CN113293161B

CN113293161B - Clone and application of gene OsZIP9 for controlling rice zinc absorption

Info

Publication number: CN113293161B
Application number: CN202010106549.4A
Authority: CN
Inventors: 练兴明; 杨猛; 张启发
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2022-06-07
Anticipated expiration: 2040-02-21
Also published as: CN113293161A

Abstract

The present invention belongs to the field of plant gene engineering technology. In particular to the clone of a gene OsZIP9 for controlling the zinc absorption of rice and application thereof. A main transporter gene OsZIP9 responsible for rice zinc absorption is cloned, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2, the protein sequence coded by the gene is shown as SEQ ID NO: 3, the nucleotide sequence of the specific promoter of the gene is shown as SEQ ID NO:1 is shown. The promoter can induce the expression of rice genes under the condition of zinc deficiency. The protein gene can enhance or regulate the expression level of the gene so as to improve the zinc accumulation in the rice. The over-expression of the OsZIP9 gene in rice can obviously improve the zinc absorption capacity of rice. The invention provides a new breeding target gene for improving the zinc absorption and utilization of rice by using genetic engineering.

Description

Clone and application of gene OsZIP9 for controlling rice zinc absorption

Technical Field

The invention relates to the technical field of plant genetic engineering. In particular to the clone of a gene OsZIP9 for controlling the zinc absorption of rice and application thereof.

Background

Zinc (Zn) is an essential trace element for the growth and development of organisms and is vital to the maintenance of the healthy and vital activities of various organisms. In animals, plants and human body, zinc is a catalytic cofactor for hundreds of enzymes (including RNA polymerase, superoxide dismutase, alcohol dehydrogenase and carbonic anhydrase) and also a structural cofactor for various zinc finger transcription factors. Zinc deficiency in plants leads to stunting, leaf greening 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.

Zinc deficiency is one of the important abiotic stresses that limit the productivity of rice worldwide and is also one of the major nutritional problems affecting human health. It is estimated that millions of hectares of farmlands in the world are affected by zinc deficiency, with about one-third of the population having insufficient zinc intake (Alloway, 2009). Because of the low zinc content of cereals, the zinc-deficient population is more concentrated in poor areas where cereals are used as staple food and the dietary structure is single, and the problems are difficult to solve through dietary diversification, nutritional supplements and food nutrition fortification and the like (Kawakami and Bhullar, 2018). In response, bioaugmentation of zinc by cereals such as rice is considered to be an economically effective and sustainable measure. In order to meet the zinc nutrition requirement of human body, the current bio-enhancement target of rice zinc concentration is 28mg/kg, but the average level of the current conventional polished rice zinc concentration is about 16mg/kg, which is far from the target (Bouis and Saltzman, 2017). Improving the accumulation of zinc in rice by understanding the genetic mechanism of zinc absorption in rice and further improving the improvement is an effective strategy.

The accumulation of zinc in rice is mainly absorbed by roots and then transported and distributed to various tissues and grains above the ground. Higher plants, including rice, have strict genetic mechanisms controlling zinc uptake, transport and balance, since excessive zinc accumulation can also be toxic to plants (Ricachenevsky et al, 2015). At present, a plurality of metal transport protein family members are found to participate in the balance and distribution of zinc in rice bodies, but the molecular mechanism responsible for the zinc absorption of rice is not clear. By researching the molecular mechanism of rice zinc absorption and further improving the rice zinc absorption capacity, the method has important significance for rice low zinc resistance and rice zinc accumulation enhancement.

The invention clones a main transport protein OsZIP9 responsible for rice zinc absorption, identifies the key effect of the transport protein OsZIP9 on rice zinc absorption, can obviously improve the zinc absorption capacity of rice by over-expressing the OsZIP9 gene in rice, and provides a new breeding target gene for improving the zinc absorption and utilization of rice by using genetic engineering.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of a zinc transport protein gene OsZIP9 in enhancing the zinc accumulation capacity of rice.

The technical scheme of the invention is as follows:

the invention separates a promoter for zinc deficiency induced expression of rice, and the nucleotide sequence of the promoter is shown as SEQ ID NO:1 is shown.

The invention separates a transport protein gene for controlling the absorption of rice zinc, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.

The invention separates a transport protein gene for controlling the absorption of rice zinc, and the protein sequence coded by the gene is shown as SEQ ID NO: 3, respectively.

The promoter of the OsZIP9 gene separated by the invention can induce the expression of the rice gene under the condition of zinc deficiency.

The protein gene separated by the invention can enhance or regulate the gene expression level so as to improve the accumulation of the zinc in the rice.

The yeast heterologous expression experiment proves that the OsZIP9 gene has extremely strong transport activity for absorbing zinc from the outside of cells into the cells, and the activity is obviously higher than that of other members of a rice ZIP family. The absorption kinetics result in yeast shows that the zinc ion transport activity of OsZIP9 is as high as 81.4pmol/min/10⁶cells, OsZIP5 transporter (activity 2.58 pmol/min/10) as a positive control⁶cells) about 32 times. Analysis of expression patterns in rice plants showed that OsZIP9 localized to the cytoplasmic membrane, expressed predominantly in the epidermal and ectodermal cells in the roots, and induced by zinc deficiency. Knockout of OsZIP9 in rice results in a very significant decrease in the zinc uptake capacity of rice. The overexpression of OsZIP9 in rice can obviously improve the zinc absorption capacity of rice. Under the condition of 0.04 mu M (1/10 of normal nutrient solution) of low-zinc nutrient solution, the roots and straws of the over-expression OsZIP9 material are respectively increased by 48-131% and 49-99% compared with wild type; at 0.4 μ M (normal nutrient) zinc concentration, the roots and stalks of the over-expressed plants increased 87-202% and 14-45% respectively compared to the wild type. In field conditions, over-expression of OThe straw and grain zinc concentration of the sZIP9 material can be significantly higher than that of the control by 20%.

The method comprises the following specific steps:

the applicant conducts preliminary analysis on the family gene of the rice zinc transport protein (ZIP) through a zinc absorption defect yeast heterologous expression system, and confirms that OsZIP9 has the transport activity of transferring zinc ions from the outside of cells into the cells, and the zinc ion transport activity of the OsZIP9 is obviously stronger than that of all other ZIP members. Subsequently, the tissue expression pattern of OsZIP9 is analyzed, and OsZIP9 is found to be specifically expressed in rice roots, mainly in epidermal and cortical cells, and plays a role in zinc ion absorption in rice roots. Verification is carried out in a rice high-zinc variety, namely, a conventional CRISPR system is utilized to knock out the OsZIP9 gene, the fact that the zinc absorption and accumulation of a rice plant with the knocked-out OsZIP9 gene are remarkably reduced is confirmed, and the OsZIP9 gene is determined to be a key gene of rice responsible for zinc absorption. The overexpression of the OsZIP9 gene can obviously increase the accumulation of zinc in roots and overground parts.

