CN114507687B

CN114507687B - Rice OsORM gene and application of protein thereof

Info

Publication number: CN114507687B
Application number: CN202210351102.2A
Authority: CN
Inventors: 李俭; 王铃艳; 姚楠; 黄焕超; 张明永
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-08-19
Anticipated expiration: 2042-04-02
Also published as: CN114507687A

Abstract

The invention discloses an application of a rice OsORM gene in improving rice cadmium resistance, wherein the rice OsORM gene comprises an OsORM1 gene and/or an OsORM2 gene; the OsORM1 gene codes an amino acid sequence shown as SEQ ID NO.2, the OsORM2 gene codes an amino acid sequence shown as SEQ ID NO.4, in the invention, a fragment with very high homology in two homologous genes of rice OsORM1 and OsORM2 is selected to design a target point, and simultaneously a target sequence of a second exon region of OsORM1 and OsORM2 is edited to obtain a gene mutation plant OsORM1OsORM2, so that the gene mutation plant OsORM2 has enhanced cadmium discharge capacity and can resist high-concentration cadmium stress. The cloning and the discovery of new functions of the OsORM gene of the rice provide new gene targets and resources for the genetic breeding of the rice.

Description

Rice OsORM gene and application of protein thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice OsORM gene and application of a rice OsORM gene protein in improvement of rice cadmium resistance.

Background

Cadmium is one of the most interesting environmental pollutants. Cadmium, a carcinogen, causes bone embrittlement and renal failure in humans, and the biological half-life of cadmium in the kidney is long (about 10-30 years), and even the continuous intake of low cadmium intake often causes chronic poisoning.

Plant-derived cadmium is the main source of dietary intake cadmium. The dietary intake of cadmium is mainly from leafy vegetables and cereals, especially cereals, which are consumed daily in asian areas in high amounts. After the cadmium in the soil is absorbed by the rice, the rice with high cadmium accumulation enters the human body, and the rice seriously threatens half of the population in the world which takes the rice as staple food. Reducing cadmium accumulation in cereals is therefore very important for human health.

At present, only a few genes involved in cadmium transport have been identified in rice. The method for developing cadmium-resistant rice germplasm by digging the cadmium-resistant gene of rice is a basis for reducing the cadmium content in rice, developing green agriculture, realizing food and environment safety and improving agricultural output value.

Disclosure of Invention

Based on the above, one of the purposes of the present invention is to provide an application of rice OsORM gene in improving rice cadmium resistance.

The specific technical scheme for realizing the aim of the invention comprises the following steps:

the application of rice OsORM genes in improving the cadmium resistance of rice, wherein the rice OsORM genes comprise OsORM1 genes and/or OsORM2 genes; the OsORM1 gene codes an amino acid sequence shown as SEQ ID NO.2, and the OsORM2 gene codes an amino acid sequence shown as SEQ ID NO. 4.

In some embodiments, the nucleotide sequence of the rice OsORM1 gene is shown in SEQ ID No.1, and/or the nucleotide sequence of the rice OsORM2 gene is shown in SEQ ID No. 3.

The invention also provides an application of the rice OsORM protein in improving rice cadmium resistance, wherein the rice OsORM protein comprises: OsORM1 protein with an amino acid sequence shown as SEQ ID NO.2 and/or OsORM2 protein with an amino acid sequence shown as SEQ ID NO. 4.

The invention also provides a rice OsORM gene mutation vector, which is constructed by using a CRISPR-Cas9 editing technology, so that the function of the rice OsORM gene is lost; the rice OsORM gene comprises an OsORM1 gene and/or an OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID No.1, and/or the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID No. 3.

In some of these embodiments, the CRISPR-Cas9 editing technique edits the target sequence of the second exon region of the rice OsORM1 gene such that the target sequence is mutated by a deletion of 1 base, and edits the target sequence of the second exon region of the rice OsORM2 gene such that the target sequence is mutated by an insertion of 1 base; the target sequence is shown as SEQ ID NO. 5.

In some of these embodiments, the sgRNA sequence for the target sequence is set forth in SEQ ID No. 6.

The invention also provides application of the rice OsORM gene mutation vector in improving rice cadmium resistance.

The invention also provides the application of the rice OsORM gene, the rice OsORM protein and the rice OsORM gene mutation vector in rice genetic breeding.

A preparation for improving cadmium resistance of rice comprises an active component of the rice OsORM gene mutation vector.

