs to the same class as the plant to be evaluated. The control plant may also be an individual that has lost the transgenic plant as a result of the segregation. Control plants as used herein refer not only to whole plants but also to plant parts, including seeds and seed parts.

As used herein, "grain" refers to the fruit or seed of a plant, also known as ear grain in rice, corn, wheat, barley, and like crops.

As used herein, said up-regulation, promotion, enhancement or enhancement means a significant up-regulation, promotion, enhancement or enhancement, such as up-regulation, promotion, enhancement or enhancement by 20%, 40%, 60%, 80%, 90% or more.

As used herein, downregulating, decreasing, inhibiting, attenuating or decreasing means a significant downregulating, decreasing, inhibiting, attenuating or decreasing, such as downregulating, decreasing, inhibiting, attenuating or decreasing by 20%, 40%, 60%, 80%, 90% or less.

GA2ox9 gene and protein coded by it

In the present invention, the GA2ox9 protein includes a homologue (homologous protein) thereof unless otherwise specified. The GA2ox9 may be a polypeptide (protein) having an amino acid sequence shown in SEQ ID NO. 2, and also includes a variant form of the sequence having the same function as that of the GA2ox9 protein.

Such variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. Any protein having a high homology to the GA2ox9 protein (e.g., 70% or more homology to the polypeptide sequence shown in SEQ ID NO: 2; preferably 80% or more homology; more preferably 90% or more homology, such as 95%,98% or 99% homology), and having the same function as the GA2ox9 protein, is also included in the present invention.

In the present invention, the "GA2ox9 protein" as mentioned above also includes homologs thereof. It will be appreciated that although the GA2ox9 protein obtained from a particular species is preferably studied in the present invention, other polypeptides or genes obtained from other species, particularly from plants of the Gramineae, which are highly homologous (e.g., have greater than 70%, more particularly greater than 80%,85%, 90%, 95%, or even greater than 98% sequence identity) to the GA2ox9 protein are also within the contemplation of the present invention.

Polypeptides derived from species other than rice that have a high homology with the polypeptide sequence of SEQ ID NO. 2 sequence or that exert the same or similar effects in the same or similar signal pathways are also included in the present invention.

The present invention also provides an isolated protein which is a fragment of the GA2ox9 protein or which is formed by adding other proteins or tags, etc., to both ends.

The invention also relates to polynucleotide sequences encoding a GA2ox9 protein of the invention, or a sequence variant thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 1 or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a polypeptide having the sequence of SEQ ID NO. 2, but differs from the sequence of the coding region shown in SEQ ID NO. 1. The present invention also relates to variants (variants) of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogues and derivatives of the polypeptides.

The invention also relates to vectors comprising said polynucleotides, and to host cells genetically engineered with said vector or polypeptide encoding nucleic acids.

In the present invention, the polynucleotide sequence encoding the polypeptide of the present invention may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements. Preferably, the expression vector may also optionally incorporate resistance elements, selection elements or reporter elements, such as Bar, GUS.

When expressed in higher eukaryotic cells, the polynucleotides will provide enhanced transcription when enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription.

Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. The transformed plant may be transformed by methods such as Agrobacterium transformation or particle gun transformation, for example, spray method, leaf disk method, rice immature embryo transformation method, etc.

Plant transformation

According to the invention, the GA2ox9 gene is identified through a large amount of systematic research and large-scale research screening, and the characters of plant seed dormancy, seed germination and the like are regulated and controlled. The protein encoded by the GA2ox9 gene improves dormancy or reduces germination in seeds by reducing the content of active gibberellins, preferably gibberellins in the endosperm of seeds, reducing alpha amylase activity, reducing the content of soluble sugars, improving the sensitivity to abscisic acid, preferably abscisic acid in the embryo. On the contrary, the down-regulation of GA2ox9 gene can increase the content of active gibberellin (preferably gibberellin in the embryo milk of the seeds), increase the activity of alpha-amylase, increase the content of soluble sugar, reduce the sensitivity of abscisic acid (preferably abscisic acid in the embryo), reduce the dormancy of plant seeds or promote germination.

