CN116790623B - Rice flower organ development regulation gene OsROXY2, protein coded by same and application thereof - Google Patents
Rice flower organ development regulation gene OsROXY2, protein coded by same and application thereof Download PDFInfo
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Abstract
Rice flower organ development regulating gene OsROXY2, and encoded protein and application thereof are provided. Mutant forms of OsROXY2 and proteins encoded thereby and uses thereof are also provided. The gene and the mutant form thereof are utilized to regulate and control the development of the rice flower organ, and have important theoretical and practical significance for deeply researching the molecular mechanism of the development of the rice reproductive organ.
Description
Technical Field
The present invention relates to plant genetic engineering technology, and is especially one kind of rice flower organ development regulating gene OsROXY2 and its encoded protein and application, and OsROXY2 mutant form and its encoded protein and application.
Background
Rice is not only a model plant of monocots, but also an important food crop. Rice is a staple food of nearly half of the world population, and flowers are not only reproductive organs, but also the basis of seed formation. The normal development of rice flowers directly affects the yield and rice quality of rice, so research on rice flower development has been the focus of attention of rice genetic breeders. The research of the rice flower development not only has important theoretical significance, but also has important guiding significance for the genetic breeding of the rice yield and quality.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.
The application provides application of a rice flower organ development regulating gene OsROXY2, wherein the gene OsROXY2 consists of a nucleotide sequence shown as SEQ ID NO. 1, and the gene OsROXY2 is applied to rice flower organ development regulation.
The application also provides a plasmid, a plant expression vector and a host cell of the rice flower organ development regulation gene OsROXY2, wherein the OsROXY2 is formed by a nucleotide sequence shown as SEQ ID NO. 1.
The application also provides application of the protein encoded by the rice flower organ development regulation gene OsROXY2, wherein the protein is composed of an amino acid sequence shown as SEQ ID NO. 2, and the protein is applied to rice flower organ development regulation.
The application also provides a mutant form of the rice flower organ development regulating gene OsROXY2, wherein the flower organ phenotype of the mutant form has the following characteristics compared with a wild type:
stamen are partially absent.
In some exemplary embodiments, the mutated version of the gene sequence has one or more of a substitution, insertion, and deletion as compared to the OsROXY2 gene sequence SEQ ID NO: 1. In some exemplary embodiments, the mutant forms have the nucleotide sequence set forth in SEQ ID NO. 3.
In some exemplary embodiments, the mutant forms have the amino acid sequence shown in SEQ ID NO. 4.
The application provides the application of the plasmid, the plant expression vector, the host cell and the protein obtained by the plasmid in the mutant form in the development regulation of the flower organ of rice.
The application provides application of the protein coded by the mutant form, wherein the protein is composed of an amino acid sequence shown as SEQ ID NO. 4, and the protein is applied to the regulation of flower organ development of rice.
SEQ ID NO. 1 sequence is as follows:
ATGCAGTACGGAGCGGCGGCCGAGCAGGCGTGGTACATGCCGGCG
GCGGCGCCGGCACCGATGGTGGAGAGCGCGGTGGCGCGGGTGGAGCG
GCTGGCGTCGGAGAGCGCGGTGGTGGTGTTCAGCGTGAGCAGCTGCTG
CATGTGCCACGCCGTGAAGCGCCTCTTCTGCGGCATGGGGGTGCACCC
GACGGTGCACGAGCTGGACCTCGACCCGCGCGGCCGCGAGCTGGAGCG
CGCCCTGGCGCGCCTCGTCGGGTACGGCGGCCCCGCCGCCGCGTCGCC
GCCCGTCGTCCCCGTCGTCTTCATCGGCGGCAAGCTCGTCGGCGCCATG
GACCGCGTCATGGCCGCGCACATCAACGGCTCCCTCGTCCCCCTCCTCA
AGGAGGCCGGCGCGCTCTGGCTCTAG
SEQ ID NO. 2 sequence is as follows
MQYGAAAEQAWYMPAAAPAPMVESAVARVERLASESAVVVFSVSSCCMCHAVKRLFCGMGVHPTVHELDLDPRGRELERALARLVGYGGPAAASPPVVPVVFIGGKLVGAMDRVMAAHINGSLVPLLKEAGALWL
