Rice environmental conditional-lethal mutant gene oses11, encoding protein and use thereof

Abstract

Disclosed are rice environmental conditional-lethal mutant gene osesl1, an encoding protein and use thereof The gene osesl1 has a nucleotide sequence shown as SEQ ID NO: 1 in the Sequence Listing. The encoding protein thereof has an amino acid sequence shown as SEQ ID NO: 2. After heading of osesl1 mutant rice, seed embryo lethal phenotype appears at 12 days after pollination, exhibiting darkening at the junction between embryo and endosperm. When an average temperature is below 22° C., a seed embryo is normal; when the average temperature is above 28° C., the seed embryo is lethal; when the temperature is between 22° C. and 28° C., the seed embryo is lethal under long daylight conditions (>13 h) and normal under short daylight conditions (<13 h). Use of the gene osesl1 in controlling seed embryo development of rice is further provided.

Claims

1. A rice environmental conditional-lethal mutant gene osesl1, having the nucleotide sequence of SEQ ID NO: 1.

2. A protein encoded by the rice environmental conditional-lethal mutant gene osesl1 according to claim 1, having the amino add sequence of SEQ ID NO: 2.

3. A method for controlling seed embryo development of rice, comprising introducing the rice environmental conditional-lethal mutant gene osesl1 according to claim 1 into a rice seed embryo using backcrossing or genetic transformation.

4. The method of claim 3, further comprising controlling an environmental temperature to remain below 22° C. during growth of the rice seed embryo to make the rice seed embryo develop normally.

5. The method of claim 3, further including controlling an environmental temperature to remain between 22° C. and 28° C. during growth of the rice seed embryo, wherein the rice seed embryo is provided a light duration of less than thirteen hours to make the rice seed embryo develop normally.

6. The method of claim 3, further including controlling an environmental temperature to remain between 22° C. and 28° C. during growth of the rice seed embryo, wherein the rice seed embryo is provided a light duration of greater than thirteen hours to kill the rice seed embryo.

7. The method of claim 3, further including controlling an environmental temperature to remain above 28° C. to kill the rice seed embryo.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates a seed embryo of wild-type Oryza sativa “Nipponbare” at 12 days after pollination;

(2) FIG. 2 illustrates a section of the seed embryo of wild-type O. sativa “Nipponbare” at 12 days after pollination;

(3) FIG. 3 illustrates an osesl1 mutant seed embryo at 12 days after pollination;

(4) FIG. 4 illustrates a section of the seed embryo of the osesl1 mutant at 12 days after pollination;

(5) FIG. 5 illustrates initial mapping of the OsESL1 mutant gene;

(6) FIG. 6 illustrates fine mapping of the OsESL1 mutant gene;

(7) FIG. 7 illustrates dCAP2-labeled detection of wild-type, osesl1 mutant, and T2 transgenic individuals;

(8) FIG. 8 illustrates phenotypes of growth and development of rice environmental conditional-lethal mutant osesl1 and T2 transgenic plants;

(9) FIG. 9 illustrates lethal phenotypes of seeds of rice environmental conditional-lethal mutant osesl1 and T2 transgenic plants; and

(10) FIG. 10 illustrates long and short daylight conditions of rice environmental conditional-lethal mutant osesl1 and wild-type plants within a critical temperature range.

DETAILED DESCRIPTION OF THE DRAWINGS

(11) The technical solution of the present invention is not limited to the specific implementations listed below, and includes any combination between the specific implementations.

(12) Specification implementation 1: A rice environmental conditional-lethal mutant gene osesl1 in the example has a nucleotide sequence shown as SEQ ID NO: 1 in the Sequencing List. The gene has five exons and four introns.

(13) Specification implementation 2: A protein encoded by the rice environmental conditional-lethal mutant gene osesl1 in the example has an amino acid sequence shown as SEQ ID NO: 2.

