Application of OsMT2b gene in regulating rice seed size

Abstract

The present invention discloses the application of OsMT2b gene in regulating rice seed size, and belongs to the field of plant molecular biology. The present invention provides the application of OsMT2b gene in regulating rice seed size, wherein the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No.1; the coding region sequence of the OsMT2b gene is shown in SEQ ID No.5. The present invention also provides the application of a recombinant vector containing the OsMT2b gene in regulating rice seed size. The present invention regulates the enlargement of rice seed grains by overexpressing the OsMT2b gene; and regulates the reduction of rice seed grains by decreasing the expression amount of the OsMT2b gene. The present invention provides gene resources for rice breeding, and the application of the OsMT2b gene in high-yield rice breeding has important application value and practical significance.

Description

Technical Field

[0001] The invention relates to the field of plant molecular biology, and in particular to application of OsMT2b gene in regulating rice seed size. Background Art

[0002] Rice is one of the most important food crops for mankind. With the continuous growth of the global population, increasing rice yield is the primary goal of rice breeding. Rice yield is mainly determined by the number of effective panicles, the number of grains per panicle and grain weight, among which grain weight is a relatively stable genetic factor. Grain weight is mainly affected by rice seed size and seed filling rate. The surface of the seed is covered by the husk, which not only protects it, but also serves as a filling container to limit the volume of the grain and restrict the development of the seed.

[0003] Several genes related to grain shape and grain weight have been isolated from rice, and almost all of them are related to glume development. Therefore, glume plays a decisive role in affecting seed size. Glume development is mainly affected by cell proliferation and cell expansion. In the early stage of glume development, glume cells expand their area by dividing and increasing the number of cells. Subsequently, cell division begins to slow down, the cell expansion ability gradually increases, the cell volume increases, and the surface area of ​​the glume increases. Studies have reported that some genes affect seed size by regulating rice glume development. For example, OsMKKK10, OsMKK4, and OsMAPK6 promote glume cell proliferation through MAPK signaling cascades to increase the number of cells in the longitudinal direction to promote seed development. OsPGL1 is a basic helix-loop-helix protein that does not bind DNA. It is a pair of antagonistic interaction factors with APG. PGL1 / APG regulates rice grain length and weight by controlling the elongation of glume cells. GRAIN SIZE 3 (GS3), encoding an atypical Gγ subunit, is the first reported major QTL for rice grain length and grain weight, negatively regulating rice seed grain length by inhibiting cell expansion in the glume.

[0004] Although many genes related to seed size have been cloned, their regulatory network is still relatively fragmented. Therefore, further exploring the genes related to rice seed size and improving the molecular regulatory pathway of rice seed size are of great significance for further understanding the rice seed development process and improving yield. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the OsMT2b gene in regulating rice seed size to solve the problems existing in the above-mentioned prior art. The present invention discloses for the first time the function of the rice OsMT2b gene in regulating rice seed size, and the results show that overexpression of the gene can make rice seeds larger and increase thousand-grain weight.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: Application of the OsMT2b gene in regulating rice seed size, the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No.1; the coding region sequence of the OsMT2b gene is shown in SEQ ID No.5.

[0008] Technical solution 2: Application of the protein encoded by the OsMT2b gene in regulating rice seed size, the amino acid sequence of the protein encoded by the OsMT2b gene is shown in SEQ ID No.6.

[0009] Technical solution three: Application of a recombinant vector containing the OsMT2b gene in regulating rice seed size.

[0010] Technical solution 4: Application of host bacteria containing the recombinant vector in regulating rice seed size.

[0011] Furthermore, when the OsMT2b gene is overexpressed, the rice seed grains become larger; when the OsMT2b gene is inhibited, the rice seed grains become smaller.

[0012] Technical Solution 5: A method for constructing transgenic rice capable of enlarging rice seed grains, comprising overexpressing the OsMT2b gene to enlarge the rice seed grains; the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No. 1.

[0013] Technical Solution 6: A method for constructing transgenic rice capable of reducing the size of rice seeds, comprising inhibiting the expression of the OsMT2b gene to reduce the size of the rice seeds; the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No. 1.

[0014] Technical Solution 7: An application of an OsMT2b gene or a protein encoded thereby in rice breeding, wherein the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No.1, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID No.6.