A method of increasing cadmium resistance in rice, said method comprising the steps of: constructing a rice OsORM gene mutation vector by using a CRISPR-Cas9 editing technology to lose the function of the rice OsORM gene; the rice OsORM gene comprises an OsORM1 gene and/or an OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID No.1, and/or the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID No. 3.

A method of breeding cadmium-resistant rice, the method comprising the steps of: constructing a rice OsORM gene mutation vector by using a CRISPR-Cas9 editing technology to lose the function of the rice OsORM gene; the rice OsORM gene comprises an OsORM1 gene and/or an OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID No.1, and/or the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID No. 3.

In some of these embodiments, the CRISPR-Cas9 editing technique edits the target sequence of the second exon region of the rice OsORM1 gene such that the target sequence is mutated by a deletion of 1 base, and edits the target sequence of the second exon region of the rice OsORM2 gene such that the target sequence is mutated by an insertion of 1 base; the target sequence is shown in SEQ ID NO. 5.

Compared with the prior art, the invention has the following beneficial effects:

in the invention, a fragment with very high homology in two homologous genes OsORM1 and OsORM2 of rice OsORM is selected to design a target spot, a CRISPR-Cas9 editing technology is utilized, and target sequences of second exon regions of OsORM1 and OsORM2 are simultaneously edited to obtain a gene mutation plant OsORm1OsORm2, and the gene mutation plant OsORm1OsORm2 has the advantages of enhanced cadmium discharge capacity and high-concentration cadmium stress resistance. The cloning and the discovery of new functions of the OsORM gene of the rice provide new gene targets and resources for the genetic breeding of the rice.

Drawings

In FIG. 1, A is the exon region information of the rice OsORM1 gene and OsORM2 gene in example 1 of the present invention, and B is the editing result of the rice OsORM gene.

FIG. 2 is a result of investigation of the resistance of wild type ZH11 and gene mutant osorm1osorm2 strain to high concentration cadmium stress in example 2 of the present invention.

FIG. 3 is a result of investigation of cadmium transport capacity of wild type ZH11 and gene mutant osorm1osorm2 strain in example 3 of the present invention, wherein Infflux indicates cadmium ion influx; efflux represents cadmium ion Efflux.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one aspect of the present invention, there is provided use of a rice OsORM gene for increasing cadmium resistance in rice, the rice OsORM gene comprising a OsORM1 gene and/or a OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID NO.1, and the coding sequence is shown as SEQ ID NO. 2; and/or the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID NO.3, and the coding sequence is shown as SEQ ID NO. 4.

The OsORM1 gene:

SEQ ID NO.1：

ATGGCGAAGCTGTACGTGCAGGCGGTGCAGCCGGCGGATCTGAACAAGAACACGGAGTGGTTCATGTACCCCGGGGTGTGGACGACCTACATCCTCATCCTCTTCTTCTCCTGGCTGCTCGTCCTCTCCGTCTTCGGCTGCACCCCCGGCATGGCGTGGACGTTCGTCAACCTCGCCCACTTCGCGATGACATACCATTTTTTTCACTGGAAGAAGGGAACTCCGTTTGCTGATGACCAGGGGATGTATAATAGATTGACTTGGTGGGAGCAAATGGACAATGGGAAGCAGCTTACTCGCAACAGAAAATTTCTGACCGTGGTTCCTTTGGTCCTATACTTGATAGCCTTGCACACGACAGATTATCAACATCCTATGCTCTTCCTCAACACCATTGCAGTTGTTGTGCTGGTTGTTGCAAAACTACCGAACATGCACAAGGTCCGGATCTTTGGAATCAATGCTGGCAACTAG

SEQ ID NO.2

MAKLYVQAVQPADLNKNTEWFMYPGVWTTYILILFFSWLLVLSVFGCTPGMAWTFVNLAHFAMTYHFFHWKKGTPFADDQGMYNRLTWWEQMDNGKQLTRNRKFLTVVPLVLYLIALHTTDYQHPMLFLNTIAVVVLVVAKLPNMHKVRIFGINAGN

