Search

CN-122012596-A - Method for cultivating low-cadmium-accumulation rice by CRISPRCas9 saturation editing of OsHMA3 promoter

CN122012596ACN 122012596 ACN122012596 ACN 122012596ACN-122012596-A

Abstract

The invention discloses a method for cultivating low-cadmium accumulated rice by CRISPRCas9 saturated editing OsHMA3 promoter, belonging to the technical field of crop molecular breeding. The method carries out gene editing by an agrobacterium-mediated transformation method aiming at a 500bp region upstream of an OsHMA3 gene of a rice variety ZH11, so that the HMA3-Pro3 mutant which can enhance the interception capability of root systems to cadmium, reduce the cadmium accumulation in overground parts and seeds and does not influence the absorption and accumulation of essential elements such as zinc, manganese, iron and the like on the premise of not influencing the agronomic characters of rice is obtained. The invention provides a high-efficiency and safe non-transgenic low-cadmium rice cultivation technology, provides technical support and excellent varieties for safe utilization of cadmium-polluted farmlands, and provides a new solution for solving the grain safety problem caused by soil cadmium pollution.

Inventors

  • LI JIAYANG
  • YU HONG
  • CHEN WEICAI
  • MENG XIANGBING
  • JING YANHUI
  • CHEN MINGJIANG
  • LIU GUIFU
  • ZHAO YANNAN

Assignees

  • 中国科学院遗传与发育生物学研究所
  • 崖州湾国家实验室

Dates

Publication Date
20260512
Application Date
20260312

Claims (10)

  1. 1. Use of a substance of a gene editing regulatory element, characterized in that said use is any of the following: A1 Application in regulating cadmium content in plants; a2 The application of the method in preparing a product for regulating and controlling the cadmium content of plants; a3 Use of the composition for growing plants with altered cadmium content; a4 Use of the composition for the preparation of a product for growing plants with altered cadmium content; a5 Use in plant breeding; the regulatory element is a regulatory element of an OsHMA3 protein coding gene, and the OsHMA3 protein is any one of the following: b1 Amino acid sequence is a protein of sequence SEQ ID NO. 2, B2 A protein with the same function, wherein the amino acid sequence shown in SEQ ID NO. 2 is subjected to substitution and/or deletion and/or addition of amino acid residues, B3 A protein having 80% or more identity with the amino acid sequences defined in B1) and B2) and having the same function, B4 A fusion protein obtained by ligating a tag to the end of the protein defined in any one of B1) to B3).
  2. 2. The use according to claim 1, wherein the regulatory element is derived from rice.
  3. 3. The use according to claim 1 or 2, wherein the regulatory element is a promoter.
  4. 4. Use according to claim 3, characterized in that the promoter is a DNA molecule represented by C1) or C2) as follows: c1 Nucleotide sequence 1495-1957 of SEQ ID NO. 1; c2 A nucleic acid molecule having more than 70% identity to the nucleotide sequence defined in C1) and having promoter activity.
  5. 5. The use according to claim 4, wherein the substance is any one of the following: D1 A sgRNA targeting the promoter; D2 Cas protein and D1) the sgRNA; d3 A gene encoding D1) the sgRNA and a gene encoding a Cas protein; d4 A biological material comprising a gene encoding D1) said sgRNA and a gene encoding a Cas protein, said biological material being a vector, an expression cassette or a recombinant microorganism.
  6. 6. The use according to claim 5, wherein the regulation of cadmium content in plants is a reduction of cadmium content in plant kernels, and the sgRNA targets the target sequence of SEQ ID NO. 1 at positions 1917-1936.
  7. 7. The use according to any one of claims 1 to 6, wherein the plant is any one of the following: n1) dicotyledonous or monocotyledonous plants; N2) gramineae plants; n3) a gramineous plant; N4) rice plants; n5) rice.
  8. 8. A method, characterized in that it is M1 or M2: M1, a method for reducing cadmium content of rice, which comprises the steps of carrying out gene editing on the regulatory element in claim 1 to obtain gene editing rice so as to reduce the cadmium content of the rice to be improved, wherein the cadmium content of the gene editing rice is lower than that of the rice to be improved; m2, breeding method for cultivating rice with reduced cadmium content, the method comprises the steps of carrying out gene editing on the rice to be improved containing the regulatory element in claim 1 to obtain the rice with reduced cadmium content, wherein the cadmium content of the rice with reduced cadmium content is lower than that of the rice to be improved.
  9. 9. The method according to claim 8, wherein said M1 and M2 comprise the step of introducing the substance according to claim 5 or 6 into said rice to be improved.
  10. 10. A genetically edited rice plant, characterized in that said genetically edited rice plant is a rice plant not comprising the regulatory element of any one of claims 1 to 4.