The dormancy and germination of seeds are regulated by a complex mechanism, and the related passages and ways are various, and the involved genes/proteins are various. Abscisic acid (ABA) and Gibberellin (GA) are two plant hormones that regulate ear germination and regulate seed dormancy and germination by antagonizing each other. The ABA mainly has two functions in the ear germination, and the first function is to inhibit the seed germination; secondly, the seeds are induced to enter a dormant state in the seed maturation process. There are also two major roles of GA in germination, one is to promote the growth potential of the embryo, and the other is to relieve the mechanical barrier formed by the endosperm during germination by increasing the activity of alpha-amylase. ABA is an inducer of seed dormancy. In plant seeds, ABA synthesis is completed by the joint participation of a plurality of enzymes, and the activity change of the enzymes can cause the change of the ABA content in the seeds, thereby regulating the dormancy of the seeds. In addition to ABA metabolic pathway, ABA signal induces the expression of downstream genes through PYR/PYL/RCAR-PP2C-SnRKs cascade reaction, thereby influencing the dormancy of seeds. GA promotes the growth of radicles in the process of seed germination, and breaks through seed coats and elongation. The seeds respond to GA signals to activate the expression of germination related genes of the seeds and induce the generation of cell wall remodeling enzymes, so that endosperm and cortex around the embryo are weakened, and the radicle is favorable for protruding the seed coat. Both GA metabolic processes and GA signal transduction processes are involved in the dormant regulation of seeds.

In the invention, the synergistic effect of the GA2ox9 gene, GA and ABA is firstly proposed, so that a novel signal channel is proposed: GA2ox 9-gibberellin-alpha amylase-soluble sugar-abscisic acid pathway.

Based on the new findings of the present inventors, there is provided the use of GA2ox9 or a regulator thereof for regulating seed dormancy and seed germination of gramineous plants. The GA2ox9 includes a homologue thereof.

It is understood that, after the regulation of the GA2ox9 in gramineae is known, the expression or activity of the GA2ox9 can be regulated as desired by various methods well known to those skilled in the art, and these methods are included in the present invention.

An up-regulator of GA2ox9 expression or activity can be used to up-regulate GA2ox9 activity. The up-regulator of the expression or activity of GA2ox9 comprises an accelerant, an agonist and an activator. The terms "up-regulation" and "promotion" include "up-regulation", "promotion" of protein activity or "up-regulation", "promotion" of protein expression. Any substance which can increase the activity of a GA2ox9 protein, improve the stability of a GA2ox9 gene or protein, up-regulate the expression of a GA2ox9 gene, and increase the effective action time of a GA2ox9 protein can be used in the present invention as a substance useful for up-regulating a GA2ox9 gene or a protein encoded thereby. They may be chemical compounds, small chemical molecules, biomolecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level.

As a preferred embodiment, there is provided a method for up-regulating the expression or activity of GA2ox9 in a plant, said method comprising: an expression construct or vector comprising the GA2ox9 gene is transferred into a plant.

Preferably, there is provided a method of producing a transgenic plant comprising:

(1) Transferring an exogenous nucleic acid encoding GA2ox9 into a plant organ or tissue to obtain a plant tissue or organ into which the nucleic acid has been transferred; and

(2) Regenerating the plant tissue or organ which is transferred with the exogenous nucleic acid and obtained in the step (1) into a plant.

As a preferred example, the method comprises the steps of:

(s 1) providing agrobacterium carrying an expression vector comprising exogenous (recombinant) GA2ox9;

(s 2) contacting the plant tissue or organ with the agrobacterium of step (s 1), thereby causing said GA2ox9 to be transferred into and integrated into the chromosome of the plant cell;

(s 3) selecting a plant cell, tissue or organ into which said GA2ox9 has been transferred.