(14) Specification implementation 3: The example describes use of the rice environmental conditional-lethal mutant gene osesl1 in controlling seed embryo development of rice.

(15) Specification implementation 4: A difference between this implementation and specific implementation 3 is that: seed embryos are normal when rice growth is controlled at an environmental temperature below 22° C., and the seed embryos are lethal when rice growth is controlled at an environmental temperature above 28° C. Others are consistent with those in specific implementation 3.

(16) Specification implementation 5: A difference between this implementation and specific implementation 3 is that: when rice growth is controlled at an environmental temperature between 22° C. and 28° C., seed embryos are lethal if sunshine duration is >13 h, and normal if the sunshine duration is <13 h. Others are consistent with those in specific implementation 3.

(17) In the following, examples of the present invention will be described in detail. The examples are implemented on the premise of the technical solution of the present invention, and the detailed examples and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

EXAMPLE 1

Obtaining and Phenotype Analysis of Rice Environmental Conditional-Lethal Mutant osesl1

(18) Rice environmental conditional-lethal mutant osesl1 was obtained from seeds of EMS-mutagenized japonica rice Oryza sativa “Nipponbare”. Treatment included the following steps: soaking seeds in ultrapure water for 24 h at room temperature until imbibition; subsequently, soaking seeds in 1.5% EMS (v/v) for 18 h; washing seeds with 75 ml of ultrapure water thrice, each for 5 min, followed by 75 ml of ultrapure water thrice, each for 20 min. Seeds were rinsed with tap water for 2 h before sowing. For detailed treatment method, refer to the following reference (Till B J, Cooper J, Tai T H, et al. Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biology, 2007, 7(1):19). The mutagenized seeds were sowed in the field and M1-generation seeds were harvested; M1 generations inbred to obtain M2 generations, which were planted in Hainan (low temperature and short day) in winter and in Heilongjiang (high temperature and long day) in summer, respectively, so as to select mutants with normal breeding under short-day and low-temperature conditions and lethal seed embryos under long-day and high-temperature conditions therefrom. A mutant, osesl1, was selected. There was no significant difference in vegetative and reproductive growth between the mutant and Oryza sativa “Nipponbare” (control); at 12 days after flowering and pollination under long-day and high-temperature conditions, programmed cell death occurred at a junction between seed embryo and endosperm, resulting in darkening of the whole seed embryo, as shown in FIGS. 1 to 4. FIG. 1 illustrates a seed embryo of wild-type O. sativa “Nipponbare” at 12 days after pollination; FIG. 2 illustrates a section of the seed embryo of wild-type O. sativa “Nipponbare” at 12 days after pollination; FIG. 3 illustrates an osesl1 mutant seed embryo at 12 days after pollination; FIG. 4 illustrates a section of the seed embryo of the osesl1 mutant at 12 days after pollination. The mutant seeds with darkened seed embryos were not dormancy-broken, but were viable at 0 day after harvest, with a germination rate of 44.67%; at 60 days after harvest (DAH60), the germination rate decreased to 12.18%, and at 90 days (DAH90), the germination rate was 0, suggesting that all seeds were almost dead. In case of breaking dormancy at 45° C. for seven days, all seed embryos would die as well. Detailed statistical results are listed in Table 1.

(19) TABLE-US-00001 TABLE 1 Statistical results of the germination rate of seed embryos. Germination DAH0 DAH 60 DAH90 DAH7 for 45° C. Wild-type  99.3% + 0.01% 99.99% + 0.01% 99.33% + 0.01% 100.00% + 0.00% osesl1 44.67% + 0.09% 12.18% + 0.10%  0.00% + 0.00%   0.00% + 0.00%