The invention has the beneficial effects that:

(1) the invention firstly proves the zinc ion transport capacity of the OsZIP9 gene in rice and the key effect of the OsZIP9 gene in rice on zinc absorption at the root of rice, and research results show that the OsZIP9 gene has extremely strong zinc ion transport capacity, the zinc ion transport activity of the OsZIP9 gene is as high as 81.4pmol/min/106cells, is about 32 times of that of a positive control OsZIP5 transport protein, and is far higher than that of all other rice ZIP members. This property also makes OsZIP9 have a wide range of potential applications.

(2) The invention provides an application of a gene for improving zinc absorption and accumulation capacity in rice. The enhancement of the expression of the OsZIP9 gene in rice can obviously improve the zinc absorption and accumulation capacity of rice. It provides a new way for improving the zinc absorption capacity of rice and other gramineous crops and cultivating zinc-rich varieties.

(3) The invention uses the high zinc variety screened from 529 cultivars with extensive genetic diversity as a genetic transformation parent, and the cultivar material can also be used as a gene resource material for cultivating zinc-rich rice.

Drawings

FIG. 1: technical route diagrams of the present invention.

FIG. 2 is a schematic diagram: the invention relates to a vector diagram. FIG. 2A is a map of the Pyes2 vector used for yeast expression. FIG. 2B is a map of the PGWB3 vector used for promoter fusion GUS expression. FIG. 2C is a map of Pm999-YFP vectors for subcellular localization. Fig. 2D is a diagram of amplification template PJE44 and expression vector pJE45/pH-Ubi-Cas9-7 involved in CRISPR material construction by the CRISPR/Cas9 system. FIG. 2E is a map of the PJC-34 vector for overexpression.

Fig. 3: analysis of yeast heterologous expression of members of the rice ZIP family. EGTA is a divalent ion chelating agent, and the invention utilizes EGTA to chelate zinc ions in the culture medium so as to reduce the concentration of the zinc ions in the culture medium.

FIG. 4: kinetic analysis of zinc ion uptake of OsZIP9 in yeast. Reference numerals indicate (positive control with OsZIP5 gene). Panel A of FIG. 4 shows the zinc uptake of OsZIP9 and OsZIP5 in yeast. Panel B of FIG. 4 is a display with OsZIP9 removed. Panel C in fig. 4 is a kinetic analysis of the zinc uptake of OsZIP5 in yeast. Panel D of FIG. 4 is the zinc uptake of OsZIP9 expressing yeast strains under a broader zinc gradient. FIG. 4E is a graph showing the kinetic analysis of OsZIP9 for zinc uptake in yeast.

FIG. 5: and analyzing the gene expression pattern of OsZIP 9. Description of reference numerals: FIG. 5A is an analysis of expression patterns in different tissues of rice. Panel B and panel C of FIG. 5 are analyses of the response of OsZIP9 expression levels to different concentrations of zinc. FIG. 5, panel D-M shows the analysis of expression pattern of OsZIP9 promoter fused with GUS gene. FIG. 5, panel N- -T, is a rice protoplast-based subcellular localization analysis of OsZIP 9.

FIG. 6: sequencing verification and expression level analysis of the CRISPR knockout material of OsZIP 9. Description of reference numerals: panel a in fig. 6 is a different rice pedigree utilizing CRISPR system knock-out material. Panel B in FIG. 6 is a different pedigree of overexpression material.

Fig. 7: hydroponic experiments of CRISPR knockout material, overexpression material and wild type material of OsZIP 9. Description of the reference numerals: panel A in FIG. 7 is a photograph of the phenotype of each material after 4 weeks of growth under 0.04 μ M and 0.4 μ M zinc conditions; panel B of FIG. 7 shows the plant height statistics for each material under 0.04 μ M zinc supply conditions. Panel C of FIG. 7 shows the plant height statistics for each material under 0.4 μ M zinc supply conditions. Panel D and panel E of FIG. 7 are the zinc ion concentration analysis in roots (panel D) and straw (panel E) for each material under 0.04 μ M zinc supply. The F graph in FIG. 7 and the G graph in FIG. 7 are the zinc ion concentration analysis in the roots (F graph) and straw (G graph) of each material under the 0.4 μ M zinc supply condition.

FIG. 8: and (3) analyzing the zinc ion concentration of the OsZIP9 straws and grains under the field planting condition. Description of reference numerals: graph a in fig. 8 is straw zinc concentration data. The B-chart in fig. 8 is brown rice zinc concentration data.

Detailed Description

Description of sequence listing:

sequence listing SEQ ID NO:1 is a promoter sequence of OsZIP9 gene.

Sequence listing SEQ ID NO: 2 is the coding sequence of the OsZIP9 gene, the sequence has the full length of 477bp, and 158 amino acids are coded.

Sequence listing SEQ ID NO: 3 is a sequence of a protein encoded by OsZIP 9.

The following examples further define the invention and describe the identification of zinc transport activity of OsZIP9 in yeast, characterization of tissue expression levels, genetic transformation, and phenotypic analysis of transgenic material. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Based on this, the applicant verifies the key effect of the OsZIP9 on controlling the zinc absorption of the rice by means of knockout, over-expression and the like in a rice high-zinc variety 'grape yellow' (the English name Putaohuang is a variety with the number of C189 in 529 germplasm materials collected by the university of agriculture in China and is found to be one of several varieties with the highest zinc accumulation in the germplasm.

Example 1 analysis of Zinc transport Activity of OsZIP9 in Yeast heterologous expression System

The invention designs a technical route shown in figure 1 for discovering transport proteins in rice and responsible for zinc absorption and providing a new gene material for improving the zinc absorption of the rice by genetic engineering. The zinc transport activity of the rice ZIP family members is primarily screened by a yeast heterologous expression system. The method can rapidly identify whether the zinc transport protein has transport activity, and is a universal identification method in the world.