OsORM2 gene

SEQ ID NO.3

ATGGCGAAGCTGTACGTGCAGGCGGTGCCGCCGCCGGATCTGAACAGGAACACGGAGTGGTTCATGTACCCGGGCGTCTGGACGACCTACATCTGCATCCTCTTCTTCTCCTGGCTCCTCGTCCTCTCCGTCTTCGGCTGCACGCCCGGCATGGCCTGGACCGTCGTCAATCTCTTCCACTTCGCGATCACATACCACTTTTTCCATTGGAAGAAGGGAACACCTTTTGCTGATGACCAGGGAATGTACAACACATTGACTTGGTGGGAGCAAATGGACAATGGCAAACAGCTTACTCGCAACAGGAAGTTCCTTGTTGTAGTTCCTGTTGTCCTGTATTTGATAGCTTCACACACTACAGACTACCAACATCCTATGCTCTTCCTCAACACCCTTGCAGTCGCAGTGCTCGTGGTCGCTAAACTACCGAACATGCACAAGGTCCGGATTTTTGGAATTAATGCTGGGAACTAG

SEQ ID NO.4

MAKLYVQAVPPPDLNRNTEWFMYPGVWTTYICILFFSWLLVLSVFGCTPGMAWTVVNLFHFAITYHFFHWKKGTPFADDQGMYNTLTWWEQMDNGKQLTRNRKFLVVVPVVLYLIASHTTDYQHPMLFLNTLAVAVLVVAKLPNMHKVRIFGINAGN

It is understood that modifications of the nucleotide sequence of the encoding gene of the present invention without changing the amino acid sequence, in view of the degeneracy of the codon and the preference of the codon for different species, are also within the scope of the present invention.

In another aspect of the present invention, there is provided a method of breeding cadmium-resistant rice, the method comprising the steps of: editing a target sequence (SEQ ID NO.5) of a second exon region of a rice OsORM1 gene under the guidance of an sgRNA sequence (SEQ ID NO.6) of the target sequence of the second exon region of the rice OsORM gene by using a CRISPR-Cas9 editing technology, so that the OsORM1 gene target sequence generates a mutation of deleting 1 base, editing the target sequence (SEQ ID NO.5) of the second exon region of the rice OsORM2 gene, and generating a mutation of inserting 1 base (B in a picture 1) in the OsORM2 gene target sequence, thereby losing the function of the rice OsORM gene and constructing the OsORM gene mutation vector.

Target sequence (SEQ ID NO. 5): 5'-TGGTGGGAGCAAATGGACAA-3'

sgRNA sequence (SEQ ID No. 6):

TTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

the present invention will be described in detail with reference to specific examples.

Example 1 construction of Rice OsORM Gene mutation vector and obtaining of Gene-mutated Rice T0 plant

In the embodiment, a rice OsORM gene mutation vector is firstly constructed, and then a gene mutation rice T0 plant is obtained. The method specifically comprises the following steps:

1. strain activation and plasmid extraction preparation

Coli (TOP10F ') carrying pYRCRISPR/Cas 9Pubi-H vector (stored in the laboratory, present in Liu Yan light academy, university of south China agriculture) was streaked on LB medium containing kanamycin (25. mu.g/ml) overnight, E.coli (TOP 10F') carrying pYRLCRNA 1-OsU a/LacZ vector (said pYRSGRNA 1-OsU a/LacZ vector is a sgRNA sequence carrying SEQ ID NO.6, OsU a promoter sequence, PUC18 vector of LacZ gene sequence, which can be constructed by a conventional technique in the art) was streaked on LCLB medium containing ampicillin (50. mu.g/ml) overnight, single colony culture seed solutions were individually picked up and then subjected to scale-up, and plasmids pYRISPR/Cas 9Pubi-H and pYRSGRNA 637-OsU/a/LacZ were extracted;

2. construction of sgRNA expression cassette

(1) First round of PCR reaction

Using 2-5ngpYLsgRNA1-OsU a/LacZ plasmid as template, U-F (SEQ ID NO.7), OsU aT1R (SEQ ID NO.8), gR-R (SEQ ID NO.9) and OsU aT1F (SEQ ID NO.10) as primers (the concentrations of the primers U-F and OsU aT1R in the reaction system are 0.2. mu.M respectively, and the concentrations of the primers gR-R and OsU aT1F in the reaction system are 0.1. mu.M respectively), and the amplification system is 10. mu.L (2x PCRbuffer for KOFXD 5. mu.l, 2mM dNTPs 2. mu.l, U-F0.2. mu.l, OsU6aT 1R.2. mu.l, gR-R0.2. mu.l, OsU6aT 1F. mu.2. mu.l, YpFX, 894. mu.l, LacZ plasmid 898/LacZ plasmid and PCR amplification products are obtained by sterilizing and carrying out on the amplification systems of the plasmid products of 2. mu.l, and the PCR products of the primers U-F, the primers U-F636 aT1 No.7 aT1R and the primers are 898 and the ultrapure water; the PCR reaction program is: 28 cycles of 94 ℃ for 10s, 58 ℃ for 15s, 68 ℃ for 20 s;