The present invention also includes plants obtainable by any of the methods described above, said plants comprising: transgenic plants transformed with said GA2ox9.

In the present invention, the GA2ox9 protein or the gene encoding the protein is any substance which can decrease the activity of the GA2ox9 protein, decrease the stability of the GA2ox9 protein or the gene encoding the protein, down-regulate the expression of the GA2ox9 protein, decrease the effective action time of the GA2ox9 protein, inhibit the transcription and translation of the GA2ox9 gene, or decrease the phosphorylation/activation level of the protein, and these substances can be used in the present invention as a substance useful for down-regulating the GA2ox9 protein. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level. For example, the down-regulating agent is: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of GA2ox9 protein or other signaling pathway genes; or a gene-editing reagent which specifically edits the GA2ox9 gene, and the like.

As a preferred mode of the present invention, there is provided a method for down-regulating a GA2ox9 protein in a plant, comprising targeted mutation, gene editing or gene recombination of the GA2ox9 protein, thereby achieving the down-regulation. As a more specific example, the GA2ox9 protein is converted into a mutant thereof by any of the methods described above, so that it no longer functions. As a more specific embodiment mode, the CRISPR/Cas9 system is adopted for gene editing. An appropriate sgRNA target site will lead to higher gene editing efficiency, so an appropriate target site can be designed and found before gene editing is initiated. After designing a specific target site, in vitro cell activity screening is also required to obtain an effective target site for subsequent experiments. Preferred gene editing reagents are provided in the examples of the present invention.

As a further alternative, the method of down-regulating the expression of GA2ox9 protein in a plant may comprise: (1) Transferring an interfering molecule interfering with GA2ox9 gene expression into a plant cell, tissue, organ or seed to obtain the plant cell, tissue, organ or seed into which the interfering molecule is transferred; (2) Regenerating the plant cell, tissue, organ or seed obtained in step (1) into which the interfering molecule has been transferred into a plant. Preferably, the method further comprises: (3) Selecting a plant cell, tissue or organ into which said vector has been transferred.

The methods may be carried out using any suitable conventional means, including reagents, temperature, pressure conditions, and the like.

Plant directional screening and molecular marking

Based on the new findings of the present inventors, the present invention provides a molecular marker suitable for identifying plant traits, namely the GA2ox9 gene; the plant traits include: seed dormancy and seed germination. The invention also relates to specific molecular markers designed for the GA2ox9 gene, and an identification strategy.

As a preferred mode, the method of the present invention for the targeted selection or identification of plants having modulated agronomic traits comprises: identifying the expression or activity of the GA2ox9 gene or the protein encoded thereby in the test plant or its seeds: (ii) a plant with increased seed dormancy or reduced germination if the test plant or seed thereof has a higher expression or activity of the GA2ox9 gene or protein encoded thereby than the average expression/activity value of the plant or seed thereof; a plant or seed thereof having reduced dormancy or improved germination of its seed if the test plant or seed thereof has an expression or activity of the GA2ox9 gene or protein encoded thereby that is significantly lower than the average expression/activity value for that plant or seed.

Based on the novel findings of the present invention, one skilled in the art can perform the analysis of nucleic acid sequences using any of a variety of techniques known in the art or being developed, and such techniques can be included in the present invention. Such methods include, for example, but are not limited to: sequencing, PCR amplification, probe, hybridization, restriction analysis, allelic polymorphism analysis (e.g., melting curve), and the like.

The invention has good application prospect in the aspects of molecular design breeding, crop variety improvement by utilizing the genetic engineering technology and the like.

After the function of the GA2ox9 gene is known, it can be used as a molecular marker for the targeted selection of plants. Substances or potential substances that can be used to directionally regulate seed dormancy and seed germination in gramineae by modulating this mechanism can also be screened based on this new finding.

