CN109295071B - Rice flower organ development regulation gene PEH1, and encoded protein and application thereof - Google Patents

Abstract

The invention provides a rice floral organ development regulation gene PEH1, and a protein coded by the gene PEH1 and application thereof, wherein the gene PEH1 has a nucleotide sequence shown as SEQ ID No. 1; 1 nucleotide sequence, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the SEQ ID No; the protein has an amino acid sequence shown as SEQ ID No. 2, and the amino acid sequence or the derivative generated by adding, substituting, inserting or deleting one or more amino acids in the amino acid sequence of the SEQ ID No. 2; the gene PEH1 and the protein coded by the gene are applied to the growth regulation of rice floral organs. The gene is used for regulating and controlling the development of rice floral organs, and has important theoretical and practical significance for deeply researching the molecular mechanism of the development of rice reproductive organs.

Description

Rice flower organ development regulation gene PEH1, and encoded protein and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the field of plant genetic engineering, in particular to a rice floral organ development regulating gene PEH1, and a protein coded by the gene and application of the gene.

[ background of the invention ]

Rice is not only a model plant for monocot genome research, but also an important food crop. Rice is the staple food of one third of the world's population, and its flowers are not only reproductive organs, but also the basis for the formation of seed particles. The normal development of rice flowers directly affects the rice yield and rice quality, so the research on the rice flower development is always the focus of attention of rice genetic breeding workers. The research on the development of rice flowers not only has important theoretical significance, but also has important guiding significance on the genetic breeding of the rice yield and quality.

In the 90 s of the 20 th century, plant developmental biologists discovered that floral organ formation of dicotyledonous plants was controlled by multiple genes, which were related to each other, through studies on homeotic mutants of Arabidopsis and grass flowers, and proposed an ABC model with milestone significance (Coen ES and Meyerowitz EM.the war of the world: genetic interactions controlling flower depth. Nature,1991,353(5): 31-37.). With the progress of the study and the increasing number of floral allotypes cloned, the ABC model has been extended to the ABCDE model (Theissen G., Development of floral organization: store from the MADS house. curr Opin Plant biol.,2001,4(1): 75-85.).

The research of rice molecular genetics finds that the part of the ABCDE flower organ regulation model obtained from the research of species such as dicotyledonous model plants Arabidopsis thaliana and snapdragon is suitable for explaining the molecular regulation of the development of the flower organ of monocotyledonous plants such as rice (Yoshida H and Nagato Y. flower definition in rice.2011, J Exp Bot.,62(14): 4719-. At present, a plurality of MADS-box genes (in New, Wangjiajun, Wangchailin, molecular mechanism of rice floral organ development, molecular plant breeding, 2013,11(4): 617-624), which have similar functions and homologous products with dicotyledonous plants Arabidopsis and antirrhinum, are cloned, however, the rice has a far difference in evolution with Arabidopsis and the like, the morphological structure difference of flowers is large, rice flowers have stamen and pistil reproductive organs similar to those of the dicotyledonous plants, but have no obvious calyx and petal structures of the dicotyledonous plants, and periandral and pistil reproductive floral organs are husk, hull and serous which are obviously different from calyx and petals of the dicotyledonous plants and other monocotyledonous plants.

With the intensive research and the isolation of new mutant genes related to rice floral development, more and more researches show that the major difference between the organ morphologies of monocotyledonous rice and dicotyledonous arabidopsis thaliana flowers still needs to be solved (Hitoshi Y and Yasuo N.flower definition in rice.J Exp Bot,2011,62(14): 4719-. Firstly, whether the palea and palea are homologues of the calyx or not is not supported by sufficient experimental evidence; secondly, the inner and outer lemma are the same organ or different organs without obtaining sufficient evidence; third, it is still unclear whether the rice glumes are organic components of a degenerated flower or an entire flower. Therefore, the genetic regulation mechanism of the rice floral development process cannot form an overall outline like that of arabidopsis thaliana, and new gene cloning and research are needed to clarify some basic problems of rice floral development.

[ summary of the invention ]

The invention aims to solve one of the technical problems and provides a rice floral organ development regulating gene PEH1 and application thereof, wherein the gene is used for regulating and controlling the rice floral organ development, and has important theoretical and practical significance for deeply researching the molecular mechanism of rice reproductive organ development.

