CN107083397B - A method for preparing rice moisture-sensitive male sterile material and related genes - Google Patents

Abstract

The invention discloses a method for preparing rice moisture-sensitive male sterile material and related genes. A method for preparing a moisture-sensitive male sterile material for rice, comprising: reducing the activity of a protein in a target plant, reducing the protein content in the target plant, inhibiting the expression of a protein-coding gene in the target plant, or knocking out a protein-coding gene in the target plant , to obtain a humidity-sensitive sterile plant; the protein is the following A1) or A2): A1) the amino acid sequence is the protein of sequence 2 in the sequence table; A2) the amino acid sequence of sequence 2 in the sequence table is passed through one or several amino acid residues substitutions and/or deletions and/or additions of proteins with the same function. The invention obtains the humidity-sensitive male sterile material of rice by controlling a glycosyltransferase OsUGT gene of rice and its encoded protein, and realizes the transformation of rice fertility under different humidity conditions.

Description

Method for preparing rice humidity sensitive male sterile material and related gene

Technical Field

The invention relates to the field of biotechnology, in particular to a method for preparing a rice humidity sensitive male sterile material and a related gene.

Background

Rice has been of great concern as a staple food for more than fifty percent of the world population, both in terms of yield and quality. The hybrid rice breeding technology is an important means for improving the yield and quality of rice, and has been a hot spot and a key point which are concerned and tracked by many scientists. In 1964, Chinese scientist Yuanping discovered 'natural male sterile plants' in Dongting early indica, and through the research on rice male sterility, a three-line method for breeding a rice male sterile line, a male sterile maintainer line and a male sterile restorer line is provided, so that the utilization of rice heterosis is realized. In 1973, the photosensitive nuclear male sterile material 'Nongzai 58S' is bred from Shimingsong in Nongzai 58, and the concept of utilizing natural dual-purpose lines is proposed, so that the problem that the sterile property of the natural sterile plant of japonica rice is difficult to maintain is solved, and the sequence of the two-line hybrid rice breeding in China is also opened. Then, a temperature-sensitive male sterile material is found in the indica rice, and the breeding technology of hybrid rice by a two-line method is further perfected. Compared with the traditional three-line hybrid rice breeding, the two-line hybrid rice breeding has the advantages of simplifying the hybrid breeding and seed production procedures, reducing the seed cost, free matching, no negative effect of sterile cytoplasm, easy transformation of new sterile lines and the like. However, the breeding technology of the two-line hybrid rice based on photo-thermo-sensitive genic male sterility is easily influenced by the photo-thermo conditions, resulting in the failure of breeding, so the seed production technology of the two-line hybrid rice needs to be enhanced and perfected urgently, and the discovery of a new conditional sterile material may bring a new breakthrough for the breeding technology of the two-line hybrid rice.

The plant growth and development not only needs illumination and temperature, but also needs proper humidity. At present, the phenomenon of humidity-sensitive male sterility is only reported in detail in arabidopsis thaliana, Preuss et al find that pollen grains of arabidopsis thaliana male sterile mutant pop1 can normally germinate in an artificial culture box, but cannot hydrate with stigma on the stigma, so that the stigma cannot germinate. However, in high humidity environments, the pollen of pop1 is able to absorb water from the environment and regain activity, thereby adhering to the stigma and germinating out of the pollen tube. Ishiguro et al found that a T-DNA insertion mutant, flaky polen 1-1(fkp1-1), showed little adhesion of pollen grains to the stigma, and also exhibited a phenotype of moisture sensitive male sterility.

At present, only in the patent with Chinese patent number CN201280006361.6, the inventor of the present invention discloses that the OsOSC8 gene function-deletion mutant of rice has the characteristic of humidity-sensitive male sterility, and the specific molecular mechanism is still to be further researched.

Glycosyltransferases (GT; EC 2.4.x.y) are present in almost all living organisms and catalyze the transfer of glycosyl groups from an activated donor molecule to an acceptor molecule, one of the most important biotransformation reactions. UDP-glycosyltransferase (UGT) is used as the largest superfamily in a glycosyltransferase family, participates in a series of physiological and biochemical reactions, and has important biological functions. The UDP-glycosyltransferase reduces or even completely eliminates the biological activity of auxin, cytokinin, abscisic acid, gibberellin, brassinolide, salicylic acid and other plant hormones by glycosylation, thereby influencing the growth and development of plants and the response to adverse conditions. By glycosylation of endogenous and exogenous toxins within cells, transport of the toxin from the interior of the cell to the exterior of the cell or outside the organism is facilitated, thereby reducing or eliminating the toxic effects of the toxin on the cell. Glycosylation can also alter the food flavor of small molecule compounds as well as the molecular properties of solubility, stability, and volatility within plant cells.

Disclosure of Invention

The invention aims to solve the technical problem of how to prepare the humidity-sensitive male sterile rice.

In order to solve the above technical problems, the present invention firstly provides a method for preparing a humidity-sensitive sterile plant.

The method for preparing the humidity-sensitive sterile plant comprises the following steps: reducing the activity of protein in a target plant, reducing the content of the protein in the target plant, inhibiting the expression of the coding gene of the protein in the target plant or knocking out the coding gene of the protein in the target plant to obtain a humidity-sensitive sterile plant;

the protein is named as OsUGT protein and is A1) or A2):

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 2 in the sequence table and has the same function.

The OsUGT protein of A2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The method can be realized by carrying out site-directed mutation on the encoding gene of the protein through a CRISPR/Cas9 system.

In the above method, the gene encoding the protein may be any of the following b1) -b 4):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 1in the sequence table;

b2) the coding sequence is cDNA molecule or DNA molecule of sequence 3 in the sequence table;

b3) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) and encoding OsUGT protein;

b4) hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and encodes a cDNA molecule or a genome DNA molecule of the OsUGT protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence of coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

The target sequence of the coding gene of the OsUGT protein used in the CRISPR/Cas9 system can be a target sequence 1, a target sequence 2 or a target sequence 3;

the target sequence 1 is H1), H2) or H3) as follows:

H1) a nucleotide sequence of 1 st to 20 th sites of a sequence 1in a sequence table;

H2) a DNA sequence derived from H1) having 75% or more than 75% identity to the DNA sequence defined by H1);

H3) a DNA sequence derived from H1) that hybridizes under stringent conditions to a DNA sequence defined by H1);

the target sequence 2 is I1), I2) or I3) as follows:

I1) a nucleotide sequence at position 267-286 of the sequence 1in the sequence table;

I2) a DNA sequence derived from I1) having 75% or more than 75% identity to a DNA sequence defined in I1);

I3) a DNA sequence derived from I1) which hybridizes under stringent conditions with a DNA sequence defined in I1);

the target sequence 3 is J1), J2) or J3) as follows:

J1) the nucleotide sequence at position 343-362 of the sequence 1in the sequence table;

J2) a DNA sequence derived from J1) having 75% or more than 75% identity to a DNA sequence defined by J1);

J3) a DNA sequence derived from J1) which hybridizes under stringent conditions with a DNA sequence defined in J1).

The method can obtain the humidity-sensitive sterile plant by introducing the sgRNA coding gene targeting the target sequence 1, the target sequence 2 or the target sequence 3 and the Cas9 coding gene into the target plant.

In the above method, the gene encoding the sgRNA can be obtained by introducing a recombinant vector containing the gene encoding the sgRNA into the target plant.

The vector may contain a DNA fragment encoding other sequences in the sgRNA except for a spacer (spacer). The DNA encoding the spacer sequence may be the target sequence 1, the target sequence 2 or the target sequence 3.

The vector can be specifically pOs-sgRNA vector.