U-F(SEQ ID NO.7)：5'-CTCCGTTTTACCTGTGGAATCG-3'

OsU6aT1R(SEQ ID NO.8)：

5'-TTGTCCATTTGCTCCCACCACGGCAGCCAAGCCAGCA-3'

gR-R(SEQ ID NO.9)：5'-CGGAGGAAAATTCCATCCAC-3'

OsU6aT1F(SEQ ID NO.10)：

5'-TGGTGGGAGCAAATGGACAAGTTTTAGAGCTAGAAAT-3'

(2) second round of PCR

1. mu.l of the first PCR reaction was taken and washed with H ₂ Diluting by 10 times with O, taking 1 mu l as a template, taking Pps-GGL (SEQ ID NO.11) and Pgs-GG2(SEQ ID NO.12) as primers, and carrying out amplification by using a high fidelity enzyme PRIMESTAR MAX PCR enzyme, wherein the amplification system is 50 mu l (2x PrimeSTAR Max Premix 25 mu l, Pps-GGL1.5 mu l, Pgs-GGR1.5 mu l, template 1 mu l and sterilized ultrapure water 21 mu l), so as to obtain an sgRNA expression cassette; the PCR reaction procedure was as follows: 20 cycles: 10s at 95 ℃, 15s at 58 ℃ and 20s at 68 ℃;

Pps-GGL(SEQ ID NO.11)

5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3'

Pgs-GGR(SEQ ID NO.12)：

5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3'

3. carrying out enzyme digestion-ligation reaction on the pYLCRISPR/Cas9Pubi-H vector obtained in the step (1) and the sgRNA expression cassette obtained in the step (2) to obtain a ligation product (namely a rice OsORM gene mutation vector); the enzyme digestion-ligation reaction is as follows: 10min at 37 ℃, 5min at 10 ℃, 5min at 20 ℃ and 3 cycles; then 3min at 37 ℃, 5min at 10 ℃ and 5min at 20 ℃ for 10 cycles; finally 5min at 37 ℃. The mass ratio of the pYLCRISPR/Cas9Pubi-H vector to the sgRNA expression cassette is 8: 1;

the ligation reaction system is shown in table 1:

TABLE 1 digestion-ligation reaction System

4. Coli competent cells transformed with ligation products

A tube of E.coli competent DH 5. alpha. was removed from the ultra-low temperature freezer and placed on ice for about 10min to be competent to thaw. Adding the ligation product obtained in the step (3) into E.coli DH5 alpha escherichia coli competent cells according to the proportion of 1:10, carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃ to destroy the bimolecular membrane structure of the competent cells, and carrying out ice bath for 5 min; then adding 1ml LB culture medium, culturing for 1-1.5 h at 37 ℃, centrifuging for 1min at 5000g, abandoning 500. mu.l of supernatant and then resuspending bacteria. The bacteria were plated (plating medium LB + 25. mu.g/ml Kan, 0.3-0.5 mM IPTG, appropriate amount of X-gal) and placed upside down in a 37 ℃ incubator overnight. Selecting a blue spot positive clone;

5. freeze-thaw transformation of agrobacterium

Amplifying and culturing the positive clone obtained in the step 4, extracting plasmid, adding the plasmid into the agrobacterium EHA105 competence, shocking at 1750V for 0.5 second, then adding 1mLYEB culture medium, culturing at 28 ℃ for 3-4 h, then coating a plate (synchronous step 4), culturing in the dark at 28 ℃ for 2-3 days, and identifying the positive agrobacterium by PCR;

6. infecting the callus of No.11 (ZH11, laboratory preservation) of the rice wild strain with the positive agrobacterium EHA105 of the step 5 to obtain a gene mutation rice T0 plant. The method comprises the following specific steps:

A. induction of Rice callus

(1) Removing the shells of seeds of the wild rice plant (ZH11) of the Zhonghua No.11 rice and keeping the integrity of rice embryos;

