Zinc-cadmium-resistant gene TmZRC1S derived from tuber melanosporum and application thereof
阅读说明：本技术 一种来源于黑孢块菌的耐锌镉基因TmZRC1S及应用 (Zinc-cadmium-resistant gene TmZRC1S derived from tuber melanosporum and application thereof ) 是由 王丽娟 姚泉洪 彭日荷 韩红娟 田永生 高建杰 许晶 付晓燕 王波 李振军 张福 于 2021-07-30 设计创作，主要内容包括：一种来源于黑孢块菌的耐锌镉基因TmZRC1S及应用,将来源于黑孢块菌的一个预测的锌转运蛋白基因序列GSTUMT00007113001按照植物密码子偏爱性进行改造,用基因合成法进行基因人工合成,获得核苷酸序列如SEQ ID No1所示的TmZRC1S基因,构建重组表达载体并转入拟南芥中,得到了具有较强锌镉抗性和锌镉富集能力的转基因拟南芥植株。经锌、镉溶液处理后,转基因株系的生长较野生型株系好,且转基因株系中的锌镉含量显著高于野生型株系,特别是地上部锌含量是野生型株系的3.74倍、镉含量是野生型株系的3.36倍,表明本发明改造合成的TmZRC1S基因具有提高植物抗和富集锌镉的能力,可用于培育富集锌镉的植物。(The invention discloses a zinc-cadmium resistant gene Tm ZRC1S derived from truffle and application. A predicted zinc transporter gene sequence GSTMTT 00007113001 derived from the truffle is transformed according to plant codon preference, gene artificial synthesis is performed by a gene synthesis method to obtain the Tm ZRC1S gene with a nucleotide sequence shown as SEQ ID No1, a recombinant expression vector is constructed and transferred into arabidopsis thaliana, and a transgenic arabidopsis thaliana plant with high zinc-cadmium resistance and zinc-cadmium enrichment capacity is obtained. After treatment with zinc and cadmium solutions, the transgenic plant grows better than a wild plant, the zinc-cadmium content in the transgenic plant is remarkably higher than that of the wild plant, particularly, the zinc content of the overground part is 3.74 times that of the wild plant, and the cadmium content is 3.36 times that of the wild plant. The transformed and synthesized Tm ZRC1S gene has the capacity of improving the zinc-cadmium resistance and enrichment capacity of the plant and can be used for cultivating the plant with enriched zinc and cadmium.)
1. Zinc-cadmium-resistant gene derived from tuber melanosporumTmAnd the nucleotide sequence of the ZRC1S is shown as SEQ ID NO 1.
2. Comprising the zinc-cadmium resistant gene of claim 1TmA recombinant expression vector of zr c 1S.
3. The recombinant expression vector of claim 2, further comprising a double 35S promoter, a GUS reporter gene, and an intron-containing kanamycin resistance marker gene.
4. The zinc-cadmium resistant gene of claim 1 derived from Tuber melanosporumTmApplication of ZRC1S in culturing zinc-cadmium resistant plants.
5. The use according to claim 4, wherein the zinc-cadmium tolerant plant is Arabidopsis thaliana.
6. The zinc-cadmium resistant gene derived from tuber melanosporumTmThe method for transferring ZRC1S into Arabidopsis thaliana comprises the following steps:
1) construction of recombinant expression vector BH440
After the PCR product was digested with BamHI and SacI, the product synthesized by the present invention was digested with T4DNA ligaseTmThe ZRC1S gene is connected with a plant expression vector pCAMBIA-1301 containing a double 35S promoter, and enzyme digestion identification and sequence determination are carried out to obtainContaining a gene of interestTmRecombinant expression vector BH440 of zr c 1S;
2) electric shock method for transforming agrobacterium
Introducing the constructed recombinant expression vector BH440 into agrobacterium tumefaciens by an electric shock method, wherein the agrobacterium tumefaciens strain is LBA 4404;
3) agrobacterium flower dipping method for transforming arabidopsis
Introducing by using Agrobacterium flower dipping methodTmThe agrobacterium tumefaciens of the ZRC1S gene is transformed into Arabidopsis, hygromycin is utilized to screen transformed plants, seedlings which normally grow on a hygromycin plate are transplanted and harvested, and positive seedling identification is carried out.
