Mets2 gene for regulating grain shape of rice, protein and application thereof

Abstract

The present invention relates to the field of genetic engineering technology and discloses a method for regulating rice grain shape. METS2 Genes, proteins and their applications, METS2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1. METS2 The present invention uses the above-mentioned gene for regulating rice grain shape. METS2 Genes, proteins and their applications, overexpressed in rice plants METS2 Genes can produce short-grain rice, which is not easy to break during processing and has high-quality finished products. It not only provides a basis for further elucidating the molecular mechanism of rice grain shape, but also provides new genetic resources and breeding resources for rice breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a grain shape regulating gene, protein and application thereof in rice. METS2 The present application relates to the technical field of genetic engineering, and in particular to a grain shape regulating gene, protein and application thereof in rice. BACKGROUND

[0002] Rice grain development determines the appearance quality (grain shape, chalkiness, etc.), and the appearance quality is a core agronomic trait determining yield and market competitiveness. A large number of grain shape influencing genes are contained in abundant rice resources, and researchers have cloned multiple grain shape regulating genes and analyzed their molecular mechanisms. Among them, GS3 encodes a transmembrane protein composed of 232 amino acids, controls rice grain weight and length, and rice grain width and grain filling degree; GW5 / qSW5 encodes a calmodulin binding protein, degrades the transcriptional repressor OsBAK1 through the ubiquitin-proteasome pathway, releases the inhibition of cell expansion, and thus increases grain width; GNP12 encodes an NB-ARC gene, affects the development of rice grain length and spike grain number; Gnp4 is a grain length regulating factor, Gnp4 / LAX2 as a component of the auxin response pathway, may play a role in the OsIAA3-OsARF25-OsERF142 pathway to regulate grain length. In addition, GS5 and GLW7 respectively affect grain shape by regulating cell cycle and cell wall loosening enzyme activity. At present, two major problems are faced by high-quality rice breeding in China: narrow genetic basis and insufficient appearance quality gene resources, and the genetic network of related traits is not clear. Therefore, mining rice grain development genes and analyzing their grain shape formation mechanisms not only provide a theoretical basis for quality genetic improvement, but also broaden the genetic resources and help molecular design breeding, which has important theoretical and practical significance for cultivating new varieties of high-quality rice. In the future, more excellent grain shape regulating genes need to be further explored and identified to broaden the gene sources and enrich the genetic background for rice breeding. SUMMARY

[0003] The present application relates to the technical field of genetic engineering, and in particular to a grain shape regulating gene, protein and application thereof in rice. METS2 The present application relates to the technical field of genetic engineering, and in particular to a grain shape regulating gene, protein and application thereof in rice. METS2 The present application relates to the technical field of genetic engineering, and in particular to a grain shape regulating gene, protein and application thereof in rice.

[0004] To achieve the above-mentioned purpose, the present application provides a grain shape regulating gene, METS2 gene, METS2The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0005] Further, the application also provides a METS2 protein for regulating grain shape of rice. METS2 The application of the gene in regulating grain shape of rice, METS2 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0006] Further, in the application, the gene is overexpressed in rice plants to reduce the grain length and grain width of rice. METS2 The gene,

[0007] Further, the application also provides a METS2 protein for regulating grain shape of rice, the amino acid sequence of the METS2 protein is shown as SEQ ID NO. 2, and the coding gene of the METS2 protein is METS2 The gene, METS2 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0008] Further, the application also provides the application of the METS2 protein in regulating grain shape of rice, the coding gene of the METS2 protein is METS2 The gene, METS2 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0009] Further, the application also provides a recombinant vector, which comprises the above-mentioned METS2 The gene, METS2 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0010] Further, the recombinant vector is a plant overexpression vector.

[0011] A biological material, which comprises the above-mentioned recombinant vector, and the biological material is a recombinant microorganism, a transgenic plant cell line or a transgenic plant tissue.

[0012] The recombinant microorganism is bacteria, yeast, algae or fungi; the bacteria is one of Escherichia (E. coli), Erwinia (E. herbicola), Agrobacterium tumefaciens, Flavobacterium (F. columnare), Alcaligenes (A. xylosoxidans), Pseudomonas (P. aeruginosa) or Bacillus (B. subtilis). Escherichia Erwinia Agrobacterium Flavobacterium Alcaligenes Pseudomonas Bacillus

[0013] Further, the application also provides the application of the above-mentioned recombinant vector in regulating grain shape of rice.