Sequence information of each ZIP member of rice is searched and downloaded on a rice RAPDB website (https:// rapdb.dna. affrc. go. jp /) according to the gene name, amplification primers for yeast heterologous expression (the primer sequences are shown in Table 1) are designed, cDNA of japonica rice Nipponbare (a well-known and commonly used rice test material, and whole genome sequencing is completed) is used as a template, fragments of each gene are amplified and constructed to a yeast heterologous expression vector pYES2 (a commonly used commercial expression vector developed by Invitrogen corporation, and the vector diagram is shown in A diagram in FIG. 2), and the primer sequences for constructing an OsZIP9 yeast vector are shown in Table 1. The vector construction adopts Nanjing NuoZan Biotechnology Co., Ltd

II kit (cat. C112-02, according to the instructions) provided one-step directed cloning technology. Subsequently transformed into a zinc uptake deficient yeast mutant strain ZHY3 (presented by Zhang hong Sheng teacher of Nanjing university of agriculture) by the yeast rapid transformation method (see details below) and the phenotype observed by gradient dilution experiments on yeast SD-Ura solid medium (0.67% YNB, 0.077% SD-Ura, 2% glucose, 50mM EDTA and adjusted pH to 6.0, solid medium plus 2% agar powder) (see FIG. 3). The results show that OsZIP9 has zinc transport activity and is significantly higher than all other members of rice ZIP. Further analyzed for zinc transport activity in yeast by absorption kinetics experiments, another ZIP member, OsZIP5, which has been shown to have zinc transport activity, served as a positive control. Analysis of the results showed (see FIG. 4) that the zinc transport activity of the positive control OsZIP5 was 2.58pmol/min/10⁶cells, and the zinc transport activity of the gene OsZIP9 of the present invention was 81.4pmol/min/10⁶cells, about 32-fold of the positive control, demonstrated that OsZIP9 is not only a functional zinc transporter, but also has very high zinc transport activity.

The method comprises the following specific steps of yeast rapid transformation:

preparation of reagents before transformation: YPAD yeast medium (2% yeast powder, 4% peptone, 4% glucose, 0.01% adenine sulfate), 50% PEG3350, 1mol/L LiAc, 2mg/ml double stranded salmon sperm DNA (s.s.carrier DNA), plasmid required for transformation (total amount 1 μ g per transformation).

A conversion step:

(1) selecting a single colony of a zinc absorption defective yeast mutant strain ZHHY 3 of a target strain, culturing the single colony in a 2 YPAD culture medium at 30 ℃ and 220-250 r/min until the concentration of bacterial liquid is 2 × 108 cells/ml (about 18 h);

(2) collecting the bacterial liquid for 2 times by using a 2ml centrifugal tube, wherein the concentration is 1.5ml each time, centrifuging at the room temperature of 14400rpm for 30sec, washing for 1-2 times by using sterilized water, centrifuging, and removing a supernatant;

(3) denaturing the subpackaged S.S.carrier DNA in a dry bath kettle at 100 ℃ for 5min, and then quickly putting on ice for later use;

(4) to the treated bacteria were added 240. mu.l of 50% PEG3350, 34. mu.l of 1M LiAc, and 50. mu.l of denatured S.S. carrier DNA in this order to transform 1. mu.g of plasmid. Adding one of the above materials, sucking, beating and mixing.

(5) And (3) carrying out water bath for 1-3 hrs at 42 ℃.

(6) Centrifuging at 14400rpm for 1min, discarding the supernatant, supplementing 500. mu.l of sterilized water to the precipitate, sucking and mixing, sucking 100. mu.l and spreading on an auxotrophic SD culture medium (mainly SD-Ura culture medium, which is purchased from clone-tech company) compatible with the carrier.

(7) Sealing with sealing glue, placing at 30 ℃ for inverted culture for 2-3 d, and picking out monoclonal antibody for verification.

TABLE 1 OsZIP9 and positive control OsZIP5 primer pair for yeast heterologous expression designed by the invention

Example 2: expression pattern analysis of OsZIP9

To fully understand the function characteristics of OsZIP9 gene, we examined the gene expression characteristics of OsZIP9 in detail in section 4.

(1) The rice variety Nipponbare is cultured under the condition of the rice total nutrient solution (the water culture nutrient solution component is 1.44mM NH)₄NO₃,0.3mM NaH₂PO4,0.5mM K₂SO₄,1.0mM CaCl₂,1.6mM MgSO₄,0.17mM NaSiO₃,50μM Fe-EDTA,0.06μM(NH₄)₆Mo₇O₂₄,15μM H₃BO₃,8μM MnCl₂,0.2μM CuSO₄,0.4μM ZnSO₄,29μM FeCl₃40.5 μ M Citric acid, pH 5.5, referenced from Yoshida et al, 1976), were sampled at seedling, vegetative and mature stages in different tissues (including roots, leaves, leaf sheaths, nodes, internodes, etc.), wrapped in tinfoil paper, and stored in liquid nitrogen. Sample RNA was extracted and reverse-transcribed into cDNA (see M-MLV reverse transcription kit Specification from Invitrogen, cat. No.28025-013 for specific procedures) and analyzed by Realtime (see Bao bioengineering, Dalian, Inc., for methods for Realtime PCR, DRR 041A). The Realtime-PCR primers are shown in Table 2. The results showed that OsZIP9 was specifically expressed in rice roots and hardly expressed in other tissues in rice at different growth stages (Panel A in FIG. 5).

(2) OsZIP9 was analyzed for its response to varying concentrations of zinc ion and to varying concentrations of cobalt, another transport substrate. The applicant analyzes the expression number of the OsZIP9 by adding zinc or cobalt with different concentrations into the hydroponic nutrient solution, and the rice variety is 'Nipponbare'. The results showed that OsZIP9 was specifically expressed in roots, and expression was inhibited by low zinc induction and high zinc (panel B in fig. 5 and panel C in fig. 5). These results indicate that the expression level of OsZIP9 is closely related to the external zinc concentration.