Technical Field
The invention belongs to the technical field of culturing zinc-cadmium-enriched plants, and particularly relates to a zinc-cadmium-resistant gene derived from tuber melanosporumTmZRC1S and application.
Background
For a long time, due to the reasons of extensive economic development mode, unreasonable industrial structure and layout and the like, the total pollutant emission amount in China is high, nearly 1/6 cultivated land is polluted by heavy metal, the problem of heavy metal pollution is gradually changed into the world, 2 hundred million hectares of cultivated land is influenced by heavy metal pollution all over the world, and the serious threat is formed to the quality safety of agricultural products and the health of human bodies. According to the global mineral product statistical data published by the national geological agency in 2013 in month 1, the Chinese cadmium yield in 2012 is 7000 tons, which accounts for 30.43% of the global total yield. The national soil pollution condition survey bulletin issued by the ministry of environmental protection and the ministry of national soil resources in 2014 shows that the point position exceeding rate of zinc and cadmium is 0.9 percent and 7 percent respectively, wherein the point position exceeding rate of the cadmium polluted soil is the main pollutant of all land utilization types (cultivated land, forest land, grassland and unused land), and zinc is the main pollutant of metal smelting industrial parks and peripheral soil thereof (northern).
The heavy metal pollution treatment in soil becomes a hot point of research. The conventional heavy metal contaminated soil remediation method comprises physical remediation and chemical remediation, which is to adopt a chemical or physical method to realize the fixation or separation of heavy metals in soil and reduce the pollution influence on the soil on the basis of the relevant characteristics of the difference between the physicochemical property of the soil and the existence of the heavy metals. These methods tend to be complicated in steps, high in cost, and liable to cause secondary pollution. The enrichment effect of some super-accumulation plants on heavy metals also draws wide attention, and the remediation of the heavy metal contaminated soil by using the plants has the advantages of low cost, easy operation, no damage to the soil structure and the like.
The transporter plays an important role in the tolerance and hyper-accumulation action mechanism of plants to heavy metals, and the transporter can not be involved in the efficient absorption and transportation of the heavy metals or the separation and detoxification of the heavy metals. Among the identified metal ion transporters, the most studied transporters for zinc ion absorption and transport are the ZIP (Zn-regulated, Iron-regulated transporter-like Protein) family and the CDF (catalysis Diffusion factor) family of proteins. Overall, the main function of the ZIP family is uptake of zinc, whereas the CDF family members are primarily involved in zinc efflux and intracellular compartmentalization for detoxification or storage. Zinc can regulate the activity of both types of transporters at both the transcriptional and translational levels to maintain zinc homeostasis at the cellular and organism levels.
TmThe ZRC1S gene belongs to the CDF family, and no research report for applying the gene to the aspect of zinc and cadmium resistance of plants is seen at present.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a bacterium (A) derived from Tuber melanosporumTuber melanosporum) The zinc-cadmium resistant geneTmZRC1S and its useWhen the gene is transferred into arabidopsis thaliana, the heavy metal zinc and cadmium resistance of a transgenic plant is improved, the accumulation amount of zinc and cadmium of the transgenic plant is improved, and the gene can be used for cultivating plant varieties with improved zinc and cadmium resistance and enrichment capacity.
In order to achieve the purpose, the invention is realized by the following technical scheme:
zinc-cadmium-resistant gene derived from tuber melanosporumTmThe ZRC1S has a nucleotide sequence shown in SEQ ID NO1, and is obtained by artificial synthesis method according to plant preferred codons, i.e., by two-step PCR using a large number of overlapping primers to perform whole gene synthesis (PTDS) (Nucleic Acids Research, 2004, 32: e 98).
Contains the zinc-cadmium resistant geneTmThe recombinant expression vector of ZRC1S further comprises a double 35S promoter, a GUS reporter gene and an intron-containing kanamycin resistance marker gene.
The invention provides the zinc-cadmium resistant gene from the tuber melanosporumTmApplication of ZRC1S in culturing zinc-cadmium resistant plants. Preferably, the zinc-cadmium tolerant plant is arabidopsis thaliana.