[0014] ​​​​​​​Furthermore, the present invention also provides a method for cultivating short-grain transgenic rice, METS2 The gene was overexpressed in rice plants, and short-grain transgenic rice was obtained through screening and cultivation; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] The method for regulating rice grain shape of the present invention METS2 The advantages and positive effects of genes, proteins and their applications are:

[0016] 1. The present invention discloses a method related to rice grain shape. METS2 Gene, through experiments, it was shown that rice grain shape-related coding genes METS2 Under the condition of enhanced function or increased expression, short-grain rice will be obtained, which proves that the rice-related coding genes METS2 The results not only provide a basis for further elucidating the molecular mechanism of rice grain shape, but also provide new genetic resources and breeding resources for rice breeding.

[0017] 2. The present invention discloses overexpression in rice METS2 The invention discloses a method for obtaining short-grain transgenic rice, wherein the obtained short-grain rice is not easy to break during processing and the finished product is of high quality.

[0018] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 In the embodiment of the present invention METS2 -OE1, METS2 -Schematic diagram of the relative expression levels of overexpressed genes in OE2 and its parent rice Guanghui 998 (abbreviated as GH998) plants;

[0020] Figure 2 is the coding gene related to rice grain shape in the embodiment of the present invention METS2 Bar graph of grain length and width of overexpressing transgenic rice, where A is grain length and B is grain width;

[0021] Figure 3 is the coding gene related to rice grain shape in the embodiment of the present invention METS2 Grain length and width phenotypes of overexpressing transgenic rice. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0024] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative work shall fall within the scope of the present application. The experimental methods in the following examples without specific conditions are generally determined according to the national standards. The experimental instruments, equipment and reagents in the following examples without sources are all commercially available raw materials.

[0025] Unless otherwise defined or explained, all professional and scientific terms used in the present application have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied to the method of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0026] Example 1

[0027] Construction of the super-expression vector of the grain shape related gene of rice:

[0028] 1. Construction of the super-expression vector:

[0029] 1.1 METS2 Obtaining of the gene:

[0030] The DNA of common wild rice Y11 (O. minuta Oryza rufipogon Griff.) collected by the Rice Institute of Guangxi Academy of Agricultural Sciences (Guangxi Germplasm Bank) was used as a template, and the target gene was obtained by PCR amplification using the following primers primer1 and primer2:

[0031] primer1: 5' - atggcgtctcacattgttgg-3' (SEQ ID NO. 3);

[0032] primer2: 5' - tcacttcgcgctagcgag-3' (SEQ ID NO. 4).

[0033] After the PCR product was recovered and purified, it was connected to the Zero sequencing vector (purchased from Beijing Quanshi Gold Company), and the DH5a competent cells were transformed. After selecting positive clones, sequencing was performed.

[0034] The sequencing result showed that the sequence of the PCR product was as shown in SEQ ID NO. 1, and the length was 2301 bp, which was named METS2 gene.

[0035]

[0036] The amino acid sequence of the METS2 protein is shown in SEQ ID NO. 2:

[0037] MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMADAGIKYIPSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGFSTYFSMARGNATVPAMEMTKWFDTNYHFIVPELGPNTKFSYSSHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKPAKGVEKSFALLSLLSSILPVYKEVIAELKAAGATWIQFDEPTLVLDLDSHQLAAFSAAYTELESALSGLNVLIETYFADIPAESYKTLTSLNSVTAYGFDLIRGFKTLDLVKSAGFPSGKYLFAGVVDGRNIWADDLAASLTTLESLEAIVGKDKLVVSTSCSLMHTAVDLVNETKLDSEIKSWLAFAAQKVVEVNALAKALAGQKDEAYFAANTAAQASRRSSPRVTNEEVQKAAAALRGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTVELRRVRREYKAKKISEDEYVSAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGSRCVKPPIIYGDVSRPNAMTVFWSKMAQSMTSRPMKGMLTGPVTILNWSFVRNDQPRFETCYQIALAIKKEVEDLEAGGIQVIQIDEAALREGLPLRKAEHAFYLDWAVHSFRITNCGVQDTTQIHTHMCYSNFNDIIHSIINMDADVITIENSRSDEKLLSVFREGVKYGAGIGPGVYDIHSPRIPSTEEIADRINKMLAVLDTNILWVNPDCGLKTRKYTEVKPALTNMVLAAKLIRTQLASAK (SEQ ID NO. 2).

