Application of OsHAN1 gene in regulating diurnal flowering time of rice

Abstract

The present invention discloses the application of OsHAN1 gene in regulating the flowering time of rice during the day, and belongs to the technical field of genetic engineering and molecular breeding. The present invention provides a JA pathway-related gene OsHAN1 for regulating the flowering time of rice during the day, and the nucleotide sequence is shown in SEQ ID NO: 1; the present invention verifies through experiments that the gene knockout mutant blooms about 2 hours earlier than the wild type ZH11, and the results of the indica-japonica hybridization experiment show that the hybridization rate of the thermosensitive sterile line GAZS and the indica rice Nan Guizhan is 10.7%, and the hybridization rate of the GAZS sterile line GS-HAN1-CAS after the flowering time modification and the indica rice Nan Guizhan is 30.6%. The seed production yield is increased by nearly 3 times. It can be seen that by editing the OsHAN1 gene, an early flowering mutant can be created for hybrid rice breeding, which is helpful to increase the yield of indica-japonica hybrid rice seed production and reduce the cost of seed production.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and molecular breeding, and in particular to the application of OsHAN1 gene in regulating the daytime flowering time of rice. Background Art

[0002] Hybrid breeding is an effective way to increase rice yield. Intra-indica hybrid rice can increase yield by 10-20% compared to conventional rice. However, hybrid rice in the past mainly used hybridization between indica rice varieties, and due to the limitation of genetic diversity of parents, it is difficult to increase yield. Hybridization between indica and japonica subspecies has stronger hybrid vigor and can increase yield by 30% on the basis of intra-subspecies hybridization. With the cloning of multiple rice indica-japonica hybrid sterility genes, inter-subspecies hybridization has become possible. Generally, indica rice has an earlier flowering time and japonica rice has a later flowering time. Indica-japonica hybridization generally uses japonica rice as the sterile line and indica rice as the restorer line. Due to the different flowering times of indica and japonica parents, the cross-pollination rate is seriously reduced and the cost of hybrid seeds is increased. This has become the main constraint on the development of indica-japonica hybrid rice. Therefore, exploring the genes that affect rice flowering time and applying genes with different flowering time characteristics to hybrid seed production will increase hybrid yield and thus reduce seed production costs.

[0003] Jasmonic acid plays an important role in the opening of rice spikelets. Jasmonic acid and its derivatives can effectively promote rice flowering. Plant hormones JAs are a class of fatty acid derivatives, including jasmonic acid and its various derivatives, such as jasmonic acid isoleucine (JA-Ile), methyl jasmonate (MeJA) and 12-hydroxyjasmonic acid (12-HSO4-JA). The synthesis of jasmonic acid begins with the release of polyunsaturated fatty acids (FAs) from chloroplast / plastid membranes by phospholipase (PLs), which are converted into oxophytodienoic acid (OPDA) under the catalysis of 13-lipoxygenase (13-LOX), 13-allene oxide synthase (AOS) and 13-allene oxide cyclase (AOC). OPDA in chloroplasts is transported to peroxisomes, reduced by OPDA reductase 3 (OPR3) and undergoes multiple β-oxidations to generate JA. After JA enters the cytoplasm, it is metabolized into a variety of derivatives under the action of various enzymes. For example, JA forms biologically active JA-Ile under the action of jasmonic acid amino acid synthase JAR1. JAs plays a crucial regulatory role in the development and opening of rice spikelets. Studying the regulatory mechanism of rice flower opening will contribute to the development of indica-japonica hybrid breeding. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the OsHAN1 gene in regulating the daytime flowering time of rice to solve the problems existing in the above-mentioned prior art. The OsHAN1 gene in rice is edited by gene editing technology, and the daytime flowering time of the obtained rice mutant is earlier than that of wild-type rice. Moreover, the seed production yield of the GAZS sterile line GS-HAN1-CAS after the flowering time modification is nearly 3 times higher than that of the original sterile line GAZS after hybridization with indica rice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of OsHAN1 gene in any of the following:

[0007] (1) Application in regulating the diurnal flowering time of rice;

[0008] (2) Application in breeding transgenic rice with early flowering time;

[0009] (3) Application in improving hybrid rice seed production yield;

[0010] The nucleotide sequence of the OsHAN1 gene is shown in SEQ ID NO: 1:

[0011]

[0012] The present invention also provides the use of the protein encoded by the OsHAN1 gene in any of the following:

[0013] (1) Application in regulating the diurnal flowering time of rice;

[0014] (2) Application in breeding transgenic rice with early flowering time;

[0015] (3) Application in improving hybrid rice seed production.

[0016] The amino acid sequence of the above protein is shown in SEQ ID NO: 2:

[0017] .

[0018] The present invention also provides the use of a recombinant vector comprising the OsHAN1 gene in any of the following:

[0019] (1) Application in regulating the diurnal flowering time of rice;

[0020] (2) Application in breeding transgenic rice with early flowering time;

[0021] (3) Application in improving hybrid rice seed production.

[0022] The present invention also provides the use of an engineered bacterium comprising the recombinant vector in any of the following:

[0023] (1) Application in regulating the diurnal flowering time of rice;

[0024] (2) Application in breeding transgenic rice with early flowering time;

[0025] (3) Application in improving hybrid rice seed production.

[0026] Preferably, the OsHAN1 gene in rice is mutated by gene editing technology, resulting in the functional loss of the protein expressed by the OsHAN1 gene, so that the daytime flowering time of the rice is advanced. The present invention includes but is not limited to using gene editing technology to induce the mutation of the OsHAN1 gene, and can also cultivate rice with early flowering time by physically or chemically inducing the gene mutation.

[0027] Preferably, the method of causing the functional loss of the OsHAN1 gene expression protein includes deletion, insertion or mutation of bases on the target sequence. The above target sequence includes but is not limited to the target sequence provided in the embodiments of the present invention, and any position that can cause the functional loss of the OsHAN1 gene expression protein can be used as the target sequence.

[0028] The present invention also provides a method for advancing the daytime flowering time of rice, comprising the following steps: editing the OsHAN1 gene in rice to make the protein expressed by the OsHAN1 gene functionally deficient, thereby advancing the daytime flowering time of the rice; the nucleotide sequence of the OsHAN1 gene is shown in SEQ ID NO: 1.

[0029] The present invention also provides a method for cultivating transgenic rice with early flowering time during the day, comprising the following steps:

[0030] The OsHAN1 gene is edited in rice cells to make the protein expression function of the OsHAN1 gene lost; then the rice cells are cultivated and rice is regenerated using the rice cells, thus obtaining transgenic rice with early daytime flowering time.

[0031] The present invention discloses the following technical effects:

[0032] The present invention provides a gene OSHAN1 for regulating the flowering time of rice during the day. The gene encodes and expresses a monooxidase that can catalyze the conversion of active jasmonic acid-L-isoleucine (JA-Ile) into inactive 12-hydroxy-jasmonoyl-L-isoleucine (12-hydroxy-JA-Ile). The mutant obtained by mutation of the gene in the ZH11 material background exhibits an early flowering of about 2 hours. The results of the indica-japonica hybridization experiment show that the hybridization rate of the thermosensitive sterile line GAZS and the indica rice Nanguizhan is 10.7%, the hybridization rate of the GAZS sterile line GS-HAN1-CAS after the flowering time modification and the indica rice Nanguizhan is 30.6%, and the hybrid seed production yield of GS-HAN1-CAS and the indica rice Nanguizhan is nearly 3 times that of the original sterile line GAZS. It can be seen that the gene OsHAN1 for regulating the daytime flowering time of rice provided by the present invention can be used to create early flowering mutants for hybrid rice breeding, which is helpful to increase the yield of indica-japonica hybrid rice seed production and reduce the cost of seed production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The figure shows the flowering phenotype and mutant sequence of the OsHAN1 knockout plant mutant; A shows the flowering phenotype of the ZH11 and HAN1 knockout lines OsHAN1-CAS4 and OsHAN1-CAS15 at 11:00am on the day of flowering; B shows the enlarged view of the inflorescence in A; C shows the statistical results of the flowering time of the ZH11 and HAN1 knockout lines OsHAN1-CAS4 and OsHAN1-CAS15 in the morning of the day of flowering; D shows the gene sequence of OsHAN1 in ZH11, OsHAN1-CAS4 and OsHAN1-CAS15; the scale bar in A indicates 20cm, and the scale bar in B indicates 2cm;