SEQ ID NO. 3 sequence is as follows
ATGCAGTACGGAGCGGCGGCCGAGCAGGCGTGGTACATGCCGGCGGCGGCGCCGGCACCGATGGTGGAGAGCGCGGTGGCGCGGGTGGAGCGGCTGGCGTCGGAGAGCGCGGTGGTGGTGTTCAGCGTGAGCAGCTGCTGCATGTGCCACGCCGTGAAGCGCCTCTTCTGCGGCATGGGGGTGCACCCGACGGTGCACGAGCTGGACCTCGACCCGCGCGGCCGCTAGCTGGAGCGCGCCCTGGCGCGCCTCGTCGGGTACGGCGGCCCCGCCGCCGCGTCGCCGCCCGTCGTCCCCGTCGTCTTCATCGGCGGCAAGCTCGTCGGCGCCATGGACCGCGTCATGGCCGCGCACATCAACGGCTCCCTCGTCCCCCTCCTCAAGGAGGCCGGCGCGCTCTGGCTCTAG (bold italic underlined sequence is the mutant sequence, i.e.in the mutant, from wild type G to T, codon GAG to TAG, encoded amino acid by E (glutamic acid)) Becomes a stop codon)
SEQ ID NO. 4 sequence is as follows
MQYGAAAEQAWYMPAAAPAPMVESAVARVERLASESAVVVFSVSS CCMCHAVKRLFCGMGVHPTVHELDLDPRGR
The application utilizes the mutant form and the constructed mutant of the rice flower organ development regulating gene OsROXY2 to carry out genetic complementation verification research on the rice flower organ development regulation, and has important theoretical and practical significance for deeply researching the molecular mechanism of the rice reproductive organ development.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. Other advantages of the present application may be realized and attained by the structure particularly pointed out in the written description and drawings.
Drawings
The accompanying drawings are included to provide an understanding of the technical aspects of the present application, and are incorporated in and constitute a part of this specification, illustrate the technical aspects of the present application and together with the examples of the present application, and not constitute a limitation of the technical aspects of the present application.
FIG. 1 shows the phenotypic characteristics of wild-type rice and a reduced stamen number mutant sl. The floret of wild rice consists of a pair of palea, two paddles, six stamens and one pistil (A in FIG. 1), the number of stamens in all florets of the mutant sl is reduced (B-D in FIG. 1), and the missing stamens is located more on the palea side (E in FIG. 1). Furthermore, the pistils of part of the florets were deleted (C-D in FIG. 1); the palea morphology is abnormal, and is characterized by a curvature of the palea, which is smaller than the palea of the wild type (F in FIG. 1), and a depression of the palea (G-I in FIG. 1).
Figure 2 shows the phenotype statistics associated with flowers in wild-type and sl mutants.
Fig. 3 is a scanning electron microscope picture of wild floret and sl mutant floret.
Figure 4 shows the map-based cloning of the sl mutant.
Fig. 5 shows the phenotype of the genetically complementary material.
Fig. 6A to 6C show genotyping and phenotyping of knockout material roxy2.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application will be described in detail hereinafter. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be arbitrarily combined with each other.
At present, three types of action mechanisms for regulating and controlling the flower organ numbers of dicotyledonous plants mainly exist: 1) ABCE model (first class) of flower organ decision, the flower organ decision gene in the path regulates the number of flower organs by regulating and controlling the feature decision of each round of flower organs; 2) The CLAVATA-WUSCHEL pathway (second class), in which genes control inflorescence and flower meristem size by regulating stem cell maintenance and differentiation in meristem, thereby affecting flower organ number; 3) Other pathways (third class), genes in which regulate the number of flower organs by regulating the initiation of flower organ primordia. Functional research on the regulation mechanism of the number of the first two floral organs in rice has already obtained a good research basis. However, molecular network analysis for regulating the number of flower organs (class III) by influencing (or regulating) the initiation of flower organ primordia is slow, and innovative research is urgently needed to develop a more comprehensive disclosure of the regulating mechanism of the number of rice flower organs.