The present invention achieves one of the above technical problems:

a rice floral organ development regulation gene PEH1, wherein the gene PEH1 has a nucleotide sequence shown as SEQ ID No. 1.

Further, the nucleotide sequence, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence of SEQ ID No. 1.

Further, the rice floral organ development regulation gene PEH 1-containing plasmid, a plant expression vector and a host cell.

Furthermore, the gene PEH1 and the nucleotide sequence, the allele or the derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence thereof are applied to the regulation of the development of the floral organs of rice.

Furthermore, the plasmid, the plant expression vector and the host cell containing the rice floral organ development regulation gene PEH1 are applied to the rice floral organ development regulation.

The second technical problem to be solved by the invention is to provide a protein encoded by a rice floral organ development regulating gene PEH1 and application thereof, wherein the protein is used for regulating and controlling rice floral organ development, and has important theoretical and practical significance for deeply researching a molecular mechanism of rice reproductive organ development.

The invention realizes the second technical problem in the following way:

a protein coded by a rice floral organ development regulation gene PEH1, which has an amino acid sequence shown in SEQ ID No. 2.

Furthermore, the amino acid sequence or the derivative generated by adding, substituting, inserting or deleting one or more amino acids in the amino acid sequence of SEQ ID No. 2.

Furthermore, the protein and the amino acid sequence thereof are added, substituted, inserted or deleted with one or more amino acids to generate the amino acid sequence or the derivative, and are applied to the regulation of the development of the floral organs of rice.

The invention has the following advantages:

the gene or protein is vital to the normal development of rice floral organs (palea, male and female organs, and the like), the mutation of the gene or protein causes the abnormal formation of rice male and female embryos, the morphological deformity of the palea and the palea to form pepper-like glume flowers, and the gene or protein can be used for modifying the rice floral organs, so that the gene or protein has important theoretical and practical significance for deeply researching the molecular mechanism of rice reproductive organ development.

[ description of the drawings ]

The invention will be further described with reference to the following examples with reference to the accompanying drawings.

FIG. 1 is a structural diagram of a mutation of the PEH1 gene of the present invention.

FIG. 2 is a sequence diagram of the PEH1 gene of the present invention, wherein a is a wild type and b is a mutant type.

FIG. 3 is a bar graph showing the expression levels of the PEH1 gene of the present invention in mutant fro1(t) and Minghui 86.

[ detailed description ] embodiments

The invention relates to a rice floral organ development regulation gene PEH1, wherein the gene PEH1 has a nucleotide sequence shown as SEQ ID No. 1.

The nucleotide sequence, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence of SEQ ID No. 1.

The gene PEH1 and the nucleotide sequence, the allele or the derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence thereof are applied to the regulation of the development of the floral organs of rice.

The invention also relates to a plasmid, a plant expression vector and a host cell containing the rice floral organ development regulating gene PEH 1.

The plasmid, the plant expression vector and the host cell containing the rice floral organ development regulation gene PEH1 are applied to the rice floral organ development regulation.

The invention also relates to a protein coded by the rice floral organ development regulatory gene PEH1, and the protein has an amino acid sequence shown as SEQ ID No. 2.

The amino acid sequence or the derivative generated by adding, substituting, inserting or deleting one or more amino acids in the amino acid sequence of SEQ ID No. 2.

The protein encoded by the rice floral organ development regulatory gene PEH1 is an amino acid sequence or a derivative generated by adding, substituting, inserting or deleting one or more amino acids in an amino acid sequence of the protein and the amino acid sequence, and is applied to the rice floral organ development regulation.

The gene obtaining and function verification steps are as follows:

PEH1 gene mapping: in order to separate genes, the invention firstly constructs a positioning population, normal plants (the genotype is wild type or heterozygote type) in the population are separated by the mutant fro1(t) and hybridized with the indica rice variety 9311 to construct the positioning population, and candidate genes are quickly positioned by adopting a segregant mixed analysis (BSA-seq) method based on high-throughput sequencing.

And (3) verifying candidate mutation sites: the information of the candidate gene is found by using the Rice Genome mutation Project, a pair of specific sequencing primers are designed at two sides of the candidate locus, the DNA of the homozygous wild type and the DNA of the mutant are respectively subjected to PCR amplification, and the products are sent to sequencing and are compared to determine the candidate gene. And analyzing the expression quantity of the wild type and the mutant on RNA by Real-time PCR to further determine the candidate gene.

