CN110699375A

CN110699375A - Genetic improvement method for specifically enhancing seed dormancy and application thereof

Info

Publication number: CN110699375A
Application number: CN201911083566.4A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2019-03-07
Filing date: 2019-11-07
Publication date: 2020-01-17
Also published as: WO2020177750A1

Abstract

The invention provides a genetic improvement method for specifically enhancing seed dormancy and application thereof, and particularly unexpectedly discovers for the first time that the seed dormancy can be remarkably enhanced and the spike germination resistance of seeds can be improved by knocking out N members (N is more than or equal to 1) of a miR156 gene family.

Description

Genetic improvement method for specifically enhancing seed dormancy and application thereof

Technical Field

The invention relates to the field of agriculture and biotechnology, in particular to a genetic improvement method for specifically enhancing seed dormancy and application thereof.

Background

During the grain harvesting season, it is often exposed to high temperature and rainy weather. This weather is very likely to cause pre-harvest sprouting (PHS), a pre-harvest sprouting phenomenon in cereals, resulting in severe losses in yield and quality. At present, PHS is an important problem to be solved urgently in agricultural production. However, there is a lack of PHS-resistant genetic loci and resources that can be efficiently utilized in crops.

The dormancy of seeds means that the seeds with vitality can not germinate under proper conditions due to embryo or seed shell. The seed dormancy prevents plant seeds from germinating at wrong time, enhances the ability of the seeds to resist ear sprouting, thereby avoiding severe growth environment, and is an adaptive mechanism formed in the evolution process. The seed dormancy can inhibit PHS and prolong the seed life, and has important practical significance in agricultural production. Primary seed dormancy (primary seed dormancy) occurs during seed maturation, which prevents premature germination of the nascent seed. After the seeds are mature, the dormancy can be gradually broken through drying and dehydration, and the seeds are promoted to germinate under proper conditions.

Currently, the mechanism of seed dormancy is mainly studied in Arabidopsis thaliana. Dormancy of seeds is affected by various factors, such as external factors affecting dormancy, e.g., temperature, humidity and light, and endogenous factors, e.g., abscisic acid (ABA), Gibberellin (GA), auxin (auxin) and Cytokinin (CK). Among them, ABA and GA are key factors determining the state of dormancy, and many factors affecting seed dormancy act by affecting ABA or/and GA pathways. ABA inhibits seed germination and induces seed dormancy. During the seed maturation process, the ABA content is gradually increased, so that the dormancy state of the seeds is induced. GA promotes seed germination and antagonizes ABA function. Therefore, the balance of ABA and GA action is a key factor in determining the state of dormancy of seeds.

There is an urgent need in the art to develop a genetically improved method for specifically enhancing rice seed dormancy.

Disclosure of Invention

The invention aims to provide a genetic improvement method for specifically enhancing the dormancy of rice seeds by regulating genes so as to improve the pre-harvest sprouting resistance of the seeds, and a new plant variety with pre-harvest sprouting resistance is obtained. More specifically, the invention provides a method for improving the pre-germination resistance of seeds by using CRISPR technology to reduce the expression level of at least one gene in miR156 family so as to enhance the dormancy capability of the seeds.

In a first aspect, the present invention provides a method of modifying a plant comprising the steps of:

(i) genetically engineering plant cells or plant tissues to mutate N members of the miR156 gene family, wherein N is greater than or equal to 1 (preferably, greater than or equal to 2, more preferably, 2-11, more preferably, 2-10, more preferably, 3-10, more preferably, 5-8);

(ii) regenerating the genetically engineered plant cells or plant tissues into plants, and performing character test on the regenerated plants, wherein the characters comprise seed dormancy and ear germination;

(iii) and selecting plants with the required character characteristics according to the character test results.

In another preferred embodiment, the desired trait feature comprises enhanced seed dormancy.

In another preferred embodiment, the desired trait further comprises increasing seed pre-spike germination resistance.

In another preferred embodiment, said enhancing seed dormancy comprises increasing the resistance of the seed to pre-harvest germination.

In another preferred embodiment, the desired trait features further comprise: reduce GA content, inhibit GA pathways, up-regulate expression of IPA 1.

In another preferred example, the miR156 gene family includes class I miR156s and class II miR156 s.

In another preferred example, the class I miR156s is selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.

In another preferred example, the class II miR156s is selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.

In another preferred example, the mutation comprises a decrease in the expression or activity of N members of the miR156 gene family.

In another preferred embodiment, the mutation comprises an insertion, deletion or substitution of one or more bases.

In another preferred embodiment, the mutation is performed by a method selected from the group consisting of: gene editing, natural variation, mutagenesis, or a combination thereof.

In another preferred example, the "decreasing" refers to decreasing the expression or activity of N members of the miR156 gene family satisfies the following condition:

the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 60 percent, more preferably less than or equal to 40 percent, and most preferably 0 to 30 percent;

wherein, A1 is the expression level or activity of N members in miR156 gene family; a0 is the expression level or activity of N members in the same miR156 gene family in wild type plants of the same type.

In another preferred example, said reduction refers to an expression level of a member of the miR156 family E1 in said plant which is 0-80%, preferably 0-60%, more preferably 0-40% of wild type compared to the expression level E0 of a member of the wild type miR156 family.

In another preferred example, the reduction of the expression or activity of N members of the miR156 gene family in plants is achieved by a method selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, gene editing techniques, or a combination thereof.

In another preferred example, the gene editing technology is selected from CRISPR technology, TALEN technology and ZFN technology.

In another preferred embodiment, said plant having the desired trait is selected from the group consisting of: mir156defghi, mir156a, mir156c, mir156b, mir156ac, mir156ab, mir156bc, mir156abc, mir156kl, mir156abcl, mir156abck, mir156abckl, mir156abcdfghikl, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of: a monocot dicot, a gymnosperm, or a combination thereof.

In another preferred embodiment, the plant is selected from the group consisting of: a graminaceous plant, a leguminous plant, a cruciferous plant, a solanaceae, an Umbelliferae, a Chenopodiaceae, or a combination thereof.