[0015] The present invention discloses the following technical effects:

[0016] The present invention discloses for the first time the function of the rice OsMT2b gene in regulating the size of rice seeds. The results show that overexpression of the gene can make rice seeds larger, thereby increasing the thousand-grain weight; interfering with the OsMT2b gene through RNAi technology can make rice seeds smaller and reduce the thousand-grain weight. In summary, the present invention provides valuable gene resources for crop breeding, and the rice OsMT2b gene can be widely used in rice breeding. The present invention provides important gene resources for regulating the size of rice seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 Expression analysis of OsMT2b in wild-type and OsMT2b overexpressing plants, UBQ gene was used for data correction (n=3); Error bars represent standard error; *** represents extremely significant difference compared with wild-type plants; SPSS analysis software t-test was used for significant difference analysis: ***P<0.001; WT: wild-type, OE22 and OE26 are overexpressing plants;

[0019] Figure 2 The expression level of OsMT2b in wild-type and OsMT2b interference plants was analyzed, and the UBQ gene was used for data correction (n=3); Error bars represent standard errors; *** represents extremely significant differences compared with wild-type plants; SPSS analysis software t-test was used for significant difference analysis: ***P<0.001; WT: wild-type, MTi10 and MTi12: RNAi plants;

[0020] Figure 3 The phenotypes of seed length and width of wild type, OsMT2b overexpression and interference lines, where A: seed length; B: seed width; scale bar is 1 cm; WT: wild type, OE22 and OE26 are overexpression plants, MTi10 and MTi12: RNAi plants;

[0021] Figure 4 Seed length, width, thickness and 1000-grain weight of wild-type, OsMT2b overexpression and interference lines, where A: seed length; B: seed width; C: seed thickness; D: 1000-grain weight; error bars represent standard error (n=30); * represents significant difference compared with wild-type plants; SPSS analysis software t-test was used for significant difference analysis: *, P<0.05, **, P<0.01 and ***, P<0.001; WT: wild-type, OE22 and OE26 overexpression plants, MTi10 and MTi12: RNAi plants. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] 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).

[0028] Example 1 Construction of target gene overexpression vector and genetic transformation

[0029] 1. Design primers (SEQ ID No.3 and SEQ ID No.4) based on the sequence (SEQ ID No.1) and mRNA sequence (SEQ ID No.2) of OsMT2b gene provided by NCBI:

[0030] Sequence of OsMT2b gene:

[0031] TGGACCCTGGATCGATCACCTCCATTCTTTCTCCGCATCTCGCCACAGTACGCTTCGCTGCTCTCCCGCCTATATATACCACCTCCTCCTCGATCATCAGTTCATCAGCAACCAAAGCAAGAAGCAATTCTTGAGCTCAATCAGCAACAACATCTATCTCCTTCTTGCTTAGTTAAGTCCTTCGCCCTCCCAAGAAGAAGAAGATGTCTTGCTGCGGCGGCAACTGCGGCTGCGGCAGCGGCTGCCAGTGCGGCGGCGGCTGCGGCGGGTAATTAACTAACTAACTAACCAACTGCTAATTAATTCACTCAAGAAACCATTGTGACACGCACAGATCGATAACTGATCAATATTAACATTAATATGCATGGATGCAGATGCAAGATGTTCCCTGATGTGGAGGCCACAGCCACCACCAAGACCTTCGTCCTCGCTGCTCCATCCAACAAGGCGTAAGTTTCATCATTAATGCTAATTAATTGATCTGTCATTCCTGTGTGTGTTCATCATTAGTGTTAATTAATTGATTGATCTGCCATTCCGAAACAAAATTGTGCTGGATTACAGGAGCTCTGGAGGAATGGAGATGGCGGTGGAGAGCGGCGAGAACGGCGGCTGCGGCTGCAACACCTGCAAGTGTGGCACCAGCTGCAGCGGCTGCTCCTGCTGCTCCTGCAACTGAATCTATCGTCGTCGTCGCCCGGCTGCATGAGGATTTATCGTATGGATGCTGCTACTGTCGATCAGAGCTTTGATCGAGGCCTTAATTTGCTTGCATTAGTACCCAGCTTATATGTAGGCAGGCCTTGCCTTTTGCTCTGACGCCTAAATAAAACCGTCGTCGTCGTTGTCAGTGTGTGCGTGTGTCGATCAATGTTGGATGGATCCCTAGCTAGCTTGGATGGATCATCTATCATCATGGTGATCTCATCATGTACTCCTGCTCCATCTCCTCATGCGTGCCAGGCTTCTTAATATAATCTACCTCTGCTTTCATCCCA(SEQ ID No.1)