[0035] Figure 2 Determination of OPDA (A), JA (B) and JA-Ile (C) contents in OsHAN1 knockout plants and wild-type ZH11;

[0036] Figure 3The flowering phenotype and mutant sequence of the GAZS japonica sterile line GS-HAN1-CAS after the flowering time modification; A is the flowering phenotype of the ZH11 and HAN1 knockout lines GS-OsHAN1-CAS1 and GS-OsHAN1-CAS2 at 11:30am on the day of flowering; B is the enlarged view of the inflorescence in A; C is the statistical result of the flowering time of the ZH11 and HAN1 knockout lines GS-OsHAN1-CAS1 and GS-OsHAN1-CAS2 in the morning of the day of flowering; D is the gene sequence of OsHAN1 in ZH11, GS-OsHAN1-CAS1 and GS-OsHAN1-CAS2; the ruler in A represents 20cm, and the ruler in B represents 2cm;

[0037] Figure 4 These are the experimental results of hybrid seed production yield between the japonica sterile line GS-HAN1-CAS of GAZS after flowering time modification and the indica rice Nan Guizhan; A is the hybrid panicle of the japonica rice sterile lines GAZS, GS-HAN1-CAS and the indica rice "Nan Guizhan"; scale: 2 cm; B is the statistical results of the hybridization rate of the japonica rice sterile lines GAZS, GS-HAN1-CAS and the indica rice "Nan Guizhan" respectively; data are mean ± standard deviation, n = 5; *, 0.01≤p<0.05; **, 0.001≤p<0.01; ***, p<0.001; C is the seeds obtained by hybridizing the panicles of the japonica rice sterile lines GAZS and GS-HAN1-CAS with the indica rice "Nan Guizhan". DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] 1. Target design

[0045] The nucleotide sequence of HAN1, a key gene in the JA biosynthesis pathway, was obtained from NCBI. The target was designed using the CRISPR / CAS9 system. The nucleotide sequence of the target is as follows:

[0046] OsHAN1-CAS Target1 (SEQ ID NO: 3): 5'-GGTCAGCTTCATCGTCCTG-3';

[0047] OsHAN1-CAS Target1 (SEQ ID NO: 4): 5'-TGATCCATGAGGCCGTGAT-3'.

[0048] 2. Construction of knockout vector, identification of transformed seedlings and phenotypic observation

[0049] 2.1 Construction of pU3-gRNA and pU6a-gRNA vectors containing Target-OsHAN1 fragment

[0050] First, synthesize a target primer with a sticky end. The target adapter primer sequence is as follows:

[0051] OsHAN1-CAS U3T1F (SEQ ID NO: 5): 5'-gccgGGTCAGCTTCATCGTCCTG-3',

[0052] OsHAN1-CAS U3T1R (SEQ ID NO: 6): 5'-aaacCAGGACGATGAAGCTGACC-3',

[0053] OsHAN1-CAS U6aT2F (SEQ ID NO: 7): 5'-gccgTGATCCATGAGGCCGTGAT-3',

[0054] OsHAN1-CAS U6aT2R (SEQ ID NO: 8): 5'-aaacATCACGGCCTCATGGATCA-3'.