In another preferred embodiment, the plant comprises: arabidopsis, wheat, barley, oats, maize, rice, sorghum, quinoa, millet, soybean, peanut, tobacco, tomato, cabbage, canola, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, lettuce, or combinations thereof.

In another preferred example, the genetic engineering comprises gene editing of a member of the miR156 gene family with one or more sgRNA-mediated Cas9 nuclease.

In another preferred example, the gene editing comprises gene editing of a gene of the miR156 gene family selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.

In another preferred example, the gene editing further comprises gene editing of a gene of miR156 gene family selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.

In another preferred example, the gene editing comprises gene editing of a gene of the miR156 gene family selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.

In a second aspect, the invention provides a genetically edited plant tissue or plant cell in which N members of the miR156 gene family are mutated, wherein N is 1 or more (preferably 2 or more, more preferably 2 to 11, more preferably 2 to 10, more preferably 3 to 10, more preferably 5 to 8).

In another preferred embodiment, the mutation is capable of enhancing dormancy of a plant tissue or plant cell.

In another preferred embodiment, the mutation increases the resistance of the seed to pre-heading.

In a third aspect, the present invention provides a method of preparing a gene-edited plant tissue or plant cell, comprising the steps of:

mutating N members of miR156 gene family in the plant tissue or plant cell to obtain the plant tissue or plant cell, wherein N is more than or equal to 1 (preferably, more than or equal to 2, more preferably, 2-11, more preferably, 2-10, more preferably, 3-10, more preferably, 5-8).

The fourth aspect of the present invention provides a method for preparing a gene-edited plant, comprising the steps of:

regenerating the gene-edited plant tissue or plant cell prepared by the method of the third aspect of the present invention into a plant body, thereby obtaining a gene-edited plant.

In a fifth aspect, the present invention provides a gene-edited plant produced by the method of the fourth aspect.

A sixth aspect of the present invention provides a method of producing grain, comprising the steps of:

(i) planting a crop in which N members of the miR156 gene family are mutated, wherein N is more than or equal to 1 (preferably, more than or equal to 2, more preferably, 2-11, more preferably, 2-10, more preferably, 3-10, more preferably, 5-8);

(ii) the grain (grain) of the crop is harvested.

In another preferred example, the crop plants include plants of the Gramineae, Leguminosae, Chenopodiaceae and Brassicaceae, and more preferably rice, corn, sorghum, wheat, quinoa, soybean.

The seventh aspect of the invention provides a use of a miR156 gene family inhibitor for (a) promoting seed dormancy; and/or (b) improving the seed anti-pre-germination trait; or for the preparation of a composition or formulation for (a) promoting dormancy of seeds; and/or (b) improving the pre-emergence trait of the seed.

In another preferred embodiment, said improving the anti-pre-germination properties of the seeds comprises increasing the anti-pre-germination properties of the seeds.

In another preferred embodiment, the inhibitor comprises a substance that decreases the expression or activity of N members of the miR156 gene family, wherein N.gtoreq.1, preferably, > 2, more preferably, 2-11, more preferably, 2-10, more preferably, 3-10, more preferably, 5-8.

In another preferred embodiment, the inhibitor is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a Crispr agent, or a combination thereof.

In another preferred example, the I I-like miR156s is selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof. In another preferred example, the regulatory effect of the miR156 gene on seed dormancy and ear germination is mediated through the GA pathway.

In another preferred example, the regulation of seed dormancy and ear germination by the miR156 gene is mediated by IPA 1-mediated regulation of expression of GA synthesis genes and degradation genes.

In another preferred example, the miR156 gene promotes seed dormancy and increases pre-harvest sprouting resistance is mediated by IPA 1-mediated down-regulation of GA synthesis genes and/or up-regulation of GA degradation genes.

The eighth aspect of the invention provides an application of miR156 gene in regulation and control of seed dormancy and/or seed pre-emergence traits, wherein the regulation and control can be realized by reducing the expression or activity of N members in the miR156 gene family, wherein N is more than or equal to 1, so as to enhance the seed dormancy or enhance the anti-pre-emergence capability of seeds, and the miR156 gene family members are as described above.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the MIR156 gene editing strategy. (A) Editing a single sgRNA expression vector of MIR156 gene. Arrows indicate promoters; the rectangular box after the arrow represents the sgRNA expression sequence; #, the PAM sequence used by the target site is CAG; base 20 of the target site did not match the sgRNA. (B) Multiple gene editing strategy of I I type MIR156 s. Arrows indicate promoters; the rectangular box after the arrow represents the sgRNA expression sequence; base 20 of the target site did not match the sgRNA. (C) MIR156 edited target site selection. The target site of Cas9 is underlined and the in-frame sequence corresponds to the mature miR156 sequence.

FIG. 2 shows the plant type analysis of mir156 mutant. (A) Maturity, phenotype comparison of wild type, mir156fg, mir156deghi and mir156 defghi. Scale, 10 cm. (B) Tiller numbers of wild type, mir156fg, mir156dehi, mir156deghi and mir156defghi were compared. (C) In the mature period, the plant heights of the wild type, mir156fg, mir156dehi, mir156deghi and mir156defghi are compared. (D) In the mature period, the diameters of second-node stems of wild type, mir156fg, mir156dehi, mir156deghi and mir156defghi are compared. (E) Comparison of the phenotypes of wild-type and mir156 abcdfghikl. Scale, 10 cm. (F) Comparison of tiller numbers for wild type, mir156dehi, mir156defghi and mir156 abcdefghikl. (G) At the seedling stage, the phenotypes of wild type and mir156abckl were compared. Scale, 3 cm. (H) Maturity, phenotype comparison of wild type and mir156 abckl. Scale, 10 cm. (I) Tillering numbers of wild type and mir156abckl were compared. P value different from wild type less than 0.001. NIP, wild type nipponbare.

