Application of rice histone deacetylase gene HDA710 in delaying leaf senescence
阅读说明：本技术 水稻组蛋白去乙酰化酶基因hda710在延迟叶片衰老中的应用 (Application of rice histone deacetylase gene HDA710 in delaying leaf senescence ) 是由 苏震 徐文英 赵楠楠 魏强 张群莲 于 2019-12-17 设计创作，主要内容包括：本发明公开了水稻组蛋白去乙酰化酶基因HDA710在延迟叶片衰老中的应用。本发明提供了水稻HDA710蛋白的新用途,即在调控植物的叶片衰老进程中的应用。过表达HDA710基因的转基因植株,叶绿素含量升高、电导率降低,符合其叶片晚衰的特征。低表达HDA710基因的转基因植株,符合叶片早衰的特征。本发明为促进表观遗传和遗传变异在作物育种中的应用提供了新的方向。(The invention discloses application of a rice histone deacetylase gene HDA710 in delaying leaf senescence. The invention provides a new application of rice HDA710 protein, namely an application in regulating and controlling the leaf senescence process of plants. The transgenic plant over expressing the HDA710 gene has the advantages of high chlorophyll content and low conductivity, and accords with the characteristics of late leaf senescence. The transgenic plant of the low expression HDA710 gene accords with the characteristics of the premature leaf senescence. The invention provides a new direction for promoting the application of epigenetic and genetic variation in crop breeding.)
1. A method of making a transgenic plant comprising the steps of: introducing a gene coding HDA710 protein into a receptor plant to obtain a transgenic plant; the transgenic plant senescence process is slowed, as compared to the recipient plant;
the HDA710 protein is (a1) or (a2) or (a3) as follows:
(a1) protein shown as a sequence 1 in a sequence table;
(a2) the protein shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to the plant senescence process and derived from the protein;
(a3) a protein derived from rice, having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in (a1), and involved in the senescence process of plants.
2. The method of claim 1, wherein: the gene encoding the HDA710 protein is (b1) or (b2) or (b3) or (b4) or (b5) as follows:
(b1) the coding region is shown as the 140 nd 1669 th nucleotide of the sequence 2 in the sequence table;
(b2) a DNA molecule shown as 140-position 2031 nucleotide of a sequence 2 in a sequence table;
(b3) DNA molecule shown in sequence 2 in the sequence table;
(b4) a DNA molecule derived from rice and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity to (b1) or (b2) or (b3) and encoding said protein;
(b5) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) or (b3) under stringent conditions and encodes the protein.
3. The method of claim 1 or 2, wherein:
the slowed aging process is manifested by high chlorophyll content and/or low conductivity.
4. The use of the HDA710 protein of claim 1 which is (c1) or (c2) or (c 3):
(c1) regulating the senescence process of plants;
(c2) promoting the aging process of plants to slow down;
(c3) promoting the late senescence of plants.
5. A method of plant breeding comprising the steps of: increasing the content and/or activity of the HDA710 protein of claim 1 in a plant, thereby promoting a slowing of the senescence process in the plant.
6. A method of making a transgenic plant comprising the steps of: introducing a nucleic acid molecule for inhibiting the expression of the HDA710 gene into a receptor plant to obtain a transgenic plant; the transgenic plant senescence progression is advanced compared to the recipient plant; the HDA710 gene is a gene encoding the HDA710 protein of claim 1.
7. A method of plant breeding comprising the steps of: inhibiting the expression of the HDA710 gene in the plant, thereby promoting the senescence process of the plant to advance; the HDA710 gene is a gene encoding the HDA710 protein of claim 1.
8. A method of plant breeding comprising the steps of: reducing the content and/or activity of HDA710 protein as defined in claim 1 in a plant, thereby promoting the senescence process of the plant in advance.
9. The method of claim 6, 7 or 8, wherein:
the senescence process is manifested in advance as a low chlorophyll content and/or a high conductivity.
10. The application of the nucleic acid molecule for inhibiting the expression of the HDA710 gene is as follows (d1) or (d 2):
(d1) advancing the aging process of the plants;
(d2) promoting the premature senility of plants;
the HDA710 gene is a gene encoding the HDA710 protein of claim 1.