The study in Arabidopsis thaliana shows that the number of petals in the Atroxy1 mutant is reduced from 4 to 2.5, and part of petals are abnormal in morphology. Further studies have found that AtROXY1 regulates initiation of petal primordia and morphogenesis of petals. Furthermore, atROXY1 has a function of inhibiting the expression of the C-group gene AGAMOUS (AG) in the outer two-round flower organs (Xing et al, 2005). AtROXY1 belongs to the family of CC-type Glutaredoxins (GRX), which are involved in biological processes such as organ development, stress response, hormonal and nitrogen signaling. 21 CC-type GRXs exist in Arabidopsis, and are named as AtROXY1-AtROXY21 (Li et al, 2009), 17 CC-type GRXs exist in rice (Xing et al, 2006), and compared with the research progress in Arabidopsis, the research on the function of the CC-type GRXs in rice is very little at present.
Wang et al (2009) found that OsROXY1 and OsROXY2 in rice wereHomologous proteins of the Arabidopsis AtROXY1, the expression patterns of which are similar to that of the AtROXY1, and both can restore the phenotype of the Atroxy1 mutant, so that the ROXYs in Arabidopsis and rice are considered to be conserved in function in terms of flower development. After overexpression of OsROXY1 or OsROXY2 in Arabidopsis, the transgenic lines showed H 2 O 2 The increased accumulation and phenotype of hypersensitivity to the necrotic pathogen Botrytis cinerea suggests that both may be involved in the disease resistance response of plants to pathogens by affecting the redox state of the cells (Wang et al 2009). However, the biological function of these two genes in rice has not been clarified so far.
The application relates to a rice flower organ development regulating gene OsROXY2, and a coded protein and application thereof. Mutant forms of OsROXY2 and proteins encoded thereby and uses thereof are also provided.
The gene OsROXY2 has a nucleotide sequence shown in SEQ ID NO. 1.
The invention also relates to a plasmid, a plant expression vector and a host cell containing the rice flower organ development regulation gene OsROXY2.
The plasmid containing the rice flower organ development regulating gene OsROXY2, the plant expression vector and the host cell are applied to the rice flower organ development regulation.
The invention also relates to a protein encoded by the rice flower organ development regulation gene OsROXY2, wherein the protein has an amino acid sequence shown as SEQ ID No. 2.
The gene of the invention is obtained and the function verification steps are as follows:
OsROXY2 Gene mapping: in order to separate the gene, the invention firstly constructs F by the hybridization of the mutant sl and rice varieties with wide land and short land 2 And (3) locating the group by generation, and locating candidate genes by adopting map cloning.
Candidate mutation site validation: and (3) finding information of candidate genes by using a RiceNet website, designing a pair of specific sequencing primers at two sides of a candidate site, respectively carrying out PCR amplification on DNA of the homozygous wild type and the homozygous mutant, sequencing and comparing products, and determining the candidate genes.
Functional analysis of OsROXY2 gene: through genetic transformation, the fragment containing the complete OsROXY2 gene is transferred into a mutant sl for performing a functional complementation test, and the transgenic rice for restoring the normal phenotype of the mutant is obtained.
The invention is based on early discovery of a rice flower organ development mutant sl controlled by unknown single recessive nuclear genes.
The mutant sl has a reduced number of stamens in all florets (B-D in FIG. 1) compared to the wild-type rice variety C418, and the deleted stamens are located more on the palea side (E in FIG. 1). Furthermore, we have found that the pistil of part of the floret is missing (C-D in FIG. 1); the palea morphology is abnormal, and is characterized by a curvature of the palea, which is smaller than the palea of the wild type (F in FIG. 1), and a depression of the palea (G-I in FIG. 1).
In addition, the present application found that the SL gene encodes the CC-type GRX OsROXY2 by map-site cloning of the stably inherited stamen number-reduced mutant SL. Phenotypic observation shows that the number of stamens of all florets in the mutant sl is reduced, and part of floret pistils are lost and the morphology of palea is abnormal. The phenotype is obviously different from the reduced petal phenotype of the arabidopsis roxy1, which shows that the function of OsROXY2 in regulating the development of flower organs in rice is different from that of the homologous gene AtROXY 1. Therefore, there is a need to develop a systematic and intensive study on the function and mechanism of action of OsROXY2 in the regulation of the number of rice flower organs (stamens).
The invention is further illustrated below with reference to specific examples.