FIG. 3 shows the mir156 mutant seed grain type analysis. (A) Grain type comparisons of wild type, mir156fg and mir156 defghi. Scale, 2 cm. (B) Seed length comparison of wild type and mir156 mutant. (C) Seed width comparisons between wild type and mir156 mutant. (D) The wild type and mir156 mutant seed grain thickness were compared. (E) Thousand kernel weight of wild type and mir156 mutant seeds were compared. P value different from wild type less than 0.001. NIP, wild type nipponbare.

FIG. 4 shows that mir156abckl mutation does not affect rice yield. (A and B) comparison of seed set rates of wild type and mir156 abckl. (C and D) comparison of seed number of main panicles between wild type and mir156 abckl. (E and F) cell yields of wild type and mir156abckl (90cm x 60cm) were compared. NIP, wild-type nipponbare; XS134, wild type xishui 134.

Fig. 5 shows that mir156 mutation enhances seed dormancy. (A) Germination of wild type and mir156abcdfghikl seeds (harvested, fully air-cured and dry stored) were compared. Scale, 2 cm. (B) Germination rates of wild type, mir156abckl and mir156abcdfghikl seeds (harvested, well air-cured and dry stored) were compared. The experiment was performed in three replicates simultaneously, with 199 seeds per material in each replicate. (C) Comparison of germination of fresh seeds (freshly matured, non-sunned or dried seeds) of wild type, mir156abcl, mir156abckl and mir156 defghi. (D) Comparison of germination rates of fresh seeds of wild type, mir156abc, mir156abcl, mir156defghi and mir156 abcdefghi. The experiment was performed in triplicate, with each material in each replicate having a seed number of no less than 199. (E) Pre-harvest pre-spike germination of wild type, mir156defghi and mir156abckl seeds. (F) Germination rates (germination rates) of wild type and mir156abckl seeds were compared after different periods of indoor storage of the seeds. P value different from wild type less than 0.001. NIP, wild-type nipponbare; XS134, wild type xishui 134.

Figure 6 shows that mir156 mutation reduces fresh seed and embryo ABA content. (A) ABA content of wild type and mir156abcdfghikl fresh seed embryos. (B) ABA content of wild type and mir156abckl fresh seeds. The assay was performed in triplicate. P value different from wild type less than 0.001. NIP, wild-type nipponbare; XS134, wild type xishui 134.

FIG. 7 shows the defect of mir156abckl in inhibiting seed dormancy of pyl mutants. (A) Comparison of germination of fresh seeds of wild type, pyl1/4/6 and pyl1/4/6-mir156 abckl. The figure is a picture of a seed soaked at 30 ℃ for two days and then germinated at 30 ℃ for 3 days. Scale, 2 cm. (B) Comparison of germination rates of fresh seeds of wild type, pyl1/4/6 and pyl1/4/6-mir156 abckl. (C) Comparison of germination of fresh seeds of wild type, pyl1/2/4/6 and pyl1/2/4/6-mir156 abckl. The figure is a picture of a seed soaked at 30 ℃ for two days and then germinated at 30 ℃ for 3 days. Scale, 2 cm. (D) Comparison of germination rates of fresh seeds of wild type, pyl1/2/4/6 and pyl1/2/4/6-mir156 abckl. NIP, wild type nipponbare. Fresh seeds are seeds which have just matured and have not been subjected to sun-curing or drying treatment. The experiment was performed in triplicate, with each material in each replicate having a seed number of no less than 199.

FIG. 8 shows mir156abcdfghikl seedling stage phenotype analysis and GA content determination. (A) Comparison of seedling stage, wild type and mir156 abcdfghikl. Scale, 3 cm. (B) GA content in wild type and in the aerial parts of mir156abcdfghikl seedlings. The assay was performed in triplicate. The material used was 2 weeks seedlings. # undetectable GA. NIP, wild type nipponbare.

FIG. 9 shows the relative expression levels of GA-related differentially expressed genes in wild type and in the aerial parts of mir156abcdfghikl seedlings.

FIG. 10 shows the relative expression levels of GA-associated differentially expressed genes in wild-type and mir156abcdfghikl fresh seed embryos. Fresh seeds are seeds which have just matured and have not been subjected to sun-curing or drying treatment.

FIG. 11 shows that mutation of MIR156 gene reduced GA content in seed embryos and sensitivity of seed germination to GA. (A) GA content of wild type and mir156abcdfghikl fresh seed embryos. Fresh seeds are seeds which have just matured and have not been subjected to sun-curing or drying treatment. # undetectable GA. NIP, wild type nipponbare. (B) Fresh seed germination pair GA₃The sensitivity of (2). Three biological replicates of the experiment were performed. XS134, wild type xishui 134.

Figure 12 shows that miR156 regulates seed dormancy and seedling development via IPA 1. (A) Wild type and P_35SIPA1 fresh seed germination comparison. The figure is a picture of a seed soaked at 30 ℃ for two days and then germinated at 30 ℃ for 3 days. Scale, 2 cm. (B) Wild type and P_35SI.e. germination rate of fresh IPA1 seeds. The experiment was performed in three biological replicates, each replicate having a seed number of no less than 209 per material. (C) Wild type andP_35Sphenotype comparison of IPA1 seedlings. Scale, 3 cm. (D) IPA1 wild type and P_35SRelative expression of aerial parts of IPA1 seedlings. (E) Wild type and P_35SIPA1 seedling aerial length comparison. (F) Wild type and P_35SFresh weight comparison of IPA1 seedlings. P_35SIPA1-1 and P_35SIPA1-2 was two IPA1 overexpression lines. NIP, wild type nipponbare.

FIG. 13 shows a ChIP-QPCR experiment of the interaction of IPA1 with the GA pathway gene promoter. 35S, 3x FLAG, and empty vector transgenic seedlings; 35S, SPL14m:3 XFLAG, SPL14 overexpression seedlings; NoAb, no FLAG antibody; α FLAG, FLAG antibody.