(3) The expression characteristics of OsZIP9 were further analyzed by GUS reporter gene. The promoter sequence (sequence shown in SEQ ID NO:1) of OsZIP9 is connected to a vector pGWB3 (a common commercial vector, present by Roger teacher of China university of agriculture), promoter amplification primers are shown in Table 2, the size of an amplification product is 2811bp, and the vector construction adopts a Gateway method and a kit (product numbers 11789020 and 11791020) of Invitrogen company. After the vector construction is completed, the transformed rice positive plants are obtained by an agrobacterium mediated transgenic method (agrobacterium EHA105, from CAMBIA laboratories, australia, genetic transformation methods are described in detail in the transgenic material section below). The basic steps of GUS staining analysis were: fresh rice samples were immersed in GUS dye solution (GUS dye solution formulation: 50mM phosphate buffer (pH7.0), 10mM EDTA, 1% Triton X-100, 1mg/ml X-Gluc, 100. mu.g/ml chloramphenicol, 1mM potassium ferricyanide, 1mM potassium ferrocyanide, 20% methanol), repeatedly evacuated 3 times for 5min each time, and then incubated in a 37 ℃ incubator for 4hrs in the dark. After incubation, the background color of the tissue was completely removed with 75% ethanol, and the tissue was photographed by observation with a microscope. The tissue needing to be sliced is sliced after being embedded by 5 percent agarose, the temperature should be controlled in the embedding process, the temperature cannot be too high, otherwise, the tissue can be scalded, the temperature cannot be too low, otherwise, the agar begins to solidify, and the embedding quality is influenced. After the embedding, if the section is not cut in time, the section should be soaked in clear water and stored at 4 ℃. The slicing was done with a vibrating microtome and the picture was taken after slicing by observation with a DIC phase contrast microscope. GUS staining and sectioning results showed that OsZIP9 was mainly expressed in lateral roots of roots, particularly in epidermal and cortical cells (see D-M in FIG. 5). This result further confirms that OsZIP9 is mainly expressed in tissues and cells responsible for mineral absorption.

(4) The subcellular localization of OsZIP9 was analyzed by rice protoplasts. PCR amplification primers are shown in Table 2, the vector is constructed on an expression vector pM999-YFP vector (a commonly used transient expression vector presented by Wang Lei doctor of university of agriculture in Huazhong, vector diagram is shown in B diagram of FIG. 2), and the processing method adopts one-step cloning technology and kit (as described above) of Nanjing Nuozhuang company. The requirement of transient expression of protoplast on plasmid purity is high, and the plasmid needs to be extracted by QIAGEN plasmid Midi Kit (Cat No.12143) plasmid extraction Kit, which refers to the Kit specification specifically, and the plasmid concentration is required to reach 1 μ g/μ l. The transformation procedure for protoplasts was previously reported (Zhang et al, 2011), and the protoplasts that were finally transformed were observed with a laser confocal microscope (TCS SP 2; Leica, Germany) and photographed. The results showed that OsZIP9 is a protein localized to the plasma membrane of cells (see FIG. 5, panel N-T). This property further substantiates the role of OsZIP9 in mineral absorption.

TABLE 2 primer set of OsZIP9 designed for expression profiling according to the present invention

Example 3: construction of OsZIP9 mutant and overexpression material

Based on the presumption that OsZIP9 is responsible for rice zinc absorption, the verification is further carried out by constructing an OsZIP9 mutant and an overexpression material. Wherein the mutant material is constructed by the CRISPR/Cas9 system. The specific process of constructing the OsZIP9-CRISPR/Cas9 mutant vector is as follows: the space amplification primer of the CRISPR vector is designed according to the coding region sequence of OsZIP9 on NEB cutter website (http:// nc2.NEB. com/NEBcut 2/) (see Table 3), the target fragment is amplified by a template pJE044(SgRNA), and the amplified fragment is connected to an expression vector pJE45/pH-Ubi-cas9-7 through a Gateway system (the vector information is shown in figure 2E). The construction of the overexpression material is that the coding region sequence of OsZIP9 is firstly amplified and then constructed into an expression vector pJC 034. The primers involved in vector construction are shown in Table 3.

Table 3 primer pair designed by the invention and used for constructing OsZIP9 transgenic material

And (3) obtaining a transformed rice positive plant by adopting the constructed CRISPR and overexpression vector and an agrobacterium-mediated transgenic method, wherein the specific transformation steps are as follows:

the obtained correctly cloned plasmid is introduced into a rice variety grape yellow (one of high zinc varieties identified in the laboratory as described above, and the invention is not limited to a specific variety) through a rice genetic transformation system mediated by agrobacterium (EHA105 provided by CAMBIA laboratories in australia), and a transformed plant is obtained through pre-culture, infection, co-culture, selection of a hygromycin-resistant callus, differentiation, rooting, seedling training and transplanting. The agrobacterium-mediated rice (japonica rice subspecies) genetic transformation system mainly adopts a method for further optimization based on Hiei et al (1994) reports.

The main steps of the genetic transformation, the culture medium and the preparation method thereof of the invention are as follows:

1) reagent and solution abbreviations

The abbreviations for the phytohormones used in the medium of the present invention are as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin ); KT (Kinetin ); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indoleacetic acid); 2,4-D (2, 4-dichlorphenoxyacetic acid, 2,4-Dichlorophenoxyacetic acid); AS (acetosyringone); CH (Casein Hydrolysate); HN (Hygromycin B, Hygromycin); DMSO (Dimethyl Sulfoxide); n6max (N6 macronutrient solution); n6mix (N6 trace element composition solution); MSmax (MS macronutrient component solution); MSmix (MS microelement component solution)

2) Solution formulation

(1) N6 medium macronutrient mother liquor (prepared as 10-fold concentrate (10 ×)):

the reagents are dissolved one by one and then the volume is up to 1000 ml by distilled water.

(2) N6 culture Medium microelement mother liquor (prepared according to 100 times of concentrated solution (100 ×))

The above reagents were dissolved at 20-25 ℃ and made up to 1000 ml with distilled water.

(3) Iron salt (Fe)₂EDTA) stock solution (prepared according to 100X concentrated solution)

3.73 grams of disodium ethylene diamine tetraacetate (Na)₂EDTA·2H₂O) and 2.78 g FeSO₄·7H₂Dissolving O respectively, mixing, fixing the volume to 1000 ml with distilled water, carrying out warm bath at 70 ℃ for 2 hours, and storing at 4 ℃ for later use.

(4) Vitamin stock solution (prepared according to 100X concentrated solution)

Adding distilled water to a constant volume of 1000 ml, and storing at 4 ℃ for later use.

(5) MS culture medium macroelement mother liquor (MSmax mother liquor) (prepared according to 10 times concentrated solution)

(6) MS culture medium microelement mother liquor (MSmin mother liquor) (prepared according to 100 times concentrated solution)

(7) Preparation of 2,4-D stock solution (1 mg/ml):

weighing 100 mg of 2,4-D, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at the temperature of 20-25 ℃.

(8) Preparation of 6-BA stock solution (1 mg/ml):

weighing 100 mg of 6-BA, dissolving for 5 minutes by using 1 ml of 1N potassium hydroxide, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at the temperature of 20-25 ℃.

(9) Preparation of Naphthaleneacetic acid (NAA) stock solution (1 mg/ml):

weighing 100 mg of NAA, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at 4 ℃ for later use.

