Novel gene OsCT1 for regulating and controlling cadmium content of rice grains, protein coded by novel gene OsCT1 and application of novel gene OsCT1

Abstract

The invention belongs to the technical field of plant breeding and biology, and particularly relates to how to reduce the cadmium content of rice grains through a gene OsCT1 and application of the gene OsCT1 to creation of new germplasm of low-cadmium-accumulation rice. The invention discloses a nucleotide sequence of a gene OsCT1 coding region, a coded protein sequence, a CRISPR-Cas9 knockout target site nucleotide sequence, a nucleotide sequence of a knockout mutant and a protein sequence of the knockout mutant, as shown in an ST.26 standard sequence nucleotide or amino acid sequence table. After the gene OsCT1 is knocked out by utilizing a CRISPR-Cas9 technology, the cadmium content of grains of the OsCT1-knocked-out transgenic plant is obviously lower than that of a wild type variety. In addition, in the seedling stage, the plant height and the root length of the OsCT1-knocked-out transgenic plant are remarkably larger than those of a wild type variety; after the OsCT1 gene is subjected to overexpression, the grain cadmium content of an overexpressed transgenic plant is slightly higher than that of a wild type variety. In addition, in the seedling stage, the plant height and the root length of the overexpressed OsCT1 transgenic plant are obviously smaller than those of a wild type variety. According to the invention, the OsCT1 gene can be utilized to create a novel rice germplasm with low cadmium content in grains.

Description

Technical Field

[0001] The present invention belongs to the background technology of plant breeding and biotechnology, and specifically relates to a new gene for regulating the cadmium content of rice grains. OsCT1 and its encoded proteins and applications. Background Art

[0002] Heavy metal cadmium (Cadmium, Cd) not only affects the normal growth of plants, but also enters the human body through the food chain, causing chronic poisoning and cancer. Cd rice pollution has become an important factor threatening my country's agricultural production and human life and health. Therefore, Cd toxicity research on rice is the key to ensuring national food safety. The analysis of Cd accumulation and transport mechanism will provide a theoretical basis for the safe production, high yield and stable yield of rice and other crops. Elucidating the Cd transport mechanism is the basis for controlling Cd accumulation in rice. Although several genes that regulate the Cd accumulation and transport mechanism of rice have been located or cloned, their mechanism of action has not been fully elucidated. Therefore, it is still necessary to continuously improve the Cd accumulation and transport mechanism of rice. Studies have found that multiple Cd transport proteins play a very critical role in regulating the Cd accumulation and transport mechanism of rice, but as an important member of the ABC transporter (ATP-binding cassette transporter) family, the mechanism of action of ABCG (WBC) transporter in the Cd transport process has rarely been reported. The new gene regulating the cadmium content of rice grains involved in the present invention OsCT1 Encodes an ABCG-WBC type transporter. OsCT1 Report on genetic regulation of cadmium accumulation in rice. Summary of the invention

[0003] In view of this, the present invention aims at the problems existing in the current research on cadmium accumulation in rice grains, and discloses a new gene for regulating cadmium content in rice grains. OsCT1 , which encodes an ABCG-WBC type transporter, is disclosed OsCT1 The 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 utilizing Os CT1 The application method of the gene and its encoded protein to regulate the cadmium content of rice grains includes the following steps: 1) conducting a subcellular localization study on the OsCT1 protein and finding that it is located on the plasma membrane of plant cells and is a membrane transport protein; 2) using CRISPR-Cas9 gene editing technology to modify the cadmium content of rice grains; OsCT1 When constructing a CRISPR-Cas9 knockout vector, select OsCT1The nucleotide forward sequence of one 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, and the nucleotide forward sequence of another target site is 5′→3′: as shown in SEQ ID No.4 in the ST.26 standard sequence nucleotide or amino acid sequence table; 3) Obtain knockout OsCT1 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. OsCT1 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, and 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. OsCT1 The nucleotide forward sequence of the gene coding region 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 is 5′→3′: as shown in SEQ ID No.8 in the ST.26 standard sequence nucleotide or amino acid sequence table. OsCT1 The transmembrane structure of the mutant protein of the gene was predicted, and no transmembrane domain was found; 4) OsCT1 Using the overexpression transgenic functional experimental technology, two homozygous overexpression transgenic lines were obtained using the conventional japonica rice variety Nipponbare as the transformation background. OsCT1 Gene transgenic homozygous strains; 5) Determination of wild type, knockout OsCT1 The phenotypes of the transgenic mutants and homozygous overexpressing transgenic lines of the gene under cadmium stress at the seedling stage, the cadmium content in the grains and agronomic traits under cadmium-contaminated soil planting environment. OsCT1 The plant height and root length of the mutant of the gene were significantly greater than those of the wild type, while the overexpression OsCT1 The height and root length of the transgenic plants were significantly smaller than those of the wild-type varieties. In addition, under the planting condition of soil cadmium content of 1.13 g / kg, the knockout OsCT1 The cadmium content in the grains of the mutant of the gene was significantly lower than that of the wild type, while the overexpression OsCT1 The cadmium content of the transgenic plants was higher than that of the wild-type varieties. OsCT1 The plant height and plant type of the mutant of the gene are not significantly different from those of the wild type. The present invention provides a new gene OsPAHX5 for regulating the cadmium content of rice grains and its encoded protein and application. OsCT1The gene encodes an ABCG-WBC type transporter located on the plasma membrane of plant cells, which has the function of transporting cadmium and positively regulates the cadmium content in rice grains. Knocking out the gene can significantly reduce the cadmium accumulation in rice grains. OsCT1 Genes change OsCT1 The protein-based method was used to create new rice germplasm with lower cadmium accumulation in grains, which can be used in rice low-heavy metal improvement 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 The figure shows the subcellular localization of the OsCT1 protein involved in the present invention. PM stands for plasma membrane, and the figure shows that the GFP fluorescence signal (green fluorescence) of OsCT1 completely overlaps with the localization fluorescence signal (red fluorescence) of the plasma membrane.

[0005] Figure 2 The present invention utilizes CRISPR-Cas9 gene editing technology to knock out OsCT1 The technical methods of the gene and the changes in the nucleotides and amino acids encoded in the coding region of the knockout mutant. OsCT1 Information on the two target sites selected in the gene coding region and the types of nucleotide mutations and amino acid changes caused by the two homozygous knockout mutants. WT is the wild type (Nipponbare), CRISPR- osct1 -1 and CRISPR- osct1 -2 are two homozygous knockout mutants; Figures B, C and D are OsCT1 protein, knockout mutant CRISPR- osct1 -1 Protein and CRISPR- osct1 -2 protein's transmembrane domain structure.

[0006] Figure 3 The knockout obtained by the technical method of the present invention OsCT1 Gene mutants, overexpression OsCT1 Phenotypes of transgenic homozygous lines and wild type under cadmium stress at the seedling stage. WT is wild type (Nipponbare, Japan), CRISPR- osct1 -1 and CRISPR- osct1 -2 are two homozygous knockout mutants, OX OsCT1 -1OX OsCT1 -2 are two homozygous overexpression lines. * and ** represent significant levels, respectively. P <0.05 and P<0.01.

[0007] Figure 4 The knockout obtained by the technical method of the present invention OsCT1 Gene mutants, overexpression OsCT1 Cadmium accumulation in grains of homozygous transgenic lines and wild type. WT is wild type (Nipponbare), CRISPR- osct1 -1 and CRISPR- osct1 -2 are two homozygous knockout mutants, OX OsCT1 -1OX OsCT1 -2 are two homozygous overexpression lines. * indicates significant level P <0.05.

[0008] Figure 5 The mutant and wild-type strains of the knockout OsPAHX5 gene obtained by the technical method of the present invention. osct1 -1 and CRISPR- osct1 -2 are two homozygous knockout mutants.

Claims

1. A new gene that regulates cadmium content in rice grains OsCT1 and the protein encoded by it and its application, characterized in that, Said OsCT1 The accession number of the gene in the rice genome is LOC_Os01g42900 , which encodes an ABCG-WBC type transporter, and the nucleic acid sequence of its coding region 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, respectively.

2. A method of using the method according to claim 1 OsCT1 The application of the gene and its encoded protein in regulating the cadmium accumulation in rice grains is characterized in that: The protein OsCT1 is a protein localized on the plasma membrane of plant cells and has six transmembrane domains.

3. A method of using the method according to claim 1 OsCT1 The application of the gene and its encoded protein in regulating the cadmium accumulation in rice grains is characterized in that: Knockout using CRISPR-Cas9 gene editing technology OsCT1 The gene can significantly reduce the cadmium content in rice grains, while overexpression OsCT1 Gene can increase the cadmium content in rice grains.

4. A method of using the method according to claim 1 OsCT1 The application of the gene and its encoded protein in regulating the cadmium accumulation in rice grains is characterized in that: Knockout using CRISPR-Cas9 gene editing technology OsCT1 After the gene was expressed, the tolerance of rice to cadmium toxicity was significantly improved at the seedling stage. OsCT1 The gene can reduce the tolerance of rice to cadmium toxicity at the seedling stage.

5. The use according to claim 2, characterized in that: Knockout OsCT1 After the gene was knocked out, there was no obvious change in the plant type and height of the knockout mutant.

6. The use according to claim 2, characterized in that: When constructing a CRISPR-Cas9 knockout vector, select OsCT1 The nucleotide forward sequence of one 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, and the nucleotide forward sequence of another target site is 5′→3′: as shown in SEQ ID No.4 in the ST.26 standard sequence nucleotide or amino acid sequence table.

7. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two OsCT1 A knockout mutant, one of which is OsCT1 The 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. Another knockout mutant OsCT1 The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 6 in the ST.26 standard sequence nucleotide or amino acid sequence table.

8. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two OsCT1 Gene knockout mutants, wherein the amino acid forward sequence of one knockout mutant is 5′→3′: as shown in SEQ ID No.7 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.8 in the ST.26 standard sequence nucleotide or amino acid sequence table.

9. The use according to claim 8, characterized in that: For both knockout OsCT1 The transmembrane domain of the mutant protein was predicted, and no transmembrane domain was found.

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