A new gene regulating rice grain length, osphax5, and the protein encoded by the gene and application thereof

Abstract

The application discloses an OsPAHX5 gene and use of the OsPAHX5 gene in regulating grain length of rice, and belongs to the field of plant breeding and biotechnology.The application discloses a coding region nucleic acid sequence of the OsPAHX5 gene, a coded amino acid sequence, a target site nucleic acid sequence of CRISPR-Cas9 knockout, a nucleic acid sequence of a knockout mutant and an amino acid sequence of the knockout mutant, as shown in a standard nucleotide or amino acid sequence table according to ST.26.The application studies the function of the OsPAHX5 gene by using molecular biology and plant transgenic technology, and finds that the grain length of a knockout transgenic plant of the OsPAHX5 gene is significantly greater than that of a wild type variety after the OsPAHX5 gene is knocked out by using CRISPR-Cas9 technology; the grain length of an overexpression transgenic plant of the OsPAHX5 gene is not significantly different from that of the wild type variety after the OsPAHX5 gene is overexpressed by using a transgenic overexpression technology.The application can create a rice variety with larger grain length by using the OsPAHX5 gene.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and biotechnology, specifically relating to a novel gene OsPAHX5 that regulates rice grain length, its encoded protein, and its applications. Background Technology

[0002] Rice grain shape, mainly including grain length, width, and thickness, is an important quantitative trait controlled by multiple genes. It not only directly affects rice yield but is also closely related to rice quality. Currently, six pathways regulating grain shape are known: the E3 ubiquitin-proteasome degradation pathway, the G protein signaling pathway, the MAPK cascade signaling pathway, the plant hormone pathway, the transcription factor pathway, and the epigenetic pathway. In recent years, based on map-based cloning and genome-wide association studies (GWAS) techniques, many genes related to grain development have been isolated in rice. Currently, over 500 QTLs regulating grain shape have been identified, located on all 12 chromosomes of the rice genome. Among them, genes controlling grain length include GLW7, GS3, GL10, and GL6; those controlling grain width include qSW5, GW5, GW2, GS5, and GWY10; those regulating grain thickness include TGW3, OsBZR1, OsMADS56, and WTG1; those promoting grain size by regulating cell proliferation include SLG7 and SMOS1; and those affecting cell division include D1, RGB1, DEP1, and OsMAPK6. These previous studies indicate that the genetic mechanism of rice grain shape is highly complex, and the understanding of its genetic basis remains insufficient. Many other genes or loci are also involved in regulating rice grain shape. The phytanoyl-CoA dioxygenase gene AtPAHX in Arabidopsis has been reported to be associated with senescence by affecting chlorophyll degradation, while the function of its homolog in rice, OsPAHX5, including the regulation of rice grain length, has not yet been reported. Summary of the Invention

[0003] In view of this, the present invention addresses the problems existing in current rice grain shape-related research and discloses a new gene, OsPAHX5, that regulates rice grain length. This gene encodes a phytyl-CoA dioxygenase. The coding region sequence of the OsPAHX5 gene and the encoded protein sequence are disclosed, as shown in SEQ ID NO.1 and SEQ ID NO.2 in the ST.26 standard sequence nucleotide or amino acid sequence listing. Attached Figure Description

[0004] The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 illustrates the technical method of knocking out the OsPAHX5 gene using CRISPR-Cas9 gene editing technology according to the present invention. It includes information on a target site selected in the coding region of the OsPAHX5 gene and the nucleotide mutation types of the two homozygous knockout mutants obtained, as well as the resulting amino acid changes. WT represents the wild type (Nipponbare), and ospahx5-1 and ospahx5-2 are two homozygous knockout mutants; Figure 2 shows the grain lengths of the OsPAHX5 gene knockout mutants and wild type obtained by the technical method of the present invention. WT represents the wild type (Nipponbare), and ospahx5-1 and ospahx5-2 are two homozygous knockout mutants. * Indicates significant (P<0.05); Figure 3 shows photographs of the main agronomic traits of the OsPAHX5 gene knockout mutant and wild type obtained by the method of this invention. WT represents the wild type (Nipponbare), and ospahx5-1 and ospahx5-2 are two homozygous knockout mutants; Figure 4 shows the grain length of the transgenic homozygous line overexpressing the OsPAHX5 gene and the wild type obtained by the method of this invention. WT represents the wild type (Nipponbare), OE represents the untagged homozygous overexpression line, OE-GFP represents the overexpression homozygous line tagged with green fluorescent protein (GFP), and OE-HA represents the overexpression homozygous line tagged with hemagglutinin (HA). Detailed Implementation

