Novel gene OsPAHX5 for regulating and controlling rice grain length, protein coded by novel gene OsPAHX5 and application of novel gene OsPAHX5

Abstract

The invention discloses an application of an OsPAHX5 gene and a protein coded by the OsPAHX5 gene in regulation and control of rice grain length, and belongs to the technical field of plant breeding and biology. The invention discloses a nucleotide sequence of a coding region of an OsPAHX5 gene, a coded amino acid sequence, a nucleotide sequence of a CRISPR-Cas9 knockout target site, a nucleotide sequence of a knockout mutant and an amino acid sequence of the knockout mutant, which are shown in an ST.26 standard sequence nucleotide or amino acid sequence table. The function of the OsPAHX5 gene is researched by utilizing molecular biology and a plant transgenic technology, and it is found that after the OsPAHX5 gene is knocked out by utilizing a CRISPR-Cas9 technology, the grain length of a knocked-out transgenic plant is remarkably larger than that of a wild type variety; after the OsPAHX5 gene is subjected to overexpression by using a transgenic overexpression technology, the grain length of an overexpressed transgenic plant is not obviously different from that of a wild type variety. According to the invention, the OsPAHX5 gene can be utilized to create a rice variety with larger grain length.

Description

Technical Field

[0001] The present invention belongs to the field of plant breeding and biotechnology, and specifically relates to a new gene for regulating rice grain length. OsPAHX5 and its encoded proteins and applications. Background Art

[0002] Rice grain shape mainly includes grain length, grain width, grain thickness, etc. It 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, there are 6 known pathways for regulating grain shape, namely E3 ubiquitin proteasome degradation pathway, G protein signaling pathway, MAPK cascade signaling pathway, plant hormone pathway, transcription factor pathway and epigenetic pathway. In recent years, based on positional cloning and genome-wide association study (GWAS) technology, many genes related to grain development have been isolated in rice. Currently, more than 500 QTLs regulating grain shape have been found, and they are located on 12 chromosomes of the rice genome. Among them, the ones that control grain length are GLW7 , GS3 , GL10 , GL6 , the particle width is controlled by Q 5 , GW5 , GW2 , GS5 , GWY10 etc.; those that adjust the grain thickness include TGW3 , ObZR1 , OsMADS56 , WTG1 Genes that promote grain size by regulating cell proliferation SLG7 , SMOS1 ; Factors that affect cell division include D1 , RGB1 , DEP1 , OsMAPK6 These previous research results show that the genetic mechanism of rice grain shape is very complex, and the genetic basis of rice grain shape is still insufficient to analyze. There are many other genes or loci that are also involved in regulating rice grain shape. AtPAHX It has been reported to be associated with senescence by affecting chlorophyll degradation, and its homologous gene in rice OsPAHX5 Its function includes regulating rice grain length, which has not been reported so far. Summary of the invention

