Application of OsGSTT3 gene in regulation and control of rice seed germination

Abstract

The invention discloses application of an OsGSTT3 gene in regulation and control of rice seed germination, and belongs to the technical field of plant molecular biology. The invention provides application of an OsGSTT3 gene in regulation and control of rice seed germination. A nucleotide sequence of the OsGSTT3 gene is shown as SEQ ID NO. 1; the sequence of a coding region of the OsGSTT3 gene is as shown in SEQ ID NO. 2. The invention also provides an application of a recombinant vector containing the OsGSTT3 gene in regulation and control of rice seed germination. According to the invention, the OsGSTT3 gene is overexpressed, so that the germination of rice seeds is inhibited; the OsGSTT3 gene is knocked out, so that the germination of the rice seeds is regulated and controlled. Gene resources are provided for rice breeding, and the OsGSTT3 gene is applied to rice breeding and has important application value and practical significance.

Description

Technical Field

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

[0002] Seed germination refers to the process in which seeds change from a dormant state to an active growth stage after absorbing water and swelling under suitable environmental conditions. It is a key developmental step in the continuation of the plant life cycle. Low seed germination efficiency is the main constraint on direct seeding of rice in the field. In-depth analysis of the key genes involved in seed germination and elucidation of their potential mechanisms will help in the breeding of direct seeding rice varieties. Seed germination is a complex process that is not only affected by environmental factors such as light, temperature and moisture, but also related to the regulation of endogenous reactive oxygen species (ROS), hormones and signal transduction. ROS, including hydrogen peroxide, superoxide ions, singlet oxygen and hydroxyl radicals, exist in all stages of seed life and play a role in seed maturation, dormancy, and germination. Studies have shown that seeds will only germinate when the ROS concentration is in the "oxidation window" of ROS signaling. ROS levels above or below the "oxidation window" may inhibit seed germination.

[0003] OsGSTT3 is a glutathione S-transferase in rice and belongs to the Theta class of the GST family. It has been reported that Arabidopsis AtGSTU7 participates in seed germination by regulating the homeostasis of glutathione and reactive oxygen species (GSH-ROS). In rice, GSTs are essential for seed development, but its function in seed germination has not yet been reported. Therefore, studying OsGSTT3 can not only clarify its role in seed germination and provide a reference for the functional study of other members of the rice GST family, but also provide new genes for direct seeding breeding in rice. Discovering more genes related to rice seed germination is of great significance for optimizing and selecting suitable direct seeding breeding in rice. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the OsGSTT3 gene in regulating rice seed germination to solve the problems existing in the above-mentioned 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 the 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: Application 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 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 the germination of rice seeds, 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 bring multiple benefits in certain situations, especially in agricultural production and seed conservation. Here are some of the main benefits:

[0014] (1) Improving seed storage resistance: 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 adverse 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, ensuring that seeds only begin to grow under suitable conditions.

[0016] (3) Controlling sowing time: By inhibiting germination, farmers can sow at the optimal time, thereby improving crop growth efficiency and yield. For example, sowing 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 growth environment of rice and reduces the need for herbicides.

[0018] (5) Improving 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] The experiment of the present invention proves that the rice gene OsGSTT3 has a significant regulatory effect on the agronomic trait of seed germination. The gene is located on chromosome 1, and its gene locus number in rice is LOC4326189. After the gene OsGSTT3 is overexpressed, the seed germination rate slows down; after the gene is knocked out, the seed germination can also be inhibited. The present invention provides gene resources for crop breeding, and the rice OsGSTT3 gene can be applied to rice breeding. The 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 the timely and neat germination of seeds and the guarantee of high and stable yield of crops. The present invention provides important gene resources for the regulation of 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 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.

[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 DNA and amino acid sequences of OsGSTT3 knockout strains;

[0028] Figure 5 The expression analysis of OsGSTT3 in OsGSTT3 overexpressing plants, the error bars represent standard errors, SPSS was used for significant difference analysis, *** represents P < 0.001;

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

[0030] Figure 7 Bar graph of germination rate of seeds of wild type and OsGSTT3 transgenic lines 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] 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.

[0033] 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.

[0034] 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.

[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 for 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 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 system was as follows: (1) pre-denaturation at 95°C for 3 min; (2) 95°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, for a total of 35 cycles; (3) extension at 72°C for 5 min. The OsGSTT3 gene is located on chromosome 1, and its gene locus number in rice is LOC4326189.

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

[0048] 3. Using the genetic transformation method mediated by Agrobacterium tumefaciens, 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 detection 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 and genetic transformation of OsGSTT3 knockout vector

[0051] 1. The whole genome sequence of OsGSTT3 (SEQ ID NO.1) was obtained through NCBI, and the target with low off-target rate was designed and selected through the target design in the website (CRISPR-GE (Genome Editing)-Liu YG Lab (http: / / skl.scau.edu.cn / ), and the corresponding target primers were designed. The primer sequences are 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 10sec, 58°C 15sec, 72°C 15sec, 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 10sec, 58°C 15sec, 72°C 20sec, 25 cycles). The second round amplified fragment was recovered. The ligation-by-cutting method was used 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 ), and obtain the OsGSTT3 knockout vector. Subsequently, the OsGSTT3 knockout vector was transformed into DH5α competent cells by heat shock method and positive colonies were screened with kanamycin resistance medium. An appropriate number of single colonies were picked from the plate for culture, and the plasmid was extracted by alkaline lysis method. The BsaI enzyme was used to detect whether there was a target size fragment inserted, and the bacterial solution with positive enzyme digestion detection was selected and sent to a sequencing company for sequencing detection, thereby obtaining a positive recombinant vector.

[0054] 4. Use the electroporation method to transform the positive plasmid into Agrobacterium competent cells to obtain positive Agrobacterium. Use the detection method in step 3 to detect the positive Agrobacterium to ensure that the Agrobacterium used for transformation is a positive strain. Use the Agrobacterium tumefaciens-mediated genetic transformation method to infect rice callus with the positive Agrobacterium obtained above, and obtain multiple plants transformed from different callus through screening, pre-differentiation and rooting.

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

[0056] 6. Screen the hygromycin-resistant and target homozygous strains from the T1 generation to obtain the knockout plant cas18 and cas24 strains ( Figure 4 ).

[0057] 7. Plant germination phenotype observation: Select seeds harvested at the same time and dried uniformly, and choose healthy and plump seeds for the experiment. Place 55±3 seeds in each culture dish, with 3 replicates for each strain, soak them in water, and culture them in the dark at 30℃. This is recorded as 0h of water absorption; after soaking for 24h, pour out the water, keep the seeds in the culture dish moist every day, and count the number of germinations and take photos when the seeds absorb water for 72h.

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

[0059] 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 OsGSTT3 gene in regulating 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.

2. The use of the protein encoded by the OsGSTT3 gene in regulating rice seed germination as claimed in claim 1, characterized in that: The amino acid sequence of the protein encoded by the OsGSTT3 gene is shown in SEQ ID NO.

5.

3. Use of a recombinant vector containing the OsGSTT3 gene described in claim 1 in regulating rice seed germination.

4. Use of a host bacteria containing the recombinant vector described in claim 3 in regulating rice seed germination.

5. The use according to any one of claims 1 to 4, 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.

6. 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.

7. 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.

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