Detailed Description

The present inventors have extensively and intensively studied and found for the first time that, surprisingly, knocking out N members (N.gtoreq.1, preferably, N.gtoreq.2, more preferably, N is 2 to 11, more preferably, N is 2 to 10, more preferably, 3 to 10, more preferably, 5 to 8) of miR156 gene family can significantly improve some traits of plants, such as seed dormancy and spike germination resistance of plants. Specifically, the invention firstly utilizes gene knockout technology (such as CRISPR/Cas9 multi-gene editing technology) to knock out rice miR156 gene, and discovers that certain required agronomic traits can be improved by knocking out different members of rice miR156 gene family, wherein mutants such as miR156defghi, miR156ac, miR156ab, miR156bc, miR156abc, miR156kl, miR156abcl miR156abckl, miR156abcdfghikl and the like can enhance seed dormancy so as to enhance the spike germination resistance of the seed, and have no influence on other traits (such as seed setting rate, number per spike, yield, plant type, grain type, seed germination rate and the like), and the invention also unexpectedly discovers that miR, 156 gene mutation can inhibit GA pathways by influencing GA synthesis, degradation and signal transduction so as to enhance seed dormancy, and further discovers that miR156 can regulate seed dormancy and seedling germination and growth through IPA 1. On this basis, the present inventors have completed the present invention.

As used herein, "MIR 156s class II" and "MIR 156s class I" refer to the subtype of the MIR156 gene family.

miR156 gene

miRNA is a non-coding RNA molecule consisting of 21-24 nucleotides. They originate from a single-stranded RNA precursor gene, are transcribed by RNA polymerase II to form a stem-loop structure, and are finally cleaved and processed to form a mature miRNA [1,2 ]. The correct spatiotemporal accumulation of some highly conserved mirnas is essential to maintain normal plant development, such as miR 156. The target gene of miR156 is a family called SPL (SQUAMOSA promoter binding protein-like). The SPL protein family is a plant-specific family of transcription factors with highly conserved DNA binding domains, the SBP domains.

Existing studies have shown that genes of the miR156 gene family are highly conserved and are critical for plant growth (especially seed dormancy).

In the invention, the miR156 gene family in rice comprises class II MIR156s (comprising MIR156a, MIR156b, MIR156c, MIR156k and MIR156l), and the seed dormancy is controlled more specifically. Mutation of class II MIR156 gene enhances seed dormancy, inhibits PHS, and has no obvious influence on plant type, rice grain type and yield potential in adult period.

Other MIR156 gene family members, such as class I MIR156s (including MIR156d, MIR156e, MIR156h, MIR156I, MIR156f, and MIR156g), can regulate plant type and grain type, and also have an effect on seed dormancy, but with a slightly lower effect than class II MIR156 genes.

In the present invention, the classification and naming of the mi156s family members in different species is not limited by the classification and naming method of the present invention, and the classification can be classified according to the functions of different subtype genes in different species. The relative classifications and nomenclature used should not be construed as limiting the invention.

Through intensive studies, the present inventors found that mir156 mutation enhances seed dormancy by inhibiting the Gibberellin (GA) pathway. The miR156 target gene Ideal Plant Architecture 1(IPA1), the expression of which is strongly induced by miR156 gene mutations, mediates the effect of miR156 mutations on seed dormancy. Biochemical studies have shown that in rice, the promoters of many genes in the GA pathway (genes that mediate GA synthesis, degradation, signaling) interact with IPA 1. These results indicate that IPA1 regulates seed dormancy by directly regulating expression of GA pathway-related genes. The research provides important gene resources and theoretical support for cultivating good varieties with PHS resistance.

In the present invention, N genes (N.gtoreq.1, preferably N.gtoreq.2, more preferably N is 2 to 10, more preferably 2 to 11, more preferably 3 to 10, more preferably 5 to 8) in the MIR156 gene family of any plant species may be knocked out, and representative plants include, but are not limited to: forestry plants, agricultural plants, such as gramineae, cruciferae, leguminosae, and the like, such as rice, millet, corn, sorghum, wheat, soybean, quinoa, or combinations thereof. It is understood that different plants may contain multiple MIR156 genes (i.e., multiple genes from the MIR156 gene). In the present invention, the MIR156 gene includes all known MIR156 genes from the plant (or species), and MIR156 genes that may be found in the future, as well as homologous genes having homology to these MIR156 genes. Wherein, the expression "having homology" means that two sequences have a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more.

In other plants, generally, the common name of the homologous gene of the MIR156 gene is the MIR156 gene, and the abbreviation of species Latin name can also be added before the name of the MIR156 gene. For example, the wheat MIR156 gene is also known as Tae-MIR 156; the maize MIR156 gene is also known as Zea-MIR 156; the soybean MIR156 gene is also called Soy-miR 156.

For example, rice is known to have at least 11 MIR156 genes, namely MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, MIR156a, MIR156b, MIR156c, MIR156k and MIR156 l. In the present invention, two or more MIR156 genes may be combined, for example, "MIR 156 ab" means MIR156a and MIR156 b.

In a preferred embodiment, the rice mir156 gene knockout pattern is as follows: mir156a, mir156c, mir156b, mir156defghi, mir156ac, mir156ab, mir156bc, mir156abc, mir156kl, mir156abcl mir156abckl, mir156abck, mir156 abcdfghikl.

Inhibitors of miR156

The invention also provides an inhibitor for miR156, and the inhibitor for miR156 can inhibit the expression or activity of miR 156. In the present invention, the inhibitor of miR156 is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a Crispr agent, or a combination thereof.

Seed dormancy

Seed dormancy refers to the phenomenon that viable seeds cannot germinate under appropriate conditions due to the embryo or seed shell, and is a complex adaptive trait influenced by both genetic and environmental factors. The seed dormancy prevents plant seeds from germinating at wrong time, enhances the ability of the seeds to resist ear sprouting, thereby avoiding severe growth environment, and is an adaptive mechanism formed in the evolution process. Dormancy does not mean that the seed is incapable of germination, and when appropriate conditions are applied, the respiration of the seed is enhanced, the glycolysis/tricarboxylic acid cycle (EMP/TCA) is shifted to the pentose phosphate cycle (PPP) to provide energy for the germination of the seed, the protein begins to degrade, the nitrogen source is provided for the seed, and the seed can begin to germinate.