The zinc-cadmium resistant geneTmThe method for transferring ZRC1S into Arabidopsis thaliana comprises the following steps:
1) construction of recombinant expression vector BH440
After the PCR product was digested with BamHI and SacI, the product synthesized by the present invention was digested with T4DNA ligaseTmThe ZRC1S gene is connected with a plant expression vector pCAMBIA-1301 containing a double 35S promoter, enzyme digestion identification and sequence determination are carried out to obtain a gene containing a target geneTmRecombinant expression vector BH440 of ZRC 1S.
2) Electric shock method for transforming agrobacterium
And (3) introducing the constructed recombinant expression vector BH440 into the agrobacterium tumefaciens by an electric shock method, wherein the agrobacterium tumefaciens strain is LBA 4404.
3) Agrobacterium flower dipping method for transforming arabidopsis
Introducing by using Agrobacterium flower dipping methodTmAgrobacterium tumefaciens of The ZRC1S gene was transformed into Arabidopsis thaliana (Clough et al, The plant journel, 1998, 16(6):735-And (4) selecting transformed plants, transplanting seedlings of the seedlings which normally grow on a hygromycin plate, harvesting the seedlings, and identifying positive seedlings.
The zinc-cadmium resistant gene derived from tuber melanosporum of the inventionTmZRC1S belongs to CDF family, and the CDF family gene from tuber melanosporum is firstly transformed, and then the zinc-cadmium resistance of the plant is researched after the gene is transformed in Arabidopsis, and the zinc-cadmium enrichment capacity of the gene is verified.
Constructing a recombinant expression vector and transferring the recombinant expression vector into arabidopsis thaliana to obtain a transgenic arabidopsis thaliana plant with stronger zinc-cadmium resistance and zinc-cadmium enrichment capacity. After being treated by zinc and cadmium solution, the transgenic plant line grows better than the wild plant line, and shows that the transgenic plant line is reformed and synthesized by the inventionTmThe ZRC1S gene has the ability of improving the resistance of plants and enriching zinc and cadmium, and can be used for cultivating plants enriched with zinc and cadmium.
Will be described in the inventionTmZRC1S gene is transferred into arabidopsis thaliana, selfing is carried out for homozygosis for 2 generations to obtain homozygosis transformant, seed is collected and sowed, seedlings are irrigated by zinc and cadmium solution, growth of contrast wild arabidopsis thaliana is greatly influenced after 10 days of treatment, and the invention containsTmThe transgenic arabidopsis plant with the ZRC1S gene grows well, and leaves are mostly green, which shows that the invention has the advantages of good growth rate, high yield and high yieldTmThe ZRC1S gene can improve the zinc-cadmium resistance of Arabidopsis.
The harvested homozygous 2-generation seeds and wild arabidopsis seeds are sterilized and then sown on an MS solid culture medium, water culture is carried out after 10-14 days, 1/2 Hoagland nutrient solution is used for culture (the nutrient solution is replaced every 3 days), zinc and cadmium solution treatment is carried out on the seedlings, then the zinc and cadmium contents of overground parts and roots of wild plants and transgenic plants are measured, the zinc and cadmium contents in the transgenic plants are obviously higher than those in the wild plants, particularly, the zinc content of the overground parts is 3.74 times that in the wild plants, and the cadmium content is 3.36 times that in the wild plants, so that the method for detecting the zinc and cadmium contents of the wild plants in the wild plants is shown to be the inventionTmThe ZRC1S gene can improve the zinc and cadmium enriching ability of Arabidopsis.
The invention has at least the following beneficial effects:
the invention synthesizes a zinc-cadmium resistant gene from tuber melanosporum by an artificial synthesis method according to plant preferred codonsTmZRC1S, and successfully transformed into arabidopsis thaliana to be efficiently expressedObtaining transgenic arabidopsis; is turned intoTmThe Arabidopsis strain with the ZRC1S gene has obvious difference with the wild type strain in zinc and cadmium resistance, and the transgenic strain can tolerate 25mM ZnSO4Solution or 5mM CdCl2Continuously irrigating the solution for three times; at 250 μ M ZnSO4Or 50. mu.M CdCl2After three days of treatment, the zinc-cadmium content in the transgenic lines is significantly higher than that of the wild type lines, particularly the overground part zinc content is 3.74 times that of the wild type lines, and the cadmium content is 3.36 times that of the wild type lines.