[0038] 1.2 Grain shape related genes of O. officinalis METS2 Construction of overexpression vector (i.e. recombinant expression vector PMDC32-OE- METS2 ):

[0039] 1) The wild rice cDNA was amplified by primer 1 and primer 2 to obtain METS2 The sequence of the gene was connected to the vector Zero to obtain a positive clone of the recombinant Zero- METS2 The recombinant vector Zero- METS2 was digested by restriction endonuclease Kpn I and Pac I to obtain the OE- METS2 fragment;

[0040] 2) The expression vector PMDC32 was digested by restriction endonuclease Kpn I and Pac I to obtain a linear expression vector PMDC32, and the linear fragment was recovered; the fragment OE- METS2 was integrated into the linear expression vector PMDC32 by the method of homologous recombination directional cloning (for details, refer to the instruction manual of PMDC32) to obtain a homologous recombination product 1 (i.e. PMDC32-OE- METS2 , the gene METS2 overexpression vector of the application), and then the homologous recombination product 1 was transformed into DH5α competent cells and cultured at 37 ℃ overnight.

[0041] 3) The recombinant vector PMDC32-OE- METS2 obtained in step 2) was sequenced, and the results showed that the nucleotide sequence as shown in SEQ ID NO. 1 was inserted in the Kpn I enzyme cutting site of the expression vector PMDC32 in a forward direction, that is, the DNA sequence between the Kpn I and Pac I recognition sites (recognition sequence) of PMDC32 was successfully replaced by the DNA sequence as shown in SEQ ID No. 1.

[0042] 1.3 Transformation:

[0043] Heat shock method was used to transform E. coli with 5 μL of the recombinant vector PMDC32-OE- METS2 , and the specific steps refer to the instruction manual of the Transgen kit.

[0044] 1.4 Bacterial liquid PCR verification:

[0045] In the super-clean bench, a single colony was picked as a template for PCR amplification, and Taq DNA polymerase PCR amplification system and program were used.

[0046] 1.5 Plasmid extraction: The plasmid was extracted and the concentration was detected according to the instruction manual of the Transgen plasmid extraction kit, and the recombinant expression vector PMDC32-OE- METS2 was obtained.

[0047] Example 2

[0048] Cultivation of rice grain shape growth and development related coding gene METS2Transgenic plants of overexpression vector of PMDC32-OE and identification of transgenic plants:

[0049] I. Cultivation METS2 Transgenic plants of gene overexpression:

[0050] The recombinant vector PMDC32-OE was introduced into Agrobacterium tumefaciens EHA105 by heat shock method to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE. METS2 GH998 indica rice was transformed by Agrobacterium tumefaciens EHA105, and the specific method was as follows:

[0051] 1. Plasmid transformation:

[0052] The recombinant vector PMDC32-OE obtained in Example 1 was introduced into Agrobacterium tumefaciens EHA105 by heat shock method to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE. METS2 The recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE was cultured at 28°C for 16 h, and the bacterial cells were collected. The bacterial cells were diluted with N6 liquid medium (Sigma, product catalog number C1416) containing 100 μM acetyl-syringone to obtain a diluted bacterial solution, and the OD600 of the diluted bacterial solution was about 0.5. METS2 METS2 2. Infection:

[0053] The mature embryo callus of GH998 indica rice cultured for one month was mixed with the diluted bacterial solution obtained in step 1 for 30 min, and then the bacterial solution was absorbed with filter paper and transferred into N6 solid co-culture medium for co-culture at 24°C for 3 d to obtain callus treated by co-culture.

[0054] 3. Screening:

[0055] 3.1 The callus treated by co-culture in step 2 was inoculated on N6 solid screening medium containing 150 mg / L hygromycin (N6 solid screening medium was obtained by adding hygromycin to N6 solid medium, and the mass concentration of hygromycin in N6 solid screening medium was 150 mg / L) for the first screening.

[0056] 3.2 The healthy callus was picked up on the 16th day of the first screening and transferred into N6 solid screening medium containing 200 mg / L hygromycin (N6 solid screening medium was obtained by adding hygromycin to N6 solid medium, and the mass concentration of hygromycin in N6 solid screening medium was 200 mg / L) for the second screening, and the callus was subcultured every 15 d for a total of 1 time to obtain resistant callus.

[0057] 4. Differentiation culture to obtain overexpression positive plants:

[0058] 4. Differentiation culture to obtain overexpression positive plants: ​

[0059] The resistant callus obtained in step 3 was transferred to a differentiation medium containing 150 mg / L of hygromycin (differentiation medium: 6-BA 2 mg, NAA 0.2 mg, N6 4 g, hydrolyzed casein 1 g, inositol 0.1 g, sucrose 25 g, sorbitol 2.4 g, agar powder 7 g, deionized water 1 L) for differentiation, and cultured at 24°C for 45 d (at this time, the height of the aerial part of the plant was about 15 cm), the bottle opening was opened for 3 d for seedling raising, and then transplanted to a greenhouse for cultivation, which was a PMDC32-OE- METS2 plant (referred to as the T0 generation). Two plants were obtained by transforming the callus, and the obtained plants were named as: METS2 -OE1, METS2 -OE2 plant.