Functional analysis of the PEH1 gene: transgenic rice with normal phenotype restored by genetic transformation was obtained by transferring a fragment containing the entire PEH1 gene into mutant fro1(t) for functional complementation test.

The invention is based on early discovery of a rice floral organ development mutant fro1(t) (Maobigang, Liuhuaqing, Chenjian, Chengjie, Pengyonghong, Wanfeng, two rice reproductive organ mutants with morphological characteristics and genetic analysis. molecular plant breeding, 2008,6(2): 233-.

Compared with the wild type rice Minghui 86, the mutant fro1(t) has the mutation phenotype mainly shown in that: the plant type is short and small, and the growth vigor is weak; the inner and outer palea slices are twisted, deformed and cohered, the appearance of the spikelet is exactly like that of hot pepper, but the spikelet is always closed and does not bloom, and finally withers; the majority of the minor ingredients are degenerated; transformation of the serosa into a thin leaf organ; the stamen filament is expanded and twisted, the surface of the anther is twisted and deformed, no pollen exists, the number of the stamens is 2-6, part of the stamens are converted into a bottom leaf shape, and the top of the stamen is provided with a pistil-shaped organ similar to a stigma; the ovary develops normally, but most of the style fuses, and 3-4 styles cluster. The mutant fro1(t) cannot fruit and can only be stored in the form of hybrid strains, and is a mutant controlled by a single recessive gene.

The present invention will be further described with reference to the following specific examples.

Example 1: acquisition of rice floral organ development regulation gene PEH1

Rice material: the rice mutant fro1(t) is from tissue-cultured progeny of indica rice variety Minghui 86, and the parent is located as indica rice variety 9311.

Constructing a positioning group: wild phenotype (genotype is wild homozygous or heterozygous) in the segregating population of the mutant fro1(t) is selected to be hybridized with indica rice variety 9311 to construct a locating population F2.

Gene localization: based on the F2 population, performing phenotype selection in the segregation progeny, selecting individuals with 'mutant phenotype' (namely the phenotype is consistent with that of the mutant parent), respectively shearing leaves, mixing the leaves in equal amount, extracting DNA, and constructing a recessive homozygous pool; randomly selecting 50 individuals with a wild phenotype (namely, the phenotype of the individuals is consistent with that of a wild parent), respectively shearing leaves, mixing the leaves in equal amount, extracting DNA, and constructing a dominant heterozygous pool; and (4) selecting a single mutant parent strain to extract DNA for sequencing. Sending the parent individual and the offspring mixed pool to a Nordhea immunogenic bioinformatics technology limited company for whole genome sequencing, and adopting an IlluminaPE (paired end) sequencing method, wherein the read length is 150bp, and the sequencing depth is about 40 multiplied. And (3) taking a Nipponbare genome sequence as a reference, and screening the variation sites based on the two-pool reading data. The mutation sites that match the desired genotype in both pools (homozygous normal genotype in the wild-type pool, homozygous mutant genotype in the mutant pool) are identified as candidate mutation sites.

The total genome was screened to 4 candidate mutant spots (shown in table 1 below), 3 of which were intergenic and therefore excluded. Another candidate mutation site occurs in the LOC _ Os08g39420 gene coding region (+800bp) with 1 base A deletion (TAAAAAT-TAAAAT), which results in a frame shift of the coding sequence, thereby causing premature termination of protein translation of the gene, specifically referring to FIG. 1 (black box indicates exon region, left white box indicates 5-UTR, right white box indicates 3-UTR, black arrow indicates mutation position, number indicates deletion site, number indicates protein translation termination). Therefore, the LOC _ Os08g39420 gene was identified as a candidate gene for the fro1(t) mutant and was designated PEH1 (i.e., abbreviation for PEPER HULL 1).

TABLE 1 annotation of candidate mutation sites and genes involved

Example 2: determination of mutation site and target gene

To verify the results of the above screening, a pair of primers (5'-TACAGAAAGCCCTCAAGGAAGC-3' (shown as SEQ ID No: 3); 5'-AACAACAAGAGGGGGAAATCAG-3' (shown as SEQ ID No: 4)) was designed on both sides of the mutation site of PEH1 gene, and the genomic DNA of Minghui 86 and the mutant were PCR-amplified and the amplified products were sequenced, specifically referring to FIG. 2, wherein the sequence a in FIG. 2 is the wild type and the sequence b in FIG. 2 is the mutant, and the sequencing results revealed that the mutation in the mutant was indeed present.

