Application of OsGSTT3 gene in regulating rice seed germination

Abstract

The present invention discloses the application of the OsGSTT3 gene in regulating rice seed germination, belonging to the technical field of plant molecular biology. The present invention provides the application of the OsGSTT3 gene in regulating rice seed germination, wherein the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1; the coding region sequence of the OsGSTT3 gene is shown in SEQ ID NO.2. Also provided is the application of a recombinant vector containing the OsGSTT3 gene in regulating rice seed germination. The present invention inhibits rice seed germination by overexpressing the OsGSTT3 gene; and regulates rice seed germination by knocking out the OsGSTT3 gene. The present invention provides genetic resources for rice breeding, and the application of the OsGSTT3 gene in rice breeding has important application value and practical significance.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and in particular to application of the OsGSTT3 gene in regulating rice seed germination. Background Art

[0002] Seed germination is the process by which seeds, after absorbing water and swelling, transition from a dormant state to an active growth phase under suitable environmental conditions. It is a critical developmental step in the plant life cycle. Low seed germination efficiency is a major constraint to direct-seeding rice in the field. In-depth analysis of the key genes involved in seed germination and elucidation of their underlying mechanisms will facilitate the breeding of direct-seeded rice varieties. Seed germination is a complex process influenced not only by environmental factors such as light, temperature, and moisture, but also by endogenous reactive oxygen species (ROS), hormones, and signaling. ROS, including hydrogen peroxide, superoxide ions, singlet oxygen, and hydroxyl radicals, are present at all stages of seed life and play a role in seed maturation, dormancy, and germination. Studies have shown that seeds will germinate only when ROS concentrations are within the "oxidation window" for ROS signaling. ROS levels above or below this "oxidation window" may inhibit seed germination.

[0003] OsGSTT3 is a glutathione S-transferase in rice, a member of the theta class of the GST family. It has been reported that Arabidopsis thaliana AtGSTU7 participates in seed germination by regulating glutathione and reactive oxygen species (GSH-ROS) homeostasis. In rice, GSTs are essential for seed development, but their function in seed germination remains largely uncharacterized. Therefore, studying OsGSTT3 will not only elucidate its role in seed germination and provide a reference for functional studies of other members of the rice GST family, but also identify new genes for direct-seeding rice breeding. Uncovering more genes involved in rice seed germination is crucial for optimizing and selecting suitable direct-seeding rice varieties. Summary of the Invention

[0004] The present invention aims to provide a method for regulating rice seed germination by using the OsGSTT3 gene, thereby overcoming the problems of the prior art. The present invention first reveals the function of the rice OsGSTT3 gene in regulating rice seed germination, and the results show that overexpression or knockout of this gene can inhibit rice seed germination.

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

[0006] Technical solution 1: Application of the OsGSTT3 gene in regulating rice seed germination, the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1; the coding region sequence of the OsGSTT3 gene is shown in SEQ ID NO.2.

[0007] Technical solution 2: Use of the protein encoded by the OsGSTT3 gene in regulating rice seed germination, the amino acid sequence of the protein encoded by the OsGSTT3 gene is shown in SEQ ID NO.5.

[0008] Technical solution three: Application of the recombinant vector of the OsGSTT3 gene in regulating rice seed germination.

[0009] Technical Solution 4: Application of the host bacteria containing the recombinant vector in regulating rice seed germination.

[0010] Furthermore, the germination of the rice seeds is inhibited by overexpressing the OsGSTT3 gene; and the germination of the rice seeds is inhibited by knocking out the OsGSTT3 gene.

[0011] Technical Solution 5: A method for constructing transgenic rice capable of inhibiting rice seed germination, comprising overexpressing the OsGSTT3 gene to inhibit the germination of the rice seeds; the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1.

[0012] Technical Solution 6: A method for constructing transgenic rice capable of inhibiting rice seed germination, comprising knocking out the expression of the OsGSTT3 gene to inhibit the germination of the rice seeds; the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1.

[0013] Inhibiting rice seed germination can offer several benefits under certain circumstances, particularly in agricultural production and seed conservation. Here are some of the key benefits:

[0014] (1) Improve seed storage: Inhibiting seed germination can extend the storage life of seeds and reduce seed loss during storage. This is very important for ensuring the quality and quantity of seeds, especially when long-term storage is required.

[0015] (2) Preventing premature germination: Under unfavorable environmental conditions (such as drought, low temperature or high humidity), seeds may germinate prematurely, resulting in seedling death. By inhibiting germination, this can be avoided and ensure that seeds only begin to grow under suitable conditions.