(10) Formulation of Indole Acetic Acid (IAA) stock solution (1 mg/ml):

weighing 100 mg of IAA, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at 4 ℃ for later use.

(11) Preparation of glucose stock solution (0.5 g/ml):

weighing 125 g of glucose, dissolving with distilled water to a constant volume of 250 ml, sterilizing and storing at 4 ℃ for later use.

(12) Preparation of AS stock solution:

0.392 g of AS is weighed, added with 10 ml of DMSO for dissolving, subpackaged into 1.5ml of centrifuge tubes and stored at 4 ℃ for standby.

(13)1N potassium hydroxide stock solution

Weighing 5.6 g of potassium hydroxide, dissolving with distilled water to constant volume of 100 ml, and storing at 20-25 ℃ for later use.

3) Culture medium formula for rice genetic transformation

(1) Induction medium

Adding distilled water to 900 ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling to 1000 ml, packaging into 50 ml triangular flask (25 ml/bottle), sealing, and sterilizing by conventional method (121 deg.C for 25 min, the following method for sterilizing culture medium is the same as that for the culture medium).

(2) Subculture medium

Adding distilled water to 900 ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling, diluting to 1000 ml, packaging into 50 ml triangular flask (25 ml/bottle), sealing, and sterilizing.

(3) Pre-culture medium

Adding distilled water to 250 ml, adjusting pH to 5.6 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved by heating and 5ml of glucose stock solution and 250. mu.l of AS stock solution were added before use and dispensed into petri dishes (25 ml/dish).

(4) Co-culture medium

(5) Suspension culture medium

Adding distilled water to 100 ml, adjusting pH to 5.4, subpackaging into two 100 ml triangular bottles, sealing, and sterilizing according to the above method.

1 ml of sterile glucose stock solution and 100. mu.l of AS stock solution were added before use.

(6) Selection medium

Adding distilled water to 250 ml, adjusting pH to 6.0, sealing, and sterilizing as above.

The medium was dissolved before use and added to 250. mu.l of HN (50 mg/ml) and 400. mu.l of CN (250 mg/ml) and dispensed into petri dishes (25 ml/dish). (Note: the concentration of carbenicillin in the first selection medium was 400 mg/L, and the concentration of carbenicillin in the second and subsequent selection media was 250 mg/L).

(7) Pre-differentiation culture medium

Adding distilled water to 250 ml, adjusting pH to 5.9 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved before use, 250. mu.l of HN (50 mg/ml) 250. mu.l of CN (250 mg/ml) and dispensed into petri dishes (25 ml/dish).

(8) Differentiation medium

Distilled water was added to 900 ml and 1N potassium hydroxide was used to adjust the pH to 6.0.

Boiling, adding distilled water to 1000 ml, packaging into 50 ml triangular flask (50 ml/bottle), sealing, and sterilizing.

(9) Rooting culture medium

Distilled water was added to 900 ml and the pH was adjusted to 5.8 with 1N potassium hydroxide.

Boiling, adding distilled water to 1000 ml, packaging into raw tube (25 ml/tube), sealing, and sterilizing.

4) Agrobacterium-mediated genetic transformation procedure (EHA105 supplied by the Australian CAMBIA laboratory)

(1) Callus induction

a. The mature medium flower 11 rice seeds were dehulled and then treated sequentially with 70% ethanol for 1 minute with 0.15% mercuric chloride (HgCl)₂) Disinfecting the surface of the seeds for 15 minutes;

b. washing the seeds with sterilized water for 4-5 times;

c. placing the seeds on an induction medium;

d. the inoculated culture medium is placed in a dark place for culturing for 4 weeks at the temperature of 25 +/-1 ℃.

(2) Callus subculture

The bright yellow, compact and relatively dry embryogenic calli were selected and placed on subculture medium for 2 weeks in the dark at 25 + -1 deg.C.

(3) Preculture

Compact and relatively dry embryogenic calli were selected and placed on pre-culture medium for 2 weeks in the dark at 25 + -1 deg.C.

(4) Agrobacterium culture

a. Agrobacterium EHA105 (a strain from Agrobacterium strain publicly used by the company CAMBIA) was pre-cultured for two days at 28 ℃ on LA medium with corresponding resistance selection (preparation of LA medium see J. SammBruke et al, 1998);

b. the Agrobacterium is transferred to a suspension medium and cultured on a shaker at 28 ℃ for 2-3 hours.

(5) Infection with Agrobacterium

a. Transferring the pre-cultured callus to a sterilized bottle;

b. adjusting the suspension of Agrobacterium to OD₆₀₀ 0.8-1.0；

c. Soaking the callus in agrobacterium tumefaciens suspension for 30 minutes;

d. transferring the callus to sterilized filter paper and sucking to dry; then placed on a co-culture medium to be cultured for 3 days at a temperature of 19-20 ℃.

(6) Callus wash and selection culture

a. Washing the callus with sterilized water until no agrobacterium is visible;

b. soaking in sterilized water containing 400 mg/L Carbenicillin (CN) for 30 min;

c. transferring the callus to sterilized filter paper and sucking to dry;

d. transferring the callus to selective medium for selective culture for 2-3 times, each time for 2 weeks.

(7) Differentiation

a. Transferring the resistant callus to a pre-differentiation culture medium and culturing for 5-7 days in a dark place;

b. transferring the pre-differentiation cultured callus to a differentiation medium, and culturing under illumination at 26 ℃.

(8) Rooting

a. Cutting off roots generated during differentiation;

b. then transferred to rooting medium and cultured for 2-3 weeks under illumination at 26 ℃.

(9) Transplanting

Residual medium on the roots was washed off and seedlings with good root system were transferred to the greenhouse while keeping the water moist for the first few days.

Verifying and screening the CRISPR mutant material obtained after transformation by sequencing, and selecting 3 homozygous independent transformation families causing amino acid frameshift mutation as subsequent further test materials (A picture in figure 6); the overexpression material obtained after transformation was identified by the realtome PCR method, and 3 independent transformed families with higher expression level were selected as further test material (fig. 6B). The Realtime PCR method was as described above with reference to the instructions for use of the TAKARA commercial kit (cat # DRR041A) from Takara, Inc., Boehringer Bio Inc.

Example 4: phenotypic analysis and effect identification of OsZIP9 transgenic material (CRISPR and overexpression)

The gene effect of OsZIP9 was verified in two ways, namely, in hydroponic conditions and in field conditions.