In order to further verify the relevance of the deletion mutation and the gene thereof to the development of rice floral organs, the total RNA of Minghui 86 and mutant strains (three plants respectively) in some glumes of the heading is extracted and then is reversely transcribed into cDNA. Primers (5'-CAGAAGTCAGCGGATGTAAGGT-3' (shown as SEQ ID No: 5) and 5'-GGAATCGGCACAGCAATCAA-3' (shown as SEQ ID No: 6)) specific to the PEH1 gene sequence are designed to carry out qRT-PCR related experiments. The qRT-PCR reaction condition is 95 ℃ and 30 s; 95 ℃ for 10 s; 20s at 55 ℃; 72 ℃ for 10 s; 40 Cycles. All reactions were performed in 3 biological and 3 technical replicates, with the reference gene for qRT-PCR being OsActin1(LOC _ Os03g50885), and the specific results are shown in fig. 3. A, B, C in FIG. 3 represents the difference of the expression level of the PEH1 gene in three different plants of the mutant fro1(t) and Minghui 86 respectively, and the result shows that the transcription level of the PEH1 in the mutant is obviously reduced compared with that of the wild type.

Example 3: complementary experiment verification of PEH1 gene function

According to the sequence of LOC _ Os08g39420 gene, finding the upstream and downstream sequences of the gene in Nipponbare BAC clone AP014964.1 containing the gene on a website (https:// blast. ncbi. nlm. nih. gov/blast. cgi), designing a whole gene amplification primer, wherein the primer pair respectively contains URS and DRS sequences of CE Entry Vector (Vazyme) and recognition sequences of two endonucleases, and one primer comprises a URS sequence and a recognition sequence of endonucleases EcoRI [5'-ggatcttccagagatgaattcGTTGAAGAACTCGCTGTCCGTG-3' (shown as SEQ ID No: 7) ]; the other primer comprises a DRS sequence and a recognition sequence of endonuclease HindIII (5'-ctgccgttcgacgataagcttGTCGTCAGTCCTCGCATTATTCA-3' (shown as SEQ ID No: 8)), a fragment 6589bp comprises a coding region of the whole gene, a promoter sequence 1320bp upstream of an initiation codon and a regulatory sequence 419bp downstream of a termination codon.

Extracting DNA of indica rice variety Minghui 86 by

HS DNA Polymerase with GC Buffer (TAKALA), PCR conditions of 94 ℃ for 5 sec; 98 ℃ for 10 sec; 60 ℃ for 30 sec; 72 ℃ for 7 min; 32 Cycles; 72 ℃ for 5 min. Using 1% agarose gel electrophoresis, the corresponding DNA fragments were recovered by scooping. This fragment was directly cloned into CE Entry Vector using the Clonexpress Entry One Step Cloning Kit (Vazyme), and the recombinant product was transformed and then subjected to monoclonal sequencing. And (3) completely digesting the clone shake bacteria quality-improving particles with correct sequencing by using endonucleases EcoRI and HindIII, and after electrophoretic separation, cutting a DNA fragment and connecting the DNA fragment to pCAMBIA1300 to construct a complementary vector pCAMBIA1300+ promoter + PEH1+ nos. The plasmid was transformed into rice by electric shock into Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain LBA 4404. Transformation of mutant fro1(t) mature embryo-induced callus by Agrobacterium-mediated genetic transformation (Sujun, Huchangquan, Dian Zhai, Yan Jing Wang, Chen Jie, Wangfeng, 2003), establishment of Agrobacterium-mediated high-efficiency stable transformation system for indica Minghui 86, Fujian agricultural science, 18(4): 209-213).

The invention obtains 23 rice plants which transfer PEH1 independently, and carries out PCR identification on the transgenic plants to find 21 positive plants and 2 negative plants. Comparing the positive PEH1 transgenic rice plant with the contemporary mutant, the spikelet shape is found to be recovered to normal, and the rice plant has 1 complete spike-axis, 2 auxiliary glumes, 1 lemma, 1 palea, 1 pair of serous discs, 6 stamens and 1 pistil. The fertility of the male and female pistils is recovered, and the fruit is normal. Thus, it is further proved by complementation experiments that the gene PEH1 is a floral organ development regulatory gene.