Example 1: obtaining of rice flower organ development regulating gene OsROXY2
Phenotypic observation and analysis of mutant sl
A stable inherited stamen-reduced mutant sl is obtained in rice variety C418. The floret of wild rice consists of a pair of palea, two paddles, six stamens and one pistil (A in FIG. 1), the number of stamens in all florets of the mutant sl is reduced (B-D in FIG. 1), and the missing stamens is located more on the palea side (E in FIG. 1). Furthermore, we have found that the pistil of part of the floret is missing (C-D in FIG. 1); the palea morphology is abnormal, and is characterized by a curvature of the palea, which is smaller than the palea of the wild type (F in FIG. 1), and a depression of the palea (G-I in FIG. 1).
In fig. 1: (A) wild rice floret contains 6 stamens and 1 pistil; (B) 5 stamens and 1 pistil are contained in the sl mutant floret; (C) 4 stamens in the sl mutant floret, pistil deletion; (D) 3 stamens in the sl mutant floret, and the pistil is lost; (E) Flower patterns of wild type (left) and sl mutant (right), 3 stamens are respectively arranged at the inner and outer palea sides of the wild type, and the stamens at the palea side of the sl mutant is reduced; (F) the palea of wild type rice; (G) The palea of the- (I) sl mutant is curved to a different extent.
Note that: in a-D of fig. 1, the upper apex downward triangle indicates stamens, the middle apex downward triangle indicates pistils, and the lower apex upward triangle indicates missing pistils. Bars=2 mm.
To gain insight into abnormalities of floral organs in sl, statistical analysis was performed on abnormal floral organs (stamen and palea). The number of stamens in sl is 2-5, wherein the number of stamens in more than half of the florets is 3 (A); more than 90% of pistils in floret are absent (B); the palea morphology is close to half of the florae (C).
In fig. 2: (a) stamen number statistics; (B) pistil number statistics; (C) palea morphology statistics of sl mutant.
Scanning electron microscope observation of initial condition of flower organ primordium in sl mutant
To further investigate the cause of reduced stamen in the sl mutant, we performed scanning electron microscope observations of the sl mutant with small flowers in early stages of wild-type development. The palea primordia, 6 stamen primordia and pistil primordia are produced in the wild type rice floret (A in FIG. 3). In the sl mutant, the lemma, palea primordia, were normally initiated and the number of palea-side stamen primordia decreased (B-D in FIG. 3), indicating that the phenotype of decreased stamen in the sl mutant was due to abnormal stamen primordia initiation.
In fig. 3: (a) the stamen primordium of wild-type floret starts normally; (B-D) stamen primordia of sl floret at different developmental stages.
Note that: triangles indicate stamen primordia, letter l represents palea primordia, and p represents palea primordia. The bar was 25. Mu.m.
Example 2: determination of mutation site and target Gene
Map-based cloning of SL
The sl mutant is a natural mutant of rice variety C418. The mutant sl is used as a female parent to be hybridized with the rice variety Guangland dwarf, F 1 The phenotype of the plant is consistent with that of wild C418, F 2 The target character separation occurs in the plant, the ratio of the mutant to the wild type is close to 1:3 (37:110), which indicates that the phenotype is a single-gene controlled recessive mutation. For F using InDel markers with polymorphisms between parents 2 A single plant of the medium mutant phenotype was subjected to genetic analysis to localize SL in the 16.5Mbp-17.5Mb region intermediate of chromosomes 2, AP005522-23210 and AP 004086-113. Fine localization was performed using SSR molecular markers, and SL was localized between chromosome 2 RM13210 and 0.7Mb in the middle of AP004086-113, which is about 100 genes, according to the recombination exchange rate (A in FIG. 4).
Candidate genes located in the target interval that may affect the number of flower organs were predicted using the RiceNet website (table 1). Sequencing of these candidate genes revealed that the mutation of GAG at positions 226-228 of the Os02g0512400 (OsROXY 2) exon into TAG resulted in a premature termination codon (B in FIG. 4).
In fig. 4: (A) map-based cloning of SL; and (B) the gene structure and site mutation of OsROXY2.
TABLE 1 list of candidate genes
Example 3: and (3) genetic complementation verification: osROXY2 restores the phenotype of sl
To confirm that the phenotype of the sl mutant was due to the OsROXY2 mutation, genetic complementation verification was performed. The coding region of OsROXY2 and a 1.5kb fragment upstream of the initiation codon are connected into pCAMBIA1301 to construct a complementary vector, and the sl mutant is transformed to obtain a transgenic material. Phenotypic observations found that transformation of empty vector failed to restore the phenotype of sl (A, B in fig. 5); after transformation of the complementing vector, the phenotype of sl was restored to normal (C, D in FIG. 5), indicating that OsROXY2 restored the phenotype of sl, thus confirming that the phenotype of reduced number of stamens in the sl mutant was caused by OsROXY2 mutation.
