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Cloning and application of the gene oszip9 controlling zinc uptake in rice

A rice and gene expression technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of unclear zinc molecular mechanism in rice, and achieve the effect of improving zinc absorption capacity, wide application potential, and improving absorption and accumulation capacity.

Active Publication Date: 2022-06-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been found that a variety of metal transporter family members are involved in the balance and distribution of zinc in rice, but the molecular mechanism responsible for zinc uptake in rice is still unclear

Method used

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  • Cloning and application of the gene oszip9 controlling zinc uptake in rice
  • Cloning and application of the gene oszip9 controlling zinc uptake in rice
  • Cloning and application of the gene oszip9 controlling zinc uptake in rice

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Analysis of zinc transport activity of OsZIP9 in yeast heterologous expression system

[0035] In order to discover the transporter responsible for zinc uptake in rice and provide new genetic material for improving zinc uptake in rice, the present invention designs figure 1 The technical route mentioned. The zinc transport activity of rice ZIP family members was initially screened by yeast heterologous expression system. This method can quickly identify whether the zinc transporter has transport activity, and is an international general identification method.

[0036] On the rice RAPDB website (https: / / rapdb.dna.affrc.go.jp / ), search and download the sequence information of each ZIP member in rice according to the gene name, and design amplification primers for heterologous expression in yeast (for primer sequences, see Table 1), using the cDNA of the japonica rice "Nipponbare" (Nipponbare, a well-known and public rice test material, which has completed the ...

Embodiment 2

[0049] Example 2: Analysis of the expression pattern of OsZIP9

[0050] In order to fully understand the characteristics of OsZIP9 gene function, we investigated the gene expression characteristics of OsZIP9 in detail in 4 parts.

[0051] (1) The rice variety "Nipponbare" was placed under the condition of rice full nutrient solution (hydroponic nutrient solution composition: 1.44mMNH 4 NO 3 ,0.3mM NaH 2 PO4, 0.5mM K 2 SO 4 ,1.0mM CaCl 2 ,1.6mM MgSO 4 ,0.17mM NaSiO 3 , 50μM Fe-EDTA, 0.06μM (NH 4 ) 6 Mo 7 o 24 ,15 μM H 3 BO 3 ,8μM MnCl 2 ,0.2μM CuSO 4 ,0.4μM ZnSO 4 ,29μM FeCl 3 , 40.5μM Citric acid, pH 5.5, referenced from Yoshida et al., 1976) planting, sampling in different tissues (including roots, leaves, leaf sheaths, nodes, internodes, etc.) at the seedling stage, vegetative growth stage and mature stage , respectively wrapped in tinfoil and stored in liquid nitrogen. Extract the sample RNA and reverse transcribe it into cDNA (for specific steps, refer to...

Embodiment 3

[0057] Example 3: Construction of OsZIP9 mutants and overexpression materials

[0058] On the basis of speculating that OsZIP9 is responsible for zinc uptake in rice, we further verified it by constructing OsZIP9 mutants and overexpression materials. The mutant material is constructed by CRISPR / Cas9 system. The specific process of constructing the OsZIP9-CRISPR / Cas9 mutant vector is as follows: on the NEB cutter website (http: / / nc2.neb.com / NEBcutter2 / ), design the space amplification primers of the CRISPR vector according to the sequence of the coding region of OsZIP9 (see Table 3), and use template pJE044 (sgRNA) to amplify the target fragment, and then connect the amplified fragment to the expression vector pJE45 / pH-Ubi-cas9-7 through the Gateway system (for vector information, see Figure 2E ). The construction of the overexpression material is first to amplify the coding region sequence of OsZIP9, and construct it into the expression vector pJC034. The primers involved ...

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Abstract

The invention belongs to the technical field of plant genetic engineering. It specifically relates to the cloning and application of a gene OsZIP9 controlling rice zinc uptake. A main transporter gene OsZIP9 responsible for rice zinc uptake was cloned, its nucleotide sequence is shown in SEQ ID NO: 2, the protein sequence encoded by the gene is shown in SEQ ID NO: 3, the specific promoter of the gene The nucleotide sequence is shown in SEQ ID NO:1. The promoter can induce the expression of rice genes under the condition of zinc deficiency. The protein gene can increase zinc accumulation in rice by enhancing or regulating gene expression level. Overexpression of OsZIP9 gene in rice can significantly improve the zinc uptake capacity of rice. The invention provides a new breeding target gene for improving rice absorption and utilization of zinc by genetic engineering.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the cloning and application of a gene OsZIP9 controlling rice zinc uptake. Background technique [0002] Zinc (Zn) is an essential trace element for the growth and development of organisms, and is essential for maintaining healthy life activities of various organisms. In animals, plants and humans, zinc is a catalytic cofactor for hundreds of enzymes (including RNA polymerase, superoxide dismutase, alcohol dehydrogenase and carbonic anhydrase) Structural cofactors. Zinc deficiency in plants can lead to stunted growth, chlorosis and reduced fertility. Zinc deficiency in humans can damage multiple organ systems including the epidermis, gastrointestinal tract, central nervous system, immune, skeletal and reproductive systems. [0003] Zinc deficiency is one of the important abiotic stresses limiting rice productivity worldwide and one of the major nut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/29C07K14/415C12N15/82A01H5/00A01H5/10A01H6/46
CPCC07K14/415C12N15/8243
Inventor 练兴明杨猛张启发
Owner HUAZHONG AGRI UNIV
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