Drawings
FIG. 1 shows a schematic diagram of the present invention includingTmConstruction schematic diagram of recombinant expression vector BH440 of ZRC1S gene
FIG. 2 is the PCR electrophoretogram of the seedling stage of transgenic Arabidopsis in example 4 of the present invention, wherein WT is a wild type plant, Tm3, Tm11, Tm12 are different strains of transgenic Arabidopsis;
FIG. 3 is a view of a rotary drumTmZRC1S gene Arabidopsis plants treated with zinc and cadmium solution for 10 days, wherein WT is wild type plant, Tm3, Tm11 and Tm12 are different strains of transgenic Arabidopsis.
Detailed Description
The invention is further described with reference to the drawings and the specific embodiments in the following description.
The test methods used in the examples are, unless otherwise specified, all conventional molecular biological methods; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 Gene Synthesis method for synthesizing Zinc-cadmium resistant Gene derived from Tuber melanosporumTmZRC1S
According to the origin of Tuber melanosporum (Tuber melanosporum) On the basis of keeping the amino acid Sequence of the predicted zinc transport protein gene Sequence (GSTUMT 00007113001, NCBI Sequence ID: XM _ 002839168.1), continuous extension PCR (Nucleic Acids Research, 2004, 32, e 98) is adopted to artificially synthesize the zinc-cadmium resistant gene according to plant preferred codonsTmZRC 1S. Design 44 pairs of primers forTmThe artificial synthesis of the ZRC1S gene comprises the following design primers:
TmZRC1S-p1
GGATCCATGGCATTCTCTCGTTCTGCACGTATCATCACTCTGCTGGTCATTGACTCTCTG
TmZRC1S-p2
AGTGGACAGAGTAACCAACGATGATCTCCAACAGGAAGAACAGAGAGTCAATGACCAGCA
TmZRC1S-p3
TTCTTCCTGTTGGAGATCATCGTTGGTTACTCTGTCCACTCTCTTGCACTTGTTGCTGAC
TmZRC1S-p4
CAACCAGCAGGGAGAAGACGTCGTTCAGCATGTGGAAGGAGTCAGCAACAAGTGCAAGAG
TmZRC1S-p5
CGTTGGTTACTCTGTCCACTCTCTTGCACTTGTTGCTGACTCCTTCCACATGCTGAACGA
TmZRC1S-p6
CAGCTTGATTGCCCACAATGCAACCAGCAGGGAGAAGACGTCGTTCAGCATGTGGAAGGA
TmZRC1S-p7
CGTCTTCTCCCTGCTGGTTGCATTGTGGGCAATCAAGCTGGCACGTCAGAAGTCCACCTC
TmZRC1S-p8
GCACGTCAGAAGTCCACCTCCTCCTACACCTACGGTTGGCAACGTGCTGAAGTCCTTGGT
TmZRC1S-p9
CTCCTACACCTACGGTTGGCAACGTGCTGAAGTCCTTGGTGCACTGATCAACGGTGTCTT
TmZRC1S-p10
GATTGCTTCCAGGAAGATGGACAGACACAGTGCAAGCAAGAAGACACCGTTGATCAGTGC
TmZRC1S-p11
CTTGCTTGCACTGTGTCTGTCCATCTTCCTGGAAGCAATCCAGCGTTTCTTCGAACCACA
TmZRC1S-p12
AGAACCAACACCAAGGACCAACCAAGGAGTAGAGATTTCCTGTGGTTCGAAGAAACGCTG
TmZRC1S-p13
GGAAATCTCTACTCCTTGGTTGGTCCTTGGTGTTGGTTCTGCTGGTCTTGCATCCAACAT
TmZRC1S-p14
ATGAGAGTGACCATGATCATGGAACAAGAACAAACCCAGGATGTTGGATGCAAGACCAGC
TmZRC1S-p15
CCTGGGTTTGTTCTTGTTCCATGATCATGGTCACTCTCATGGTGGTAACTCCCATGAACA