The recombinant vector can be pOs-sgRNA-OsUGT-spacer1, pOs-sgRNA-OsUGT-spacer2 or pOs-sgRNA-OsUGT-spacer3, the pOs-sgRNA-OsUGT-spacer1, the pOs-sgRNA-OsUGT-spacer2 and the pOs-sgRNA-OsUGT-spacer3 are recombinant vectors obtained by inserting the target sequence 1, the target sequence 2 and the target sequence 3 into the Bsa I recognition sequence of the pOs-sgRNA vector respectively, the pOs-sgRNA-OsUGT-spacer1, the pOs-sgRNA-OsUGT-spacer2 and the pOs-sgRNA-OsUGT-spacer3 respectively express sgRNA1, sgRNA2 and sgRNA3, the sgRNA1 contains an RNA sequence encoded by the target sequence 1, the sgRNA2 contains an RNA sequence encoded by the target sequence 2, and the sgRNA3 contains an RNA sequence encoded by the target sequence 3.

In the above method, the gene encoding Cas9 can be obtained by introducing a recombinant vector containing the gene encoding Cas9 into the plant of interest.

In the above method, the sgRNA encoding gene and the Cas9 encoding gene may be obtained by introducing a recombinant vector containing the sgRNA encoding gene and the Cas9 encoding gene into the target plant. The recombinant vector may specifically be a pH-Ubi-cas9-OsUGT-spacer1, a pH-Ubi-cas9-OsUGT-spacer2 and a pH-Ubi-cas9-OsUGT-spacer3, the pH-Ubi-cas9-OsUGT-spacer1, the pH-Ubi-cas9-OsUGT-spacer2 and the pH-Ubi-cas9-OsUGT-spacer3 are recombinant LR vectors obtained by reacting the pOs-sgRNA-OsUGT-spacer1, the pOs-sgRNA-OsUGT-spacer2 and the pOs-sgRNA-OsUGT-spacer3 with an expression vector pH-Ubi-cas9, respectively. The pH-Ubi-Cas9-OsUGT-spacer1 expresses Cas9 and the sgRNA1, the pH-Ubi-Cas9-OsUGT-spacer2 expresses Cas9 and the sgRNA2, the pH-Ubi-Cas9-OsUGT-spacer3 expresses Cas9 and the sgRNA 3.

In the above method, the humidity-sensitive sterile plant may be a humidity-sensitive male sterile plant. The humidity-sensitive male sterile plant may be a humidity-sensitive pollen sterile plant.

The water loss rate of the humidity sensitive sterile plant pollen is higher than that of the target plant pollen. The water loss rate can be more than or equal to 60% RH and more than or equal to 30%, and specifically can be the water loss rate under the humidity condition that RH is less than 30%.

In order to solve the technical problems, the invention also provides a method for preparing the humidity-insensitive fertility plant.

The method for preparing the humidity-insensitive fertility plant comprises the steps of increasing the activity of OsUGT protein in a receptor plant, increasing the content of the OsUGT protein in the receptor plant or promoting the expression of a coding gene of the OsUGT protein in the receptor plant to obtain the humidity-insensitive fertility plant;

the recipient plant is a humidity-sensitive sterile plant.

In the above method for producing a humidity-insensitive fertility plant, the increase in the content of the OsUGT protein in the recipient plant may be achieved by introducing a gene encoding the OsUGT protein into the recipient plant.

In the above method, the gene encoding the OsUGT protein may be obtained by introducing a recombinant vector containing the gene encoding the OsUGT protein into the recipient plant.

The vector may be pCAMBIA1301 or pU 1301. pU1301 is a vector obtained by replacing the DNA fragment between HindIII and BamHI recognition sequences of pCAMBIA1301 with the Ubiqutin promoter shown at positions 7-2016 in SEQ ID No. 5.

The recombinant vector can be pCAMBIA1301-OsUGT_ProOsUGT or pU1301-OsUGT, said pCAMBIA1301-OsUGT_ProOsUGT is a recombinant vector obtained by replacing a recognition sequence between Pst I and EcoR I of pCAMBIA1301 with a DNA molecule shown in sequence 3, and pU1301-OsUGT is a recombinant vector obtained by replacing a DNA fragment between Sma I and EcoR I recognition sequences of pU1301 with pCAMBIA1301-OsUGT_Pro-a DNA fragment between the recognition sequences of SmaI and EcoRI of OsUGT. The pCAMBIA1301-OsUGT_ProOsUGT and the pU1301-OsUGT can both express OsUGT protein shown in sequence 2.

The humidity-sensitive sterile plant may be a humidity-sensitive male sterile plant. The humidity-sensitive male sterile plant may be a humidity-sensitive pollen sterile plant.

The water loss rate of the humidity-insensitive fertile plant pollen is lower than that of the humidity-sensitive sterile plant pollen. The water loss rate can be more than or equal to 60% RH and more than or equal to 30%, and specifically can be the water loss rate under the humidity condition that RH is less than 30%.

In order to solve the problems, the invention also provides a product for regulating and controlling the humidity sensitivity fertility of the plants.

The product for regulating and controlling the humidity sensitivity fertility of the plant comprises the following M1) or M2):

m1) preparing a humidity sensitive sterile plant product having the following functions: reducing the activity of OsUGT protein in the plant, reducing the content of OsUGT protein in the plant, inhibiting the expression of the coding gene of OsUGT protein in the plant or knocking out the coding gene of OsUGT protein in the plant;

m2) preparing a humidity insensitive fertile plant product having the following functions: increasing the activity of OsUGT protein in the plant, increasing the content of OsUGT protein in the plant or promoting the expression of the coding gene of OsUGT protein.

M1) the product can be a reagent and/or an instrument required for site-directed editing of the coding gene of the OsUGT protein by using a CRISPR/Cas9 system.

The reagent may be reagent 1, reagent 2, reagent 3, reagent 4, or reagent 5:

the reagent 1 is any one of R1) -R3):

r1) sgRNA targeting the target sequence 1;

r2) sgRNA targeting the target sequence 2;

r3) sgRNA targeting the target sequence 3;

the reagent 2 is a composition consisting of the reagent 2 and Cas 9;

the reagent 3 is a recombinant vector containing a coding gene of any one of RNA of R1) -R3);

the reagent 4 is a composition consisting of the reagent 3 and a recombinant vector containing a gene encoding Cas 9;

the reagent 5 is a recombinant vector containing a coding gene of any one of RNA from R1) to R3) and a coding gene of Cas 9.

The recombinant vector of the reagent 3 can be the pOs-sgRNA-OsUGT-spacer1, the pOs-sgRNA-OsUGT-spacer2 or the pOs-sgRNA-OsUGT-spacer 3.

The recombinant vector of the reagent 5 may be the pH-Ubi-cas9-OsUGT-spacer1, the pH-Ubi-cas9-OsUGT-spacer2 or the pH-Ubi-cas9-OsUGT-spacer 3.

M2) the product may be a coding gene of the OsUGT protein or a recombinant vector containing the coding gene of the OsUGT protein. The recombinant vector can be the pCAMBIA1301-OsUGT_Pro-OsUGT or said pU 1301-OsUGT.

In order to solve the technical problems, the invention also provides application of the OsUGT protein or the coding gene of the OsUGT protein in regulating and controlling plant humidity sensitive fertility.

In the present invention, the plant, the recipient plant and the target plant may be any of the following 1) or 2):

1) a monocot or dicot;

2) a rice plant.

In the invention, the humidity-sensitive sterile rice can be fertile under the humidity condition of more than 60 percent, and specifically can be fertile under the high humidity condition (RH is more than or equal to 80 percent), sterile under the natural condition (RH is more than or equal to 30 percent) and sterile under the humidity condition of RH is less than 30 percent. The temperature of each humidity condition and the natural condition can be 27-32 ℃.