[0060] two, METS2 PCR identification of transgenic plants:

[0061] The genomic DNA of the T1 seedlings of the above METS2 -OE1, METS2 -OE2 plants and the recipient parent rice GH998 plant seedling leaves was extracted, and primer hyg-F and hyg-R were used for PCR molecular detection to identify positive seedlings. After PCR verification, the single plant with a hyg positive band was identified as a transgenic single plant. METS2 -OE1, METS2 -OE2.

[0062] hyg-F: 5' -AAAAGTTCGACAGCGTCTCCGACC-3' (SEQ ID NO. 5),

[0063] hyg-R: 5' -TCTACACAGCCATCGGTCCAGACG-3' (SEQ ID NO. 6).

[0064] The PCR system is shown in Table 1:

[0065] Table 1 PCR system

[0066] ;

[0067] The amplification program is shown in Table 2:

[0068] Table 2 Amplification program

[0069] ;

[0070] three, METS2 identification of gene expression levels in transgenic plants METS2 :

[0071] Extracting the above METS2 -OE1, Actin -OE2 plant T1 generation seedlings and recipient parent rice GH998 plant leaf RNA, setting the internal reference as Actin , using internal reference primers Actin -F and METS2 -R, and METS2 gene-specific quantitative primers METS2 -qRT-F and METS2 -qRT-R to perform fluorescent quantitative PCR reaction to detect the expression level changes of different transgenic plants Figure 1 genes.

[0072] The results show that (1) in the positive plants into the recombinant vector PMDC32-OE- METS2 , the expression levels of METS2 genes are significantly higher than the expression levels of METS2 genes of the control strain (GH998), and the primers are as follows: METS2

[0073] METS2 -qRT-F: 5' -GCGCTGAGGATTTGGAGAAG-3' (SEQ ID NO. 7)

[0074] Actin -qRT-R: 5' -ATAGCAGGGACAGTGGCATT-3' (SEQ ID NO. 8)

[0075] Actin- -F: 5' - ATTTGGCACCACACATTCTAC-3' (SEQ ID NO. 9)

[0076] METS2 R: 5' -ATAACCTTCGTAGATTGGGACT-3' (SEQ ID NO. 10)

[0077] Four, METS2 phenotypic identification of transgenic plants:

[0078] Respectively METS2 -OE1, METS2 -OE2 and GH998 plants were planted in the transgenic test greenhouse of Guangxi Academy of Agricultural Sciences, and the seeds were harvested after maturity and the grain shape phenotypic differences of METS2 -OE1, Figure 2 -OE2 and GH998 were observed. The measurement observation results are shown in Tables 3, Figure 3 and METS2 . ​

[0079] Table 3 Genes encoding rice grain shape, growth and development METS2 Grain shape expression in overexpression plants

[0080] ;

[0081] Compared with GH998 plants, PMDC32-OE- METS2 Plants ( METS2 -OE1, METS2 -OE2) showed a phenotype of significantly reduced rice grain length and width compared with the control group (GH998) ( P <0.05), thus proving METS2 Genes involved in controlling rice grain shape, i.e. METS2 The gene is related to rice grain shape and is overexpressed METS2 The gene can significantly reduce rice grain length and width.

[0082] Therefore, the present invention adopts the above-mentioned method for regulating rice grain shape METS2 Genes, proteins and their applications, overexpressed in rice plants ​ Genes can produce short-grain rice, which is not easy to break during processing and has high-quality finished products. It not only provides a basis for further elucidating the molecular mechanism of rice grain shape, but also provides new genetic resources and breeding resources for rice breeding.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. METS2 The application of genes in regulating rice grain shape is characterized by: Overexpression in rice plants METS2 Genes that reduce rice grain length and width; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. METS2 The amino acid sequence of the METS2 protein encoded by the gene is shown in SEQ ID NO.

2.

2. Contains METS2 The application of a recombinant gene vector in regulating rice grain shape is characterized by: Overexpression METS2 The recombinant vector of the gene is introduced into rice, METS2 Overexpression of the gene in rice reduces grain length and width; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A method for cultivating short-grain transgenic rice, characterized by: Will METS2 The gene was overexpressed in rice plants, and short-grain transgenic rice was obtained through screening and cultivation; METS2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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