[0003] In view of this, the present invention aims to solve the problems existing in the current research on rice grain shape and discloses a new gene for regulating rice grain length. OsPAHX5 , which encodes a phytanoyl-CoA dioxygenase, is disclosed OsPAHX5The coding region sequence of the gene and the encoded protein sequence are shown in SEQ ID NO.1 and SEQ ID NO.2 in the ST.26 standard sequence nucleotide or amino acid sequence table. The present invention also discloses a method for using OsPAHX5 The application method of the gene and its encoded protein to regulate rice grain length comprises the following steps: 1) using CRISPR-Cas9 gene editing technology to modify OsPAHX5 When constructing a CRISPR-Cas9 knockout vector, select OsPAHX5 The nucleotide forward sequence of a target site in the gene coding region is 5′→3′: as shown in SEQ ID No. 3 in the ST.26 standard sequence nucleotide or amino acid sequence table; 2) Obtaining knockout OsPAHX5 After the knockout vector was constructed, a conventional japonica rice variety Nipponbare was selected as the transformation background for genetic transformation, and two homozygous knockout mutant transgenic lines were obtained, one of which was a knockout mutant. OsPAHX5 The nucleotide forward sequence of the gene coding region is 5′→3′: as shown in SEQ ID No.4 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the amino acid forward sequence is 5′→3′: as shown in SEQ ID No.6 in the ST.26 standard sequence nucleotide or amino acid sequence table; another knockout mutant OsPAHX5 The nucleotide forward sequence of the gene coding region is 5′→3′: as shown in SEQ ID No.5 in the ST.26 standard sequence nucleotide or amino acid sequence table. The amino acid forward sequence is 5′→3′: as shown in SEQ ID No.7 in the ST.26 standard sequence nucleotide or amino acid sequence table; 3) Obtain OsPAHX5 Using the overexpression transgenic functional experimental technology, three homozygous transgenic lines were obtained using the conventional japonica rice variety Nipponbare as the transformation background. OsPAHX5 Gene transgenic functional complementation homozygous strains; 4) Determination of wild type, knockout OsPAHX5 Grain length and main agronomic traits of transgenic mutants and homozygous overexpressing transgenic lines of the gene. OsPAHX5 The grain length of the mutant of the gene is significantly larger than that of the wild type, while the agronomic traits such as plant height and plant type are not significantly different from those of the wild type. OsPAHX5 The grain length of the transgenic homozygous strain is not significantly different from that of the wild type. The present invention provides a new gene for regulating rice grain length. OsPAHX5 and its encoded protein and application, OsPAHX5 The gene negatively regulates rice grain length, and knocking out the gene can significantly increase rice grain length. OsPAHX5The gene can then be used to change the OsPAHX5 protein to create new rice germplasm with larger grain length, which can be used to improve rice grain shape and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 shows the knockout of the gene using CRISPR-Cas9 gene editing technology in the present invention. OsPAHX5 Genetic technology methods. Included in OsPAHX5 Information on a target site selected in the gene coding region and the types of nucleotide mutations of the two homozygous knockout mutants obtained, as well as the resulting amino acid changes. WT is the wild type (Nipponbare), ospahx5-1 and ospahx5-2 There are two homozygous knockout mutants.

[0005] Figure 2 shows the knockout obtained by the technical method of the present invention. OsPAHX5 The grain length of the gene mutant and wild type. WT is the wild type (Nipponbare), ospahx5-1 and ospahx5-2 are two homozygous knockout mutants. * indicates significant ( P <0.05).

[0006] Figure 3 shows the knockout obtained by the technical method of the present invention. OsPAHX5 Photos of the main agronomic traits of the gene mutant and wild type. WT is the wild type (Nipponbare), ospahx5-1 and ospahx5-2 There are two homozygous knockout mutants.

[0007] Figure 4 The overexpression obtained by the technical method of the present invention OsPAHX5 Grain length of transgenic homozygous strains and wild type. WT is wild type (Nipponbare), OE is overexpression homozygous strain without tag, OE-GFP is overexpression homozygous strain with green fluorescent protein (GFP) tag, and OE-HA is overexpression homozygous strain with hemagglutinin (HA) tag.

Claims

1. A new gene that regulates rice grain length OsPAHX5 and the protein encoded by it and its application, characterized in that, The accession number of the OsPAHX5 gene in the rice genome is LOC_Os05g39650 The nucleic acid sequence of the coding region and the encoded protein sequence are shown as SEQ ID NO.1 and SEQ ID NO.2 in the ST.26 standard sequence nucleotide or amino acid sequence table, respectively.

2. A method of using the method according to claim 1 OsPAHX5 The application of the gene and its encoded protein in regulating rice grain length is characterized in that: Knockout using CRISPR-Cas9 gene editing technology OsPAHX5 Gene can significantly increase rice grain length.

3. The use according to claim 2, characterized in that: Knockout OsPAHX5 After knocking out the gene, other major agronomic traits of the mutant had no significant changes.

4. The use according to claim 2, characterized in that: When constructing a CRISPR-Cas9 knockout vector, select OsPAHX5 The nucleotide forward sequence of a target site on the gene coding region is 5′→3′: as shown in SEQ ID No. 3 in the ST.26 standard sequence nucleotide or amino acid sequence table.

5. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two OsPAHX5 A knockout mutant, one of which is OsPAHX5 The nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 4 in the ST.26 standard sequence nucleotide or amino acid sequence table. Another knockout mutant OsPAHX5 The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 5 in the ST.26 standard sequence nucleotide or amino acid sequence table.

6. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two OsPAHX5 A gene knockout mutant, wherein the amino acid forward sequence of one knockout mutant is 5′→3′: as shown in SEQ ID No.6 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the amino acid forward sequence of another knockout mutant is 5′→3′: as shown in SEQ ID No.7 in the ST.26 standard sequence nucleotide or amino acid sequence table.

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