(20) Using a backcross method, an osesl1 site was introduced into conventional japonica rice and indica rice cultivars, such as O. sativa “Minghui 63” (MH63 .sup.osesl1), O. sativa “Chenghui 448” (CH448 .sup.osesl1), O. sativa “7001S” (7001S .sup.osesl1), etc. The osesl1 site had no effect on rice yield, suggesting that the osesl1 site could be used in different background. The osesl1 site was introduced into O. sativa “Kongyu 131” (KY131, a local japonica rice cultivar in Northeast China) to obtain KY131.sup.osesl1 by backcrossing; the KY131.sup.osesl1 was planted in long-day and high-temperature regions; vegetative and reproductive growth thereof were observed, and it was not significantly different from that of wild-type. Yield-related traits of KY131.sup.osesl1, i.e., 1,000-grain weight and seed setting rate, were analyzed statistically and they were found that: differences in 1,000-grain weight and seed setting rate were not significantly different between KY131.sup.osesl1 and wild-type KY131, there was an increase in number of seeds per plant, and lethality rate reached 93.3% (Table 2). By means of these characteristics, osesl1 genotype can be introduced into O. sativa “Kongyu 131” (KY131.sup.osesl1, a patented rice cultivar in North China), and seeds thereof can be multiplied in Southern China by means of the lethal characteristic of embryo, providing variety protection for rice production in Northern China.

(21) TABLE-US-00002 TABLE 2 Analysis of yield-related traits of KY131.sup.osesl1 and KY131 Number of seeds per 1,000-Grain weight Seed setting rate Cultivar plant (g) (%) Lethality (%) KY131.sup.osesl1 455.6 ± 119.6 20.3 ± 1.35 93.2% ± 1.30% 93.3% ± 1.30% KY131 374.4 ± 92.56 21.9 ± 1.21 93.8% ± 1.70% 0.00% ± 0.00%

EXAMPLE 2

Genetic Analysis and Map-Based Cloning of Rice Environmental Conditional-Lethal Mutant osesl1

(22) (1) Genetic Analysis

(23) Osesl1 mutant was hybridized with Oryza sativa “Minghui 63” (MH63) to obtain F1 seeds; 278 F2 population were obtained after F1 selfing, and phenotypes of F2 populations were investigated to calculate a segregation ratio, where the ratio of wild-type to mutant type was 216:62, and chi-square test satisfied a 3:1 segregation ratio; F2 populations backcrossed by osesl1 mutant and wild-type O. sativa “Nipponbare” were further investigated, and the result was also consistent with the 3:1 segregation ratio, suggesting that the phenotype of the mutant was regulated by a single recessive gene.

(24) (2) Gene Mapping

(25) a) Initial mapping is conducted with an SSR molecular marker. First, rice SSR primers were synthesized according to the SSR sequence information published on RiceData; next, DNAs were extracted from parents and F2 generations (osesl1×MH63) using the CTAB method; finally, PCR amplification and polyacrylamide gel electrophoresis (PAGE) were conducted. With parents osesl1 and MH63 as controls, molecular markers analysis shows differences in F2 populations.

(26) The CTAB method was used. Brief steps were as follows: placing approximately 0.1 g of leaves in a 2 ml EP tube, precooling in liquid nitrogen with steel balls, grounding under vibration, mixing well with 700 μl of DNA extract preheated at 65° C. carefully, incubating in a water bath at 65° C. for 40 min, mixing with isometric chloroform vigorously, and centrifuging (at 12,000 rpm) for 10 min; precipitating supernatant with isometric isopropanol for 30 min, centrifuging (at 12,000 rpm) for 10 min; washing precipitates with 70% absolute alcohol, centrifuging (at 12,000 rpm) for 5 min, discarding supernatant, and drying invertedly in the air; dissolving the precipitates with 50 μl of water; storing in a refrigerator at −20° C.

(27) The total volume of a PCR system was 10 μl: rice genomic DNA template 1 μl (approximately 200 ng); 2× Master Mix 5 μl; 10 μM primers, each for 0.5 μl; diluting to 10 μl with ddH.sub.2O. The reaction program was as follows: 35 cycles of denaturation at 94° C. for 5 min, 94° C. for 30 s, 58° C. for 30 s, and 72° C. for 20 s; and extension at 72° C. for 10 min.