[0055] Heat the target linker at 90℃ for 30s on a PCR instrument, then cool it at room temperature to complete the annealing step. Enzyme digestion of gRNA vector: Take about 1μg of pYL-U3 / U6a-gRNA plasmids, and digest them at 37℃ for 30min on a PCR instrument according to the enzyme digestion system in the table below.

[0056] Enzyme digestion system (20 μL) see Table 1:

[0057] Table 1 Enzyme digestion system

[0058]

[0059] The digestion product was directly recovered using the GenStar gel recovery kit. The recovered digestion vector was ligated with the corresponding adapters. The ligation reaction system was shown in the following table. The reaction was carried out on a PCR instrument at 16°C for 30 minutes.

[0060] The ligation reaction system is shown in Table 2:

[0061] Table 2 Ligation reaction system

[0062]

[0063] 2.2 Construction of pCRISPR / Cas9 vector containing Target-HAN1 fragment

[0064] The vector connected in the previous step (sgRNA expression cassette with linker target sequence) was amplified by PCR.

[0065] PCR amplification reaction system (25 μL) is shown in Table 3:

[0066] Table 3 PCR amplification reaction system

[0067]

[0068] Reaction program: pre-denaturation at 95°C for 1 min, 28 PCR cycles (95°C for 10 s, 58°C for 15 s, 72°C for 20 s), extension at 72°C for 1 min.

[0069] The PCR product was gel-purified according to the instructions of the GenStar recovery kit and then ligated to the pCRISPR / Cas9 vector containing the Cas9 expression cassette using T4 ligase.

[0070] Cutting and ligating system (15 μL) is shown in Table 4:

[0071] Table 4 Edge cutting and connection system

[0072]

[0073] Ligation procedure: Use PCR instrument to perform enzyme digestion and ligation reaction: 37°C for 10 min; 10-12 cycles (37°C for 5 min, 10°C for 3 min, 20°C for 5 min); 37°C for 3 min.

[0074] 2.3 Obtaining transgenic seedlings:

[0075] The target-containing pCRISPR / Cas9 vector was sent to a biological company for genetic transformation (the japonica rice variety ZH11 and the japonica rice sterile line GAZS were used). After the transformed seedlings were obtained, they were planted in the Qilin North experimental field of South China Agricultural University, and the positive transgenic plants were identified by hygromycin resistance.

[0076] 2.4 Identification of mutation sites

[0077] The DNA of the positive plants was extracted and amplified with the following primers: CAS-HAN1-F (SEQ ID NO: 9): 5'-GCATGGGCATCTTCAACGTC-3'; CAS-HAN1-R (SEQ ID NO: 10): 5'-ATCCTCCGGTAGTCGTCGAG-3'. The DNA was sent to the company for sequencing and the mutation was analyzed at http: / / skl.scau.edu.cn / dsdecode / . The results are as follows: Figure 1 As shown in D, it is the gene sequence of OsHAN1 in ZH11, OsHAN1-CAS4 and OsHAN1-CAS15.

[0078] 2.5 Observation of mutant phenotypes

[0079] The OsHAN1 gene was edited using the CRISPR / CAS9 system, and knockout mutants named OsHAN1-CAS4 and OsHAN1-CAS15 were obtained in the japonica rice variety ZH11. All rice was grown in the fields of South China Agricultural University (average summer temperature 28-30°C in Guangzhou).

[0080] The results are as follows Figure 1 As shown in AC, through field observation, it was found that the knockout mutant of this gene bloomed about 2 hours earlier than the wild type ZH11. At 9:00 in the morning on the day of flowering, OsHAN1-CAS4 and OsHAN1-CAS15 plants began to bloom, while ZH11 did not bloom.

[0081] 2.6 Determination of JA content in mutants

[0082] The contents of OPDA, JA and JA-Ile in ZH11 and OsHAN1-CAS material slurries were tested. Figure 2 As shown, it was found that the OPDA, JA and JA-Ile contents of the knockout mutant OsHAN1-CAS were significantly increased compared with the wild-type ZH11.