In conclusion, the invention utilizes early screened rice mutant to construct a mixed pool, performs whole genome sequencing, rapidly locates and determines a target gene PEH1, and the gene controls the development of floral organs. Through the function reading of the PEH1 and the relationship of genes at the upstream and the downstream thereof, the method lays a foundation for further exploring the molecular regulation mechanism of the monocotyledon floral development.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Sequence listing

<110> institute of biotechnology of academy of agricultural sciences of Fujian province

<120> rice floral organ development regulation gene PEH1, and protein coded by same and application thereof

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Ala Leu Glu Ser Thr Ile Ile Cys His Gly Met Pro Tyr Pro Lys Asn

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Val Pro Ala Thr Ile Ala Ile Leu Asn Gly Val Pro His Val Gly Leu

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Ser Gly Glu Gln Leu Lys Ser Leu Ala Val Ser Gly Arg Gln Phe Gln

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Lys Thr Ala Arg Arg Asp Ile Ala His Val Val Ala Ser Gly Gly Asn

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Gly Ala Thr Thr Val Ser Ala Thr Met Phe Phe Ala His Lys Val Gly

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Ile Pro Ile Phe Val Thr Gly Gly Ile Gly Gly Val His Arg Asn Gly

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Glu Gln Thr Met Asp Ile Ser Ser Asp Leu Thr Glu Leu Gly Lys Thr

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Pro Val Thr Val Ile Ser Ala Gly Val Lys Ser Ile Leu Asp Ile Pro

165 170 175

Arg Thr Leu Glu Tyr Leu Glu Thr Gln Gly Val Thr Val Ala Ala Tyr

180 185 190

Lys Thr Asn Glu Phe Pro Ala Phe Phe Thr Glu Val Ser Gly Cys Lys

195 200 205

Val Pro Cys Arg Val Asp Ser Pro Glu Glu Cys Ala Lys Ile Ile Tyr

210 215 220

Ala Asn Lys Asn Leu His Leu Gly Ser Gly Ile Leu Ile Ala Val Pro

225 230 235 240

Ile Pro Lys Glu His Ala Ala Ser Gly Asn Ala Ile Glu Ser Ala Ile

245 250 255

Gln Lys Ala Leu Lys Glu Ala Glu Asp Lys Asn Ile Ile Gly Asn Ala

260 265 270

Ile Thr Pro Phe Met Leu Asp Arg Val Lys Val Leu Thr Gly Arg Ser

275 280 285

Ser Leu Glu Ala Asn Ile Ala Leu Val Lys Asn Asn Ala Leu Val Gly

290 295 300

Ala Lys Ile Ala Val Ala Leu Ser Asp Leu His Gln Arg Val Thr Asn

305 310 315 320

Arg Phe Arg Arg Ser Ala Leu

325

<210> 3

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 3

tacagaaagc cctcaaggaa gc 22

<210> 4

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 4

aacaacaaga gggggaaatc ag 22

<210> 5

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 5

cagaagtcag cggatgtaag gt 22

<210> 6

<211> 20

<212> DNA

<213> (Artificial sequence)

<400> 6

ggaatcggca cagcaatcaa 20

<210> 7

<211> 43

<212> DNA

<213> (Artificial sequence)

<400> 7

ggatcttcca gagatgaatt cgttgaagaa ctcgctgtcc gtg 43

<210> 8

<211> 44

<212> DNA

<213> (Artificial sequence)

<400> 8

ctgccgttcg acgataagct tgtcgtcagt cctcgcatta ttca 44

Claims (3)

1. The application of a rice floral organ development regulation gene PEH1 is characterized in that: the gene PEH1 consists of a nucleotide sequence shown as SEQ ID No. 1, and the gene PEH1 is applied to the regulation of the development of rice floral organs.

2. A plasmid, a plant expression vector and a host cell of a rice floral organ development regulation gene PEH1 are applied to the rice floral organ development regulation, and the gene PEH1 consists of a nucleotide sequence shown as SEQ ID No. 1.

3. The application of the protein coded by the rice floral organ development regulatory gene PEH1 is characterized in that: the protein consists of an amino acid sequence shown as SEQ ID No. 2, and is applied to the growth regulation of rice floral organs.

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