In fig. 5: (a) turning empty palea parcela depressions; (B) transferring 4 stamens from empty florets, wherein the stamens are absent; (C) normal palea of the genetically complementary material; (D) the number of male and female stamen of the genetic complement is normal.
Note that: arrows in fig. 5 (B) and (D) indicate stamens, triangles in fig. 5 (B) indicate absent pistils, and triangles in fig. 5 (D) indicate normal pistils.
The phenotype of the knockout material of OsROXY2 is similar to sl
To further confirm that the phenotype of the sl mutant was due to the OsROXY2 mutation, the knockout material roxy2 for OsROXY2 was obtained using the CRISPR-cas9 technique. After sequencing, it was found that an A was inserted at position 41 of the gene sequence of OsROXY2 (FIG. 6A), resulting in a frameshift mutation. Phenotypic observations revealed a reduction in the number of stamens in the knockdown material roxy2 to 3-5, with loss of pistils in part of the material (fig. 6C). This result further confirms that the phenotype of reduced numbers of stamens in the sl mutant is caused by the OsROXY2 mutation.
In fig. 6A-6C: (FIG. 6A) sequencing results show that an A is inserted into the DNA sequence of OsROXY2 in the knockdown material; (FIG. 6B) 6 stamens and 1 pistil in the control material; (FIG. 6C) the number of stamens in the knockdown material roxy2 was reduced to 3-5, with loss of pistils in part of the material.
Note that: the upper apex downward triangle indicates stamens, the lower apex upward triangle indicates missing pistils, and the lower apex downward triangle indicates normal pistils. Bar is 2mm.
In conclusion, by phenotypic observation of mutant sl, genetic complement and knockout material, it was confirmed that the OsROXY2 gene in rice has a function related to the reduction of pistil and stamen numbers. The function can be applied to intelligent emasculation molecular breeding and quickens the breeding process of sterile lines.
Claims (5)
1. Rice flower organ development regulation and control geneOsROXY2Wherein the mutant form encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO. 4; and saidThe floral organ phenotype of the mutant form has the following characteristics compared to the wild type:
stamen are partially absent.
2. The mutant form according to claim 1, wherein the mutant form has the nucleotide sequence set forth in SEQ ID No. 3.
3. The mutant form according to claim 2, wherein the nucleotide sequence of the mutant form is identical toOsROXY2The gene sequence SEQ ID NO. 1 has one or more of substitution, insertion and deletion.
4. Use of a plasmid, plant expression vector, host cell, and protein obtained therefrom in the regulation of floral organ development in rice in a mutant form as claimed in any one of claims 1 to 3.
5. Use of a protein encoded by a mutant form according to any one of claims 1 to 3, wherein the protein consists of the amino acid sequence shown in SEQ ID No. 4, for use in the regulation of floral organ development in rice.
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| CN109295071A (en) * | 2018-10-12 | 2019-02-01 | 福建省农业科学院生物技术研究所 | Protein and the application of a kind of rice flower organ developmental regulation gene PEH1 and its coding |
| CN113122552A (en) * | 2021-04-21 | 2021-07-16 | 西北农林科技大学 | Cucumber glutaredoxin gene CsGRX4 and application thereof to susceptibility of botrytis cinerea |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109295071A (en) * | 2018-10-12 | 2019-02-01 | 福建省农业科学院生物技术研究所 | Protein and the application of a kind of rice flower organ developmental regulation gene PEH1 and its coding |
| CN113122552A (en) * | 2021-04-21 | 2021-07-16 | 西北农林科技大学 | Cucumber glutaredoxin gene CsGRX4 and application thereof to susceptibility of botrytis cinerea |
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| Conserved functions of Arabidopsis and rice CC-type glutaredoxins in flower development and pathogen response;Wang Z等;《Mol Plant》;323-335 * |
| Oryza sativa Japonica Group ROXY2 (ROXY2) mRNA, complete cds;NCBI;《Genbank》;FJ463034.1 * |
| 李鲁华,王忠妮,徐如宏.水稻AGL17-like亚家族研究进展.《农业生物技术学报》.2022,1606-1613. * |
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