(2) treating the shelled seeds with 70% ethanol for 1-2 minutes, shaking gently for multiple times, washing with sterilized distilled water twice, adding a proper amount of 20% bleaching solution (one drop of Tween 20 is added per 50 ml), shaking slowly at 120rpm on a constant-temperature shaking table at 22 ℃ for 45-60 minutes, and washing gently with sterilized distilled water for about 10 times to remove the bleaching solution on the surfaces of the seeds (the operation is completed in an ultra-clean workbench);

(3) uniformly placing the sterilized seeds on the surface of a flat plate containing 2mg/ml NB medium (callus induction medium) by using autoclaved tweezers; the formulation of NB medium is shown in Table 2.

TABLE 2 formulation of culture medium used in Rice callus formation and Agrobacterium-mediated transformation of Rice

(4) Sealing the flat plate by using a sealing film, and then placing the flat plate in a sealed lightproof carton at 25 ℃ for about 15 days;

(5) and stripping the callus from the rice plantlet, and subculturing on the same induction medium until the light yellow callus with strong activity is obtained.

B. Transformation of Agrobacterium

(1) The single clone containing the objective plasmid (positive Agrobacterium EHA105 in step 5) was selected and inoculated into 5ml of a liquid bacterial medium containing rifampicin at 50. mu.g/ml and kanamycin at 50mg/L, and cultured at 28 ℃ and 200rpm to OD ₆₀₀ About 0.5 or so;

(2) adding into 100ml bacterial culture medium containing 50 μ g/ml rifampicin and 50mg/L kanamycin at a ratio of 1:100, culturing at 28 deg.C and 200rpm overnight, and allowing Agrobacterium to grow to OD ₆₀₀ ＝0.8-1.0；

(3) Centrifuging at 4000rpm for 10min to collect thallus, removing supernatant, suspending thallus with AAM culture medium of the same volume, centrifuging under the same conditions, and removing supernatant; the formula of the AAM culture medium is shown in the table 2;

(4) resuspending the cells to OD in AAM Medium ₆₀₀ About 0.4 and transferred into a fresh sterilized 250ml flask;

(5) and selecting light yellow and non-fragile embryogenic callus with the diameter of about 5mm, soaking the embryogenic callus in the agrobacterium liquid for about 30 minutes, and occasionally gently shaking to ensure that the callus is uniformly contacted with the liquid.

(6) Placing the infected calluses on sterilized filter paper, sucking the residual bacteria liquid, slightly drying, uniformly placing the calluses on the surface of a prepared 2N6-AS solid plate, placing about 25 calluses on each plate, sealing the plate by using a sealing film, and placing the plate at 25 ℃ for dark culture for 2-3 days. During co-cultivation, care was taken to check to prevent overgrowth of Agrobacterium; the formula of 2N6-AS medium is shown in Table 2.

C. Selection culture and regeneration of plants

(1) Transferring the co-cultured callus to a new sterilized 250ml triangular flask, washing with sterilized distilled water containing 500mg/l cephalosporin and 200mg/l carbenicillin for at least 10 times, each time with 50-75ml water, slightly shaking the triangular flask during washing, finally adding 100ml sterilized distilled water (containing the same antibiotic), slowly shaking at 25 ℃, 120rpm for two hours, pouring off the water, transferring the callus to sterilized filter paper to absorb excess water, and slightly drying;

(2) transferring the callus to a new selective culture medium, adding 250mg/l of cephalosporin, 250mg/l of carbenicillin and 50mg/l of hygromycin into the culture medium, placing the culture dish in a lightproof paper box, culturing for three weeks at 25 ℃, frequently checking the pollution condition, and timely transferring the non-polluted callus in the same culture dish to the new culture medium; the selection medium formulation is shown in table 3.

TABLE 3 formulation of the Medium for selection and regeneration of resistant calli in Agrobacterium-mediated transformation of Rice

(3) Cutting off the resistant callus and transferring the cut resistant callus to a new solid pre-regeneration culture medium through selective culture for 2-3 weeks, and continuously culturing for 2-3 weeks under the conditions of 25 ℃, 16h of illumination and 8h of darkness; transferring the callus to a regeneration culture medium flat plate, and regenerating the callus into rice seedlings; the pre-regeneration medium and regeneration medium formulations are shown in table 3.