4.1 Water culture validation test.

Soaking the rice seeds for 3-4 days until the rice seeds are exposed to the white, accelerating germination for one day, and then sowing the seeds in the wet yellow sand. The yellow sand is repeatedly washed by tap water, mainly to remove impurities and neutralize the pH value. When the plant after sowing grows to a period of one leaf and one core, the seedling is wrapped by sponge and fixed on a water culture plate with holes and is placed in a water culture nutrient solution for culture. The nutrient solution was set at two zinc concentrations of 0.04 μ M (zinc sulfate, low zinc condition) and 0.4 μ M (zinc sulfate, normal zinc level). After approximately 4 weeks or so of hydroponic growth, the rice material phenotype and zinc element concentration were analyzed.

The results of the phenotypic analysis (see panels a to C in fig. 7) show that under the zinc deficiency condition, the growth vigor of the mutant is significantly weaker than that of the wild type (i.e. non-transgenic type, the same below), while the over-expressed material has no significant difference from that of the wild type; when the zinc is supplied at a normal zinc level, the growth vigor of the mutant, the wild type and the over-expression material has no obvious difference. These results indicate that under low zinc conditions, the growth and development of plants are significantly affected after OsZIP9 mutation, and the effect on growth vigor is relieved with the increase of zinc concentration supply. This demonstrates that OsZIP9 is critical for rice growth under low zinc conditions. In addition, the results also show that overexpression of OsZIP9 does not adversely affect the growth of rice plants.

The concentration of zinc element in the transgenic plant is analyzed by the following method: separately sampling the roots and straws of the plants, drying the roots and the straws for 2-3 days at 80 ℃ to constant weight in time after sampling, and cleaning the rice root samples for 3 times by using single distilled water (distilled water) during sampling. After being crushed and accurately weighed, the sample is treated with 120-DEG C180-DEG C gradient digestion for 45min by 65 percent nitric acid and is treated in a microwave digestion instrument (MARS6, CEM company), the digested sample is diluted by deionized water and then is measured, and the measuring instrument is an inductively coupled plasma mass spectrometer (ICP-MS; Agilent 7700series, USA).

Analysis of the results on zinc element concentration showed that at 0.04. mu.M (low zinc condition) zinc concentration, the roots and aerial parts of the mutant plants were reduced by 21-42% and 24-37%, respectively, compared to the wild type (FIG. 7, panels D and E); at 0.4. mu.M (normal nutrition) zinc concentration, roots and aerial parts of mutant plants were reduced by 31-59% and 9-12%, respectively, compared to wild type (FIG. 7, panels F and G). These results indicate that the OsZIP9 mutation significantly reduces the zinc uptake in rice plants. Therefore, OsZIP9 is proved to be a key gene responsible for zinc absorption of rice. In addition, at 0.04. mu.M (low zinc condition) zinc concentration, the roots and aerial parts of the over-expressed plants increased by 48-131% and 49-99%, respectively, compared to the wild type (FIG. 7, panels D and E); at 0.4. mu.M (normal nutrition) zinc concentration, the roots and aerial parts of the over-expressed plants increased 87-202% and 14-45%, respectively, compared to the wild type (FIG. 7, panels F and G). These results indicate that overexpression of OsZIP9 significantly increases the zinc uptake capacity of rice.

4.2 field verification test.

To further verify the effect of OsZIP9, applicants planted transgenic material and wild type material in a closed transgenic rice field, harvested about 40 days after heading, and measured the accumulation of zinc in straw and brown rice.

The results show that the zinc concentration of OsZIP9 mutant straw and brown rice is respectively reduced by 40-54% and 40-76 (A picture and B picture in figure 8) compared with the wild type, and further confirm the important function of OsZIP9 on the zinc absorption of rice. The zinc concentration of the OsZIP9 overexpression plant and brown rice was also significantly increased compared to the wild type (a and B panels in fig. 8), with the most amplified transgenic line having about 20% increased zinc concentration.

The main references:

j. SammBruk, E.F. Fritchi, T. Mannich Abetis. molecular cloning guide (second edition), golden winter swallow et al (translation), Beijing, scientific Press 1998, 908-;

2.Alloway BJ.Soil factors associated with zinc deficiency in crops and humans.Environ Geochem Health,2009,31:537-48；

3.Bouis HE,Saltzman A.Improving nutrition through biofortification:A review of evidence from HarvestPlus,2003through 2016.Global Food Security-Agriculture Policy Economics and Environment,2017,12:49-58.

4.Hambidge M.Human zinc deficiency.J Nutr,2000,130:1344S-9S；

5.Hiei Y,Ohta S,Komari T,Kumashiro T.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J,1994,6:271-82；

6.Kawakami Y,Bhullar NK.Molecular processes in iron and zinc homeostasis and their modulation for biofortification in rice.J Integr Plant Biol,2018,60:1181-98；

7.Ricachenevsky FK,Menguer PK,Sperotto RA,Fett JP.Got to hide your Zn away:Molecular control of Zn accumulation and biotechnological applications.Plant Sci,2015,236:1-17；

8.Yoshida S,Forno DA,Cock JH,Gomez KA.Laboratory Manual for Physiological Studies of Rice,3rd ed.International Rice Research Institute,Manila.,1976；

9.Zhang Y,Su J,Duan S,Ao Y,Dai J,Liu J,Wang P,Li Y,Liu B,Feng D,Wang J,Wang H.A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes.Plant Methods,2011,7:30。

sequence listing

<110> university of agriculture in Huazhong

<120> cloning and application of gene OsZIP9 for controlling zinc absorption of rice

<141> 2020-02-19

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2811

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(2811)

