Application of waxy synthetic gene of soybean cuticle or protein thereof
阅读说明：本技术 大豆角质层蜡质合成基因或其蛋白的应用 (Application of waxy synthetic gene of soybean cuticle or protein thereof ) 是由 冯献忠 杨素欣 高金珊 宋晓峰 于 2019-11-14 设计创作，主要内容包括：本发明涉及遗传育种技术领域,尤其涉及大豆角质层蜡质合成基因或其蛋白的应用。本发明研究表明,来源于大豆的编码甘油三磷酸激酶的GmGK1基因能够调控大豆叶表面角质层蜡质的发育,因此可以用于实现提高大豆抗旱及抗病能力。研究表明,过量表达GmGK1基因的gmgk1突变体植株叶片皱缩恢复正常,与野生型大豆叶片形态一致。利用GmGK1作为标志物,能够实现对植物种质,特别是大豆种质抗旱性能的鉴定。(The invention relates to the technical field of genetic breeding, in particular to an application of a waxy synthetic gene of soybean cuticle or a protein thereof. The research of the invention shows that the GmGK1 gene which is derived from soybean and codes glycerol triphosphate kinase can regulate and control the development of the cuticle wax of the soybean leaf surface, thereby being used for realizing the improvement of the drought resistance and disease resistance of the soybean. Research shows that the shriveling of the leaf of the GmGK1 mutant plant with the overexpression of the GmGK1 gene is recovered to be normal and is consistent with the shape of the wild soybean leaf. The GmGK1 is used as a marker, so that the identification of the drought resistance of plant germplasm, particularly soybean germplasm, can be realized.)
1. The application of at least one of the following I) to V) in regulating and controlling the formation of plant wax;
I) GmGK1 protein;
II) protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of GmGK1 protein and has the same or similar function with GmGK 1;
III), nucleic acid molecules encoding the proteins of I) or II);
IV), a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of the nucleic acid molecule of III) and can encode the same or similar functional protein;
v), a substance capable of modulating the level or activity of at least one of I) to V).
2. The use according to claim 1,
the amino acid sequence of the GmGK1 protein is shown as SEQ ID NO. 1;
the sequence of the nucleic acid molecule for coding the protein shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2.
3. Use according to claim 1, wherein the plant is a leguminous plant.
4. The use of any one of claims 1 to 3, wherein the modulation of plant wax formation comprises: regulating waxy crystal accumulation, regulating alkane substance formation and/or regulating triterpene substance formation.
5. An agent for promoting waxy formation in plants, comprising at least one of the following i) to v):
i) a GmGK1 protein or a nucleic acid molecule encoding a GmGK1 protein;
ii) an expression vector comprising a nucleic acid encoding a GmGK1 protein;
iii) a recombinant host comprising ii);
iv) a promoter or enhancer that enhances expression of the GmGK1 gene;
v) an inducer promoting the expression of the GmGK1 gene;
vi) agents that increase the activity of GmGK1 protein.
6. Use of the formulation of claim 5 to improve drought and/or disease resistance in a plant.
7. Method for increasing the drought and/or disease resistance of plants, characterized in that the preparation of claim 5 is used to increase the level and/or activity of endogenous GmGK1 protein in plants, or to express GmGK1 protein in plants that do not contain GmGK1 or GmGK1 inactivated.
8. A formulation for characterizing wax formation in plants, comprising:
a preparation for detecting the transcription level of the GmGK1 gene;
and/or detecting the expression level or active agent of the GmGK1 protein.
9. Use of the formulation of claim 8 to identify drought and/or disease resistance in plant germplasm.
10. A method for identifying drought resistance and/or disease resistance of plant germplasm, which is characterized by detecting the transcription level of GmGK1 gene of plant germplasm or detecting the expression level or activity of GmGK1 protein by using the preparation of claim 8.
Technical Field
The invention relates to the technical field of genetic breeding, in particular to an application of a waxy synthetic gene of soybean cuticle or a protein thereof.