(4) Transferring the regenerated plantlets to a new MS culture medium, and culturing under the conditions of 25 ℃, 16h of illumination and 8h to ensure that the plantlets grow to be of a size capable of being transplanted; the MS medium formulation is shown in table 2.

(5) Transplanting the seedlings with the height of about 10cm into a small box filled with soil, and transferring the seedlings into a greenhouse for normal growth to obtain the gene mutation rice T0 plant.

7. Detection of targeting effect of gene mutation rice T0 plant

The DNA of a gene mutation rice T0 plant (seedling) is used as a template, SEQ ID NO.13(5'-TCCCTATTGCCACTGCCTACT-3') and SEQ ID NO.14(5'-TGGAACTTGGATTTGGGGCTA-3') aiming at a target sequence (the position is shown as A in figure 1) of a second exon region of an OsORM1 gene are used as primers, SEQ ID NO.15(5'-CAAACACAAGTAAAGCCA-3') and SEQ ID NO.16(5'-TGTGGAATACTACCGTTA-3') aiming at the target sequence (the position is shown as A in figure 1) of the second exon region of the OsORM2 gene are used as primers, PCR amplification is carried out, and the amplification system is 20 mu l (10 mu l of 2 xTaq Master Mix (Vazyme), 10 mu l of SEQ ID NO.151 mu l, 161 mu l of SEQ ID NO.161 mu l, 1 mu l of DNA template and 7 mu l of sterilized ultrapure water). The PCR reaction procedure was as follows: 95 ℃ for 5min,30 cycles: 30s at 95 ℃,30 s at 55 ℃ and 30s at 72 ℃; 5min at 72 ℃. Sequencing the amplified product.

Through sequencing detection, the gene mutation rice T0 plant obtained in the embodiment can generate 1 base deletion mutation shown as B in figure 1 at the second exon target position of the OsORM1 gene, and generate 1 base insertion mutation shown as B in figure 1 at the second exon target position of the OsORM2 gene, and is named as a gene mutation rice strain orm1 orm 2.

Example 2 resistance of Gene-mutated Rice orm1 orm2 line to high concentrations of cadmium

This example investigated the resistance of the genetically mutated rice orm1 orm2 strain to high concentrations of cadmium.

After disinfecting and germinating the surfaces of wild type ZH11 seeds and gene mutation rice orm1 orm2 seeds, culturing the seedlings to two weeks old by using a rice nutrient solution, adding 300 mu M cadmium chloride into the hydroponic solution, continuously performing hydroponic culture for 5 days, pouring two leaves, three leaves and four leaves of the rice into the water culture solution, and observing the phenotype, wherein the results are shown in figure 2.

As can be seen from FIG. 2, compared with wild type ZH11 rice seedlings, the gene mutant rice orm1 orm2 strain has low curling and yellowing degree of leaves and shows stronger resistance to high-concentration cadmium stress.

Example 3 transport of cadmium by Gene-mutated Rice line orm1 orm2

This example investigated the transport ability of the gene-mutated rice orm1 orm2 strain for cadmium.

Disinfecting and germinating the surfaces of wild ZH11 seeds and gene mutation rice orm1 orm2 seeds, culturing the seedlings to two weeks old by using a rice nutrient solution, and evaluating the cadmium transport capacity of the wild ZH11 seedlings and the gene mutation rice orm1 orm2 seedlings by using a non-damage micro-detection system. The method comprises the following specific steps of measuring the cadmium ion flow rate by a non-damage micrometering system:

1. the reagent bottle containing the cadmium ion LIX is quickly dipped with the LIX carrier to fill the tip of the reagent bottle.

2. A glass flow rate sensor was taken and electrolyte was injected from the back end using an electrolyte fill syringe to create a10 mm liquid column.

3. The flow rate sensor tip is opposed to the LIX carrier tip on the same horizontal plane. And (3) enabling the LIX to enter the ion selective flow velocity sensor due to the capillary action, and adjusting the length of the LIX to 6-10 mm by pushing and pulling a pressure adjusting device of the flow velocity sensor to obtain the manufactured cadmium ion flow velocity sensor.

4. And placing the prepared cadmium ion correction liquid on an objective table, and starting to correct the cadmium ion flow velocity sensor.

5. The primary root tips of two-week-old ZH11 and orm1 orm2 were cut and the cadmium ion flux was measured at the meristematic site for 5 minutes.