Technical Field
The invention relates to application of a rice histone deacetylase gene HDA710 in delaying leaf senescence.
Background
Epigenetic alterations can reprogram the transcriptome in a variety of biological processes. Deacetylation of histones plays an important regulatory role in plant development and response to various adversity stresses. In rice, 18 histone deacetylase genes have been reported. Wherein HDA710/OsHDAC2 belongs to type I histone deacetylase.
Leaf senescence is an important stage in plant development, and in agricultural production, early leaf senescence limits the plant photosynthesis cycle, thereby affecting crop yield. Rice, as a main grain crop in the world, mainly comprises two subspecies, and previous researches show that the leaf senescence in indica rice and japonica rice is different, and the leaf senescence of the indica rice is earlier than that of the japonica rice, but the specific mechanism research of the leaf senescence is not clear.
Genetic variation of different varieties provides valuable resources for improving agronomic traits of crops, and can widen the sources of phenotypic variation of crops. With the completion of 3K rice genome project, abundant genetic resources can help us to better establish the association between genes and phenotypes, and the method is greatly helpful for researching agronomic traits such as aging and the like.
Disclosure of Invention
The invention aims to provide application of a rice histone deacetylase gene HDA710 in delaying leaf senescence.
The HDA710 protein is also called HDA710/OsHDAC2 protein. The HDA710 protein is (a1) or (a2) or (a3) as follows:
(a1) protein shown as a
(a2) the protein shown in the
(a3) a protein derived from rice, having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in (a1), and involved in the senescence process of plants.
The HDA710 gene is a gene encoding HDA710 protein. The HDA710 gene is also called HDA710/OsHDAC2 gene.
The HDA710 gene is (b1) or (b2) or (b3) or (b4) or (b5) as follows:
(b1) the coding region is shown as the 140 nd 1669 th nucleotide of the
(b2) a DNA molecule shown as 140-position 2031 nucleotide of a
(b3) DNA molecule shown in
(b4) a DNA molecule derived from rice and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity to (b1) or (b2) or (b3) and encoding said protein;
(b5) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) or (b3) under stringent conditions and encodes the protein.
The stringent conditions are hybridization and washing of the
The invention provides a method for preparing a transgenic plant, which comprises the following steps: introducing HDA710 gene into a receptor plant to obtain a transgenic plant; the transgenic plant senescence process is slowed compared to the recipient plant. The transgenic plant has a late senescence phenotype compared to the recipient plant.
The HDA710 gene may be specifically introduced into a recipient plant by a recombinant expression vector. The recombinant expression vector can be transformed into a recipient plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.
The recombinant expression vector containing the HDA710 gene can be constructed by using the existing plant expression vector.
The slowed aging process is manifested by high chlorophyll content and/or low conductivity.
The slowing of the senescence process is manifested by high leaf chlorophyll content and/or low leaf conductivity.
The late senescence is manifested as high chlorophyll content and/or low conductivity.
The delayed senescence is manifested by high leaf chlorophyll content and/or low leaf conductivity.
The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of HDA710 protein in the plant, thereby promoting a slowing of the senescence process in the plant. Thereby promoting the late senescence of the plants.
The invention also protects the application of the HDA710 protein, which is (c1) or (c2) or (c 3):
(c1) regulating the senescence process of plants;
(c2) promoting the aging process of plants to slow down;
(c3) promoting the late senescence of plants.
The invention also provides a method for preparing a transgenic plant, which comprises the following steps: introducing a nucleic acid molecule for inhibiting the expression of the HDA710 gene into a receptor plant to obtain a transgenic plant; the transgenic plant senescence progresses earlier than the recipient plant. The transgenic plant has a premature senescence phenotype compared to the recipient plant.
The nucleic acid molecule that inhibits the expression of the HDA710 gene may specifically be an antisense gene of the HDA710 gene. The gene that produces antisense RNA by transcription is called antisense gene (antisense gene).
The nucleic acid molecule that inhibits the expression of the HDA710 gene can be specifically introduced into a recipient plant by a recombinant expression vector. The recombinant expression vector can be transformed into a recipient plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.