The invention provides a method for screening substances (potential substances) for adjusting the characteristics of gramineous plants, which comprises the following steps: (1) Adding the candidate substance to a system containing the GA2ox9 gene or a protein encoded by the gene; (2) Detecting said system and observing the expression or activity of the GA2ox9 gene or of the protein encoded by it; if the candidate substance up-regulates the GA2ox9 gene or the protein encoded by the gene, the candidate substance is a regulator for improving the dormancy or reducing the germination of the seeds; if the candidate substance down-regulates the expression or activity of the GA2ox9 gene or the protein encoded thereby, this is an indication that the candidate substance is a modulator for reducing dormancy or promoting germination of a plant seed.

Methods for targeting proteins or genes or specific regions thereof to screen for substances that act on the target are well known to those skilled in the art and all of these methods can be used in the present invention. The candidate substance may be selected from: peptides, polymeric peptides, peptidomimetics, non-peptidic compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the kind of substance to be screened, it is clear to the skilled person how to select a suitable screening method.

The interaction between proteins and the strength of the interaction can be detected by various techniques known to those skilled in the art, such as GST-sink technique (GST-Pull Down), bimolecular fluorescence complementation assay, yeast two-hybrid system or co-immunoprecipitation technique.

Through large-scale screening, a substance which specifically acts on GA2ox9 protein or a coding gene thereof and has a regulating effect on the character improvement of gramineous plants can be obtained.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, or according to the manufacturer's recommendations.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Sequence information

CDS sequence of OsGA2ox9 gene (SEQ ID NO: 1):

OsGA2ox9 protein sequence (SEQ ID NO: 2):

ATTERYSVAYFLCPSYDSPIGTCREPSPYKAFTFGEYRRRVQEDVKKTGKKTGLSNFLV*

example 1 deletion of OsGA2ox9 Gene and alteration of dormancy and Germination of seeds overexpressing transgenic Material

1. Preparation of transgenic Material deficient in OsGA2ox9 Gene

The gene editing technology is utilized to specifically edit OsGA2ox9 in a genome, a sgRNA (Single guide RNA) capable of specifically recognizing OsGA2ox9 exon is selected, the Cas9 protein can be brought to the target position of the OsGA2ox9 genome, the Cas9 protein cuts double-stranded DNA and generates a gap, the non-homologous end connection repair can be carried out on the gap in a plant body, and finally the insertion or deletion of a basic group is caused

The sequence of sgRNA is: cctcaacgactcctaccgct (SEQ ID NO: 3).

In order to form sgRNA, two knockout primers are respectively designed, phosphorylated, annealed and paired, and then connected to pOs-sgRNA, then the pOs-sgRNA and pUbi-Cas9 are mixed, the pOs-sgRNA is transferred to the pUbi-Cas9 vector by LR reaction, and the vector is identified by colony PCR and sequencing method, and finally the GA2ox9-CRISPR-Cas9 binary vector is obtained.

A GA2ox9-CRISPR-Cas9 binary vector is introduced into agrobacterium EHA105 (purchased from Invitrogen corporation, USA), rice callus is transformed, and a positive transgenic strain is obtained through resistance screening. Sequencing the T0 generation and the T1 generation of the transgenic plant to obtain 6OsGA2ox9 function-deletion homozygous transgenic materials with different editions, which are named as OsGA2ox9-Cas 9-1-6 (plants L1-L6) as shown in figure 1. The inventors selected L2 and L4 for subsequent experiments.

2. Preparation of overexpression transgenic Material of OsGA2ox9 Gene

A1351 bp OsGA2ox9 upstream promoter sequence (1351 bp before the ATG initiation codon) and a 1077bp CDS sequence of OsGA2ox9 are amplified by utilizing PCR, the promoter and CDS of the OsGA2ox9 gene are connected with a skeleton fragment of a pCAMBIA1300 vector by utilizing T4 ligase, escherichia coli DH5 alpha is transformed, and plasmids are further extracted, enzyme digestion verification and sequencing are carried out. The recombinant plasmid pGA2ox9-GA2ox9-pCAMBIA1300 containing the OsGA2ox9 gene confirmed by sequencing is introduced into agrobacterium tumefaciens EHA105 to transform rice callus, and a positive transgenic strain is obtained through resistance screening. 8 independent OsGA2ox9 Overexpression (OE) homozygous transgenic lines are obtained by planting T1 and T2 generations and identifying by using resistance and PCR, and are named as OsGA2ox 9-OE-1-8.