The results are shown in fig. 3, and it is understood from fig. 3 that the cadmium excretion ability of the mutant rice strain orm1 orm2 is enhanced as compared with the wild-type ZH11 rice seedling.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Sequence listing

<110> south China plant garden of Chinese academy of sciences

<120> rice OsORM gene and application of protein thereof

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcgaagc tgtacgtgca ggcggtgcag ccggcggatc tgaacaagaa cacggagtgg 60

ttcatgtacc ccggggtgtg gacgacctac atcctcatcc tcttcttctc ctggctgctc 120

gtcctctccg tcttcggctg cacccccggc atggcgtgga cgttcgtcaa cctcgcccac 180

ttcgcgatga cataccattt ttttcactgg aagaagggaa ctccgtttgc tgatgaccag 240

gggatgtata atagattgac ttggtgggag caaatggaca atgggaagca gcttactcgc 300

aacagaaaat ttctgaccgt ggttcctttg gtcctatact tgatagcctt gcacacgaca 360

gattatcaac atcctatgct cttcctcaac accattgcag ttgttgtgct ggttgttgca 420

aaactaccga acatgcacaa ggtccggatc tttggaatca atgctggcaa ctag 474

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Met Ala Lys Leu Tyr Val Gln Ala Val Gln Pro Ala Asp Leu Asn Lys

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Asn Thr Glu Trp Phe Met Tyr Pro Gly Val Trp Thr Thr Tyr Ile Leu

20 25 30

Ile Leu Phe Phe Ser Trp Leu Leu Val Leu Ser Val Phe Gly Cys Thr

35 40 45

Pro Gly Met Ala Trp Thr Phe Val Asn Leu Ala His Phe Ala Met Thr

50 55 60

Tyr His Phe Phe His Trp Lys Lys Gly Thr Pro Phe Ala Asp Asp Gln

65 70 75 80

Gly Met Tyr Asn Arg Leu Thr Trp Trp Glu Gln Met Asp Asn Gly Lys

85 90 95

Gln Leu Thr Arg Asn Arg Lys Phe Leu Thr Val Val Pro Leu Val Leu

100 105 110

Tyr Leu Ile Ala Leu His Thr Thr Asp Tyr Gln His Pro Met Leu Phe

115 120 125

Leu Asn Thr Ile Ala Val Val Val Leu Val Val Ala Lys Leu Pro Asn

130 135 140

Met His Lys Val Arg Ile Phe Gly Ile Asn Ala Gly Asn

145 150 155

<210> 3

<211> 474

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcgaagc tgtacgtgca ggcggtgccg ccgccggatc tgaacaggaa cacggagtgg 60

ttcatgtacc cgggcgtctg gacgacctac atctgcatcc tcttcttctc ctggctcctc 120

gtcctctccg tcttcggctg cacgcccggc atggcctgga ccgtcgtcaa tctcttccac 180

ttcgcgatca cataccactt tttccattgg aagaagggaa caccttttgc tgatgaccag 240

ggaatgtaca acacattgac ttggtgggag caaatggaca atggcaaaca gcttactcgc 300

aacaggaagt tccttgttgt agttcctgtt gtcctgtatt tgatagcttc acacactaca 360

gactaccaac atcctatgct cttcctcaac acccttgcag tcgcagtgct cgtggtcgct 420

aaactaccga acatgcacaa ggtccggatt tttggaatta atgctgggaa ctag 474

<210> 4

<211> 157

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Lys Leu Tyr Val Gln Ala Val Pro Pro Pro Asp Leu Asn Arg

1 5 10 15

Asn Thr Glu Trp Phe Met Tyr Pro Gly Val Trp Thr Thr Tyr Ile Cys

20 25 30

Ile Leu Phe Phe Ser Trp Leu Leu Val Leu Ser Val Phe Gly Cys Thr

35 40 45

Pro Gly Met Ala Trp Thr Val Val Asn Leu Phe His Phe Ala Ile Thr

50 55 60

Tyr His Phe Phe His Trp Lys Lys Gly Thr Pro Phe Ala Asp Asp Gln

65 70 75 80

Gly Met Tyr Asn Thr Leu Thr Trp Trp Glu Gln Met Asp Asn Gly Lys

85 90 95

Gln Leu Thr Arg Asn Arg Lys Phe Leu Val Val Val Pro Val Val Leu

100 105 110

Tyr Leu Ile Ala Ser His Thr Thr Asp Tyr Gln His Pro Met Leu Phe

115 120 125

Leu Asn Thr Leu Ala Val Ala Val Leu Val Val Ala Lys Leu Pro Asn

130 135 140

Met His Lys Val Arg Ile Phe Gly Ile Asn Ala Gly Asn

145 150 155

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggtgggagc aaatggacaa 20

<210> 6

<211> 530

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttttttcctg tagttttccc acaaccattt tttaccatcc gaatgatagg ataggaaaaa 60