[0016] (3) Controlling sowing time: By inhibiting germination, farmers can sow seeds at the optimal time, thereby improving crop growth efficiency and yield. For example, sowing seeds before the end of the dry season ensures that the seeds can germinate and grow normally when the rainy season arrives.

[0017] (4) Reduce weed competition: Inhibiting seed germination can reduce competition from weeds in the field, as weed seeds may also germinate at the same time. This helps improve the growing environment of rice and reduces the need for herbicides.

[0018] (5) Improve stress resistance: Inhibiting germination can enhance the stress resistance of seeds, making them better able to survive under adverse conditions (such as saline-alkali soil, drought or disease). This helps to improve the overall adaptability and stability of crops.

[0019] (6) Optimizing breeding and genetic research: In breeding and genetic research, inhibiting seed germination can help researchers better control experimental conditions and ensure that all seeds germinate under the same conditions, thereby obtaining more reliable data.

[0020] (7) Ease of transportation and distribution: Seeds with inhibited germination are easier to transport and distribute because they will not begin to grow during transportation. This is particularly important for large-scale seed distribution and international trade.

[0021] The present invention discloses the following technical effects:

[0022] Experiments in the present invention demonstrate that the rice gene OsGSTT3 significantly regulates the agronomic trait of seed germination. This gene is located on chromosome 1, and its locus number in rice is LOC4326189. Overexpression of the OsGSTT3 gene slows seed germination; knocking out the gene also inhibits seed germination. The present invention provides a genetic resource for crop breeding, and the rice OsGSTT3 gene can be applied in rice breeding. This gene affects the germination rate of rice seeds and has important theoretical and practical significance for plant breeding and application. This is of great significance for timely and uniform seed germination and ensuring high and stable crop yields. The present invention provides an important genetic resource for regulating rice seed germination. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0024] Figure 1 Figure 1 is a map of the overexpression vector;

[0025] Figure 2 This is a map of the CRISPR / gRNA vector;

[0026] Figure 3 This is a map of the pYLCRISPR / Cas9-Mtmono binary vector;

[0027] Figure 4 Analysis of mutation sites in the DNA and amino acid sequences of OsGSTT3 knockout strains;

[0028] Figure 5 The expression level of OsGSTT3 in OsGSTT3-overexpressing plants was analyzed. Error bars represent standard errors. SPSS was used for significant difference analysis. *** represents P < 0.001.

[0029] Figure 6 Phenotypes of wild-type plants and OsGSTT3 transgenic plants at 72 h after germination; scale bar is 2 cm;

[0030] Figure 7 Bar graph of germination rate of wild-type and OsGSTT3 transgenic seeds after 72 h of water absorption (n=3); error bars represent standard error; SPSS was used for significant difference analysis, ** represents P<0.01. DETAILED DESCRIPTION

[0031] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Plant materials: The rice variety used in the test was the japonica rice variety Zhonghua 11, namely rice (Oryza sativa L. ssp. japonica. cv. Zhonghua 11).

[0037] Example 1 Construction of target gene overexpression vector and genetic transformation

[0038] 1. Primers (SEQ ID NO.3 and SEQ ID NO.4) were designed based on the DNA sequence (SEQ ID NO.1) and coding region sequence (SEQ ID NO.2) of the OsGSTT3 gene provided by NCBI. The amino acid sequence of OsGSTT3 is shown in SEQ ID NO.5.

[0039] DNA sequence of the OsGSTT3 gene:

[0040]

[0041] Coding region sequence of OsGSTT3 gene:

[0042]

[0043] Amino acid sequence of OsGSTT3 protein:

[0044] MSTAAAGRKKKGGTPEASDAAAAAPARTLGCGVGSATAALPFSRGGRGVGLAPPPRSLGRGGGPAVAAIPPGGFPSYSASMDGFPFPPPFDGSYRGGFPSSSAWLDASGGDESSPGSW DKDVHPRGGFMSYFGNHAQNSHLVGAPIYIADASSPPEVEILQGNDDGNGNVRTEKRILWTEEEDIILMSAWIEHSTDSTCGADKGGGQYWGEVVESYNKTTPPLRKRRNLKQCKDR WHKINRWTDLFECAYVKARRIFTSGYSNQMWIDAAHKFYVDDNKEAKLGPFVLMEVWKICREVSKWKTYNDNLRNARKRKSFHLEGDSEEADDTFDEMPKRPMGQKAAKKAALDAKIK SNGLGSSDDGHSKESPIQLDKFDRYSKFQEENNDKRMKLLDRQEKISSEKLEATKIAHLTAQEYKEGRKLEKESKMMETYNSLISQDTSSMSAEEKAQRVSMMKCLMKTLFPESD(SEQ ID NO.5); F:TTACTTCTGCAGGGTACCATGTCCACGGCGGCTGCAGG (SEQ ID NO.3);

[0045] R: ACGCGTACTAGTAAGCTTTCAATCAGATTCAGGGAAAA (SEQ ID NO. 4).