(28) Analysis result found that a mutant gene of the osesl1 mutant was initially mapped between markers M5 and M6 in linkage group 8 (FIG. 5).

(29) b) Fine mapping was conducted by resequencing. F2 (osesl1×Nipponbare) populations were investigated and classified according to the presence of mutant phenotype; representative wild-type and mutant-type F2 individuals were screened to establish a wild-type pool and a mutant pool, respectively; DNAs were extracted by the CTAB method, respectively; a DNA pool was established for resequencing, thereby enabling fine mapping of mutant genes (FIG. 6); the DNA had been registered in The Rice Annotation Project (RAP) (accession number: Os08g0484500).

(30) Flanking primers of the gene were designed according to the fine mapping result, followed by PCR amplification and sequencing. The total volume of a PCR system was 50 rice genomic DNA template 1 μl (approximately 200 ng); 2× PCR buffer for KOD Fx 25 μl; 2 mM dNTP 10 μl; KOD Fx (1U/μl) 1 μl; 10 μM primers, each for 1.5 μl; diluting to 50 μl with ddH.sub.2O.

(31) TABLE-US-00003 Primer OsESL1 F: (SEQ ID NO. 4) 5′-ATGCCCCTCGCGCCATGCCC-3′ Primer OsESL1 R: (SEQ ID NO. 5) 5′-GAGCCCCAAAGATGGGATGT-3′

(32) The reaction program was as follows: 30 cycles of denaturation at 98° C. for 10 s, 60° C. for 90 s, and 68° C. for 2 min; and extension at 78° C. for 10 min. PCR products were recovered and analyzed by Sanger sequencing.

(33) It was found that Os08g0484500 gene was an enzyme gene of shikimate pathway, i.e., OsDAHPS, where exon 1 has an SNP locus, which was a substitute for G-T, resulting in a missense mutation in Val(gtg)-leu(ttg). Therefore, the gene was named OsESL1. A mutant gene of the OsESL1, CDS (Coding Sequence), had a sequence shown as SEQ ID NO: 1; an encoding protein had a sequence shown as SEQ ID NO: 2; a genome had a sequence shown as SEQ ID NO: 3.

Example 3: Functional Marker Analysis

(34) According to SNP loci of the sequencing result, dCAPS markers were designed by dCAPS Finder 2.0; genotypes of F2 (osesl1×Nipponbare) populations were screened and recognized by the dCAP2 markers.

(35) dCAP2 primer had an upstream primer sequence shown as SEQ ID NO: 4 and a downstream primer sequence shown as SEQ ID NO: 5, with a genome DNA as template. The total volume of a PCR system was 10 μl: rice genomic DNA template 1 μl (approximately 200 ng); 2× Master Mix 5 μl; 10 μM dCAP2 primers, each for 0.5 μl; diluting to 10 μl with ddH.sub.2O. The reaction program was as follows: 35 cycles of denaturation at 94° C. for 5 min, 94° C. for 30 s, 58° C. for 30 s, and 72° C. for 20 s; and extension at 72° C. for 10 min. Enzyme digestion was conducted with NcoI overnight at 37° C. Digested products were detected on 4% agarose. The wild type was restricted as 102 bp, while mutant types could not be restricted as 127 bp (FIG. 7). The method can be further useful in detecting osesl1 genotype introduced materials.

(36) Detection results of F2 generations found a wild type/heterozygous type/mutant type separation ratio was 1:2:1, consistent with the Mendelian genetic law, suggesting that the SNP was tightly linked to the mutant phenotype of F2 generation, and that the mutant phenotype was determined by the mutation site.