Background
Soybean (Glycine max) is an ancient crop, has important and special economic value, is sensitive to water shortage and has weak high-temperature resistance. The soybean encounters abiotic adversity stresses such as drought, high temperature, salt and alkali and the like in the growth period, which is an important influence factor causing yield reduction of the soybean. Therefore, improving the drought resistance of soybean has become one of the key problems in modern breeding work.
The most important role of the cuticle on plants is to deal with the drought threat, and the cuticle also has the functions of defending against mechanical damage, resisting against the invasion of germs and insects, blocking ultraviolet rays and the like. The microstructure of the cuticle is divided into two layers of cutin and wax, and the wax forms wax crystals on the outermost layer of the cutin and permeates into the cutin with a net-shaped structure, so that the cutin is called as inner wax. The main task of the stratum corneum of the land plant is to prevent the non-porous evaporation of water in the body, so that the plant can dynamically regulate gas exchange, water potential and the like through the opening and closing of pores. Plants derive a series of other functions in the process of continuous evolution, such as: is the first physical barrier of plants against the stress of pathogenic bacteria and insects; the wax crystals on the outermost surface can prevent dust and the like from attaching to the leaf skin; shielding ultraviolet rays and reducing the damage of the ultraviolet rays; as an adjunct to epidermal cells, it plays a role in determining boundaries in organ development.
The regulation of stratum corneum biosynthesis is complex and involves many signaling pathways including: response to environmental stress, response to pathogens, and feedback regulation of the stratum corneum itself based on structure and integrity. In addition, the components of the stratum corneum are synthesized by epidermal cells, which differ from other cells as part of the regulation of stratum corneum synthesis. Previous researches show that the mutation of the Glyma.07G028600 gene causes great change of the proportion of waxy components in the horny layer and influences the development of the horny layer.
Researches on related genes influencing the synthesis and regulation of cutin wax and action mechanisms of the genes, not only can fully recognize the formation and regulation process of plant cutin, but also provides gene resources for further improving the drought resistance and disease resistance of soybeans.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide the application of the waxy synthetic gene of soybean cuticle or its protein in the development process of soybean hypocuticle and leaf.
The invention provides an application of at least one of the following I) to V) in regulation and control of plant wax formation;
I) GmGK1 protein;
II) protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of GmGK1 protein and has the same or similar function with
III), nucleic acid molecules encoding the proteins of I) or II);
IV), a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of the nucleic acid molecule of III) and can encode the same or similar functional protein;
v), a substance capable of modulating the level or activity of at least one of I) to V).
In the invention, the amino acid sequence of the GmGK1 protein is shown as
the sequence of the nucleic acid molecule for coding the protein shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2.
In the present invention, the plant is a leguminous plant; the plants used for verifying the function of GmGK1 in the invention are leguminous plants, in particular soybeans Williams82 and lotus 12. The study shows that the protein and the nucleic acid sequence of GmGK1 in Williams82 and lotus bean 12 are completely consistent.
In the present invention, the regulation of plant wax formation comprises: regulating waxy crystal accumulation, regulating alkane substance formation and/or regulating triterpene substance formation. In the invention, the regulation comprises two aspects of positive regulation or negative regulation. The Glyma.07G028600 gene of the GmGK1 mutant phenotype, which deletes TCTTTTATCC bases at an exon and inserts an A base research, shows that the cuticle layer of the GmGK1 gene mutant is thinner than that of the wild type Nelumbo 12, the density is lower, the wax crystal accumulation on the leaf surface is sparse, and the leaf is shriveled. And the shriveling of the leaf blade of the gmGK1 mutant plant over expressing the GmGK1 gene is recovered to be normal. Therefore, the fact that the GmGK1 gene is over-expressed, the transcription level of the GmGK1 gene is promoted, the protein level of the GmGK1 is increased, or the activity of the GmGK1 protein is promoted is proved to be beneficial to accumulation and accumulation of wax on the leaf surface, the formation of alkane substances is promoted, and the content of triterpenoids is reduced. Knocking out or knocking down the GmGK1 gene, inhibiting the transcription level of the GmGK1 gene, reducing the protein level of the GmGK1 or inhibiting the activity of the GmGK1 protein can inhibit the accumulation and accumulation of wax on the surface of the leaf and inhibit the formation of alkane substances.