[0046] The OsGSTT3 gene was cloned from the rice variety Zhonghua 11 using conventional PCR. The amplification protocol was as follows: (1) pre-denaturation at 95°C for 3 minutes; (2) 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds; and (3) extension at 72°C for 5 minutes. The OsGSTT3 gene is located on chromosome 1, and its locus in rice is LOC4326189.

[0047] 2. Select the KpnI and HindIII restriction sites on the pOx vector (pOx and the vectors mentioned below were provided by the research group of Academician Liu Yaoguang of South China Agricultural University) and digest the vector with enzymes. The enzyme digestion system is: 1 μL Kpn I, 1 μL Hind III, 1 ug pOx vector, 3 μL 10x enzyme digestion buffer, ddH2O is added to 30 μL system, incubated at 37°C for 30 min, and inactivated at 70°C for 10 min ( Figure 1 The cloned OsGSTT3 gene was ligated to the pOx vector after restriction enzyme digestion using homologous recombination. The ligation system was as follows: 120 ng of pOx linearized vector, 60 ng of the OsGSTT3 target fragment, 5 μL of Super Fusion Cloning Mix (2×), and ddH2O supplemented to 10 μL of the system. The reaction was incubated at 50°C for 30 min. The ligation product was then transformed into DH5α competent cells using the heat shock method and positive colonies were screened using a medium containing kanamycin resistance. An appropriate number of single colonies were picked from the plate for culture, and the plasmid was extracted using the alkaline lysis method. The plasmid was double-digested with KpnI and HindIII to detect the presence of the target size fragment. The bacterial solution that tested positive for restriction enzyme digestion was selected for sequencing to obtain a positive recombinant vector. The positive plasmid was transformed into Agrobacterium competent cells using the electroporation method to obtain positive Agrobacterium. The Agrobacterium was then tested using the same detection method to ensure that the Agrobacterium used for transformation contained the target gene.

[0048] 3. Using the Agrobacterium tumefaciens-mediated genetic transformation method, the positive Agrobacterium obtained above is used to infect rice callus tissue, and multiple different positive plants are obtained through screening, pre-differentiation, differentiation and rooting.

[0049] 4. The expression of OsGSTT3 gene in transgenic plants was detected by qRT-PCR (two-step method) using primers F1: CTATGAGAGTCCAAAGCCGA (SEQ ID NO.6) and R1: AGGCCAAGATCATTCTCCTC (SEQ ID NO.7). Two different overexpression lines, OE2 and OE22, were selected. The expression of OsGSTT3 in these two lines was significantly increased ( Figure 5 ).

[0050] Example 2 Construction of OsGSTT3 knockout vector and genetic transformation

[0051] 1. The complete genome sequence of OsGSTT3 (SEQ ID NO. 1) was obtained from NCBI. Target design was performed using the CRISPR-GE (Genome Editing) - Liu YG Lab website (http: / / skl.scau.edu.cn / ). Target sites with low off-target rates were selected and primers were designed accordingly. The primer sequences were: F2: GTTGAAACATACAATGATAACCTG (SEQ ID NO. 8); R2: AAACCAGGTTATCATTGTATGTTT (SEQ ID NO. 9).

[0052] 2. Use BsaI to cut the U3 gRNA vector, and connect the target to the U3 gRNA expression cassette using T4 ligase to construct the CRISPR / gRNA vector ( Figure 2 ).