According to the invention, a glycosyl transferase OsUGT gene of rice and a coding protein thereof are controlled to obtain the humidity-sensitive male sterile material of rice, so that the conversion of rice fertility under different humidity conditions is realized. The rice CRISPR mutant obtained by the invention has no obvious difference with a wild type in the vegetative growth stage; in the reproductive growth stage, the morphology and the integrity of floral organs and the activity of pollen are similar to those of the wild type, but eventually show sterility. Intensive research shows that under natural conditions (Tm is 27-32 ℃, RH is 30-60%), pollen grains of the mutant rapidly lose water and shrink in the air, and cannot adhere to stigma, so that the maturing rate of the mutant is less than 10%; under high humidity conditions (Tm is 27-32 ℃, RH is more than or equal to 80%), pollen grains can normally adhere to stigma, and the maturing rate can be recovered to more than 80%. The humidity sensitive male sterile material can be used as a female parent to produce hybrid seeds by matching with dominant varieties. The discovery and utilization of the material not only provides a new sterile line material for the two-line method cross breeding of rice, but also lays a theoretical foundation for the cross breeding of other gramineous crops.

Drawings

Fig. 1 is sequence change of the OsUGT gene in 5 strains CRISPR homozygous mutant. Underlined sequences are drawn as designed target sequences; the sequence within the rectangular box is a PAM motif; nucleotide sequence representing deletion

Fig. 2 shows the adhesion of pollen grains to stigma under natural and moisturizing conditions. A is the adhesion condition of pollen grains of the wild type medium flower No. 11 on the stigma under natural conditions; b is the adhesion condition of pollen grains of the mutant cugt on the stigma under the natural condition; c is the adhesion condition of pollen grains of the wild type medium flower No. 11 on the stigma after the moisture preservation treatment; d is the adhesion condition of pollen grains of the mutant form cugt on the stigma after the moisturizing treatment. Scale 500 μm.

FIG. 3 shows the adhesion of pollen grains after crossing the mutant line cugt with the wild type, flower No. 11. And (3) hybridizing and pollinating the wild type medium flower No. 11 and the mutant cugt for 1h, and then taking the stigma to dye and observe with water-soluble aniline blue. A: cubt is male parent and is multiplied by ZH 11; b: cubt is male parent and ZH11 female parent; cross-pollination is accomplished by artificial pollination. Scale 500 μm.

Fig. 4 is a phenotype of CRISPR homozygous mutants. Wherein ZH11 is Zhonghua No. 11, and cugt is homozygous mutant. The rice ears pointed by the arrows are subjected to moisture preservation treatment, and the rest rice ears are blossomed and fruited under natural conditions.

FIG. 5 is an analysis of expression pattern of OsUGT gene.

FIG. 6 shows the identification of a part of transgenic plants. Wherein A is pCAMBIA1301-UGT_Pro-identification of OsUGT transgenic plants, lanes 1-11, 13-17, 19 and 22 are all positive plants, the remainder are negative plants; b is the identification result of the pU1301-OsUGT transgenic plant, and lanes 1-12, 14-16 and 18-22 are all positive plants; m is DNA molecular weight standard, P is positive control, N is negative control.

FIG. 7 shows phenotypic identification of OsUGT transgenic plants. A is the water loss rate of the pollen, and cugt represents cugt 1-8; b and C are the phenotype and the setting percentage of CTP and pCAMBIA1301 control plants under natural conditions (namely, moisture retention treatment is not carried out), pCAMBIA1301 is the pCAMBIA1301 control plant, and CTP1, CTP2 and CTP3 are three positive strains of CTP; d and E are the phenotype and seed set respectively of OE and pU1301 control plants under natural conditions (i.e. without moisturizing treatment), pU1301 are pU1301 control plants, and OE1, OE2 and OE3 are the three positive lines of OE.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The pOs-sgRNA vector (Jin Miao et al, Targeted mutagenesis using CRISPR-Cas system, Cell Research (2013)23:1233-1236.) in the following examples was obtained from the applicant after approval of the Dianthus caryophyllus professor at Beijing university, and the biomaterial was used only for repeating the experiments related to the present invention and was not used for other purposes.

The expression vector pH-Ubi-Cas9 in the following examples is pH-Ubi-Cas9-7 in the literature (Jin Miao et al, Targeted administration in rice CRISPR-Cas system, Cell Research (2013)23:1233-1236.) which was publicly available from the applicant after approval of professor Dianthus Pieris university, Beijing university, and which was used only for repeating the experiments related to the present invention and was not used for other purposes.

The Zhonghua No. 11 (ZH11) in the following examples is Zh11 in the literature (Peng, et al, Ectopic expression of OsLFL1in rice expressions Ehd1by binding on patients promoter, Biochemical and biological Research Communications 360(2007)251 and 256), which is publicly available from the Applicant and is used only for repeating the experiments related to the present invention and is not used for other purposes.

The expression vector pCAMBIA1301 in the following examples is a product of Beijing Huayuyo Biotechnology Co., Ltd, the expression vector pU1301 is a vector obtained by replacing a DNA fragment between HindIII and BamH I recognition sequences of pCAMBIA1301 with the Ubiqutin promoter shown in positions 7-2016 of sequence 5, and positions 1-6 and 2017-2022 of sequence 5 are recognition sequences of HindIII and BamH I, respectively.

Example 1 obtaining of moisture-sensitive Male sterile Rice

The inventor of the invention finds a gene related to humidity-sensitive male sterility in rice, namely OsUGT gene. Wherein, the coding sequence of the OsUGT gene is the 2 nd-1486 th position of the sequence 1in the sequence table, the sequence of the protein coded by the OsUGT gene (namely the OsUGT protein) is the sequence 2, and the genome sequence of the OsUGT gene is the sequence 3.

In the embodiment, the rice OsUGT gene is knocked out by a CRISPR/Cas9 gene editing technology to obtain the mutant cugt with the humidity-sensitive male sterility phenotype. The specific operation method comprises the following steps:

(1) target sequences

The target sequences used were target sequence 1, target sequence 2 and target sequence 3, target sequence 1 was 5'-CATGCAGTCGCCAGAGAATG-3' (positions 1-20 of sequence 1), target sequence 2 was 5'-TCCCCAGCCGCGAGCTCGCG-3' (positions 267-286 of sequence 1) and target sequence 3 was 5'-GCGCAGACCCGCCGACGCCG-3' (positions 343-362 of sequence 1).

(2) Construction of intermediate vectors

Two primers of each target sequence form a dimer structure through annealing reaction, and are connected with a vector framework of pOs-sgRNA vector after being cut by BsaI enzyme, so that a correct recombinant vector containing the rice OsUGT gene target sequence is obtained.

