A method for engineering cereal crops to recognize nodulation factors and increase the number of rhizobia colonization

A nodulation factor, rhizobia technology, applied in the fields of biotechnology and botany, can solve the problems of energy and resource waste, environmental burden, and increase production costs

Active Publication Date: 2022-07-12
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than half of the food in the world is obtained through the application of chemical fertilizers (especially nitrogen fertilizers), and 40-60% of crop yields (even up to 90% in tropical soils) are also attributed to the use of nitrogen fertilizers. Excessive use of nitrogen fertilizers not only increases Production costs, but also a heavy burden on the environment
Every year, about 40% of the nitrogen fertilizer that is not used by plants is directly deaminated to form N 2 return to the atmosphere, resulting in a serious waste of energy and resources; secondly, the N produced by nitrogen fertilizer metabolism 2 O is an important "greenhouse effect" gas, and its GWP (100-year Average Global-Warming Potential) is CO 2 296 times
Furthermore, nitrogen fertilizers flowing into rivers, lakes and seas with irrigation and rainwater will also cause water eutrophication and other hazards

Method used

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  • A method for engineering cereal crops to recognize nodulation factors and increase the number of rhizobia colonization
  • A method for engineering cereal crops to recognize nodulation factors and increase the number of rhizobia colonization
  • A method for engineering cereal crops to recognize nodulation factors and increase the number of rhizobia colonization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] Example 1. Analysis of mycorrhizal symbiosis level of Osmyr1 mutants

[0203] Through CRISPR / Cas9 site-directed mutagenesis technology, the inventors isolated and obtained three OsMYR1 homozygous mutant lines Osmyr1-1, Osmyr1-2 and Osmyr1-3. Their mutations are as follows ( figure 1 A):

[0204] Osmyr1-1: Deletion of 1 base, resulting in premature termination of protein coding in the LysM1 domain.

[0205] Osmyr1-2: Deletion of 1 base results in a frameshift of the OsMYR1 gene during protein translation and premature termination in the extracellular domain.

[0206] Osmyr1-3: Added 1 base, resulting in a frameshift of the OsMYR1 gene during protein translation and premature termination in the intracellular domain.

[0207] Quantitative PCR analysis showed that the transcript levels of OsMYR1 in Osmyr1-1 and Osmyr1-2 were down-regulated to 0.22±0.03 and 0.25±0.05, respectively, while the transcript levels of OsMYR1 in Osmyr1-3 did not change significantly (0.93±0.20) ...

Embodiment 2

[0209] Example 2. Detection of responsiveness of Osmyr1 and Oscerk1 mutants to Myc factor CO4

[0210] Calcium oscillations induced by Myc factors in root epidermal nuclei are a hallmark event of the activation of mycorrhizal symbiotic signaling. In order to study the response of OsMYR1 to Myc factors, the inventors introduced the calcium ion genetic probe YC3.6 (transgenic material stably expressing the YC3.6 element in the nucleus) into the Osmyr1-1 homozygous mutant by genetic hybridization, thereby To observe the calcium oscillation response of Osmyr1-1 mutants to Myc factors. Myc factors, including Myc-LCO and CO4 / CO5, both induce calcium oscillations in rice non-root hair cells and promote root morphogenesis. However, since there are commercial high-purity CO4 products currently on the market, the sources of which are more stable and convenient, the inventors used CO4 as a representative to study the response of rice to Myc factors. The inventors found that 10 -8 MCO4...

Embodiment 3

[0212] Example 3. Study on the protein interaction between OsMYR1 and OsCERK1

[0213] First, the present inventors used yeast two-hybrid (Yeast-2-hybrid, Y2H) and Pull-down technology to detect the interaction between OsMYR1 and OsCERK1 in vitro. The OsMYR1 and OsCERK1 intracellular domains were cloned into pGBKT7 (BD-OsMYR1 KD ) and pGADT7(AD-OsCERK1 KD ) vector, and then the above vector was combined with pGBKT7 and pGADT7 empty vector, and then transferred into yeast cells, and then screened and cultured on the nutrient deficiency medium SD-LWH (-Leu / -Trp / -His). The inventors found that only co-expression of BD-OsMYR1 KD and AD-OsCERK1 KD of yeast cells can grow on auxotrophic medium, which means that OsMYR1 KD and OsCERK1 KD able to interact in yeast cells ( image 3 A). MBP-tagged OsMYR1 fused in in vitro Pull-down experiments KD (MBP-OsMYR1 KD ) can effectively bind to His-OsCERK1 KD protein( image 3 B), further demonstrating that the intracellular domains o...

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Abstract

The present invention relates to a method for engineering cereal crops to recognize Nod factors and increase the number of rhizobia colonization. The inventors have identified and obtained a new rice Myc factor receptor, namely the MYR1 gene for the first time; on this basis, the present invention discloses a method for modifying cereal crops to recognize Nod factors and increase the colonization of rhizobia number method.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and botany; more particularly, the present invention relates to a method for engineering cereal crops to recognize Nod factors and increase the number of rhizobia colonization. Background technique [0002] Root nodule symbiosis (RNS) is another mutually beneficial symbiosis between legumes and rhizobia. Rhizobium can invade the plant cortex and stimulate the division of cortical cells to form enlarged organ nodules. In the anaerobic microenvironment of root nodules, rhizobia can use nitrogenase to remove N in the air 2 reduced to NH 4 + Used by plants, thereby reducing the plant's dependence on nitrogen fertilizers. [0003] Nitrogen is a macronutrient essential for plant growth and a limiting factor in agricultural production. At present, more than half of the world's food is obtained through the application of chemical fertilizers (especially nitrogen fertilizers), and 40 to 60% of cr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/82C07K19/00
CPCC12N9/12C12N15/8261C07K14/415C07K2319/00Y02A40/146
Inventor 王二涛何江曼张晓伟戴慧玲刘欢董文涛
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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