Description

Method for cultivating low-cadmium-accumulation rice by CRISPRCas9 saturation editing of OsHMA3 promoter Technical Field The invention belongs to the technical field of crop molecular breeding and environmental remediation, and particularly relates to a method for cultivating low-cadmium-accumulation rice by CRISPRCas9 saturation editing of an OsHMA3 promoter. Background Cadmium (Cd) is a heavy metal element with strong toxicity, and the pollution of soil cadmium becomes a global environmental problem, thus forming a serious threat to crop production and human health. Worldwide, soil cadmium pollution results in crop losses of up to 15,000 tons per year. As staple food for more than half of the population of the world, rice has stronger absorption and accumulation capacities on cadmium in soil, and eating rice polluted by cadmium is one of main ways of taking cadmium by human bodies, and long-term taking can cause various diseases such as pain, renal dysfunction and even cancer. Therefore, cultivating rice varieties with low cadmium accumulation in seeds is a key way for solving the grain safety problem caused by cadmium pollution in soil. The transfer process of cadmium from soil to rice seeds mainly comprises three key steps of root system absorption, root-stem transportation and seed distribution. Research shows that the rice root system absorbs cadmium in soil mainly through natural resistance related macrophage protein 5 (OsNramp), after cadmium is absorbed, one part of the cadmium is isolated in vacuoles under the mediation of heavy metal ATPase 3 (OsHMA 3) positioned by vacuole membranes, the deficiency of gene functions can cause the accumulation of overground parts and seed cadmium, the excessive expression can enhance the interception capability of the root system to cadmium and reduce the cadmium content of seeds, and the other part of cadmium is transported to the overground parts through the mediation of homologous gene OsHMA2 of the OsHMA 3. Disclosure of Invention The invention aims to provide a high-efficiency and safe non-transgenic low-cadmium accumulation rice cultivation method, which remarkably reduces the accumulation of cadmium in seeds on the premise of not influencing the agronomic characters of rice by CRISPR/Cas9 mediated editing of an OsHMA3 promoter and provides technical support and excellent varieties for safe utilization of cadmium-polluted farmlands. In order to achieve the above object, the present invention provides the use of a substance of a gene editing regulatory element, which can be any of the following: A1 Application in regulating cadmium content in plants; a2 The application of the method in preparing a product for regulating and controlling the cadmium content of plants; a3 Use of the composition for growing plants with altered cadmium content; a4 Use of the composition for the preparation of a product for growing plants with altered cadmium content; a5 Use in plant breeding; The regulatory element is a regulatory element for regulating and controlling an OsHMA3 protein coding gene, and the OsHMA3 protein is any one of the following: b1 Amino acid sequence is a protein of sequence SEQ ID NO. 2, B2 A protein with the same function, wherein the amino acid sequence shown in SEQ ID NO. 2 is subjected to substitution and/or deletion and/or addition of amino acid residues, B3 A protein having 80% or more identity with the amino acid sequences defined in B1) and B2) and having the same function, B4 A fusion protein obtained by ligating a tag to the end of the protein defined in any one of B1) to B3). In the above application, the regulatory element is derived from rice. In the application, the plant breeding index is the cadmium content of the plant, and the plant breeding aim is to cultivate the plant with the changed cadmium content in the seeds and/or the changed cadmium content in the root system. The alteration may be an increase or decrease in cadmium content in the grain and/or root system. The OsHMA3 protein can be synthesized artificially or can be obtained by synthesizing the encoding gene and then biologically expressing. In the present invention, the protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc. In the present invention, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap