CN116218888A - Gene OsECS for regulating and controlling rice to form haploid induction line and application thereof - Google Patents

Abstract

The invention discloses a rice haploid induction gene OsECS and application thereof in preparation of rice haploid plants. Knocking out or silencing an OsECS gene in a rice genome to obtain a rice haploid induction line, selfing the rice haploid induction line, and obtaining a selfing offspring haploid after phenotype screening. After mutation of the coding region, the OsECS gene in rice can generate haploid induction capacity in the selfing process. The haploid induction gene OsECS has important application potential in the aspects of breeding novel induction genes with high induction rate and improving the haploid breeding efficiency of crops.

Description

Gene OsECS for regulating and controlling rice to form haploid induction line and application thereof

The invention belongs to the field of:

the invention relates to the field of rice haploid breeding, in particular to a rice haploid induction gene OsECS and application thereof in preparing rice haploid plants, and a method for preparing rice haploids by using the OsECS.

The background technology is as follows:

rice is one of the important grain crops in the world, and more than 60% of people in China take rice as main food. The selection of the excellent rice inbred line is the basis and key of the selection of excellent hybrid seeds by utilizing heterosis of rice. The phenomenon that the 1st generation of a hybrid of conventional hybrid rice breeding is superior to the parent in all aspects is widely used for improving productivity and yield of crops. However, hybrid vigour is lost after the 1st generation of the hybrid selfing due to genetic segregation. At the same time, the high cost of hybrid seed production also hampers the use of heterosis in many crops. In addition, the traditional breeding method needs 7 to 8 generations to obtain a stable inbred line. In recent years, haploid doubling breeding technology through haploid induction is a revolutionary pure line breeding method, and a homozygous line can be obtained only by 2 generations, so that the trait evaluation of genetic materials in early breeding stage is facilitated, and the breeding efficiency can be greatly improved. Therefore, haploid breeding technology is applied to a plurality of industrial companies at home and abroad in large scale as a method for rapidly obtaining pure lines, and is one of three core technologies of modern breeding which are comparable with transgenic technology and molecular marker assisted breeding technology.

The haploid doubling breeding technology mainly comprises two steps: firstly, generating a haploid plant by using a haploid induction system; and secondly, doubling the chromosome to recover the ploidy of the chromosome to obtain a homozygous plant. Among these, the biggest limiting factor is the acquisition of haploid lines. The current rice haploid induction needs to be subjected to an in vitro anther culture process, so that the time is long, the operation difficulty is high, and the rice haploid induction has dependence on varieties. The intraspecific hybridization method using endogenous haploid inducer has the characteristics of low labor intensity and relatively low cost, and has become a key technology for economic crop breeding gradually. However, only 1 rice haploid inducer line has been reported at present, i.e., inducer lines created by inactivating the osmal gene using gene editing mutations (Yao, l., zhang, y., liu, c., liu, y., wang, y., liang, d., liu, j., sahoo, g., and keliher, t.osmal mutation induces haploid seed formation in indica price.nat.plants, 2018,4,530-533). Therefore, the discovery of a new rice haploid induction line is significant for promoting the breeding progress of rice molecules.

OsECS (Os 01g 08330.1) is an AtECS1/2 homologous gene encoding aspartic protease, which is expressed predominantly in egg cells (ricenode). The sequence was confirmed by PCR amplification of rice Japanese genome and ovary cDNA and Sanger sequencing, and the OsECS coding region contained 1365 bases, and the transcriptional and translational proteins comprised 454 amino acids. However, the function of the rice in the growth and development is not known.

Chinese patent CN202210009291.5 discloses a method for preparing haploid by arabidopsis ovum cell specific expression gene AtECS1/2 and application thereof. The ability to induce haploids is generated by inserting the Arabidopsis AtECS1 and AtECS2 genes into T-DNA and deleting ECS1/2, but the homozygous ECS1ECS double mutant line needs to be obtained through 2-generation crossing, the haploid obtaining time needs to be further shortened, and whether the knockout ECS genes have haploid induction ability in other monocotyledonous crops is unclear.

The invention comprises the following steps:

an object of the present invention is to provide a gene OsECS for regulating the formation of haploid inducer lines in rice.

Still another object of the present invention is to provide a method for preparing a rice haploid inducer line.

The third object of the invention is to provide an application of the OsECS gene in preparing a rice haploid inducer.

The invention discloses a gene OsECS for regulating and controlling a rice haploid induction line, the nucleotide sequence of the gene OsECS is shown as SEQ ID NO. 1, the expressed protein sequence of the gene OsECS is shown as SEQ ID NO. 2, and the gene OsECS is derived from Nipponbare of rice.

The rice (Nipponbare) genome and ovary cDNA were amplified using primers SEQ ID NO. 3 (gOsECS-F: 5'-ATGGCGGCCATGGCGC-3') and SEQ ID NO. 4 (gOsECS-R: 5'-CTAGGATGAGCCGGCGCAGTC-3'), and the presence of the OsECS gene in rice Nipponbare was confirmed by Sanger sequencing, as shown in the gene sequence SEQ ID NO. 1. Obtaining a rice haploid induction line by knocking out or silencing an OsECS gene in a rice genome, then carrying out selfing on the rice haploid induction line to obtain a selfing offspring, and screening the rice haploid plant by a haploid identification method. The OsECS gene in the rice gene can be knocked out or silenced to form a haploid plant.

Preferentially, the gene OsECS for regulating and controlling the formation of the haploid induction line of the rice disclosed by the invention is characterized in that: the sequence of which is mutated or deleted at positions 120-139 and/or 301-320.

The invention also discloses a method for preparing the rice haploid induction line, which comprises the following steps of knocking out or silencing an OsECS gene in a rice genome to obtain the rice haploid induction line, and then selfing the rice haploid induction line to obtain a selfing offspring, namely the rice haploid; the nucleotide sequence of the OsECS gene is shown as SEQ ID NO. 1.

Preferably, the knockout allows for the knockout of the OsECS gene in the rice genome to be CRISPR/Cas9 or TELLEN or T-DNA insertion or EMS mutagenesis; the OsECS gene in the silent rice genome may be RNAi interference (including, but not limited to, single stranded RNA interference, such as miRNA, siRNA, dsRNA, shRNA, etc.);

secondly, selfing the rice haploid induction line to obtain a selfing offspring, and obtaining the rice haploid through haploid phenotype identification and screening; the nucleotide sequence of the OsECS gene is shown as SEQ ID NO. 1.

Furthermore, the mode of deletion mutation, insertion mutation or base substitution of the OsECS gene in the genome of the rice is CRISPR/Cas9, and the target sequence is 120 th to 139 th or 301 th to 320 th in SEQ ID NO. 1.

Still further, the method further comprises the steps of: and carrying out haploid character identification and/or leaf ploidy identification on the selfing offspring single plant.

The haploid character identification method can be carried out according to the following method: if the plant to be detected has short plant, narrower leaf blade and compact plant type, the plant is or is candidate to be haploid; if the plant to be detected has the characteristics of high plant height, large leaf width, shawl and the like, the plant is or is candidate to be diploid.

The leaf ploidy identification method can be carried out according to the following method: extracting cell nuclei of tender leaves of plants to be detected, and taking diploid rice leaves as a control; the signal was again detected with a flow cytometer, and the diploid nuclear signal was first detected and the diploid nuclear signal peak position was set to 100 (since the genetic material within the diploid cell was twice that within the haploid cell, the haploid nuclear signal peak position appeared around 50). If the nuclear signal peak of the plant to be detected appears near 50, the plant is or is candidate to be a haploid; if the signal peak of the plant to be detected appears near 100 and is the same as the signal intensity enrichment position of the diploid cell nucleus, the plant is or is candidate to be diploid.

The invention also discloses application of the gene OsECS for regulating and controlling the rice to form a haploid induction line in preparation of rice haploids.

The invention has the advantages that:

compared with the prior art, the invention has the following advantages:

1. the invention proves that the rice OsECS has the function of regulating and controlling the formation of haploid plants of rice, and haploid rice plants can be obtained by knocking out or silencing the OsECS genes in rice genome.

2. Compared with the method that Arabidopsis is inserted into ECS genes through T-DNA to cause deletion induction to generate haploids, double-target gene editing is performed in rice by using a CRISPR/Cas9 system to obtain a large number of OsECS knockout lines, T0 generation homozygous knockout lines can be screened out from the lines, and relatively efficient haploid induction lines can be obtained, so that the time cost for obtaining haploids is greatly shortened.