Wherein, the name of the recombinant vector containing the target sequence 1 is pOs-sgRNA-OsUGT-spacer1, and the sequences of two primers of the target sequence 1 are as follows:

OsUGT-SgRNA1-F：5’-GGCACATGCAGTCGCCAGAGAATG-3’

OsUGT-SgRNA1-R：5’-AAACCATTCTCTGGCGACTGCATG-3’

the recombinant vector containing the target sequence 2 is named pOs-sgRNA-OsUGT-spacer2, and the sequences of two primers of the target sequence 2 are as follows:

OsUGT-SgRNA2-F：5’-GGCATCCCCAGCCGCGAGCTCGCG-3’

OsUGT-SgRNA2-R：5’-AAACCGCGAGCTCGCGGCTGGGGA-3’

the recombinant vector containing the target sequence 3 is named pOs-sgRNA-OsUGT-spacer3, and the sequences of two primers of the target sequence 3 are as follows:

OsUGT-SgRNA3-F：5’-GGCAGCGCAGACCCGCCGACGCCG-3’

OsUGT-SgRNA3-R：5’-AAACCGGCGTCGGCGGGTCTGCGC-3’

(3) construction of expression vectors

And (3) catalyzing the recombinant vector containing the rice OsUGT gene target sequence and the expression vector pH-Ubi-cas9 in the step (2) by using invitrogen LR clone II to carry out LR reaction respectively to obtain the recombinant vectors with correct sequences, namely pH-Ubi-cas9-OsUGT-spacer1, pH-Ubi-cas9-OsUGT-spacer2 and pH-Ubi-cas9-OsUGT-spacer 3. pH-Ubi-Cas9-OsUGT-spacer1 expresses Cas9 and sgRNA1, and the sgRNA1 contains an RNA sequence coded by a target sequence 1; pH-Ubi-Cas9-OsUGT-spacer2 expresses Cas9 and sgRNA2, and the sgRNA2 contains an RNA sequence coded by a target sequence 2; pH-Ubi-Cas9-OsUGT-spacer3 expresses Cas9 and sgRNA3, and the sgRNA3 contains an RNA sequence encoded by a target sequence 3.

An invitrogen LR clone II is used for catalyzing pOs-sgRNA vector to carry out LR reaction with an expression vector pH-Ubi-cas9 to obtain a recombinant vector pH-Ubi-cas9-sgRNA as a control vector.

Respectively introducing pH-Ubi-cas9-OsUGT-spacer1, pH-Ubi-cas9-OsUGT-spacer2 and pH-Ubi-cas9-OsUGT-spacer3 into agrobacterium EHA105 to obtain recombinant bacteria EHA105-pH-Ubi-cas9-OsUGT-spacer1, EHA105-pH-Ubi-cas9-OsUGT-spacer2 and EHA105-pH-Ubi-cas9-OsUGT-spacer 3. The pH-Ubi-cas9-sgRNA was introduced into Agrobacterium EHA105 to obtain recombinant strain EHA105-pH-Ubi-cas9-sgRNA as a control strain.

(4) Obtaining of humidity sensitive male sterile rice material

Impregnating mature embryo embryogenic callus of rice with agrobacterium to obtain humidity-sensitive male sterile rice, wherein the strains used in the step (3) are EHA105-pH-Ubi-cas9-OsUGT-spacer1, EHA105-pH-Ubi-cas9-OsUGT-spacer2 and EHA105-pH-Ubi-cas9-OsUGT-spacer3, each strain independently transforms the mature embryo embryogenic callus of rice, and the strain is EHA105-pH-Ubi-cas9-sgRNA to obtain empty vector control rice:

selecting full and mature seeds of Zhonghua No. 11, removing glumes, placing the seeds in 70% ethanol for surface disinfection for 5min, then disinfecting the seeds in 0.1% HgCl for 10min (or disinfecting the seeds in 20% NaClO for 20min), washing the seeds with sterile water for several times after the disinfection is finished, enabling the surfaces of the seeds not to have residual disinfection substances as far as possible, and placing the treated seeds in the sterile water for soaking for 5-15 h. In a clean bench, the embryo is cut and placed on NB2 induction medium, the section is contacted with the medium, about 20 seeds are placed in each culture dish, and dark culture is carried out for 4-5 weeks at 25 ℃. Transferring the induced callus onto NB1 subculture medium, and culturing at 25 deg.C in dark for 2-3 weeks; the light yellow dense embryogenic callus was picked and transferred to new NB1 subculture medium for 2-3 weeks. The well-grown yellow compact embryogenic callus was selected and cut to 2mm size, transferred to new NB1 subculture medium and cultured in the dark for 4-6 days at 25 ℃ for transformation. Soaking callus in fresh AAM-AS resuspended OD₆₀₀About 0.5 of Agrobacterium for about 20min, with shaking from time to time. And pouring out the bacterial liquid, sucking the bacterial liquid by using a liquid transfer gun, putting the callus blocks on a culture dish paved with filter paper for 5-10min, sucking residual liquid on the surface of the callus blocks, transferring the callus blocks to an NB2C co-culture medium covered with a layer of filter paper, and culturing for 3-4 days at 25 ℃. Transferring the co-cultured callus to NB1S1 culture medium for screening and culturing for two weeks, transferring to NB1S2 screening culture medium for dark culturing for two generations after two weeks, wherein 15 days each generation is about, most of the callus is browned about 10 days after screening, and then milky resistant callus grows on the edge of the browned tissue again. The bright yellow resistant callus is transferred to RE1 differentiation culture medium for differentiation culture, and the dark culture is firstly carried out for 1 week, and then the dark culture is carried out for 2-3 weeks under light. Transferring the callus with adventitious bud to RE2 differentiation medium, culturing for 2 weeks, and if the state is not good, continuing to induce differentiation on RE 2. Transferring the differentiated plant to 1/2MS rooting medium when the plant grows to 2-3cm, culturing for about two weeks, opening the container sealing film when the plantlet grows to about 10cm, hardening the plantlet for 5-7 days, and transplanting to culture room or field to obtain T₀And (5) plant generation.

The culture medium and the components thereof are as follows:

NB2 induction medium: n6 culture medium +2 mg/L2,4-D +30g/L sucrose +300mg/L hydrolyzed casein, 7g/L agar powder, pH 5.8;

NB1 subculture medium: n6 culture medium +0.5mg/L2,4-D +30g/L sucrose +300mg/L hydrolyzed casein, 7g/L agar powder, pH 5.8;

NB2C co-medium: 2 mg/L2,4-D, 30g/L sucrose, 300mg/L hydrolyzed casein, 7g/L agar powder, 10g/L glucose, 100 mu mol/L Acetosyringone (AS) and pH5.2, wherein the culture medium is N6; NB1S1 medium: n6 culture medium +0.5mg/L2,4-D +30g/L sucrose +300mg/L hydrolyzed casein +50mg/L hygromycin +250mg/L ticarcillin, 7g/L agar powder, pH5.8; NB1S2 medium: n6 culture medium, 0.5mg/L2,4-D, 30g/L sucrose, 300mg/L hydrolyzed casein, 100mg/L hygromycin, 200mg/L ticarcillin, 7g/L agar powder, and pH is 5.8;

AAM-AS: AAM salt and amino acid + MS vitamin +100 μmol/L Acetosyringone (AS), pH 5.2;

RE1 differentiation medium: MS salt and vitamin +300mg/L hydrolyzed casein +1 mg/L6-BA +0.5mg/L KT +0.2mg/L ZT +0.25mg/L NAA +100mg/L hygromycin +250mg/L ticarcillin +30g/L sucrose +30g/L sorbitol, 10g/L agar powder, pH 5.8;

RE2 differentiation medium: MS salt and vitamin +300mg/L hydrolyzed casein +1 mg/L6-BA +0.5mg/L KT +0.2mg/L ZT +0.25mg/L NAA +50mg/L hygromycin +30g/L sucrose +30g/L sorbitol, 10g/L agar powder, pH 5.8;

1/2MS rooting culture medium: 1/2MS culture medium +0.5mg/L NAA, 7g/L agar powder, pH 5.8.

(5) Identification of Positive plants

Converting T of step (4)₀And extracting a genome from the plant to perform PCR and sanger sequencing identification, wherein the used primers are OsUGT-CRISPR-F: 5'-GCCAAGAAATGCCATAAGC-3', respectively; OsUGT-CRISPR-R: 5'-CCGACGACCCACACGAAGTT-3' are provided. If homozygous mutation occurs, the plant is an effective gene knockout plant and can be at T₀Directly observing the phenotype; if the mutation is heterozygous, it is also required to be at T₁The phenotype of the homozygous mutant was observed after progeny segregation.