The invention also provides a preparation for promoting the formation of plant wax, which comprises at least one of the following i) to v):
i) a GmGK1 protein or a nucleic acid molecule encoding a GmGK1 protein;
ii) an expression vector comprising a nucleic acid encoding a GmGK1 protein;
iii) a recombinant host comprising ii);
iv) a promoter or enhancer that enhances expression of the GmGK1 gene;
v) an inducer promoting the expression of the GmGK1 gene;
vi) agents that increase the activity of GmGK1 protein.
The accumulation of the waxy layer of the plant epidermis is useful for combating drought stress, protecting against mechanical damage, combating bacterial or insect infestation, and for blocking ultraviolet light. The preparation can be used for improving the level and/or activity of endogenous GmGK1 protein of a plant or germplasm, or enabling a plant without GmGK1 to express GmGK1 protein, so that the effects of improving the drought stress resistance of the plant, improving the disease resistance and improving the mechanical damage defense capability of the plant can be achieved.
In the preparation, the amino acid sequence of the GmGK1 protein is shown as SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule for coding the GmGK1 protein is shown as SEQ ID NO. 2.
The backbone vector of the expression vector containing the nucleic acid encoding the GmGK1 protein was a plant expression vector, and in the present invention, the pCAMBIA3301H vector was used for functional verification.
The recombinant host containing the expression vector is agrobacterium tumefaciens, and the method for promoting the formation of the plant wax adopts an agrobacterium-mediated method to transform the expression vector containing the nucleic acid molecule for coding the GmGK1 protein into a plant explant. The explant used as recipient material was hypocotyl cells.
The preparation provided by the invention is applied to improving the drought resistance and/or disease resistance of plants.
The invention also provides a method for improving the drought resistance and/or disease resistance of plants, which adopts the preparation provided by the invention to improve the level and/or activity of endogenous GmGK1 protein of the plants, or to enable the plants without GmGK1 or GmGK1 to express GmGK1 protein.
In the invention, the improvement of the drought resistance and/or disease resistance of the plant refers to the breeding improvement of the existing germplasm so as to obtain a new variety with better drought resistance and/or disease resistance. Or applying an inducer capable of promoting the expression of the GmGK1 gene or a preparation for improving the activity of the GmGK1 protein to the plant so as to improve the drought resistance and/or disease resistance of the plant.
In some embodiments, the method of inactivating GmGK1 in a plant expressing GmGK1 protein comprises:
constructing a vector containing a nucleic acid for encoding GmGK1 protein, and transforming the vector into agrobacterium; infecting a plant seed or explant with the agrobacterium.
In addition, GmGK1 can also be used as a marker for identifying wax formation ability in plant breeding screening.
The invention also provides a preparation for identifying the wax forming capability of a plant, which comprises the following components:
a preparation for detecting the transcription level of the GmGK1 gene of plant germplasm;
and/or detecting the expression level or active agent of the GmGK1 protein.
The preparation provided by the invention is applied to identification of drought resistance and/or disease resistance of plant germplasm.
The invention also provides a method for identifying drought resistance and/or disease resistance of plant germplasm, which detects the transcription level of GmGK1 gene or the expression level or activity of GmGK1 protein by using the preparation provided by the invention.
The assay of the present invention includes the measurement of the expression level or activity level. For example, Western blot is adopted for detecting the expression level of the GmGK1 protein; the transcription level of the GmGK1 gene is detected by adopting a real-time PCR mode. By using the identification method, the drought resistance and/or disease resistance of the germplasm can be predicted only by detecting the young tissues of the plant, and the breeding period is shortened.