3. The method can realize scale and high efficiency of rice haploid induction and break through the limitation of varieties, thereby bringing great value to rice breeding.

4. The invention expands the induction method of the dicotyledonous female parent haploid into monocotyledonous crop rice for the first time, and opens up a brand new way for establishing a monocotyledonous crop haploid breeding technology system.

Drawings

FIG. 1 is an OsECS pure and mutant gene sequence obtained by CRISPR/Cas9 gene editing. T1 and T2 represent two target positions, respectively.

Figure 2 is a diploid and haploid rice plant 3 weeks after sowing, left for diploid rice and right for haploid pseudo-rice, bar=1 cm.

FIG. 3 shows haploid detection by flow cytometry, with upper representation of diploid rice peaks and lower representation of haploid rice peaks.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were mainly used as reagents, and other reagent consumables were purchased from conventional biochemical reagent companies (national pharmaceutical group chemical reagent Co., ltd., shanghai Bioengineering Co., etc.). The seeds of Paddy Nipponbare were derived from the national emphasis experiments on hybrid rice (university of Wuhan).

Example 1: identification of Rice OsECS Gene

1) After 2 weeks from germination of wild type Japanese sunny seeds, about 100mg of the leaf was ground with liquid nitrogen, and the rice genome was extracted by the CTAB method.

2) The flowering rice ovaries were emasculated artificially, and RNA from unfertilized ovaries was extracted by FastPure Universal Plant Total RNAIsolation Kit (Vazyme), and further by

II 1stStrandcDNASynthesis Kit (Vazyme) kit reverse transcribes RNA into cDNA.

3) The rice genomic and ovary cDNA obtained in the above procedure was PCR amplified using the following primers and high fidelity enzyme (2X PhantaMax Master Mix, vazyme):

gOsECS-F:5’-ATGGCGGCCATGGCGC-3’

gOsECS-R:5’-CTAGGATGAGCCGGCGCAGTC-3’

4) The PCR product (about 1.3 kb) was recovered using a gel recovery kit (Tiangen), and the sequence thereof was confirmed to be shown as SEQ ID NO:1 by Sanger sequencing (Shanghai Biotechnology Co.).

Example 2: rice OsECS gene knocked out by CRISPR/Cas9 system

The CRISPR/Cas9 system is utilized to knock the rice OsECS gene, so as to obtain the transgenic rice OsECS gene mutant. The method comprises the following specific steps:

1) selection of sgRNA sequences:

design of website rice OsECS gene candidates by using crispr/cas9 target sites of agricultural university in China

The sgRNA target site selects 2 high-molecular target sites (SEQ ID NO:5 and SEQ ID NO: 6) with the length of 20bp. Target site 1 is located at positions 120-139 (SEQ ID NO: 5) of sequence 1, and target site 1 sequence is 5'-CCGTGACTCACCCAGGTCGC-3'.

Target site 2 was located at positions 301-320 (SEQ ID NO: 6) of sequence 1, and target site 2 was 5'-TACCTCATGACGGTCAACCT-3'.

2) Construction of CRISPR/Cas9 vector:

the CRISPR/Cas9 vector is obtained by inserting the encoding DNA molecule of the sgRNA in the step 1) into the pYLCRISPRCas9Pubi-H-osu vector

pYLCRISPRCas9Pubi-H-osu3-osecs。

3) Obtaining transgenic rice:

transferring the CRISPR/Cas9 vector obtained in the step 2) to an agrobacterium competent cell GV3101 through heat shock transformation to obtain recombinant bacteria GV3101/CRISPR/Cas9.

And then the recombinant bacterium GV3101/CRISPR/Cas9 is transformed into the young embryo of the rice by adopting an agrobacterium infection method (the recombinant agrobacterium is subjected to 28 ℃ for propagation, and the bacterial liquid after propagation is used for infecting the young embryo of the rice), and the T0 generation transgenic rice plant is obtained after screening, differentiation and rooting.

4) Identification of transgenic Rice mutated in OsECS Gene:

collecting the T0 generation transgenic rice plant leaves obtained in the step 3), extracting genome DNA as a template, and carrying out PCR amplification by using the following primers to obtain PCR amplification products of different strains.