3. Deletion of OsGA2ox9 gene and character analysis of overexpression transgenic material

Homozygous transgenic material of OsGA2ox9-Cas9 and OsGA2ox9-OE was planted in the experimental field, harvested after normal growth and fructification, and planted with wild type Zhonghua 11 as control.

Seeds from day 27-29 after pollination were taken and 50 in groups placed in dishes with 3 replicates per material. Adding sterilized water to soak the seeds, placing the round dish in a light incubator with the temperature of 28 ℃ and the light of 12 hours/the dark of 12 hours for 5 days, changing the water every day and counting the germination quantity of the seeds.

The result shows that the germination rate of the OsGA2ox9-Cas9 transgenic material is higher than that of a wild type, the germination rate reaches 80% when the water is absorbed for 4 days, and the mature seeds have the sprouting phenomenon; while the germination rate of the OsGA2ox9 overexpression material is slower, and the germination rate is less than 40% after absorbing water for 4 days, as shown in figure 2.

The above results indicate that OsGA2ox9 plays an important role in the dormancy and germination of seeds.

Therefore, osGA2ox9 helps to put plant seeds in dormancy, reducing sprouting of ears, which is advantageous when longer-term conservation is required. While down-regulation of OsGA2ox9 in plants can promote germination and ear sprouting of plant seeds.

Example 2 altered levels of active Gibberellin (GA) in OsGA2ox9 transgenic material, altered alpha-amylase activity and soluble sugar levels in seeds

1. OsGA2ox9 functions in the form of GA2 oxidase

The present inventors selected a tissue having a high active GA content-rice seedling to determine the function of the protein encoded by the OsGA2ox9 gene, and observed whether it is a GA2 oxidase (which can convert an active GA precursor into an inactive state).

Seedlings of OsGA2ox9-Cas9 and OsGA2ox9-OE grown for 14 days were taken and subjected to GA content determination by high performance liquid chromatography.

The determination result shows that the content of the OsGA2ox9-Cas9 transgenic strain is increased compared with that of the wild active GAs, namely GA4, and the content of the inactive GAs, namely GA34 and GA51, is reduced; and the content of the OsGA2ox9-OE transgenic line is reduced compared with that of the wild active GAs, namely GA1 and GA3, and the content of the inactive GAs, namely GA51 is increased.

This result indicates that OsGA2ox9 functions as GA2 oxidase, changing the presence of active GA and inactive: downregulating OsGA2ox9 increases the active GA content in plants, and upregulating OsGA2ox9 decreases the active GA content in plants.

2. Amylase Activity

The activity of the active GA can enhance the activity of alpha amylase, and according to the principle that the starch turns blue when meeting iodine, a proper amount of starch is added into a culture medium, if the activity of the alpha amylase in seeds is stronger, the decomposition of the starch can be catalyzed, and the occurrence of white spots is caused. The present inventors placed the embryo-removed seeds of wild type and OsGA2ox9-Cas9 transgenic material on agar medium containing 0.2% soluble starch, treated in the dark at 30 ℃ for 48 hours, and after removing the seeds, stained the medium with iodine/potassium iodide solution.

The results show that the seeds of the OsGA2ox9-Cas9 transgenic material cause more remarkable 'white spot' phenomenon, which indicates that the seeds of the OsGA2ox9-Cas9 transgenic material have higher alpha amylase activity than the wild type, as shown in FIG. 3.