[0053] 3. In the first round of PCR, primers UF: CTCCGTTTTACCTGTGGAATCG (SEQ ID NO. 10); gRNAR: CGGAGGAAAATTCCATCCAC (SEQ ID NO. 11) were used to clone the ligation product in step 2 (95°C for 10 sec, 58°C for 15 sec, 72°C for 15 sec, 25 cycles). 0.1 μL of PCR product was used as a template and primers B1: TTCAGAGGTCTCTACCGACTAGTCACGCGTATGGAATCGGCAGCAAA (SEQ ID NO. 12); BL: AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGC (SEQ ID NO. 13) were used to perform a second round of PCR to clone the U3 gRNA expression cassette (95°C for 10 sec, 58°C for 15 sec, 72°C for 20 sec, 25 cycles). The second-round amplified fragment was recovered. The ligation was performed to connect it to the pYLCRISPR / Cas9-Mtmono binary vector (37°C for 5 min, 10°C for 5 min, 20°C for 5 min, 13 cycles; 37°C for 5 min) Figure 3 ) to obtain the OsGSTT3 knockout vector. The OsGSTT3 knockout vector was then transformed into DH5α competent cells using a heat shock method, and positive colonies were screened using kanamycin-resistant medium. An appropriate number of single colonies were selected from the plates for culture, and the plasmids were extracted using alkaline lysis. The plasmids were digested with BsaI to detect the insertion of the target size fragment. The bacterial cultures that tested positive for enzyme digestion were sent to a sequencing company for sequencing, thereby obtaining positive recombinant vectors.

[0054] 4. Transform the positive plasmid into competent Agrobacterium cells using electroporation to obtain positive Agrobacterium. Test the positive Agrobacterium using the method described in step 3 to ensure that the Agrobacterium used for transformation is a positive strain. Using Agrobacterium tumefaciens-mediated genetic transformation, infect rice callus with the positive Agrobacterium obtained above. Through screening, predifferentiation, and rooting, multiple plants transformed from different calli are obtained.

[0055] 5. Design specific primers F3: TTTCTTGTGGATTGGATGAAGC (SEQ ID NO. 14); R3: CTGCCTTTTTAGCGGCCTTC (SEQ ID NO. 15) to amplify the target position by conventional PCR and sequence, detect the mutation site and determine the sequence mutation (mutation type).

[0056] 6. Screening of hygromycin-resistant and target homozygous lines from the T1 generation to obtain knockout plants of cas18 and cas24 ( Figure 4 ).

[0057] 7. Observation of Plant Germination Phenotype: Seeds harvested and dried uniformly at the same time were selected, and healthy, plump seeds were selected for the experiment. 55 ± 3 seeds were placed in each Petri dish, with three replicates for each strain. The seeds were soaked in water and incubated in the dark at 30°C. This was marked as the 0th hour of water absorption. After 24 hours of soaking, the water was discarded. The seeds in the Petri dish were kept moist daily. After 72 hours of water absorption, the number of germinations was counted and photographed.

[0058] qRT-PCR results showed that the expression level of OsGSTT3 in overexpression plants was significantly increased compared with that in wild-type plants ( Figure 5 The germination experiment was conducted on the seeds of wild type, overexpression strains (OE2, OE22) and knockout strains (cas18, cas24). The seeds germinated for 72 hours and were photographed, and the difference in germination rate was clearly seen ( Figure 6 Statistical analysis results showed that the germination rates of seeds of overexpression lines OE2, OE22 and knockout lines cas18, cas24 were slower than those of the wild type after 72 hours of water absorption ( Figure 7 ).

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of the OsGSTT3 gene in inhibiting rice seed germination, characterized in that: The nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1; the coding region sequence of the OsGSTT3 gene is shown in SEQ ID NO.2; The germination of the rice seeds is inhibited by overexpressing the OsGSTT3 gene; and the germination of the rice seeds is inhibited by knocking out the OsGSTT3 gene.

2. Use of a recombinant vector containing the OsGSTT3 gene according to claim 1 in inhibiting rice seed germination, characterized in that: The germination of the rice seeds is inhibited by overexpressing the OsGSTT3 gene; and the germination of the rice seeds is inhibited by knocking out the OsGSTT3 gene.

3. Use of a host bacteria containing the recombinant vector according to claim 2 in inhibiting rice seed germination, characterized in that: The germination of the rice seeds is inhibited by overexpressing the OsGSTT3 gene; and the germination of the rice seeds is inhibited by knocking out the OsGSTT3 gene.

4. A method for constructing transgenic rice capable of inhibiting rice seed germination, characterized in that: The method comprises overexpressing the OsGSTT3 gene to inhibit the germination of the rice seeds; the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1; and the coding region sequence of the OsGSTT3 gene is shown in SEQ ID NO.

2.

5. A method for constructing transgenic rice capable of inhibiting rice seed germination, characterized in that: The method comprises knocking out the expression of the OsGSTT3 gene to inhibit the germination of the rice seeds; the nucleotide sequence of the OsGSTT3 gene is shown in SEQ ID NO.1; and the coding region sequence of the OsGSTT3 gene is shown in SEQ ID NO.2.

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