Transgenic T₀Generating 49 strains in total, amplifying by PCR, saThe identification and analysis are carried out by the nger sequencing, wherein 5 strains are homozygous mutants, 31 strains are heterozygotes, 13 strains have no change in target sequence, and the sequence changes of 5 strains (cugt) (cugt1-7, cugt1-8, cugt1-16, cugt2-1 and cugt2-13) of the OsUGT gene homozygous mutant are shown in a figure 1. Wherein the cugt1-7, the cugt1-8 and the cugt1-16 are mutants obtained by mutation at a target sequence 1, 5 nucleotides (AAAAA) are inserted into the cugt1-7 at the target sequence 1 to cause the protein coded by the OsUGT gene to have frame shift mutation, 16 nucleotides are deleted from the cugt1-8 at the target sequence 1 to cause the protein coded by the OsUGT gene to have frame shift mutation, and 3 nucleotides are deleted from the cugt1-16 at the target sequence 1 to cause the protein coded by the OsUGT gene to have deletion mutation; the cugt2-1 and the cugt2-13 are mutants obtained by mutation at a target sequence 2, the cugt2-1 is deleted for 45 nucleotides at the target sequence 2, so that the loss mutation occurs on the protein coded by the OsUGT gene, and the cugt2-13 is inserted for 1 nucleotide at the target sequence 2, so that the frame shift mutation occurs on the protein coded by the OsUGT gene.

Phenotypic analysis shows that 5 OsUGT gene homozygous mutant lines have no obvious difference with wild type flower No. 11 in vegetative growth stage, flowering time and the like, also have a set of complete normal flower organs and normal pollen activity, but finally show male sterility (figure 4). The germination experiment of the stigma of the pollen shows that pollen grains of 5 OsUGT gene homozygous mutant strains can not normally adhere to the stigma, but can recover the adhesion to the stigma under the high-humidity condition (figure 2). The experimental method for pollen stigma germination is as follows: carrying out self-pollination on the wild type medium flower No. 11 and the mutant cugt for 1h, and then taking the stigma to dye with water-soluble aniline blue for observation. And (3) natural growth conditions: tm is 30-39 deg.C, RH is 40-70%; and (3) moisturizing treatment: the fresh-keeping film wraps the inflorescence in the flowering period, and the humidity can reach 80-100%.

Under natural conditions, 5 OsUGT gene homozygous mutant lines and wild type middle flower No. 11 are mutually crossed to observe the germination condition of pollen on stigma, and the fact that pollen grains of the wild type can normally adhere to the stigma of the homozygous mutant line (B in figure 3), but the pollen grains of the homozygous mutant line cannot adhere to the stigma of the wild type (A in figure 3) is found, and the homozygous mutant line belongs to male sterility.

The partially tillered tassel of the homozygous mutant line is subjected to moisture-keeping treatment at the flowering stage, and the rest tillered tassel is not subjected to any treatment (namely, natural conditions, Tm is 27-32 ℃, and RH is 30-60%). The moisturizing treatment method comprises the following steps: in the flowering period of rice, a preservative film or a preservative bag is used for wrapping the whole inflorescence, so that the humidity of the environment where the flower spike is located is maintained at 80-100%, the temperature is maintained at 27-32 ℃, the time for carrying out moisture preservation treatment on the flower spike is 7 days, and the maturing rate under different conditions is counted after the seeds are mature, as shown in fig. 4. Statistical results show that the setting rate (96.25 +/-0.02%) of the wild-type rice spike (ZH11) without any treatment and the setting rate (81.91 +/-0.08%) of the moisturizing rice spike have no obvious difference, the setting rate (10 rice spikes, the average setting rate is 80.06 +/-4.53%) of the moisturizing rice spike in the mutant has no obvious difference with the wild-type rice spike, the setting rate (n ═ 10) of the non-moisturizing rice spike is far lower than the setting rate (the average setting rate of 10 rice spikes without moisturizing is 8.25 +/-4.18%) of the moisturizing rice spike, the setting rate without moisturizing treatment between mutant homozygosis has no obvious difference, the setting rate with moisturizing treatment between homozygous mutants has no obvious difference, and the phenotype and the setting rate of the moisturizing treatment between the empty vector control rice and the wild-type rice have no obvious difference whether the treatment or not. The results show that the rice has a humidity-sensitive male sterility phenotype after OsUGT gene mutation, and the rice is sterile under natural conditions and fertile in high humidity environment.

Example 2 expression analysis of Rice moisture-sensitive Male sterility-related Gene OsUGT

Transcript analysis was performed on anthers and mature pollen of wild type flower No. 11 developed to 9-10, 11-12, 13-14 and other 15 tissue sites, and it was found that OsUGT was specifically expressed in anthers, particularly anthers developed to 9-10, and that little expression was detected in other tissue sites (FIG. 5).

Example 3 functional analysis of Rice moisture-sensitive Male sterility-related Gene OsUGT Gene

In order to determine whether the OsUGT gene controls the characters of the rice mutant cubt humidity-sensitive male sterility, the inventor further verifies through a transgenic experiment. In the embodiment, the normal OsUGT gene and the regulatory sequence thereof are transferred into the mutant cugt by using a transgenic technology and expressed, and whether the humidity-sensitive male sterility character of the mutant can be recovered or not is observed. The specific experimental steps are as follows:

(1) cloning of the fragment of interest:

extracting genome DNA of young leaves of No. 11 flowers in wild rice as a template, and amplifying the full-length sequence of the gene by using a primer pair specific to the OsUGT gene; the promoter region with the length of about 1500bp before the OsUGT gene ATG is amplified by using the specific primer of the OsUGT gene promoter segment. The primer pairs used were as follows:

OsUGT gene full-length amplification primers:

OsUGT-F：5’-GGGGTACCCCATGCAGTCGCCAGAGAATGC-3’

OsUGT-R：5’-CCGGAATTCCGGCTCTTCATTAAACCTCACATTGAATA-3 '(the primer is located on the 3' UTR)

OsUGT promoter segment amplification primers:

OsUGT_Pro-F：5’-AACTGCAGAACCAATGCATTGGTTGCGACTTGATTTATGCT-3’

OsUGT_Pro-R：5’-GGGGTACCCCGGCGGCACCGACCCGCTC-3’

(2) construction of expression vector:

cutting the amplified OsUGT gene full-length sequence with correct sequence by Kpn I and EcoR I, cutting the amplified promoter segment (sequence 4 in the sequence table) with correct sequence by Pst I and Kpn I, connecting the two cut DNA segments with the expression vector pCAMBIA1301 by the vector skeleton obtained by cutting the expression vector pCAMBIA1301 by Pst I and EcoR I to obtain a recombinant vector, and naming the recombinant vector with correct sequence as pCAMBIA1301-OsUGT_Pro-OsUGT。pCAMBIA1301-OsUGT_ProOsUGT can express OsUGT protein shown in the sequence 2, and the promoter is an OsUGT gene promoter segment.

Mixing pCAMBIA1301-OsUGT_Pro-DNA fragment containing OsUGT gene obtained by cutting OsUGT with SmaI and EcoRI enzyme andthe expression vector pU1301 is connected with a vector framework obtained after enzyme digestion of SmaI and EcoRI to obtain a recombinant vector containing a complete Ubiqutin promoter, the recombinant vector is named as pU1301-OsUGT, the pU1301-OsUGT can express OsUGT protein shown in a sequence 2, and the promoter is the Ubiqutin promoter.

(3) Obtaining of transgenic plants:

mixing pCAMBIA1301-UGT_ProOsUGT, pU1301-OsUGT, pCAMBIA1301 (as empty vector control) and pU1301 (as empty vector control) are respectively introduced into agrobacterium EHA105 to obtain recombinant strain EHA105-pCAMBIA1301-UGT_ProOsUGT, EHA105-pU1301-OsUGT, EHA105-pCAMBIA1301 and EHA105-pU 1301.