The research of the invention shows that the GmGK1 gene which is derived from soybean and codes glycerol triphosphate kinase can regulate and control the development of the cuticle wax of the soybean leaf surface, thereby being used for realizing the improvement of the drought resistance and disease resistance of the soybean. Research shows that the shriveling of the leaf of the GmGK1 mutant plant with the overexpression of the GmGK1 gene is recovered to be normal and is consistent with the shape of the wild soybean leaf. The GmGK1 is used as a marker, so that the identification of the drought resistance of plant germplasm, particularly soybean germplasm, can be realized.
Drawings
FIG. 1 shows the genomic and transcribed sequence of the soybean GmGK1 gene, wherein: the exon sequences are in bold, the intron sequences are in italics,
FIG. 2 shows a GFP-tagged GmGK1 sense expression vector pCAMBIA3301H-
FIG. 3 shows the leaf discs of wild type Nelumbo 12 and the gmgk1 mutant,
FIG. 4 shows a first pair of true leaves TB staining of isolated soybean at 17:00 PM 17 p.m. after planting, with wild type at the top and mutant at the bottom, with scale of 3 cm;
FIG. 5 shows that the shriveling of the leaf of the gmGK1 mutant plant which overexpresses the GmGK1 gene is recovered to be normal, wherein, the
FIG. 6 shows that the GmGK1 gene affects the thickness of the stratum corneum and the bulk density of waxy crystals, wherein 6-A is the transmission electron microscope of the upper and lower epidermis of lotus bean 12 and GmGK1 mutant leaves, and the scale is 500 nm; 6-B is a scanning electron microscope for the epidermis of the lotus bean 122 and the gmgk1 mutant leaves, and the scale is 500 mu m;
FIG. 7 shows the results of waxy analysis of stratum corneum for wild type and gmgk1 mutants, wherein A shows the total amount of waxy for wild type and gmgk1 mutants and B shows the waxy composition for wild type and gmgk1 mutants;
FIG. 8 shows the water loss rate of the wild type and the gmgk1 mutant leaves ex vivo.
Detailed Description
The invention provides the application of the waxy synthetic gene of the soybean cuticle or the protein thereof, and a person skilled in the art can realize the application by properly improving the process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The nucleic acid molecule coding the GmGK1 protein comprises genomic DNA, cDNA, recombinant DNA or mRNA, hnRNA coding the GmGK1 protein; or a nucleic acid molecule which is reverse complementary to the above DNA, cDNA, recombinant DNA or mRNA.
The above-described nucleic acid molecules can be modified or optimized according to the actual needs in order to make the gene expression more efficient, for example ① can be modified according to the codon preference of the recipient plant while maintaining the amino acid sequence of the GmGK1 gene of the invention in order to meet the preference of the recipient plant. ② or the gene sequence adjacent to the initiation methionine is modified in order to make the translation efficiently initiated, for example, by modifying with sequences known to be efficient in plants. ③ is linked to various plant-expressed promoters, which can include constitutive, inducible, time-sequential, developmental, chemical, tissue-preferred and tissue-specific promoters, the choice of which will vary with the time and space of expression and also depending on the target species, ④ introduces enhancer sequences, such as intron sequences (e.g.from Adhl and bronzel) and viral leader sequences (e.g.from TMV, MCMV and AMV).
In the present invention, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The host may be a fungus, a bacterium, an alga or a cell.
For plants not containing GmGK1, the gene fragment of GmGK1 may be introduced into plant cells by chemical methods, shotgun methods, microinjection, electroporation, or the like, or the gene fragment of GmGK1 may be introduced into plant cells by homologous recombination, zinc finger nucleases, TALENs, CRISPRs, or the like.
The invention is further illustrated by the following examples:
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