TmZRC1S-p16
AGCAGCAGACTCCTCACCAACAAGAGAGGACTCAAGATCGTGTTCATGGGAGTTACCACC
TmZRC1S-p17
CGATCTTGAGTCCTCTCTTGTTGGTGAGGAGTCTGCTGCTGCTGGTCATGAGCACCACAA
TmZRC1S-p18
AGCAGGATCATGGATGTGACCACGCAGACCACGAGTGTGTTTGTGGTGCTCATGACCAGC
TmZRC1S-p19
ACACACTCGTGGTCTGCGTGGTCACATCCATGATCCTGCTGAAGATGATCGTGGTGACAT
TmZRC1S-p20
TGCACGTTTACGACCAACGATGTCAGGCAGGATGTCGTCGATGTCACCACGATCATCTTC
TmZRC1-p21
CGACGACATCCTGCCTGACATCGTTGGTCGTAAACGTGCATACTCTCGTTCCTGCCATCA
TmZRC1S-p22
AGAGGACTTGTTCTCCTTTGGCTTTGCATGGTTGTGGTTCTGATGGCAGGAACGAGAGTA
TmZRC1S-p23
GAACCACAACCATGCAAAGCCAAAGGAGAACAAGTCCTCTCACTCCCATCAGAACCTGAA
TmZRC1S-p24
CAGTGCATCACCAAGAACATGCAGGAAGACACCACGCATGTTCAGGTTCTGATGGGAGTG
TmZRC1S-p25
CATGCGTGGTGTCTTCCTGCATGTTCTTGGTGATGCACTGGGTAACGTTGGTGTCATGTC
TmZRC1S-p26
CCAGATGGTCTCAGGCAGAAGAAGCAGAGCACCAGCAACGGACATGACACCAACGTTACC
TmZRC1S-p27
CGTTGCTGGTGCTCTGCTTCTTCTGCCTGAGACCATCTGGTGGCGTCATCTGCTGGACCC
TmZRC1S-p28
AGAGGAGAAGATGATCATGGTGATCAGCAGAGAGATGGATGGGTCCAGCAGATGACGCCA
TmZRC1S-p29
ATCCATCTCTCTGCTGATCACCATGATCATCTTCTCCTCTGCACTGCCACTGTGCAAGTC
TmZRC1S-p30
GATACCCTTAGGAACACCTTGAAGCAAGATTTTGGATGCAGACTTGCACAGTGGCAGTGC
TmZRC1S-p31
TGCATCCAAAATCTTGCTTCAAGGTGTTCCTAAGGGTATCTCTCTGGAGGAAGTCAAAGA
TmZRC1S-p32
TTCGTGGACGGATTCGACACCTTGGATGGATGCGATGTCTTCTTTGACTTCCTCCAGAGA
TmZRC1S-p33
AGACATCGCATCCATCCAAGGTGTCGAATCCGTCCACGAACTGCACATCTGGCAACTGTC
TmZRC1S-p34
TGCGATCTGGATGTGCAGAGATGCGATCATCTTGACGTCGGACAGTTGCCAGATGTGCAG
TmZRC1S-p35
CGACGTCAAGATGATCGCATCTCTGCACATCCAGATCGCATTCGATCCTGAGTGCGAAGG
TmZRC1S-p36
GGTACGGACTGCGTTTGCAAGTTGCATGTAACGACCACCACCTTCGCACTCAGGATCGAA
TmZRC1S-p37
TGGTGGTCGTTACATGCAACTTGCAAACGCAGTCCGTACCTGCCTTCATGCATACGGTAT
TmZRC1S-p38
CTCTTCTTTGGTGTACTCAGGCTGAATGGTGGAGGAGTGGATACCGTATGCATGAAGGCA
TmZRC1S-p39
CCACTCCTCCACCATTCAGCCTGAGTACACCAAAGAAGAGGAAGCTGCTGCATCTCGTAC
TmZRC1S-p40
ACATGCAGCCTCTTCTTCAGTGCCACCACGAGTAGAGCCAGTACGAGATGCAGCAGCTTC
TmZRC1S-p41
TGGCTCTACTCGTGGTGGCACTGAAGAAGAGGCTGCATGTCTGTTGGAATGTGGTGATGG
TmZRC1S-p42
AGCACCAGAACCAGGTGCACAGCACTTACCGTCAACGCAACCATCACCACATTCCAACAG
TmZRC1S-p43
TTGCGTTGACGGTAAGTGCTGTGCACCTGGTTCTGGTGCTCCTGGTGATGAAGTCATCGG
TmZRC1S-p44
GAGCTCTTAACCGATGACTTCATCACCAGGAGCACCAGAACCAGGTGCACAGCACTTACC
performing zinc transport protein gene amplification by using PCR, performing PCR amplification on the zinc transport protein gene in a 100 microliter reaction system,TmZRC1S-p2 toTmThe total amount of 44 inner primers, ZRC1S-p43 was 2ng, and the outer primers were addedTmZRC1S-p1 andTmthe addition amount of the ZRC1S-p44 is 30ng, and the amplification conditions are as follows: preheating at 94 deg.C for 1 min; 94 ℃ 30s, 50 ℃ 30s, 72 ℃ 2min, using KOD FXtaq enzyme as Taq DNA polymerase (Toyobo Co., Japan) for 25 cycles.