For the mature seeds, the dormancy of the seeds is beneficial to the propagation and the uniform distribution of space and time of the seeds, particularly avoids adverse conditions, prolongs the storage time of the seeds and improves the utilization value of the seeds;

sprouting (sprouting before harvest)

In the grain harvest season, when it is exposed to rainy weather or humid environmental conditions at high temperature for many days, the phenomenon of germination on the parent plant, called pre-harvest germination, is the ear of grain, and often causes severe loss of grain yield and quality. The main crops with prominent ear sprouting phenomena are: rice, wheat, millet, quinoa, sorghum and the like. Research shows that the germination rate of the ears is increased by 10 percent, and the yield loss is increased by 2.0 to 2.6 percent. The phenomenon of ear sprouting is related to various aspects of the grain characteristics, ear structure, seed water content, alpha-amylase activity, soluble sugar, endogenous hormone, environment and the like. The current method for controlling the sprouting of the ear mainly comprises the following steps: 1. regulating the sprouting of the spikes in a cultivation way, for example, selecting a field block with convenient irrigation and drainage, sufficient sunshine and moderate N content for seed production, reasonably and densely planting plant groups and the like; 2. regulating and controlling the sprouting of the panicle by a chemical way, such as spraying the nona-diox, the paclobutrazol, the exogenous ABA, the panicle sprouting inhibitor and the like; 3. the genetic approach regulates and controls the ear germination, for example, the sterile line with lighter ear germination is selected and used as the female parent for breeding to reduce the ear germination.

Genetic engineering modification

In the present invention, the genetic engineering method includes, but is not limited to, methods using gene mutagenesis or gene editing, etc. Methods for gene mutagenesis include, but are not limited to, physical mutagenesis (e.g., ultraviolet mutagenesis), chemical mutagenesis (e.g., acridine dyes), biological mutagenesis (e.g., viral, phage mutagenesis), and the like. In a preferred embodiment, the genetic engineering of the invention comprises gene editing of a member of the miR156 gene family with one or more sgRNA-mediated Cas9 nuclease.

Method for improving plants

In the present invention, there is also provided a method of improving a plant, comprising the steps of:

(i) genetically engineering plant cells or plant tissues to mutate N members of the miR156 gene family, wherein N is greater than or equal to 1 (preferably, N is greater than or equal to 2, more preferably, N is from 2 to 11, more preferably, N is from 2 to 10, more preferably, N is from 3 to 10, more preferably, N is from 5 to 8);

(ii) regenerating the genetically engineered plant cells or plant tissues into plants, and testing the regenerated plants for traits, wherein the traits comprise seed dormancy;

In the present invention, the mutation comprises a reduction in the expression level or activity of N members of the miR156 gene family.

In a preferred embodiment, the "decreasing" refers to decreasing the expression level or activity of N members of the miR156 gene family satisfies the following condition:

wherein, A1 is the expression or activity of N members in miR156 gene family; a0 is the expression or activity of N members in the same miR156 gene family in wild-type plants of the same type.

In a preferred embodiment, the reduction refers to an expression level E1 of the member of the miR156 gene family in the plant that is 0-80%, preferably 0-60%, more preferably 0-40% of wild type compared to the expression level E0 of the member of the wild type miR156 gene family.

In a preferred embodiment, said reducing the expression or activity of N members of the miR156 gene family in a plant is effected by a means selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, criprpr techniques, or a combination thereof.

In the present invention, plants with poor traits are excluded according to the results of the trait test.

In the present invention, the method further comprises a step (iv) of further screening the plants with the desired shape characteristics selected in the step (iii), thereby screening out plants capable of balancing the characteristics of seed dormancy, seed setting rate, seed number per ear, yield, plant type, grain type and the like, wherein the comprehensive character performance of the plants is optimal.

Method for producing grain

The invention also provides a method for producing grains, which comprises the following steps:

(i) planting crops in which N members of the miR156 gene family are mutated, wherein N is more than or equal to 1 (preferably, N is more than or equal to 2, more preferably, N is 2-11, more preferably, N is 2-10, more preferably, N is 3-10, more preferably, N is 5-8);

(ii) the grain (grain) of the crop is harvested.

The main advantages of the invention include:

(a) according to the invention, the knockout of miR156 gene family members of different plants (such as rice) is firstly found, under the condition of not influencing other properties, the dormancy of seeds can be obviously enhanced, the germination rate of the ears of the seeds in the harvest season is reduced (by about 7-10 times compared with that of the wild type), the harvest time of the seeds is widened, the service life and the storage time of the seeds are prolonged, and the germination rate of the seeds is not influenced.

(b) The invention discovers that GA pathway inhibition is realized by knocking out miR156 gene family members of different plants (such as rice) to influence GA synthesis (to reduce expression of synthetase genes), degradation (to up-regulate expression of degradation gene enzymes) and signal conduction, so that seed dormancy is enhanced.

(c) The method tests the knockout characters of miR156 gene family members of different plants (such as rice) for the first time, and picks out plants with required character characteristics (such as enhanced seed dormancy).

(d) The mutant mir156a, mir156c, mir156b, mir156defghi, mir156ac, mir156ab, mir156bc, mir156abc, mir156kl, mir156abcl, mir156abck, mir156abckl and mir156abcdfghikl can enhance seed dormancy for the first time.

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. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: a laboratory manual (New York: Cold Spring Harbor laboratory Press,1989) or a Plant Molecular Biology-laboratory manual (Plant Molecular Biology-A laboratory Manual, catalog S.Clark, Springer-verlag Berlin Heidelberg,1997), or according to the conditions suggested by the manufacturer.

Unless otherwise indicated, percentages and parts are by weight.