[0005] This invention discloses a method for increasing rice grain length using the OsPAHX5 gene and its encoded protein, comprising the following steps: 1) Knocking out the OsPAHX5 gene using CRISPR-Cas9 gene editing technology. When constructing the CRISPR-Cas9 knockout vector, the forward nucleotide sequence of a target site in the coding region of the OsPAHX5 gene is selected as 5′→3′: as shown in SEQ ID No. 3 of the ST.26 standard sequence nucleotide or amino acid sequence listing; 2) Obtaining homozygous transgenic mutant lines with the OsPAHX5 gene knocked out. After constructing the knockout vector, a conventional japonica rice variety, Nipponbare, was selected as the transformation background for genetic transformation, resulting in two homozygous knockout mutant transgenic lines. One knockout mutant has a 5′→3′ forward nucleotide sequence in the OsPAHX5 gene coding region, as shown in SEQ ID No. 4 of the ST.26 standard sequence nucleotide or amino acid sequence listing, and a 5′→3′ forward amino acid sequence, as shown in SEQ ID No. 6 of the ST.26 standard sequence nucleotide or amino acid sequence listing. The other knockout mutant has a 5′→3′ forward nucleotide sequence in the OsPAHX5 gene coding region, as shown in SEQ ID No. 5 of the ST.26 standard sequence nucleotide or amino acid sequence listing, and a 5′→3′ forward amino acid sequence, as shown in SEQ ID No. 7 of the ST.26 standard sequence nucleotide or amino acid sequence listing. 3) Obtain homozygous transgenic lines overexpressing the OsPAHX5 gene. Using overexpression transgenic functional experiments, three homozygous OsPAHX5 gene-modified homozygous lines were obtained using the conventional japonica rice variety Nipponbare as the transformation background; 4) Grain length and major agronomic traits were measured in wild-type, OsPAHX5 gene-knockout transgenic mutants, and overexpression homozygous lines. It was found that the grain length of the OsPAHX5 gene-knockout mutant was significantly larger than that of the wild type, while agronomic traits such as plant height and plant type showed no significant difference from the wild type. The grain length of the OsPAHX5 gene-modified homozygous lines showed no significant difference from the wild type. This invention provides a novel rice grain length regulating gene OsPAHX5, its encoded protein, and its applications. The OsPAHX5 gene negatively regulates rice grain length; knocking out this gene can significantly increase rice grain length. This invention discloses the creation of new rice germplasm with larger grain lengths by knocking out the OsPAHX5 gene and thereby altering the OsPAHX5 protein, which has significant application value for rice grain shape improvement.

Claims

1. The application of the gene OsPAHX5 and its encoded protein in increasing rice grain length, characterized in that, The accession number of the OsPAHX5 gene in the rice genome is LOC_Os05g39650, and its coding region nucleic acid sequence and encoded protein sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A method for increasing rice grain length by regulating the expression of the OsPAHX5 gene as described in claim 1, characterized in that, This includes using gene editing technology to knock out the OsPAHX5 gene in rice to obtain rice plants with increased grain length.

3. The application according to claim 2, characterized in that, in, The gene editing was CRISPR-Cas9 editing, and the target site on the coding region of the OsPAHX5 gene selected when constructing the knockout vector had a nucleotide forward sequence of 5′→3′, as shown in SEQ ID NO.

3.

4. The application according to claim 2, characterized in that, Two OsPAHX5 gene knockout mutants were obtained using CRISPR-Cas9 gene editing technology. The forward nucleotide sequence of the OsPAHX5 gene coding region of one knockout mutant is 5′→3′, as shown in SEQ ID No.

4. The forward nucleotide sequence of the OsPAHX5 gene coding region of the other knockout mutant is 5′→3′, as shown in SEQ ID No.

5.

5. The application according to claim 2, characterized in that, Two OsPAHX5 gene knockout mutants were obtained using CRISPR-Cas9 gene editing technology. One knockout mutant has a forward amino acid sequence of 5′→3′, as shown in SEQ ID No. 6, and the other knockout mutant has a forward amino acid sequence of 5′→3′, as shown in SEQ ID No. 7.

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