The recombinant expression vector containing the nucleic acid molecule that inhibits the expression of the HDA710 gene can be constructed using existing plant expression vectors.
The invention also provides a plant breeding method, which comprises the following steps: inhibit the expression of the HDA710 gene in the plant, thereby promoting the senescence process of the plant. Thereby promoting the premature senility of the plants.
The invention also provides a plant breeding method, which comprises the following steps: reduce the content and/or activity of HDA710 protein in the plant, thereby promoting the aging process of the plant to advance. Thereby promoting the premature senility of the plants.
The invention also protects the application of the nucleic acid molecule for inhibiting the expression of the HDA710 gene, which is (d1) or (d 2):
(d1) advancing the aging process of the plants;
(d2) promoting plant senilism.
The senescence process is manifested in advance as a low chlorophyll content and/or a high conductivity.
The senescence process is manifested in advance as a low chlorophyll content of the leaves and/or a high electrical conductivity of the leaves.
The premature senility is characterized by low chlorophyll content and/or high electrical conductivity.
The premature senility is characterized by low chlorophyll content and/or high leaf conductivity.
Any of the above recipient plants is a monocot or a dicot.
Any of the above plants is a monocot or a dicot.
The monocotyledon may be a gramineous plant.
The gramineous plant may be a plant of the genus oryza.
The plant of the genus oryza may be japonica rice, for example, nipponlily.
When constructing a recombinant expression vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter may be added in front of its transcription initiation nucleotide, either alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, may be used in the construction of recombinant expression vectors, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the expression vector used may be processed, for example, by adding a gene expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance, or a chemical-resistant marker gene, etc. From the viewpoint of transgene safety, the transformed plants can be directly screened for phenotypes without adding any selectable marker gene.
The plant expression vector can be specifically a vector pCAMBIA 1300.
The invention provides a new application of rice HDA710 protein, namely an application in regulating and controlling the leaf senescence process of plants. The aging of plant leaves is mainly embodied by chlorophyll content and electric conductivity. The transgenic plant over expressing the HDA710 gene has the advantages of high chlorophyll content and low conductivity, and accords with the characteristics of late leaf senescence. The transgenic plant of the low expression HDA710 gene (namely, the transgenic plant inhibiting the expression of the HDA710 gene by introducing the antisense gene) accords with the characteristics of the premature leaf senescence. Namely, the over-expression of the HDA710 gene can promote the senescence process of plant leaves to be obviously delayed.
The inventor of the invention checks sequence polymorphism of rice histone deacetylase gene HDA710/OsHDAC2 through a rice metagenome browser (RPAN), finds that the difference of the HDA710 gene in indica rice and japonica rice is extremely obvious, and two fragments of the HDA710 gene are deleted in a gene region and a gene downstream region in the indica rice. Further, the inventors of the present invention performed correlation analysis on the varieties (Nipponbare, 9311 and Teqing) and phenotypes using the corresponding phenotypic traits, and found that the varieties with deletions tend to have premature leaf senescence.
The invention provides a new direction for promoting the application of epigenetic and genetic variation in crop breeding.
Drawings
FIG. 1 is a schematic diagram of the elements of
FIG. 2 shows the relative expression level results of HDA710 gene in example 1.
FIG. 3 shows the results of the plant phenotype in example 1.
FIG. 4 is the result of the chlorophyll content in example 1.
FIG. 5 shows the results of the conductivity in example 1.
FIG. 6 shows the sequence difference of rice deacetylase gene HDA710 in indica and japonica rice varieties.
FIG. 7 is a graph showing the senescence phenotype of the cultivars corresponding to the two HDA710 fragment deletions shown in FIG. 6.
FIG. 8 shows the results of the plant phenotype in example 2.
FIG. 9 shows the results of the chlorophyll content in example 2.
FIG. 10 shows the results of the conductivity in example 2.
Note: in fig. 4, 5, 9 and 10, p-value <0.001 is indicated by x, p-value <0.01 is indicated by x, and p-value <0.05 is indicated by x.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, four replicates were set up and the results averaged. The rice Nipponbare belongs to japonica rice.
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