[0032] CGCATCTCGCCACAGTACGCTTCGCTGCTCTCCCGCCTATATATACCACCTCCTCCTCGATCATCAGTTCATCAGCAACCAAAGCAAGAAGCAATTCTTGAGCTCAATCAGCAACAACATCTATCTCCTTCTTGCTTAGTTAAGTCCTTCGCCCTCCCAAGAAGAAGAAGATGTCTTGCTGCGGCGGCAACTGCGGCTGCGGCAGCGGCTGCCAGTGCGGCGGCGGCTGCGGCGGATGCAAGATGTTCCCTGATGTGGAGGCCACAGCCACCACCAAGACCTTCGTCCTCGCTGCTCCATCCAACAAGGCGAGCTCTGGAGGAATGGAGATGGCGGTGGAGAGCGGCGAGAACGGCGGCTGCGGCTGCAACACCTGCAAGTGTGGCACCAGCTGCAGCGGCTGCTCCTGCTGCTCCTGCAACTGAATCTATCGTCGTCGTCGCCCGGCTGCATGAGGATTTATCGTATGGATGCTGCTACTGTCGATCAGAGCTTTGATCGAGGCCTTAATTTGCTTGCATTAGTACCCAGCTTATATGTAGGCAGGCCTTGCCTTTTGCTCTGACGCCTAAATAAAACCGTCGTCGTCGTTGTCAGTGTGTGCGTGTGTCGATCAATGTTGGATGGATCCCTAGCTAGCTTGGATGGATCATCTATCATCATGGTGATCTCATCATGTACTCCTGCTCCATCTCCTCATGCGTGCCAGGCTTCTTAATATAATCTACCTCTGCTTTCATCCCAAAAAAAAAAAAAAA(SEQ ID No.2)

[0033] OE-F:GCAAGAAGCAATTCTTGAGC(SEQ ID No.3);

[0034] OE-R:CATCCAACATTGATCGACAC(SEQ ID No.4)。

[0035] The OsMT2b gene was cloned from the rice variety Zhonghua 11 by conventional PCR method. The amplification system was as follows: 2×PCRMix, 15 μL; OE-F, 1 μL; OE-R, 1 μL; cDNA, 1 μL; ddH 2 O, 12 μL. The PCR program was 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min.

[0036] The coding region sequence of the OsMT2b gene is shown in SEQ ID No.5, and the protein sequence encoded by the gene is shown in SEQ ID No.6. The gene is located on chromosome 5, and its gene locus number in rice is LOC_Os05g02070 (Os05g0111300).

[0037] Coding region sequence of OsMT2b gene:

[0038] ATGTCTTGCTGCGGCGGCAACTGCGGCTGCGGCAGCGGCTGCCAGTGCGGCGGCGGCTGCGGCGGATGCAAGATGTTCCCTGATGTGGAGGCCACAGCCACCACCAAGACCTTCGTCCTCGCTGCTCCA TCCAACAAGGCGAGCTCTGGAGGAATGGAGATGGCGGTGGAGAGCGGCGAGAACGGCGGCTGCGGCTGCAACACCTGCAAGTGTGGCACCAGCTGCAGCGGCTGCTCCTGCTGCTCCTGCAACTGA(SEQ ID No.5);

[0039] Protein sequence encoded by OsMT2b gene:

[0040] MSCCGGNCGCGSGCQCGGGCGGCKMFPDVEATATTKTFVLAAPSNKASSGGMEMAVSGENGGCGCNTCKCGTSCSGCSCCSCN (SEQ ID No. 6).

[0041] 2. Select the restriction enzyme sites (EcoRI and EcoRV) added in the amplification, and digest the amplified fragment and the overexpression vector pCAMBIA1380 (which has been connected to the Ubi promoter). The enzyme digestion system is vector / fragment, 0.5-1μg; endonuclease 1, 1μL; endonuclease 2, 1μL; FastDigest Buffer, 3μL; ddH 2O, make up to 30μL. The reaction conditions are 37℃, 30min. After the enzyme digestion is completed, the linearized fragment and vector are recovered by the kit. The fragment is connected to the overexpression vector by T4 ligase. The connection system is 10×T4 ligase buffer, 1μL; linearized vector, 30ng; fragment, 20ng; T4 ligase, 0.3μL; ddH 2 O, make up to 10 μL. The connection conditions are 22°C, 2h. The ligation product is transformed into DH5α competent cells and PCR is performed using OE-F and OE-R primers. Positive colonies are preliminarily screened, and after successful sequencing and sequence alignment, the overexpression vector is obtained. The positive plasmid is transformed into Agrobacterium EHA105 competent cells by electroporation, and the Agrobacterium is identified. PCR is performed using OE-F and OE-R primers to obtain positive Agrobacterium, ensuring that the Agrobacterium used for transformation contains the target gene.

