CN-119709791-B - Novel gene OsPAHX for regulating grain length of rice, protein coded by novel gene OsPAHX and application of novel gene

Abstract

The invention discloses OsPAHX gene and application of coded protein thereof in regulating and controlling rice grain length, belonging to the field of plant breeding and biotechnology. The invention discloses a coding region nucleic acid sequence of OsPAHX genes, a coded amino acid sequence, a CRISPR-Cas9 knockdown target site nucleic acid sequence, a knockdown mutant nucleic acid sequence and a knockdown mutant amino acid sequence, which are shown as a ST.26 standard sequence nucleotide or amino acid sequence table. The invention researches the functions of OsPAHX genes by utilizing molecular biology and plant transgenic technology, and discovers that the grain length of a transgenic plant knocked out is obviously longer than that of a wild type variety after the OsPAHX genes are knocked out by utilizing CRISPR-Cas9 technology, and the grain length of a transgenic plant overexpressed by utilizing transgenic overexpression technology is not obviously different from that of the wild type variety after the OsPAHX genes are overexpressed by utilizing transgenic overexpression technology. The invention can utilize OsPAHX gene to create rice variety with larger grain length.

Inventors

• ZHANG PENG
• TONG HANHUA
• HUANG ZENGYING

Assignees

• 中国水稻研究所

Dates

Publication Date

20260512

Application Date

20250108

Claims (5)

1. 1. The application of the gene OsPAHX and the encoded protein thereof in increasing the grain length of rice is characterized in that the accession number of the OsPAHX gene in the genome of the rice is LOC_Os05g39650, and the nucleic acid sequence of the encoding region and the encoded protein sequence are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. 2. A method for increasing grain size of rice by controlling OsPAHX gene expression according to claim 1, which comprises knocking out OsPAHX gene in rice by gene editing technique to obtain rice plant with increased grain size.
3. 3. The use according to claim 2, wherein the gene editing is CRISPR-Cas9 editing and the nucleotide forward sequence of a target site on the coding region of OsPAHX gene selected when constructing the knockout vector is 5'→3' as shown in SEQ ID No. 3.
4. 4. The use according to claim 2, wherein two OsPAHX gene knockout mutants are obtained by CRISPR-Cas9 gene editing technique, wherein the forward sequence of the nucleotide of the OsPAHX gene coding region of one knockout mutant is 5'→3': as shown in SEQ id No.4, and the forward sequence of the nucleotide of the OsPAHX gene coding region of the other knockout mutant is 5'→3': as shown in SEQ id No. 5.
5. 5. The use according to claim 2, wherein two OsPAHX gene knockout mutants are obtained by using CRISPR-Cas9 gene editing technology, wherein the amino acid forward sequence of one knockout mutant is 5'→3' as shown in SEQ ID No.6 and the amino acid forward sequence of the other knockout mutant is 5'→3' as shown in SEQ ID No. 7.

Description

Novel gene OsPAHX for regulating grain length of rice, protein coded by novel gene OsPAHX and application of novel gene Technical Field The invention belongs to the background technology in the field of plant breeding and biotechnology, and particularly relates to a novel gene OsPAHX for regulating and controlling rice grain length, and a coded protein and application thereof. Background The rice grain type mainly comprises grain length, grain width, grain thickness and the like, is an important quantitative character controlled by multiple genes, directly influences the yield of rice and is closely related to the quality of the rice. There are 6 pathways known at present for regulating the granulocyte type, namely the E3 ubiquitin proteasome degradation pathway, the G protein signal pathway, the MAPK cascade signal pathway, the plant hormone pathway, the transcription factor pathway, the epigenetic pathway and the like. In recent years, a number of genes related to grain development have been isolated in rice based on map-based cloning and whole genome association analysis (GWAS) techniques, and more than 500 QTLs regulating grain types have been found, which are located on 12 chromosomes of the rice genome, respectively. Wherein GLW7, GS3, GL10 and GL6 are used for controlling grain length, qSW, GW5, GW2, GS5, GWY10 and the like are used for controlling grain width, TGW3, osBZR1, osMADS56, WTG1 and other genes are used for regulating grain thickness, SLG7 and SMOS1 are used for promoting grain size by regulating cell proliferation, D1, RGB1, DEP1, osMAPK and the like are used for influencing cell division. The results of these previous studies indicate that the genetic mechanism of rice grain type is very complex, the genetic basis for analyzing rice grain type is still insufficient, and many other genes or loci are involved in regulating rice grain type. Phytyl-coa dioxygenase encoding gene AtPAHX in arabidopsis is reported to be related to senescence by affecting chlorophyll degradation, while its function of homologous gene OsPAHX5 in rice has not been reported until the aim of regulating the rectangular area of rice grain. Disclosure of Invention In view of the above, the present invention discloses a novel gene OsPAHX for regulating rice grain length, which codes for a phytantyl-CoA dioxygenase, and discloses the coding region sequence of OsPAHX gene and the encoded protein sequence, which are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table of ST.26 standard sequence nucleotide or amino acid sequence, aiming at the problems existing in the related research of the rice grain type. Drawings The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the attached drawings, FIG. 1 is a technical method for knocking out OsPAHX genes by using CRISPR-Cas9 gene editing technology. Including information on a target site selected in the OsPAHX gene coding region and the types of nucleotide mutations of the two homozygous knockout mutants obtained, and the resulting amino acid changes. WT is wild type (Japanese sunny), ospahx-1 and ospahx-2 are two homozygous knockout mutants, and FIG. 2 shows the knockout OsPAHX5 gene mutant and wild type grain length obtained by the technical method of the present invention. WT is wild type (Nitazengqing), ospahx-1 and ospahx-2 are two homozygous knockout mutants. * Shows remarkable (P < 0.05), and FIG. 3 is a photograph of the major agronomic traits of the knock-out OsPAHX gene mutant and wild type obtained by the technical method of the present invention. WT is wild type (Japanese sunny), ospahx-1 and ospahx-2 are two homozygous knockout mutants, and FIG. 4 shows the grain length of the transgenic homozygous strain of the overexpressed OsPAHX5 gene obtained by the technical method of the invention and the wild type. WT is wild-type (japan-all), OE is a non-tagged over-expressed homozygous strain, OE-GFP is a Green Fluorescent Protein (GFP) -tagged over-expressed homozygous strain, and OE-HA is a Hemagglutinin (HA) -tagged over-expressed homozygous strain. Detailed Description The invention discloses an application method for increasing rice grain length by utilizing OsPAHX genes and coded proteins thereof, which comprises the following steps of 1) knocking out OsPAHX genes by utilizing a CRISPR-Cas9 gene editing technology. When the CRISPR-Cas9 knockout vector is constructed, a nucleotide forward sequence of a target site on a OsPAHX gene coding region is selected to be 5 '. Fwdarw.3', as shown in a sequence table of ST.26 standard sequence nucleotides or amino acids SEQ ID No.3, and 2) a transgenic mutant homozygote strain for knocking out OsPAHX gene is obtained. After constructing the knockout vec