EXAMPLE 4

Complementation Test for Phenotype of Rice Environmental Conditional-Lethal Mutant osesl1

(37) (1) Construction and Genetic Transformation of Complementary Vector

(38) To confirm whether the mutant phenotype was caused by the missense mutation in the base, genomic DNAs of “Nipponbare” and osesl1 mutant were used as templates, respectively, and Os08g0484500F and Os08g0484500R as primers,

(39) TABLE-US-00004 Primer-Os08g0484500 F: (SEQ ID NO. 6) 5′-TTACCCGGGATGCCCCTCGCGCCATGCCC-3′; Primer--Os08g0484500 R: (SEQ ID NO. 7) 5′-CCGTCTAGAGAGCCCCAAAGATGGGATGT-3′.

(40) Approximately 3,000 bp DNA fragment of wild-type (WT) and mutant (MU) genomic sequences of the OsESL1 gene were cloned. The total volume of a PCR system was 50 μl: rice genomic DNA template 1 μl (approximately 200 ng); 10× PCR buffer for KOD 5 μl; 25 mM MgSO.sub.4 3 μl; 2 mM dNTPs 5 μl; 105 μM primers, each for 1.5 μl; 1 U KOD (TOYOBO); diluting to 50 μl with ddH.sub.2O. The reaction program was as follows: 35 cycles of denaturation at 94° C. for 5 min, 94° C. for 40 s, 55° C. for 40 s, and 68° C. for 4 min 30 s; and extension at 68° C. for 5 min.

(41) The pCAMBIA 2300 vector and the above PCR amplified products were digested with restriction endonucleases SmaI and XbaI; a molar ratio of digested vector fragment to PCR product fragment was allowed to be 1:3; at room temperature, 1 μl of T4 DNA ligase was added, and finally the volume was diluted to 10 μl with water. After flicking the outer wall and mixing well, rapid centrifugation was conducted briefly, followed by incubation overnight at 16° C. The resulting ligation product was transformed into Escherichia coli DH5α; recombinant vectors pCAMBIA 2300-WT and pCAMBIA 2300-MU were obtained by restriction enzyme digestion and sequencing; subsequently, pCAMBIA 2300-WT and pCAMBIA 2300-MU were verified by sequencing. If correctly verified by sequencing, both vectors may be used for downstream experiment.

(42) (2) Obtaining of Transformation Lines and Phenotypic Identification Thereof

(43) The well-constructed recombinant vectors pCAMBIA 2300-WT and pCAMBIA 2300-MU were transformed into EHA105 Agrobacterium strain, and further into osesl1 mutants using the Agrobacterium-mediated genetic transformation method (Hiei et al. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 1994, 6:271-282). Screened positive T0 transgenic plants were planted in the paddy field to obtain T1 seeds; T1 generations selfed to obtain T2 seeds. OsESL1 genes with both OsESL1 genotype and overexpressed wild-type and mutant-type were identified from T2 generations, and phenotypical reversion of these plants was observed. It was indicated that: mutant and transgenic plants were not significantly different from the wild-type during growth and development (FIG. 8); transformation of T2 transgenic plants with mutated OsESL1-MU vector still exhibited a seed embryonic lethal phenotype, but transformation of T2 transgenic plants with OsESL1-WT (wild-type gene) vector restored the wild-type phenotype (FIG. 9), suggesting that the embryo lethal phenotype was caused by the missense mutation in the above single base.

EXAMPLE 5

Study of Illumination and Temperature on osesl1 Mutant Gene

(44) In two-line male sterile lines, the fertility of thermo-sensitive male sterile line had a critical transition temperature of 23.5° C.: sterile at >23.5° C.; otherwise, fertile. Differences in genetic background and research method led to different thermo-sensitive phases of different male sterile lines. The fertility of photoperiod sensitive male sterile line and fertility alteration thereof were influenced by photoperiod. In the sensitive period, the photoperiod was 13.45 h; when the photoperiod was <13.45 h, pollen fertility began to restore gradually. However, the photoperiod only induced the fertility thereof in a particular period, i.e., sensitive period; the temperature played a dominant role in the fertility of photo-thermo-sensitive male sterile line; under long-day and low-temperature conditions, the photo-thermo-sensitive male sterile line was partially fertile; under short-day and high-temperature conditions, the fertility decreased in varying degrees, suggesting that photoperiod-sensitive sterility was influenced by both illumination and temperature.