According to the method of the embodiment step (2), the four recombinant bacteria are used for transforming the cugt1-8 of the embodiment 2 to obtain a transgenic plant, and the EHA105-pCAMBIA1301-UGT is used_ProPlants obtained from OsUGT and EHA105-pU1301-OsUGT are named CTP plant and OE plant respectively,

transgenic plants derived from EHA105-pCAMBIA1301 and EHA105-pU1301 were designated pCAMBIA1301 control plants and pU1301 control plants, respectively.

(4) Plant identification and phenotype confirmation:

CTP plants are identified by CAPS marker analysis, the primer pairs are 5'-CTTTCGTGCTGTTACCTGTCG-3' (located in the promoter region of OsUGT gene) and 5'-CCGACGACCCACACGAAGTT-3' (located in the coding region of OsUGT gene), and the length of the amplification product is 1286 bp. After cleavage with KpnI, two fragments (A in FIG. 6) of 965bp and 321bp in length were obtained using pCAMBIA1301-UGT_ProOsUGT vector as positive control, and pCAMBIA1301 vector as negative control. When identifying OE plants, a primer is respectively designed on an overexpression vector and a gene sequence, the specific sequences are 5'-CATGGCTGACGAGGCGATG-3' (located in an OsUGT gene coding region) and 5'-TGATGGCATTTGTAGGTG-3' (located on a pU1301 vector), the amplification length is 852bp (B in figure 6), the pU1301-OsUGT vector is used as a positive control, and the pU1301 vector is used as a negative control.

Detecting T₀Substituted CTP positive plant and T₀Loss of water in air for pollen of OE-positive plantsRate and using pCAMBIA1301 control plants, pU1301 control plants, cugt1-8 of example 2 and middle flower No. 11 (wild type, ZH11) as controls, the assay was as follows:

by using an Olympus CX21 microscope and Olympus measuring software USB2.0, the volume change caused by pollen dehydration can be continuously photographed at regular time, dehydrated pollen can be shrunken and is easily distinguished from un-dehydrated pollen, and the proportion of the dehydrated pollen in the total pollen at each time point is counted, so that the water loss rate of the pollen is calculated. The whole process detects the pollen in the same visual field, namely the visual field which can not be observed movably before all the pollen loses water completely. At least 3 repeats are guaranteed per material.

Partial results are shown in FIG. 7, A, and the results show, T₀Substituted CTP positive plant and T₀The water loss rate of pollen of an OE-generation positive plant under natural conditions (Tm is 27-32 ℃, and RH is 30-60%) is basically consistent with that of a wild type, while the water loss rate of pollen of a pCAMBIA1301 control plant, a pU1301 control plant and cugt1-8 under natural conditions is basically consistent, which shows that the mutation of the OsUGT gene can lead to the acceleration of the water loss rate of the pollen, and the OsUGT gene can lead to the loss of the phenotype of the acceleration of the water loss rate of the pollen of the mutant cugt 1-8.

Selecting three T₀The tillers of the CTP-substituted positive plants were randomly divided into two groups, one group was not treated, and the other group was subjected to moisturizing treatment according to the moisturizing treatment method in example 1, and pCAMBIA1301 control plants and medium flower No. 11 (wild type) were used as controls.

Selecting three T₀The OE positive plants were divided randomly into two groups, one group was left untreated, and the other group was treated with moisturizing treatment as in example 1, using pU1301 control plants and Zhonghua No. 11 (wild type) as controls.

The results show that the fruit setting rates of CTP positive plants, OE positive plants and Zhonghua No. 11 under the moisture preservation treatment and the moisture preservation treatment are not obviously different, the fruit setting rates of pCAMBIA1301 control plants under the moisture preservation treatment are not obviously different from those of CTP positive plants, the fruit setting rates under the moisture preservation treatment (8.78 +/-1.28%) are far lower than those of CTP positive plants (the fruit setting rates of moisture preservation treatment tillers are 81.48 +/-1.17%, 79.92 +/-3.79% and 79.90 +/-1.68% respectively), the fruit setting rates of pU1301 control plants under the moisture preservation treatment are not obviously different from those of OE positive plants, and the fruit setting rates under the moisture preservation treatment (8.56 +/-1.22%) are far lower than those of OE positive plants (the fruit setting rates of moisture preservation treatment tillers are 68.48 +/-5.78%, 82.76 +/-5.33% and 74.54 +/-1.17% respectively). The OsUGT gene can make the mutant cugt1-8 lose the phenotype of humidity-sensitive male sterility.

<110> institute of plant of Chinese academy of sciences

<120> method for preparing rice humidity sensitive male sterile material and related gene