Example 1 mutation of class II MIR156 Gene does not significantly affect the adult plant type and grain type

There are 11 MIR156 genes (MIR156 a-MIR156 i, MIR156k and MIR156l) in the rice genome, and 12 pri-miR156 genes are produced through transcription, wherein the pri-miR156h and pri-miR156j are from the same gene, and the gene is called MIR156h in the research. To knock out MIR156 gene, we first constructed 6 single sgRNA expression vectors specifically targeting MIR156 (fig. 1A, 1B, 1C), in which vector I (expressing sgRNA1) targets 6 genes (MIR156 d-MIR 156I); vector II (expressing sgRNA2) targets MIR156f and MIR156 g; sgRNA expressed by vectors II I and IV (sgRNA3 and sgRNA4) can target three genes of MIR156 a-MIR156 c; vectors vector V (expressing sgRNA5) and vector VI (expressing sgRNA6) target MIR156k and MIR156l, respectively. The transgenic receptor of the vectors is japonica rice variety Nipponbare and Xiushui 134. All the materials were analyzed in the field survey in Shanghai and Hangzhou.

Using Agrobacterium-mediated transgenic approach, we obtained four combinations of mir156dehi, mir156deghi, mir156defghi and mir156fg through vectors vector1 and vector 2. In the context of Japanese sunny and elegant water 134, there are at least 18 independent lines per combination of mutations. The tiller number of these mutants showed a different reduction compared to the wild type nipponlily, with mir156dehi, mir156deghi and mir156defghi also showing phenotypes of tall plants, stout stalks, etc. (fig. 2A-2D). The seed length and thousand kernel weight of mir156dehi, mir156deghi, mir156defghi and mir156fg were significantly increased compared to the wild type, while the grain width and grain thickness were not significantly changed (FIGS. 3A-3E). In the context of xishui 134, these mutants exhibited similar phenotypes.

By co-transforming the above 6 vectors with the gene gun, we obtained two lines of MIR156abcdfghikl mutant (10 MIR156 genes were simultaneously mutated except MIR396 e). In addition, by crossing mir156abckl with mir156defghi, we also obtained another mir156abcdfghikl line. By careful phenotypic observation we found that mir156abcdfghikl had fewer tillers than mir156dehi, but slightly more mir156defghi (fig. 2F). The mir156abcdfghikl strain was otherwise similar to mir156defghi (FIG. 2E). This indicates that five genes such as MIR156a, MIR156b, MIR156c, MIR156k and MIR156l have little effect on plant type regulation.

Through other four vectors (expressing sgRNA 3-sgRNA 6 respectively), mutants such as mir156a, mir156b, mir156c, mir156ac, mir156bc, mir156abc, mir156k and mir156l are obtained. These mutants had no significant difference from the wild type in the plant type and seed grain type at adulthood. To further study the functions of these five genes, we constructed a multigene editing vector VII that simultaneously expresses sgRNA 3-sgRNA 6 (fig. 1B). From this vector we obtained a large number of mir156abcl and mir156abckl mutants. In the context of Japanese Qinghai and Xiuhui 134, mir156abcl and mir156abckl exhibited similar traits. Approximately before the trefoil stage, mir156abcl and mir156abckl were slightly shorter than the wild type, but then the plant type and seed grain type of mir156abcl and mir156abckl were not significantly different from the wild type (FIGS. 2G-2I and 3B-3E). mir156abckl had no significant effect on seed set rate, number of seeds per ear and cell yield (fig. 4A-4F).

The above analysis results show that MIR156 d-MIR 156i regulates plant type and rice grain type. For convenience we named these 6 genes as class I MIR156 genes. The other 5 genes were named class II MIR156 genes, and these 5 genes had no significant effect on rice plant type and rice grain type (FIGS. 2G-2I and 3B-3E).

Example 2 mutation of class II MIR156 Gene specifically enhances seed dormancy

During the study of the mir156 mutants, we found that the seeds of the mir156 type II mutants (seeds that were well dehydrated and dry stored after harvest) germinated significantly slower than the wild type (fig. 5B), while the seeds of the mir156 type I mutants (seeds that were well dehydrated and dry stored after harvest) did not have a significant difference in germination rate from the wild type. mir156abcdfghikl seeds (seeds that had been extensively dehydrated and dryly stored after harvest) had slower germination rates than the class II mutants (fig. 5A and 5B). However, these mutants did not differ significantly from the wild type in seed germination rate (germination rate). These phenomena suggest that our miR156 might be involved in the regulation of seed dormancy.

To explore the role of miR156 on seed dormancy, we performed germination experiments using fresh seeds (seeds that have just matured and have not been sun-cured or dried). Mutants (including mir156abcdfghikl, mir156defghi, mir156abckl, mir156abcl and mir156abc) were found to germinate significantly slower than the wild type (fig. 5C), in particular, the mir156abcdfghikl, mir156abckl mutants had little or very low germination at the same time. Under the same germination rate, the dormancy time of mir156abckl is prolonged by about 8-10 days compared with that of a wild type. The germination rate of each plant is in the following order: wild type > mir156defghi > mir156abc > mir156abcl > mir156abckl > mir156 abcdefghikl (fig. 5D). Mir156kl, mir156bc, mir156ac and mir156 ab.

The results show that miR156 negatively regulates seed dormancy, and I I genes have more remarkable effect on seed dormancy than I genes. The reduction or inhibition of the expression of the miR156 gene can obviously increase the dormancy time of the seeds, broaden the harvest period of the seeds and has no influence on the germination rate of the seeds after drying treatment.

Example 3 mutation of class II MIR156 Gene inhibits Pre-harvest Germination (seed ear Germination) and prolongs seed longevity

To explore the potential of mir156 mutants in production, we analyzed pre-harvest germination (PHS) of wild type and mutant seeds in 2018 in a hangzhou field survey. The wild type variety Nipponbare is sown at the beginning of 6 months and matured at the end of 9 months. The Hangzhou at the bottom of 9 months is in a high-temperature rainy season, and the climate is very easy to cause the germination of rice seeds before harvesting. At the end of 9 months, our survey showed that wild type seeds had approximately 14% PHS probability, whereas mir156abckl seeds had only approximately 1.5% germination (fig. 5E). mir156defghi has a PHS probability significantly higher than mir156abckl, but slightly lower than wild-type (fig. 5E). These results show that MIR156 gene mutation can inhibit PHS of seed and reduce seed ear germination rate, and compared with I gene mutation, I I gene mutation has better effect and can reduce ear germination rate by about 7-10 times.