The sequences of the OsECS mutant sequence amplification detection primers are as follows (SEQ ID NO:7 and SEQ ID NO: 8):

OsECS-F:5’-CGCCATTAGCAGCGTGAGGAG-3’

OsECS-R:5’-GCCGCCGATGAGGTGTCG-3’

the PCR amplified products of the different strains were subjected to Sanger sequencing, and according to the sequencing result, were compared with the OsECS gene in the wild rice Nipponbare.

The genotype of the strain is a heterozygous genotype (the OsECS genes on 1 chromosome in 2 homologous chromosomes are mutated and the OsECS genes on the other 1 chromosome are not mutated) when the sequence with bimodal characteristics is from the target site sequence, and the strain is a T0 generation transgenic rice OsECS gene mutation heterozygous strain; the sequence with specific unimodal characteristics from the target site sequence is compared with the OsECS gene sequence of wild rice Nipponbare, if the sequence is the same, the genotype of the strain is wild type, namely the OsECS gene sequence is not mutated, and the following analysis does not take the mutation into consideration; if mutation exists, the genotype of the strain is homozygous genotype (mutation occurs in the OsECS genes on 2 homologous chromosomes), and the strain is a T0 generation transgenic rice OsECS gene mutation homozygous strain.

By identification, 2 strains are homozygous mutant lines of the OsECS gene. Further selection of homozygous mutant individuals resulted in frame shift mutations (deletions were not multiples of 3) for phenotypic identification, designated Osecs-1 and Osecs-2, as shown in FIG. 1.

Example 3: identification of haploid in mutant selfing offspring obtained by knocking rice OsECS gene through CRISPER/Cas9 system

The T1 generation transgenic rice OsECS gene mutation homozygous strain obtained in the example 2 is sown in a greenhouse, the single plant phenotype of the offspring is observed, the haploid has a plant dwarf, the leaf is narrower, and the diploid is expressed as a plant tall and big, and the leaf width is shown in the figure 2.

Diploid wild type rice Nippon sunny was used as a control.

And (3) carrying out genome ploidy detection on the plant with the phenotype of haploid character obtained by the identification of the steps by using a flow cytometer. 1-2 rosette leaves are placed in a culture dish, 100 mu l of nuclear extract (Partec CyStain) is added to the leaves, the leaves are rapidly and uniformly cut up by a blade, 400 mu l of nuclear extract is added, the leaves are gently beaten by a liquid-transferer, the mixture is kept stand for 5 minutes at room temperature, 1ml of dye liquor (Partec CyStain UV Precise-Kit) is added, and the mixture is uniformly mixed and incubated for 1 minute at room temperature. The mixture was passed through a flow tube with a 40 μm nylon screen (FALCON, 352235) and samples were haploid identified using a Beckmann CytoFlex flow cytometer. First, a wild-type sample is detected as a reference value, a diploid nuclear signal is detected, and the initial peak signal of the diploid is 100 (fig. 3), and the initial peak of the haploid is 50 (fig. 3). The error of the flow cytometer is small, so that the measured plant can be accurately distinguished into haploid and diploid after the measurement. The results show that the haploid plants identified by phenotype are haploid after detection by a flow cytometer.

Claims (6)

1. A nucleotide sequence of the gene OsECS for regulating and controlling the formation of a haploid induction line of rice is shown as SEQ ID NO. 1, an expressed protein sequence of the gene OsECS is shown as SEQ ID NO. 2, and the gene OsECS is derived from rice.

2. The gene OsECS according to claim 1, characterized in that: the sequence of which is mutated or deleted at positions 120-139 and/or 301-320.

3. A method for preparing a rice haploid induction line comprises the following steps of knocking out or silencing an OsECS gene in a rice genome to obtain the rice haploid induction line, and then selfing the rice haploid induction line to obtain a selfing offspring, namely the rice haploid; the nucleotide sequence of the OsECS gene is shown as SEQ ID NO. 1.

4. A method according to claim 3, characterized in that: the mode of causing the OsECS gene in the target rice genome to generate deletion mutation, insertion mutation or base substitution is CRISPR/Cas9, and the target sequence is 120 th to 139 th and/or 301 th to 320 th in SEQ ID NO. 1.

5. A method according to claim 3, characterized in that: the method further comprises the steps of: and carrying out haploid character identification and/or leaf ploidy identification on the selfing offspring.

6. The use of the gene OsECS for regulating and controlling a rice haploid inducer line according to claim 1 for preparing rice haploids.

Images