<160>5

<170>PatentIn version 3.5

<210>1

<211>1486

<212>DNA

<213> Rice

<400>1

catgcagtcg ccagagaatg cggcaccgcg cgtgtacttc atcccgttcc cgacgccggg 60

gcacgcgctg ccgatgtgcg acctcgcgcg cctcttcgcg tcccgcggcg ccgacgcgac 120

gctcgtcctc acgcgcgcca acgccgccag gctcggcggc gccgtcgccc gcgccgccgc 180

cgcgggatca cggatccgcg tccacgcgct cgccctgccc gccgaggccg cggggctcac 240

gggcggccac gagagcgccg acgacctccc cagccgcgag ctcgcggggc ccttcgccgt 300

cgccgtcgac ctcctcgcgc cgctcttcgc cgacctcctg cggcgcagac ccgccgacgc 360

cgtggtgttc gacggcgtcc tcccgtgggc tgccaccgcg gcggccgagc tccgcgtccc 420

gcggtacgcg ttcacgggga cggggtgctt cgcgctctcg gtgcagcgag cactgctgct 480

ccacgccccg caggacggcg tcgcgtcgga cgacgagccg ttcctcgtgc cgggcctccc 540

cgacgcggtg cggctcacca agtcgaggct cgccgaggcg accctccccg gcgcgcactc 600

gcgggagttc ttgaaccgaa tgttcgacgg cgagcgcgcc acgacggggt gggtggtcaa 660

ctccttcgcc gacctagagc agaggtacat cgagcactac gagaaggaga cggggaagcc 720

cgtgttcgcc gtcgggccgg tctgcctcgt caacggcgac ggcgacgacg tcatggagcg 780

cgggcgcggc ggggaaccgt gcgccgccac ggacgccgcg cgcgcgctgg cgtggctcga 840

cgcgaagccc gcgcggtcgg tggtgtacgt ctgcttcggc agcctcaccc ggttcccgga 900

cgagcaggtc gcggagctcg gcgcggggct cgcgggctcc ggcgtgaact tcgtgtgggt 960

cgtcgggggc aagaacgcgt cggcggcgcc gctgctcccg gacgtcgtgc acgccgccgt 1020

gtcgtccggc cgcggccacg tgatcgcggg gtgggcgccg caggtggcgg tgctgaggca 1080

cgcggcggtg ggcgcgttcg tgacgcactg cgggtggggc gcggtgacgg aggcggcggc 1140

ggccggggtg ccggtgctgg cgtggccggt gttcgcggag cagttctaca acgaggcgct 1200

ggtggtcggg ctcgccggca cgggcgccgg cgtcggcgcg gagagggggt acgtgtgggg 1260

aggcgaggag tccggcgggg tggtggtgtg cagggagaag gtggcggaga gggtgcgcgc 1320

cgccatggct gacgaggcga tgcggcggcg ggcggaggag gtcggcgagc gcgcgcgccg 1380

cgccgtcgag gtgggcgggt cgtcgtacga cgccgtcggc gcgctgctgg aggatgtgcg 1440

gcgccgggag atggcggcgg atccacggaa cgtgaaggaa gtatga 1486

<210>2

<211>494

<212>PRT

<213> Rice

<400>2

Met Gln Ser Pro Glu Asn Ala Ala Pro Arg Val Tyr Phe Ile Pro Phe

1 5 10 15

Pro Thr Pro Gly His Ala Leu Pro Met Cys Asp Leu Ala Arg Leu Phe

20 25 30

Ala Ser Arg Gly Ala Asp Ala Thr Leu Val Leu Thr Arg Ala Asn Ala

35 40 45

Ala Arg Leu Gly Gly Ala Val Ala Arg Ala Ala Ala Ala Gly Ser Arg

5055 60

Ile Arg Val His Ala Leu Ala Leu Pro Ala Glu Ala Ala Gly Leu Thr

65 70 75 80

Gly Gly His Glu Ser Ala Asp Asp Leu Pro Ser Arg Glu Leu Ala Gly

85 90 95

Pro Phe Ala Val Ala Val Asp Leu Leu Ala Pro Leu Phe Ala Asp Leu

100 105 110

Leu Arg Arg Arg Pro Ala Asp Ala Val Val Phe Asp Gly Val Leu Pro

115 120 125

Trp Ala Ala Thr Ala Ala Ala Glu Leu Arg Val Pro Arg Tyr Ala Phe

130 135 140

Thr Gly Thr Gly Cys Phe Ala Leu Ser Val Gln Arg Ala Leu Leu Leu

145 150 155 160

His Ala Pro Gln Asp Gly Val Ala Ser Asp Asp Glu Pro Phe Leu Val

165 170 175

Pro Gly Leu Pro Asp Ala Val Arg Leu Thr Lys Ser Arg Leu Ala Glu

180 185 190

Ala Thr Leu Pro Gly Ala His Ser Arg Glu Phe Leu Asn Arg Met Phe

195 200 205

Asp Gly Glu Arg Ala Thr Thr Gly Trp Val Val Asn Ser Phe Ala Asp

210 215220

Leu Glu Gln Arg Tyr Ile Glu His Tyr Glu Lys Glu Thr Gly Lys Pro

225 230 235 240

Val Phe Ala Val Gly Pro Val Cys Leu Val Asn Gly Asp Gly Asp Asp

245 250 255

Val Met Glu Arg Gly Arg Gly Gly Glu Pro Cys Ala Ala Thr Asp Ala

260 265 270

Ala Arg Ala Leu Ala Trp Leu Asp Ala Lys Pro Ala Arg Ser Val Val

275 280 285

Tyr Val Cys Phe Gly Ser Leu Thr Arg Phe Pro Asp Glu Gln Val Ala

290 295 300

Glu Leu Gly Ala Gly Leu Ala Gly Ser Gly Val Asn Phe Val Trp Val

305 310 315 320

Val Gly Gly Lys Asn Ala Ser Ala Ala Pro Leu Leu Pro Asp Val Val

325 330 335

His Ala Ala Val Ser Ser Gly Arg Gly His Val Ile Ala Gly Trp Ala

340 345 350

Pro Gln Val Ala Val Leu Arg His Ala Ala Val Gly Ala Phe Val Thr

355 360 365

His Cys Gly Trp Gly Ala Val Thr Glu Ala Ala Ala Ala Gly Val Pro

370 375380

Val Leu Ala Trp Pro Val Phe Ala Glu Gln Phe Tyr Asn Glu Ala Leu

385 390 395 400

Val Val Gly Leu Ala Gly Thr Gly Ala Gly Val Gly Ala Glu Arg Gly

405 410 415

Tyr Val Trp Gly Gly Glu Glu Ser Gly Gly Val Val Val Cys Arg Glu

420 425 430

Lys Val Ala Glu Arg Val Arg Ala Ala Met Ala Asp Glu Ala Met Arg

435 440 445

Arg Arg Ala Glu Glu Val Gly Glu Arg Ala Arg Arg Ala Val Glu Val

450 455 460

Gly Gly Ser Ser Tyr Asp Ala Val Gly Ala Leu Leu Glu Asp Val Arg

465 470 475 480

Arg Arg Glu Met Ala Ala Asp Pro Arg Asn Val Lys Glu Val

485 490

<210>3

<211>1590

<212>DNA

<213> Rice

<400>3

atgcagtcgc cagagaatgc ggcaccgcgc gtgtacttca tcccgttccc gacgccgggg 60

cacgcgctgc cgatgtgcga cctcgcgcgc ctcttcgcgt cccgcggcgc cgacgcgacg 120

ctcgtcctca cgcgcgccaa cgccgccagg ctcggcggcg ccgtcgcccg cgccgccgcc 180

gcgggatcac ggatccgcgt ccacgcgctc gccctgcccg ccgaggccgc ggggctcacg 240

ggcggccacg agagcgccga cgacctcccc agccgcgagc tcgcggggcc cttcgccgtc 300

gccgtcgacc tcctcgcgcc gctcttcgcc gacctcctgc ggcgcagacc cgccgacgcc 360

gtggtgttcg acggcgtcct cccgtgggct gccaccgcgg cggccgagct ccgcgtcccg 420

cggtacgcgt tcacggggac ggggtgcttc gcgctctcgg tgcagcgagc actgctgctc 480

cacgccccgc aggacggcgt cgcgtcggac gacgagccgt tcctcgtgcc gggcctcccc 540

gacgcggtgc ggctcaccaa gtcgaggctc gccgaggcga ccctccccgg cgcgcactcg 600

cgggagttct tgaaccgaat gttcgacggc gagcgcgcca cgacggggtg ggtggtcaac 660

tccttcgccg acctagagca gaggtacatc gagcactacg agaaggagac ggggaagccc 720

gtgttcgccg tcgggccggt ctgcctcgtc aacggcgacg gcgacgacgt catggagcgc 780

gggcgcggcg gggaaccgtg cgccgccacg gacgccgcgc gcgcgctggc gtggctcgac 840

gcgaagcccg cgcggtcggt ggtgtacgtc tgcttcggca gcctcacccg gttcccggac 900

gagcaggtcg cggagctcgg cgcggggctc gcgggctccg gcgtgaactt cgtgtgggtc 960

gtcgggggca agaacgcgtc ggcggcgccg ctgctcccgg acgtcgtgca cgccgccgtg 1020

tcgtccggcc gcggccacgt gatcgcgggg tgggcgccgc aggtggcggt gctgaggcac 1080

gcggcggtgg gcgcgttcgt gacgcactgc gggtggggcg cggtgacgga ggcggcggcg 1140

gccggggtgc cggtgctggc gtggccggtg ttcgcggagc agttctacaa cgaggcgctg 1200

gtggtcgggc tcgccggcac gggcgccggc gtcggcgcgg agagggggta cgtgtgggga 1260

ggcgaggagt ccggcggggt ggtggtgtgc agggagaagg tggcggagag ggtgcgcgcc 1320

gccatggctg acgaggcgat gcggcggcgg gcggaggagg tcggcgagcg cgcgcgccgc 1380

gccgtcgagg tgggcgggtc gtcgtacgac gccgtcggcg cgctgctgga ggatgtgcgg 1440

cgccgggaga tggcggcgga tccacggaac gtgaaggaag tatgaaacca tatgaatcac 1500

aaactgaaat gttaaaatgt ttcataacta aaaatttgga ccgattacca aacgagtgta 1560

tatcgggctt aaattgatga ctacttattt 1590

<210>4

<211>1514

<212>DNA

<213> Rice

<400>4

ttgcgacttg atttatgcta aaatgttttt taagtgtggc atattttttt atatttacat 60

aaaaaattta actaagacaa atggtcaaac atttgtcaga aagttaatgg tatcaagtat 120

ttaaaaaact aaggtagtaa ctgaaatata gagagtgtga tctgtagcaa aagtgatcag 180

tactacaact gtttggaaat gtaatcaaac agcgttgatg aaatactgaa gctggctttg 240

atcattgcaa acgctaacag aatgctatag cagaacgcaa cccttctcat gttgttggta 300

agagccaaga aatgcgttag gcatgtcctg ctgcttaaca acttaaatta attgattaca 360

taaaaacgtt aactcatcca ggtatagcaa agtattattg aaagggatta ccaatgtaaa 420

tattaaaatt gtaatataat tatagtgtaa cttacacata actataatat aatttgttta 480

acacattatt tccactaaag gagtacacgt gaccaaattg catcacccgt ggttgcgcta 540

tgattttttc ctttcccgct tgcagagtct taaagtaaat ataacagatt aaaaatacat 600

aaaaagttac atacatatta tattataatt acatacaagt tataatattt tttgaacgac 660

tcgcataaga cggtgcgaag ttctattgat agagcagaag aaaaattata agattacaac 720

cctggaagtt gtaactagga aaaagggaaa aatataccac cacccacaca ccgacaacgc 780

caaaacactg aacaaggaga aggctagcac cggaccggcc accactaacc gtggccgacc 840

tccgctaggc caacaaagaa aacacggtcg ggatcgccca aaacccaact cttacaatca 900

acacaaagcc ccgctctatc caagtattca ggagttaagg agagagggtg agagagaaga 960

atggaactcc tctcccctta ggccctccga catggccagc ttgtagagac tagttaggat 1020

gccgccacaa gaggatgtaa tctagagtga gtttgtacta attggttggg tggttttagc 1080