GA induces the expression of alpha amylase, which cleaves the alpha-1, 4-glucan bond in starch, hydrolyzing the starch to soluble sugars. The inventor utilizes a liquid chromatography technology to detect the content of four soluble sugars (trehalose, glucose, fructose and sucrose) in mature seeds of OsGA2ox9-Cas9 transgenic materials.

The results show that the content of glucose, fructose and sucrose in the mature seeds of the OsGA2ox9-Cas9 transgenic material is increased compared with the wild type, as shown in figure 4.

Example 3 alteration of ABA Signal in OsGA2ox9 transgenic Material affecting dormancy and Germination of seeds

1. Sensitivity assay for ABA

The inventors treated seeds 27-29 days after pollination of wild type and OsGA2ox9-Cas9 transgenic material with different concentrations of ABA (0, 5. Mu.M, 10. Mu.M) and cultured under the same conditions as those in germination rate determination in example 1.

The results show that the sensitivity of the OsGA2ox9-Cas9 transgenic material seeds to ABA is reduced compared with the wild type.

2. qPCR analysis of key factors of ABA signaling pathway

An increase in soluble sugar content in seeds inhibits the expression of the key factors ABI3 and ABI5 of the ABA signaling pathway. Therefore, the present inventors analyzed the expression of key factors of ABA signaling pathway in OsGA2ox9-Cas9 transgenic material by qPCR.

qPCR showed that the expression of OsABI3 and OsABI5 was down-regulated in OsGA2ox9-Cas9 transgenic material seeds, as shown in FIG. 5.

Therefore, osGA2ox influences the activity of alpha amylase and the content of soluble sugar by regulating the content of active GA, and further changes the signal of ABA (sensitive to ABA) in the embryo and the dormancy of seeds.

Example 4 analysis of the conservation of OsGA2ox9 in different species

The inventors analyzed the conservation of OsGA2ox9 in different species. The results show that there are homologous genes in various plants such as Arabidopsis, wheat, maize, etc., as shown in FIG. 6.

The pre-harvest sprouting of the crops not only affects the yield, but also seriously reduces the quality of the crop seeds. The method has the advantages that the variety with strong dormancy resistance is resistant to pre-harvest germination, key control genes of seed dormancy are separated, and the method has important significance for mining gene resources resistant to pre-harvest germination. Homology analysis showed that OsGA2ox9 has a homologous gene in various plants such as Arabidopsis, wheat, and maize.

The results of the enhanced seed dormancy and delayed germination of the transgenic material overexpressing OsGA2ox9 suggest that OsGA2ox9 or its homologous gene is useful for improving the seed dormancy of plants (putting seeds in a dormant state) and reducing the germination of ears.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims. Also, all documents referred to herein are incorporated by reference in this application as if each had been individually incorporated by reference.

Claims (10)

1. A method for regulating dormancy and germination of seeds of a graminaceous plant comprising: modulating expression or activity of a GA2ox9 gene or a protein encoded thereby in a plant, thereby modulating seed dormancy and seed germination in a plant.

2. The method of claim 1, wherein the method is selected from the group consisting of:

(a) Up-regulating the expression or activity of the GA2ox9 gene or its encoded protein, thereby improving seed dormancy or reducing germination; or

(b) Down-regulating the expression or activity of the GA2ox9 gene or the protein encoded thereby, thereby reducing the dormancy of plant seeds or promoting germination.

3. The method of claim 2, wherein up-regulating the expression or activity of a GA2ox9 gene or a protein encoded thereby comprises: transferring the GA2ox9 gene or an expression construct or vector comprising the gene into a plant; or performing GA2ox9 gene function gain mutation on a plant with low GA2ox9 gene expression or activity; or

Said down-regulating the expression or activity of the GA2ox9 gene or its encoded protein comprises: knocking out or silencing a GA2ox9 gene in a plant, or inhibiting the activity of a GA2ox9 protein; preferably, the method comprises the following steps: performing gene editing by using a CRISPR system to knock out a GA2ox9 gene, silencing the GA2ox9 gene by using an interference molecule which specifically interferes with the expression of the GA2ox9 gene, and knocking out the GA2ox9 gene by using a homologous recombination method or performing loss-of-function mutation on the GA2ox9 gene; preferably, the method of down-regulation comprises: modifying the GA2ox9 gene to cause the gene to generate frame shift or a terminator in advance; preferably, the modification is targeted to 367 to 410 th of the nucleotide sequence shown in SEQ ID NO. 1; preferably, the nucleotide sequence of sgRNA used for gene editing is shown in SEQ ID NO. 3.