Seed dormancy often extends seed longevity. To examine the effect of mir156abckl mutation on seed longevity, germination experiments were performed with seeds stored for different periods of time. The results showed that after 14 months of storage (Hangzhou room temperature conditions) the germination rate (germination rate) was only about 40% for wild type, whereas the germination rate for mir156abckl was about 80% -85% (FIG. 5F). Indicating that the mutation of the MIR156 gene in class II is beneficial to seed storage.

Example 4 mir156abckl and mir156abcdfghikl mutations reduce ABA content in seeds and embryos

ABA inhibits seed germination and induces seed dormancy. To explore the relationship between ABA and miR156 in seed dormancy, the ABA content of wild-type and miR156abcdfghikl fresh seed (seed which is just mature and is not aired or dried) embryos is detected. The results show that mir156abcdfghikl fresh seed embryos had a severe decrease in ABA content compared to wild type (fig. 6A). We also determined ABA content of wild type and mir156abckl fresh seeds (containing embryo and endosperm). The ABA content of mir156abckl fresh seeds was also significantly reduced compared to wild type (fig. 6B). These results suggest that the regulation of seed dormancy by miR156 is not achieved by the accumulation of ABA.

Example 5 mutation of MIR156 Gene class II inhibits the defect of pyl mutant seed dormancy (higher germination)

PYLs is ABA receptor coding gene. In previous studies, we obtained a number of PYL mutants, of which mutants of a PYL subfamily (comprising PYL 1-PYL 6 and PYL12) showed severe seed dormancy defects. To explore the relationship between miR156 and ABA pathway in controlling seed dormancy, combined mutants of MIR156s and PYLs are constructed, and homozygous pyl1/4/6-miR156abckl (8 mutations) and pyl1/2/4/6-miR156abckl (9 mutations) are obtained at present. Seed dormancy experiments showed that mir156abckl significantly inhibited the defect in seed dormancy of pyl1/4/6 and pyl1/2/4/6 (FIGS. 7A-7D). Experimental results of ABA content determination and mir156-pyl genetic interaction suggest that the promoting effect of mir156 mutation on seed dormancy is not achieved through ABA synthesis or signaling pathway.

Example 6 miR156 regulates seedling growth through GA pathway

Although mir156abcdfghikl showed a similar plant type to mir156defghi at maturity, mir156abcdfghikl showed severe growth retardation at seedling stage: seedlings were extremely short (FIG. 8A) and leaves were dark green. This phenotype is similar to the GA-deficient mutant, so we examined GA content in wild-type and mir156abcdfghikl seedling aerial parts (seedlingshot). The results showed that the active GA includes GA as compared with the wild type₃、GA₄And GA₇The content of (a) was significantly reduced in mir156abcdfghikl (fig. 8B). These results suggest that miR156 can control seedling development by regulating GA accumulation.

To further elucidate the regulation of GA content by miR156, we analyzed the transcriptome data comparing wild-type and the aerial parts of miR156abcdfghikl seedlings and found that the expression of most of the genes mediating GA synthesis and signaling was down-regulated by the miR156abcdfghikl mutation. The transcriptome analysis detects 10955 differentially expressed genes (Ratio is more than or equal to 2 or less than or equal to 0.5, P is less than 0.05) in total, wherein 5921 and 5034 genes with respectively up-regulated (Ratio is more than or equal to 2) and down-regulated (Ratio is less than or equal to 0.5) expression in mir156abcdfghikl are included. Such many gene expression changes indicate that the miR156 and target genes SPLs thereof play a wide and important role in rice gene expression regulation. 5034 expression downregulation genes contained five key genes for GA synthesis (CPS1, KAO, KO2, GNP1/GA20ox1 and GA20ox4) and 14 putative GA receptor genes (highly homologous to known GA receptor genes) (Table 1). From this transcriptome analysis, we also found that the expression of one GA-degrading enzyme gene, GA2ox10, and two negative regulatory genes for GA signaling, SLR1 and SPY, were up-regulated by mir156abcdfghikl mutation. The results of QPCR validation were consistent with the transcriptome (figure 9). These results demonstrate that the GA pathway mediates the regulation of miR156 on seedling growth.

TABLE 1 expression profiles of GA-related differentially expressed genes in aerial parts of wild type and mir156abcdfghikl seedlings

Remarking: data were extracted from transcriptome analysis results of 2-week seedlings; NIP, wild type nipponbare.

Example 7 miR156 regulates seed dormancy through GA pathway

To explore the reason why miR156 regulates seed dormancy, we analyzed transcriptome data comparing wild type and miR156abcdfghikl fresh seed embryos, and identified 1132 differentially expressed genes (Ratio is more than or equal to 2 or less than or equal to 0.5, and P is less than 0.05) which comprise 729 genes with up-regulated expression in miR156abcdfghikl and 403 genes with down-regulated expression in miR156 abcdfghikl. Among these differentially expressed genes, we found that the expression of 2 putative GA receptor genes (LOC _ Os06g20200 and LOC _ Os09g28630), three key GA synthetase genes (GNP1/GA20ox1, SD1/GA20ox2 and KAO) and one putative GA oxidase gene (LOC _ Os03g42130) was significantly down-regulated by mir156abcdfghikl mutation; whereas the expression of the GA-degrading enzyme gene GA2ox6 was significantly up-regulated by mir156abcdfghikl mutation. When the criteria for differentially expressed genes were reduced to Ratio ≥ 1.5 or ≤ 0.75(P <0.05), we also found that expression of three additional putative GA receptor genes (LOC _ Os08g37040, LOC _ Os02g35940 and LOC _ Os09g28730) was significantly down-regulated by mir156abcdfghikl mutation; GA-degrading enzyme genes GA2ox8 and EUI1 were significantly up-regulated by mir156abcdfghikl mutation; the expression of the GA signaling negative regulator gene SLR1 was also significantly upregulated by mir156abcdfghikl mutation (table 2). The results of QPCR validation were consistent with the transcriptome (figure 10). These results indicate that negative regulation of seed dormancy by miR156 is also achieved through the GA pathway.