[0042] 3. The overexpression vector is introduced into rice callus tissue by using genetic transformation mediated by Agrobacterium tumefaciens. The specific steps are as follows: select callus tissue with good growth status, infect and co-cultivate it with Agrobacterium; inoculate the co-cultivated callus tissue to a screening medium with antibiotics for screening; select tissue with good growth status in the screening medium and transfer it to a differentiation medium for differentiation; after the callus tissue differentiates into green adventitious buds, continue to culture it until it develops into seedlings; transfer it to a rooting medium to obtain multiple plants transformed from different callus tissues.

[0043] 4. The leaves were soaked in hygromycin solution to screen positive plants. The expression of OsMT2b gene in transgenic plants was detected by qRT-PCR. Two different overexpression strains OE22 and OE26 were selected. The results showed that the expression of OsMT2b in these two strains was significantly increased ( Figure 1 ).

[0044] 5. Phenotypic observation of seeds of positive plants was performed and photographs were taken. The results showed that the length and width of seeds of OE22 and OE26 were significantly larger than those of wild type ( Figure 3 ). The length, width and thickness of seeds were measured and the thousand-grain weight was weighed with an analytical balance. According to the statistical data, the length, width and thousand-grain weight of the OsMT2b overexpression strain were significantly higher than those of the wild type ( Figure 4 ).

[0045] Example 2 Construction and genetic transformation of OsMT2b interference vector

[0046] 1. Design primers (SEQ ID No.7 and SEQ ID No.8), use the genomic DNA of Zhonghua 11 (SEQ ID No.1) as template, and amplify the promoter region of OsMT2b by conventional PCR method. The PCR amplification system is 2×PCR mix, 15 μL; PRO-F, 1 μL; PRO-R, 1 μL; DNA, 1 μL; ddH 2 O, 12 μL. The PCR amplification program was 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min. HindIII and EcoRV were selected as restriction sites to digest the pCAMBIA1380 empty load and amplified fragments. The restriction digestion system was vector / fragment, 0.5-1 μg; endonuclease 1, 1 μL; endonuclease 2, 1 μL; 10×FastDigest Buffer, 3 μL; ddH 2 O, make up to 30μL. The reaction conditions are 37℃, 30min. After the digestion is completed, the digested fragments and vectors are recovered by the kit. The fragments are connected to the pCAMBIA1380 empty load by T4 ligase. The connection system is 10×T4 ligase buffer, 1μL; linearized vector, 30ng; fragment, 60ng; T4 ligase, 0.3μL; ddH 2 O, make up to 10 μL. The connection conditions are 22°C, 2h. The fragment is connected to the pCAMBIA1380 empty vector to form the pCAMBIA1380-promoter intermediate vector. The primer sequences are as follows:

[0047] PRO-F:AAAAAAGCTTGAGATGCTAATCAAGTCTCTCTG (SEQ ID NO.7);

[0048] PRO-R:AAAAGATATCAGATGTTGTTGCTGATTGAGCTC (SEQ ID No. 8).

[0049] 2. In order to form the stem-loop structure of RNAi, the following three primers were designed, with the genomic DNA of Zhonghua 11 (SEQ ID No.1) as a template, using primers RNAi-F1 (SEQ ID NO.9) and RNAi-R2 (SEQ ID NO.10) as primer pairs, and amplifying to obtain fragment 1 according to the PCR system and amplification program of step 1. Select EcoRI and EcoRV as restriction sites, and digest fragment 1 and the pCAMBIA1380-promoter vector obtained in step 1 according to the restriction system and restriction conditions of step 1, and recycle them by a recovery kit. According to the connection method and connection system of step 1, fragment 1 is connected to the promoter downstream in the pCAMBIA1380-promoter vector to form a pCAMBIA1380-promoter-fragment 1 intermediate vector.