[0083] 2.7 Creation of early flowering mutants in japonica sterile lines

[0084] According to the flowering characteristics of the hybrid parents, the present invention uses the CRISPR / CAS9 method to knock out OsHAN1 in the late-flowering japonica sterile line GAZS. All the transformed rice seedlings obtained through the above genetic transformation were planted in the fields of South China Agricultural University (in Guangzhou, the average summer temperature is 28-30°C). The knockout mutants obtained by planting in the experimental field were named GS-HAN1-CAS1 and GS-HAN1-CAS2. Figure 3 As shown in AC, through field observation, it was found that the knockout mutant of this gene bloomed about 1.5 hours earlier than the wild-type GAZS. At 10:00 a.m. on the day of flowering, GS-HAN1-CAS1 and GS-HAN1-CAS2 plants began to bloom, while GAZS did not.

[0085] Figure 3 Center D is the gene sequence of OsHAN1 in GAZS, GS-HAN1-CAS1 and GS-HAN1-CAS2.

[0086] 2.8 Indica-Japonica hybrid seed production experiment

[0087] The results of the above-mentioned japonica sterile line GAZS knockout mutant showed that it can promote early flowering of rice, that is, it can make the flowering time of hybrid parents similar, which is beneficial to improve the yield of hybrid seed production. Therefore, the GAZS japonica sterile line GS-HAN1-CAS with modified flowering time was used for hybrid seed production with indica rice 'Nanguizhan'. The results are as follows Figure 4 shown.

[0088] The results of the indica-japonica hybrid seed production test showed that the hybridization rate of the japonica sterile line GAZS and the indica Nanguizhan was 10.7%, and the hybridization rate of the GAZS sterile line GS-HAN1-CAS after flowering time modification and the indica Nanguizhan was 30.6%. The seed production yield of GS-HAN1-CAS is nearly 3 times that of the original sterile line GAZS.

[0089] The above results indicate that early flowering mutants can be created by editing the OsHAN1 gene for use in hybrid rice breeding, which will help increase the yield of indica-japonica hybrid rice seed production and reduce seed production costs.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of knocking out the OsHAN1 gene in any of the following: (1) Application in promoting early flowering time of rice; (2) Application in breeding transgenic rice with early flowering time; (3) Application in improving hybrid rice seed production yield; the hybrid rice seed production is to use the GAZS japonica sterile line GS-HAN1-CAS after flowering time modification and the indica rice 'Nan Guizhan' for hybrid seed production, and the GAZS japonica sterile line GS-HAN1-CAS after flowering time modification is obtained by knocking out OsHAN1 in the japonica sterile line GAZS; in, The nucleotide sequence of the OsHAN1 gene is shown in SEQ ID NO:

1.

2. Use of the protein encoded by the knockout OsHAN1 gene according to claim 1 in any of the following: (1) Application in promoting early flowering time of rice; (2) Application in breeding transgenic rice with early flowering time; (3) Application in improving hybrid rice seed production; the hybrid rice seed production is to use the GAZS japonica sterile line GS-HAN1-CAS after flowering time modification and the indica rice 'Nan Guizhan' for hybrid seed production, and the GAZS japonica sterile line GS-HAN1-CAS after flowering time modification is obtained by knocking out OsHAN1 in the japonica sterile line GAZS.

3. A method for advancing the flowering time of rice, characterized in that: The method comprises the following steps: editing the OsHAN1 gene in rice to make the protein expressed by the OsHAN1 gene lack its function, thereby promoting the early flowering time of the rice; the nucleotide sequence of the OsHAN1 gene is shown in SEQ ID NO:

1.

4. A method for cultivating transgenic rice with early flowering time during the day, characterized in that: The following steps are involved: The OsHAN1 gene is gene-edited in rice cells to render the protein-expressing function of the OsHAN1 gene ineffective; the rice cells are then cultivated and rice is regenerated using the rice cells, thereby obtaining transgenic rice with an early flowering time; the nucleotide sequence of the OsHAN1 gene is shown in SEQ ID NO: 1.

Images