4. The method of claim 1, wherein the GA2ox9 gene or protein encoded thereby is reduced by reducing the content of active gibberellin, reducing alpha amylase activity, reducing the content of soluble sugars, increasing abscisic acid sensitivity, increasing seed dormancy or reducing germination; or

By reducing GA2ox9 gene or protein coded by the gene, the activity of alpha amylase is improved by improving the content of active gibberellin, the content of soluble sugar is improved, the sensitivity of abscisic acid is reduced, and the dormancy of plant seeds is reduced or the germination is promoted.

5. Use of a GA2ox9 gene, its encoded protein, or modulators thereof, for modulating seed dormancy and seed germination in gramineae.

6. Use according to claim 5, wherein the modulator is a GA2ox9 gene or a protein encoded thereby, is an expression construct or vector comprising a GA2ox9 gene, for increasing seed dormancy or reducing germination; or

The regulator is a down-regulator of GA2ox9 gene or protein coded by the gene, and is used for reducing dormancy of plant seeds or promoting germination.

7. A method of screening for a modulator that modulates seed dormancy and seed germination in graminaceous plants, said method comprising:

(1) Adding the candidate substance to a system containing the GA2ox9 gene or a protein encoded by the gene;

(2) Detecting said system and observing the expression or activity of the GA2ox9 gene or of the protein encoded by it; if the candidate substance up-regulates the GA2ox9 gene or the protein encoded by the gene, the candidate substance is a regulator for improving the dormancy or reducing the germination of the seeds; if the candidate substance down-regulates the expression or activity of the GA2ox9 gene or the protein encoded thereby, it is an indication that the candidate substance is a modulator for reducing dormancy or promoting germination in plant seeds.

8. A method for the targeted selection of a graminaceous plant or a seed thereof with modulated seed dormancy and seed germination, said method comprising: identifying the expression or activity of the GA2ox9 gene or the protein encoded thereby in the test plant or its seeds: a test plant or seed thereof is a plant with increased seed dormancy or reduced germination if the test plant or seed thereof has a higher expression or activity of the GA2ox9 gene or protein encoded thereby than the average expression/activity value of such plant or seed thereof; a plant or seed thereof having reduced seed dormancy or improved germination is identified if the test plant or seed thereof has an expression or activity of the GA2ox9 gene or protein encoded thereby that is significantly lower than the average expression/activity value for that plant or seed.

9. The method or use of any one of claims 1 to 8, wherein the GA2ox9 is derived from or comprises a plant of the family poaceae: rice (Oryza sativa), millet (Setaria italica), wheat (Triticum aestivum), maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), millet (Panicum miliceum), rye (Secale cereale), oats (Avena sativa l.), brachypodium distachyon (brachyphidium distachyum).

10. The method or use according to any one of claims 1 to 8, wherein the protein encoded by the GA2ox9 gene is selected from the group consisting of:

(a) 2, and the amino acid sequence shown in SEQ ID NO;

(b) 2, and (b) protein which is formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2 and has the protein function of (a) and is derived from (a);

(c) Protein with the amino acid sequence being more than 80 percent of the same as the amino acid sequence limited by the (a) and having the function of the protein of the (a); or (d) a fragment of SEQ ID NO 2 having (a) a protein function; or

(d) 2, or a protein formed by adding a signal peptide sequence to the N-terminal or the C-terminal of the protein of the amino acid sequence shown in SEQ ID NO.

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