<400> 1

acgtcctatt tccatgagta cctgcagaaa attgggctag tatatttttg agattgatgg 60

ggttacaatg ggtgccttgc tatcgacgta tatgttgtat aacaacacta aacgaataat 120

tagccactaa tatgagtacc gtgtgaagtt tagaatttga acctggatat gcatgttccg 180

taacaagaaa aggagcaccg tgtgaagttt agaattcaaa cctagatatg catgttccat 240

aacaagaaaa gaaaaacgag atttggagga agagatgtcc ctccaaacat cttttaaaaa 300

aaagttataa gtatgttggg gattaacacg ggaccttaga tttgaaatca cacacccctt 360

accactacta tcaaatgcct cttcacgttc catcacaaca aacctaatta attagaccca 420

taccgccgat tcaaaatgcg cgcagtacaa tttgagggtc ctagcgtgtt tctcgtgtag 480

ctatcgtgaa ttctctatta tcgccttatt ggtcactata tatgtgtcaa caagttttaa 540

aaggctctat ttgacattac gagaattatt ttacaaagga aaaaagaaag aaagaagatg 600

gcattacaaa ggagtagtaa tatgcaactc aaagtcaaat ccattttaaa ttactatagc 660

aacctggggt ctcatcgtca tgacccaatc aaatgaagcc accgtacgta tcaatgaatt 720

ctttatctct gcagtcgtgt taatttgcag ctgctaaaca cgattgcact aaagatggcg 780

acatatgtgt gctcatatga ggacacatgc caccgttttc gagatgagta gtttggataa 840

aatactttct atctgcagct tctcgagagc tagggcacac ttcatgcggc cggtgtgctt 900

cttgcctggt cagggttaac agaaacagac acatcgtcaa ctaatataat actaaactga 960

tgaactcgcc catggcaact catctcacta catgttgcaa taatgtgtca cactctacat 1020

acattcatag tgtcgacatc tgaaaaaatc tatagacttg tagagtacta caactactcc 1080

atccgtttcg ttttatttga cgccgttgac tttttatcac atgtttgacc attcgtctta 1140

tttaaaaact tttatgaata tgtaaaatta tacgtatgca taaaagtata tttaacgatg 1200

aatcaaatga taggaaaaga attaataatt atttaaattt tttgaataag acaagcggtc 1260

aaacatgcgc taaaaagtca acggtgtcaa ataaaatgaa acggagggag tatttatttg 1320

caaagccaac accgggtttt gcgaaagatt gatcgtgatg acagtgtttt atgtgattct 1380

ttaatactac tggtgaattt ggttgtgtga tgacagtgtt cagtgttgta gctggtatgc 1440

agggttcaac tagctgggaa aacatatgtg atttaattgt gtgctttatt agatggagta 1500

ctaaagatta ttctgcattc caaatgatct aatacgattc accatgttct aatattaact 1560

ttaactgtat atatggtgat aagttgcttg atgggaaaat ctctcttgtg gagaaaagtg 1620

aggagatgtt cacgtacgta catcaaatta tacgtacggt aaaaaatgaa tatgctttgt 1680

taatttaatt ggattaaaaa atttgcatgt acttccattt cgtcgatatc tcttaaaaaa 1740

gattatcacc aatgcatgca ataatcggtt gttttgctgg aaaatatact gctccccatg 1800

gcacaagctt ctagatttgt tatctcgagt tgtcaacatg gcatgcatga tattgttaac 1860

aatactctca tgattagaaa tatgcatttc atacatttaa gttgttaatt ctaaattatt 1920

ggcacaaaat aatcaattat acatatcaat atgatgagca cattgtacgt aaattgcggg 1980

gcccatcaaa cacgtatttg gtgggggccc accatgtgcg ttcatcggtt gacaactatg 2040

ccaaccaaac acgtatatag taggccccat caaatatgtt cctatttgac atctattctc 2100

atatgactta ctctattttg ttagatttac gaactactaa tattataaaa agctatacgt 2160

tgctatccac ttgtctttat atttctcgga ttggttggca cttgttgtat caatacttac 2220

aaacttattt tatattttca cagatcttag ggtaatttat tttatatttt cactagcatc 2280

ttagggtaac tcttagggtt ttctttttct tatccagcct ataaaactat tttatatcaa 2340

tatatatgct cctctactta ggacagtccc aacccatagt gttcataagc agtgtctatg 2400

gtgccatgtt aataagacat cacaatagaa actacactct ctacaaccca tagtttctta 2460

aagtggacca ttaataaata catcatctct cttttctacc aatcatattt atttttcatc 2520

taatatgaag acactattct ctcccaatgc aaaacttgat agtgtctagt gcataggttc 2580

tcatgttgaa gctgtatttt gcatgagacc cagtttcttt ctcttttcac tctctctctt 2640

aattaatata gtaccacata agctaaaagt attacatggt aatgaagtta atgccataga 2700

caccattcta gatggagggt tgagactgcc cttatatcca tataactgta cggatactat 2760

ctccacattg gttgttcgat catcaccaat ctttggccac aggctaaaaa g 2811

<210> 2

<211> 1089

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> CDS

<222> (1)..(1089)