taggggatgc ctaaacgatg gatcatagca aaaccgttac ttgaatactg aagctacctt 1140

tgaatctttg atcatggcaa aagcttaaca gaaggctaga gcaggataca accctttcgt 1200

gctgttacct gtcggtacga gccaacttag ccaagaaatg ccataagcat gtcatgttgt 1260

ttaacgaatt cactagctta gtagtagctt aattaactgc ctaaaagata aaaaggctaa 1320

taaaacggtt aactcattcg gagacaaact cgtactgcaa agcctacgtg aaaatcgcgt 1380

ttcccgcgct ctcgtggcgc accttctttg tctgtacaag aagtctatta tatacccccc 1440

atcctggcca agaccacacc tcgccgtcgc caccacctcc ctcgccgccg gcagccgagc 1500

gggtcggtgc cgcc 1514

<210>5

<211>2022

<212>DNA

<213> Artificial sequence

<400>5

aagcttgcat gcctgcagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc 60

attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt 120

gcagtttatc tatctttata catatattta aactttactc tacgaataat ataatctata 180

gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta 240

aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt 300

gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta 360

catccattta gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt 420

ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta 480

ataatttaga tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta 540

agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt 600

aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc 660

aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg 720

ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac 780

gtgagccggc acggcaggcg gcctcctcct cctctcacgg cacggcagct acgggggatt 840

cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 900

ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 960

ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 1020

ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact 1080

tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac 1140

acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 1200

gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 1260

tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 1320

tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 1380

ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 1440

ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 1500

aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 1560

tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 1620

agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 1680

gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 1740

taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 1800

tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 1860

tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 1920

agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 1980

tgttgtttgg tgttacttct gcaggtcgac tctagaggat cc 2022

Claims (9)

1. A method of making a moisture-sensitive sterile plant, comprising: reducing the activity of protein in a target plant, reducing the content of the protein in the target plant, inhibiting the expression of the coding gene of the protein in the target plant or knocking out the coding gene of the protein in the target plant to obtain a humidity-sensitive sterile plant;

the protein is shown as a sequence 2 in a sequence table;

the plant is rice.

2. The method of claim 1, wherein: the method is realized by performing site-directed mutation on a coding gene of the protein through a CRISPR/Cas9 system.

3. The method of claim 2, wherein: the encoding gene of the protein is any one of the following b1) -b 3):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 1in the sequence table;

b2) the coding sequence is cDNA molecule or DNA molecule of sequence 3 in the sequence table;

b3) a cDNA molecule or a genomic DNA molecule encoding the protein of claim 1;

and/or the presence of a gas in the gas,

the target sequence of the encoding gene of the protein used in the CRISPR/Cas9 system is target sequence 1, target sequence 2 or target sequence 3;

the target sequence 1 is a nucleotide sequence from 1 st to 20 th sites of a sequence 1in a sequence table;

the target sequence 2 is a nucleotide sequence at the 267-position 286 of the sequence 1in the sequence table;

the target sequence 3 is a nucleotide sequence at position 343-362 of the sequence 1in the sequence table.

4. The method of claim 3, wherein: the method comprises the step of introducing a sgRNA coding gene targeting the target sequence 1, the target sequence 2 or the target sequence 3 and a Cas9 coding gene into the target plant to obtain the humidity-sensitive sterile plant.

5. The method according to any one of claims 1-4, wherein: the humidity-sensitive sterile plant is a humidity-sensitive male sterile plant;

and/or the rate of water loss of the pollen of the moisture-sensitive sterile plant is higher than the rate of water loss of the pollen of the target plant.

6. A method for producing a humidity-insensitive fertile plant, comprising increasing the activity of the protein of claim 1in a recipient plant, increasing the content of the protein of claim 1in a recipient plant, or promoting expression of a gene encoding the protein of claim 1in a recipient plant, to obtain a humidity-insensitive fertile plant;

the recipient plant is a humidity-sensitive sterile plant;

the plant is rice.

7. The method of claim 6, wherein: increasing the content of the protein of claim 1in a recipient plant by introducing into the recipient plant a gene encoding the protein of claim 1;

and/or, the humidity sensitive sterile plant is a humidity sensitive male sterile plant;

and/or the rate of water loss of pollen of said humidity insensitive fertile plant is lower than the rate of water loss of pollen of said humidity sensitive sterile plant.

8. A product for regulating moisture sensitive fertility of a plant, comprising the following M1) or M2):

m1) preparing a humidity sensitive sterile plant product having the following functions: reducing the activity of the protein of claim 1in a plant, reducing the content of the protein in a plant, inhibiting the expression of a gene encoding the protein in a plant, or knocking out a gene encoding the protein in a plant;

the product is a reagent and/or an apparatus required for site-directed editing of a coding gene of the protein of claim 1by using a CRISPR/Cas9 system;

the reagent may be reagent 1, reagent 2, reagent 3, reagent 4, or reagent 5:

the reagent 1 is any one of R1) -R3):

r1) sgRNA targeting the target sequence 1 of claim 3;

r2) sgRNA targeting the target sequence 2 of claim 3;

r3) sgRNA targeting the target sequence 3 of claim 3;

the reagent 2 is a composition consisting of the reagent 1 and Cas 9;

the reagent 3 is a recombinant vector containing a coding gene of any one of RNA of R1) -R3);

the reagent 4 is a composition consisting of the reagent 3 and a recombinant vector containing a gene encoding Cas 9;

the reagent 5 is a recombinant vector containing a coding gene of any one of RNA from R1) to R3) and a coding gene of Cas 9;

m2) preparing a humidity insensitive fertile plant product having the following functions: increasing the activity of the protein of claim 1in a plant, increasing the content of the protein in a plant, or promoting expression of a gene encoding the protein;

the product is a gene encoding the protein of claim 1 or a recombinant vector containing the gene encoding the protein of claim 1;

the plant is rice.

9. Use of the protein of claim 1 or the gene encoding the protein of claim 3 for regulating moisture sensitive fertility in a plant; the plant is rice.

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