tatccaagtg aacagtattc ctataaaatt cccgtaaaaa gcctgcaatc cgaatgagcc 120

ctgaagtctg aactagccgg tcacctgtac aggctatcga gatgccatac aagagacggt 180

agtaggaact aggaagacga tggttgattc gtcaggcgaa atcgtcgtcc tgcagtcgca 240

tctatgggcc tggacggaat aggggaaaaa gttggccgga taggagggaa aggcccaggt 300

gcttacgtgc gaggtaggcc tgggctctca gcacttcgat tcgttggcac cggggtagga 360

tgcaatagag agcaacgttt agtaccacct cgcttagcta gagcaaactg gactgcctta 420

tatgcgcggg tgctggcttg gctgccggtt ttagagctag aaatagcaag ttaaaataag 480

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 530

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctccgtttta cctgtggaat cg 22

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttgtccattt gctcccacca cggcagccaa gccagca 37

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggaggaaaa ttccatccac 20

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tggtgggagc aaatggacaa gttttagagc tagaaat 37

<210> 11

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttcagaggtc tctctcgact agtatggaat cggcagcaaa gg 42

<210> 12

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agcgtgggtc tcgaccgacg cgtatccatc cactccaagc tc 42

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tccctattgc cactgcctac t 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tggaacttgg atttggggct a 21

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caaacacaag taaagcca 18

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

tgtggaatac taccgtta 18

Claims

1. The application of the rice OsORM gene in improving the cadmium resistance of rice is characterized in that the rice OsORM gene is OsORM1 gene and OsORM2 gene; the OsORM1 gene codes an amino acid sequence shown as SEQ ID NO.2, and the OsORM2 gene codes an amino acid sequence shown as SEQ ID NO. 4.

2. The use of a rice OsORM gene as claimed in claim 1 for increasing cadmium resistance in rice, wherein the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID No. 3.

3. The application of the rice OsORM protein in improving the cadmium resistance of rice is characterized in that the rice OsORM protein is: OsORM1 protein with an amino acid sequence shown as SEQ ID NO.2 and OsORM2 protein with an amino acid sequence shown as SEQ ID NO. 4.

4. The application of the rice OsORM gene mutation vector in improving the rice cadmium resistance is characterized in that the gene mutation vector is constructed by using a CRISPR-Cas9 editing technology, so that the function of the rice OsORM gene is lost; the rice OsORM gene is OsORM1 gene and OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID NO. 3.

5. The use of claim 4, wherein the CRISPR-Cas9 editing technique edits the target sequence of the second exon region of the rice OsORM1 gene to make a1 base deletion mutation, and edits the target sequence of the second exon region of the rice OsORM2 gene to make a1 base insertion mutation; the target sequence is shown in SEQ ID NO. 5.

6. The use according to claim 5, wherein the sgRNA sequence for the target sequence is shown in SEQ ID No. 6.

7. A method of increasing cadmium resistance in rice, said method comprising the steps of: constructing a rice OsORM gene mutation vector by using a CRISPR-Cas9 editing technology to lose the function of the rice OsORM gene; the rice OsORM gene is OsORM1 gene and OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID NO. 3.

8. A method of growing cadmium-resistant rice, said method comprising the steps of: constructing a rice OsORM gene mutation vector by using a CRISPR-Cas9 editing technology to lose the function of the rice OsORM gene; the rice OsORM gene is OsORM1 gene and OsORM2 gene; the nucleotide sequence of the rice OsORM1 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the rice OsORM2 gene is shown as SEQ ID NO. 3.

9. The method for breeding cadmium-resistant rice as claimed in claim 8, wherein the CRISPR-Cas9 editing technique edits the target sequence of the second exon region of rice OsORM1 gene to make a 1-base deletion mutation of the target sequence, and edits the target sequence of the second exon region of rice OsORM2 gene to make a 1-base insertion mutation of the target sequence; the target sequence is shown as SEQ ID NO. 5.