TABLE 2 expression of GA-related genes in wild type and mir156abcdfghikl fresh seed embryos

Remarking: data are extracted from transcriptome analysis results; NIP, wild type nipponbare.

GA content in wild type and mir156abcdfghikl fresh seed embryos was also examined and it was found that the content of bioactive GA including GA3 and GA7 was significantly reduced in mir156abcdfghikl (fig. 11A). And mir156abckl fresh seed germination was less sensitive to GA3 than wild type (fig. 11B).

The above results demonstrate that MIR156 gene mutation inhibits GA pathway by affecting GA synthesis, degradation and signaling, thereby enhancing seed dormancy.

Example 8 miR156 regulates seed dormancy and seedling growth by IPA1

There are 19 SPL genes in the rice genome, and 11 of them (SPL 2-SPL 4, SPL7, SPL 11-SPL 14 and SPL 16-SPL 18) are target genes of miR 156. From the 11 target genes, we found that IPA1 (i.e., SPL14) was expressed in the highest amount in wild type, mir156abckl and mir156abcdfghikl fresh seed embryos by transcriptome analysis (tables 3 and 4); in particular, in mir156abckl and mir156abcdfghikl fresh seed embryos, the expression level of IPA1 was far superior to that of the other 10 SPL genes (tables 3 and 4). This suggests that miR156 might regulate seed dormancy primarily through IPA 1. In the aerial part of seedlings, IPA1 was expressed in miR156abcdfghikl with the greatest up-regulation amplitude (table 5), suggesting that IPA1 may also play an important role in the regulation of miR156 on seedlings.

To verify the effect of IPA1 on seed dormancy and seedling development, IPA1 overexpressing plants (P) were observed_35SIPA1) seed dormancy and seedling development. The results show P_35SSeed dormancy of IPA1 is significantly enhanced (fig. 12A and 12B); p in comparison with the wild type_35SSPL14 also phenotypically produces growth retardation at seedling stage (FIGS. 12C-12F). These results demonstrate that IPA1 mediates the effect of mir156 mutations on seed dormancy and seedling development.

TABLE 3 expression profiles of miR156 target genes in wild-type and miR156abcdfghikl fresh seed embryos

TABLE 4 expression profiles of miR156 target genes in wild-type and miR156abckl fresh seed embryos

TABLE 5 expression profiles of miR156 target genes in wild-type and miR156abcdfghikl two-week aerial seedlings

Example 9 in vivo interaction of IPA protein with promoter of GA pathway-associated Gene

The IPA1 transcription factor broadly regulates gene expression through GTAC and TGGGCC/T elements. By analyzing the promoter sequence of GA pathway genes (1.5 kb upstream of the start codon), we found that GTAC and TGGGCC/T elements are widely present in the promoter of GA pathway genes. The results of the CHIP-seq study of IPA1 published by li-seyi academy in 2013, which showed that IPA1 protein interacts with the promoters of several GA-related genes (including GSR1, EUI1, SLR1, GA2ox6 and two putative GA receptor genes LOC _ Os02g35940 and LOC _ Os06g20200), but the biological significance was unknown. To further reveal the interaction of IPA1 with the GA pathway gene promoter, we performed the ChIP-QPCR experiment. In transgenic seedlings overexpressing IPA1-Flag, ChIP-QPCR showed that IPA1 interacted with the promoters of the 14 GA-related differentially expressed genes identified previously. These 14 genes included 5 key enzyme genes mediating GA synthesis (CPS1, KO2, KAO, GNP1/GA20ox1 and SD1/GA20ox2), 5 putative GA receptor genes (LOC _ Os09g28630, LOC _ Os06g20200, LOC _ Os02g35940, LOC _ Os08g37040 and LOC _ Os09g28730), 3 GA degradase genes (GA2ox6, GA2ox8 and EUI1) and SLR1 (FIGS. 13A-13N). These results indicate that IPA1 regulates seed dormancy and seedling growth by directly regulating expression of genes involved in GA synthesis, degradation, and signaling.

The above results demonstrate that miR156 controls seed dormancy and seedling growth through regulation of GA content and signaling by IPA 1.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method of modifying a plant comprising the steps of:

(i) carrying out genetic engineering transformation on plant cells or plant tissues so as to mutate N members in the miR156 gene family, wherein N is more than or equal to 1;

2. The method of claim 1, wherein the desired trait feature comprises enhanced seed dormancy.

3. The method of claim 1, wherein the miR156 gene family comprises class I miR156s and class II miR156 s.

4. The method of claim 3, wherein the class I miR156s is selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.

5. The method of claim 3, wherein the class II miR156s is selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.

6. The method of claim 1, wherein the mutating comprises reducing the expression or activity of N members of the miR156 gene family.

7. A genetically engineered plant tissue or plant cell comprising N members of the miR156 gene family mutated in the plant tissue or plant cell, wherein N is greater than or equal to 1.

8. A method of producing genetically engineered plant tissue or plant cells comprising the steps of:

n members in miR156 gene family in plant tissues or plant cells are mutated, so that the genetically engineered plant tissues or plant cells are obtained, wherein N is more than or equal to 1.

9. A method of producing a transgenic plant comprising the steps of:

regenerating the genetically engineered plant tissue or plant cells produced by the method of claim 8 into a plant body, thereby obtaining a transgenic plant.

10. A method of producing grain, comprising the steps of:

(i) planting crops, wherein N members in a miR156 gene family in the crops are mutated, wherein N is more than or equal to 1;

(ii) the grain (grain) of the crop is harvested.

11. Use of a miR156 gene family inhibitor for (a) promoting seed dormancy; and/or (b) improving the seed anti-pre-germination trait; or for the preparation of a composition or formulation for (a) promoting dormancy of seeds; and/or (b) improving the pre-emergence trait of the seed.