[0050] 3. Using the cDNA of Zhonghua 11 (SEQ ID No. 2) as a template, RNAi-F1 (SEQ ID NO. 9) and RNAi-R3 (SEQ ID NO. 11) as primers, amplify the gene fragment of OsMT2b. Fragment 2 was amplified by the PCR method of step 1, and EcoRI and SpeI were selected as restriction sites. Fragment 2 and the pCAMBIA1380-promoter-fragment 1 intermediate vector obtained in step 2 were digested according to the restriction system and method of step 1. The linearized vector and fragment were recovered by a recovery kit, and fragment 2 was connected to the intermediate vector according to the connection method of step 1 to form a pCAMBIA1380-promoter-fragment 1-fragment 2 vector.

[0051] RNAI-F1:AAAAGAATTCGCTGCTCCATCCAACAAGG (SEQ ID NO.9);

[0052] RNAI-R2:AAAAGATATCTCAGTTGCAGGAGCAGCA (SEQ ID NO. 10);

[0053] RNAI-R3: AAAAACTAGTTCAGTTGCAGGAGCAGCA (SEQ ID NO. 11).

[0054] 4. Transform the ligation product into DH5α competent cells, and perform PCR amplification using the PCR method in step 1 with PRO-F and RNAI-R3 as primers to screen positive colonies. RNAi interference vector strains were obtained by sequencing and sequence alignment. The positive plasmid was transformed into Agrobacterium EHA105 competent cells by electroporation, and the Agrobacterium was tested. PCR amplification was performed using PRO-F and RNAI-R3 as primers to obtain positive Agrobacterium, ensuring that the Agrobacterium used for transformation contains the target fragment.

[0055] 5. The interference vector is introduced into rice callus tissue by using genetic transformation mediated by Agrobacterium tumefaciens. The specific steps are as follows: select callus tissue with good growth status, infect and co-cultivate it with Agrobacterium; inoculate the co-cultivated callus tissue to a screening medium with antibiotics for screening; select tissue with good growth status on the screening medium and transfer it to a differentiation medium for differentiation; after the callus tissue differentiates into green adventitious buds, continue to culture it until it develops into seedlings; transfer it to a rooting medium to obtain multiple plants transformed from different callus tissues.

[0056] 6. Soak the leaves in hygromycin solution to screen positive plants. Use qRT-PCR to detect the expression of OsMT2b gene in transgenic plants. Select two different interference strains, MTi10 and MTi12, and the expression of OsMT2b in these two strains is significantly reduced ( Figure 2 ).

[0057] 7. Phenotypic observation of seeds of positive plants was performed and photographs were taken. The results showed that the length and width of seeds of MTi10 and MTi12 were significantly smaller than those of wild type ( Figure 3 ). The length, width and thickness of seeds were measured, and the thousand-grain weight was weighed with an analytical balance. According to the statistical data, the length, width and thousand-grain weight of the OsMT2b interference strain were significantly lower than those of the wild type ( Figure 4 ).

[0058] In summary, the present invention discloses for the first time the function of the rice OsMT2b gene in regulating the size of rice seeds. Overexpression of the gene makes the rice seeds larger and the thousand-grain weight increases. Interference of the OsMT2b gene by RNAi technology makes the rice seeds smaller and the thousand-grain weight decreases. The present invention provides gene resources for rice breeding, and the OsMT2b gene can be applied to high-yield rice breeding, which has important application value and practical significance.

[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 OsMT2b gene in regulating rice seed size, characterized in that: The nucleotide sequence of the OsMT2b gene is shown in SEQ ID No.1; the coding region sequence of the OsMT2b gene is shown in SEQ ID No.

5.

2. The use of the protein encoded by the OsMT2b gene in regulating rice seed size as claimed in claim 1, characterized in that: The amino acid sequence of the protein encoded by the OsMT2b gene is shown in SEQ ID No.

6.

3. Use of a recombinant vector containing the OsMT2b gene of claim 1 in regulating rice seed size.

4. Use of a host bacteria containing the recombinant vector according to claim 3 in regulating rice seed size.

5. The use according to any one of claims 1 to 4, characterized in that: When the OsMT2b gene is overexpressed, the rice seed grains become larger; when the OsMT2b gene is inhibited, the rice seed grains become smaller.

6. A method for constructing transgenic rice capable of enlarging rice seeds, characterized in that: The method comprises overexpressing the OsMT2b gene to enlarge the grains of the rice seeds; the nucleotide sequence of the OsMT2b gene is shown in SEQ ID No.

1.

7. A method for constructing transgenic rice capable of reducing the size of rice seeds, characterized in that: The method comprises inhibiting the expression of the OsMT2b gene to reduce the size of the rice seeds; the nucleotide sequence of the OsMT2b gene is shown as SEQ ID No. 1.

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