(45) Osesl1 mutant also had a seed embryo conditional-lethal feature similar to the fact that the fertility of the male sterile line was controlled by illumination and temperature. The mutant exhibited as follows: in osesl1 homozygous mutants, seed embryos were lethal in Northern China in summer under long-day and high-temperature conditions, whereas seed embryos returned to wild-type phenotypes in Southern China in winter under short-day and low-temperature conditions.

(46) Osesl1 mutants were treated at different temperatures (22° C., 24° C., 26° C., 28° C., and 30° C.) for different photoperiods (10 h, 13 h, 14 h, and 15 h). It was found that: the critical temperature ranged between 24° C. and 28° C. for seed embryonic lethality of osesl1 mutants. Within this critical temperature range, when the photoperiod lasted for >13 h per day, seed embryos were dead; when the photoperiod lasted for <13 h per day, seed embryos developed normally (Table 4). At a temperature of <24° C., regardless of photoperiod, all seed embryos developed normally; at a temperature of >28° C., the seed embryonic lethality was not regulated by the photoperiod (FIG. 10).

(47) TABLE-US-00005 TABLE 4 Analysis of lethality of osesl1 mutant gene under different illumination-temperature conditions Critical temperature (° C.) Photoperiod (h) Lethality (%) 24 11  4.78% ± 3.33% 24 13 99.58% ± 1.04% 24 15   100% ± 0.00% 26 11 25.20% ± 31.00% 28 11  6.45% ± 14.88% 28 13 96.74% ± 4.30% 28 15   100% ± 0.00% Extreme temperature (° C.) Photoperiod (h) Lethality (%) >30 10  99.5% ± 1.25% >30 14  99.5% ± 0.00% 22 10  0.00% ± 0.00% 22 14  0.00% ± 0.00%

(48) In view of the fact that illumination-temperature-related lethal conditions of the osesl1 mutant are wider than the critical temperature and photoperiod of the photo-thermo-sensitive male sterile line, such recessive trait further guarantees the breeding of two-line hybrid rice. If the OsESL1 gene is introduced into the two-line male sterile line, seed embryo lethal phenotype of the OsESL1 genotype appears at the late stage of pollen fertility; process of embryo development is accompanied by a gradual increase in environmental temperature. Even if the male sterile line becomes fertile due to temperature changes at the early stage, temperature rising at the late stage kills selfed seed embryos to guarantee safe seed production. Table 5 summarizes illumination-temperature conditions of safe seed production and multiplication. However, if the OsESL1 gene is introduced into restorer lines, seed multiplication will be conducted along with male sterile lines in long-day and high-temperature regions; because the seed embryonic lethality of the OsESL1 genotype omits the independent harvest of parent restorer lines, together with male sterile lines with the same growth period, mixed sowing and harvesting can be allowed, and fully mechanized farming can be realized to improve the seed production efficiency of hybrid rice. Either male sterile line or restorer line can be subjected to seed multiplication under short-day and low-temperature conditions.

(49) TABLE-US-00006 TABLE 5 Illumination-temperature conditions of safe seed production and multiplication of male sterile and restorer lines of the OsESL1 genotype Male sterile or Low High restorer line of temperature Low temperature High temperature temperature osesl1 (<22° C.) (<24° C.) (>24° C.) (>30° C.) Short day (<13 h) Safe seed Seed Not suitable for Safe seed multiplication multiplication seed production or production multiplication Long day (>13 h) Safe seed Guaranteed seed Seed production Safe seed multiplication production production