<400> 2

atg gct ttc gat ctc aag cta acc gca tgc ctc ctc ctc gcc gtc ttc 48

Met Ala Phe Asp Leu Lys Leu Thr Ala Cys Leu Leu Leu Ala Val Phe

1 5 10 15

tcc ctc gcc gcc gcc gcc gac tgc gag tgc cag ccc agc gac gaa ggc 96

Ser Leu Ala Ala Ala Ala Asp Cys Glu Cys Gln Pro Ser Asp Glu Gly

20 25 30

cac gac gcg gcc aag tcc cgg acg ctc aag gtc atc gcc atc ttc tgc 144

His Asp Ala Ala Lys Ser Arg Thr Leu Lys Val Ile Ala Ile Phe Cys

35 40 45

atc ctc gtg ggc agc tca gcg ggg tgc gcc att ccg tcg ctc ggc cgg 192

Ile Leu Val Gly Ser Ser Ala Gly Cys Ala Ile Pro Ser Leu Gly Arg

50 55 60

agg ttc ccg gcg ctc cgc ccg gac acg agc ctc ttc ttc gcc ctc aag 240

Arg Phe Pro Ala Leu Arg Pro Asp Thr Ser Leu Phe Phe Ala Leu Lys

65 70 75 80

gcg ttc gcc gcc ggc gtc atc ctc gcc acg gcg ttc gtg cac atc ctc 288

Ala Phe Ala Ala Gly Val Ile Leu Ala Thr Ala Phe Val His Ile Leu

85 90 95

ccg gtg tcc ttc gac aag ctc ggc tcg ccg tgc ctc gtg gac ggg ccg 336

Pro Val Ser Phe Asp Lys Leu Gly Ser Pro Cys Leu Val Asp Gly Pro

100 105 110

tgg cgg aag tac ccg ttc acg ggg ctc gtc gcc atg ctc gcc gcc gtg 384

Trp Arg Lys Tyr Pro Phe Thr Gly Leu Val Ala Met Leu Ala Ala Val

115 120 125

gcc acg ctc ctc ctc gac acg atc gcc acg ggc tac ttc ctg cag cgc 432

Ala Thr Leu Leu Leu Asp Thr Ile Ala Thr Gly Tyr Phe Leu Gln Arg

130 135 140

gcg cag gac agc cgc ggc gcc gtc gcc gcc gtc gcc gcg tgc ggc ggg 480

Ala Gln Asp Ser Arg Gly Ala Val Ala Ala Val Ala Ala Cys Gly Gly

145 150 155 160

gac gcg tcg tcg tcg cac gac cac gag cgc ggg aac gcg cac ggc gtg 528

Asp Ala Ser Ser Ser His Asp His Glu Arg Gly Asn Ala His Gly Val

165 170 175

tcg tcg gcc gtg atc gcg tcg gcg acg atg ccg aac gac gcc gcc gac 576

Ser Ser Ala Val Ile Ala Ser Ala Thr Met Pro Asn Asp Ala Ala Asp

180 185 190

gac tgc gac gac gcc gag gac cgc gcg aag ctc gtc cgc cac cgc gtc 624

Asp Cys Asp Asp Ala Glu Asp Arg Ala Lys Leu Val Arg His Arg Val

195 200 205

atc tca cag gtg ttt gag ctg ggt atc ata gtg cat tcg att atc atc 672

Ile Ser Gln Val Phe Glu Leu Gly Ile Ile Val His Ser Ile Ile Ile

210 215 220

ggg ata tca cta ggt gca tcc gag agc cca agc acg atc aga ccg ctc 720

Gly Ile Ser Leu Gly Ala Ser Glu Ser Pro Ser Thr Ile Arg Pro Leu

225 230 235 240

gtg gcc gcg tta aca ttt cat cag ttc ttc gaa ggg ata ggg ctg gga 768

Val Ala Ala Leu Thr Phe His Gln Phe Phe Glu Gly Ile Gly Leu Gly

245 250 255

gga tgc att gtt cag gct agg ttc cat ctt aag tct gca gtg aca atg 816

Gly Cys Ile Val Gln Ala Arg Phe His Leu Lys Ser Ala Val Thr Met

260 265 270

gcc atc ttc ttc tcg cta acc aca ccg gtt ggg att atg atc ggt atc 864

Ala Ile Phe Phe Ser Leu Thr Thr Pro Val Gly Ile Met Ile Gly Ile

275 280 285

ggc atc tcc tcc gcc tac aat gag aat agc ccc acg gcc ctg atc gtg 912

Gly Ile Ser Ser Ala Tyr Asn Glu Asn Ser Pro Thr Ala Leu Ile Val

290 295 300

gaa ggc att ctc gac gca gcg gct gct gga atc ctc aac tac atg gcg 960

Glu Gly Ile Leu Asp Ala Ala Ala Ala Gly Ile Leu Asn Tyr Met Ala

305 310 315 320

ctc gtc gac ctt cta gct gaa gat ttc atg aac cct agg gtg cgg aag 1008

Leu Val Asp Leu Leu Ala Glu Asp Phe Met Asn Pro Arg Val Arg Lys

325 330 335

agc gga agg ttg cag ctc atc atc agc atc ttg ctg ctg gtt ggg ata 1056

Ser Gly Arg Leu Gln Leu Ile Ile Ser Ile Leu Leu Leu Val Gly Ile

340 345 350

gct ttg atg tcc ttg ctt ggt att tgg gct tga 1089

Ala Leu Met Ser Leu Leu Gly Ile Trp Ala

355 360

<210> 3

<211> 362

<212> PRT

<213> Rice (Oryza sativa)

<400> 3

Met Ala Phe Asp Leu Lys Leu Thr Ala Cys Leu Leu Leu Ala Val Phe

1 5 10 15

Ser Leu Ala Ala Ala Ala Asp Cys Glu Cys Gln Pro Ser Asp Glu Gly

20 25 30

His Asp Ala Ala Lys Ser Arg Thr Leu Lys Val Ile Ala Ile Phe Cys

35 40 45

Ile Leu Val Gly Ser Ser Ala Gly Cys Ala Ile Pro Ser Leu Gly Arg

50 55 60

Arg Phe Pro Ala Leu Arg Pro Asp Thr Ser Leu Phe Phe Ala Leu Lys

65 70 75 80

Ala Phe Ala Ala Gly Val Ile Leu Ala Thr Ala Phe Val His Ile Leu

85 90 95

Pro Val Ser Phe Asp Lys Leu Gly Ser Pro Cys Leu Val Asp Gly Pro

100 105 110

Trp Arg Lys Tyr Pro Phe Thr Gly Leu Val Ala Met Leu Ala Ala Val

115 120 125

Ala Thr Leu Leu Leu Asp Thr Ile Ala Thr Gly Tyr Phe Leu Gln Arg

130 135 140

Ala Gln Asp Ser Arg Gly Ala Val Ala Ala Val Ala Ala Cys Gly Gly

145 150 155 160

Asp Ala Ser Ser Ser His Asp His Glu Arg Gly Asn Ala His Gly Val

165 170 175

Ser Ser Ala Val Ile Ala Ser Ala Thr Met Pro Asn Asp Ala Ala Asp

180 185 190

Asp Cys Asp Asp Ala Glu Asp Arg Ala Lys Leu Val Arg His Arg Val

195 200 205

Ile Ser Gln Val Phe Glu Leu Gly Ile Ile Val His Ser Ile Ile Ile

210 215 220

Gly Ile Ser Leu Gly Ala Ser Glu Ser Pro Ser Thr Ile Arg Pro Leu

225 230 235 240

Val Ala Ala Leu Thr Phe His Gln Phe Phe Glu Gly Ile Gly Leu Gly

245 250 255

Gly Cys Ile Val Gln Ala Arg Phe His Leu Lys Ser Ala Val Thr Met

260 265 270

Ala Ile Phe Phe Ser Leu Thr Thr Pro Val Gly Ile Met Ile Gly Ile

275 280 285

Gly Ile Ser Ser Ala Tyr Asn Glu Asn Ser Pro Thr Ala Leu Ile Val

290 295 300

Glu Gly Ile Leu Asp Ala Ala Ala Ala Gly Ile Leu Asn Tyr Met Ala

305 310 315 320

Leu Val Asp Leu Leu Ala Glu Asp Phe Met Asn Pro Arg Val Arg Lys

325 330 335

Ser Gly Arg Leu Gln Leu Ile Ile Ser Ile Leu Leu Leu Val Gly Ile

340 345 350

Ala Leu Met Ser Leu Leu Gly Ile Trp Ala

355 360

Claims

1. A promoter for inducing expression of rice by zinc deficiency, wherein the nucleotide sequence of the promoter is shown as SEQ ID NO:1 is shown.

2. The use of the promoter of claim 1 for inducing gene expression in rice under zinc deficiency conditions.