After the PCR was completed, 1% agarose gel was recovered, and 10. mu.l of agarose gel was directly connected to a T/A cloning vector (Dalianbao Bio Inc.). Ligation was performed overnight at 4 ℃ and efficiently transformed into DH 5. alpha. competence. Obtaining positive clone, and making sequence determination and BLAST comparison to make the gene synthesized by gene synthesis method completely identical with amino acid sequence coded by GSTUMT00007113001 gene in tuber melanosporum so as to obtain the invented productTmThe nucleotide sequence of the ZRC1S gene is shown as SEQ ID NO1, and the coded amino acid sequence is shown as SEQ ID NO 2.
Example 2 containsTmConstruction of ZRC1S Gene plant expression vector
Carrying out double enzyme digestion by using BamHI and SacI respectively, recovering DNA fragments, and carrying out T4DNA ligaseTmThe ZRC1 enzyme gene is connected with pCAMBIA-1301 carrier containing double 35S promoter, enzyme digestion identification and sequence determination are carried out to obtain the product containing ZRC1 enzyme geneTmRecombinant plasmid BH440 of ZRC1S gene, as shown in FIG. 1. The expression vector also contains a GUS reporter gene and an intron kanamycin resistance marker gene.
EXAMPLE 3 Agrobacterium culture and electrotransformation
The agrobacterium strain is agrobacterium tumefaciens LBA4404 strain. The recombinant expression vector BH440 obtained in example 2 was introduced into Agrobacterium LBA4404 by electroporation. Selecting single bacteria, culturing in 25ml YEB culture medium (50 mg/l rifampicin) overnight, transferring 5ml bacteria solution into 100ml YEB culture medium (50 mg/l rifampicin), culturing to OD600=0.7-0.8, standing on ice for 10min, centrifuging at 5000rpm for 10min, collecting bacteria at 4 ℃, adding 100ml sterile double distilled water, and washing twice. The cells were suspended in 4ml of 10% glycerol and transferred to a 50ml centrifuge tube. Centrifuge at 5500rpm for 10min, 4 ℃. The cells were collected, 500. mu.l of 10% glycerol was added to suspend the cells, and the cells were transferred to a 1.5ml centrifuge tube to obtain Agrobacterium competent cells. Taking 70 mul of competent cells, adding 1 mul of recombinant expression vector BH440, uniformly mixing with a decapitated yellow gun head, and transferring into a 0.1cm electric shock cup. Electric shock parameters: 200 Ω, 1.7KV, 2.5F, and 800. mu.l SOC culture medium was added immediately after the shock. After 1 hour of incubation, 200. mu.l of the coated plate was incubated overnight at 28 ℃ to select a strain into which the recombinant expression vector BH440 was successfully introduced.
Example 4 Arabidopsis floral Dipper transformation
The selected single colony of Agrobacterium strain was inoculated in 5ml LB medium containing the corresponding antibiotic and cultured at 28 ℃ for 2 days. 5ml of the cell suspension was transferred to 500ml of liquid LB medium and cultured at 28 ℃ for 16 to 24 hours (OD =1.5 to 2.0), and the cells were collected by centrifugation at room temperature and centrifuged at 5000rpm for 10 minutes. Suspended with an equal volume of 5% fresh sucrose solution. Adding 0.02% Silwet-77, mixing uniformly, and transferring into a beaker to obtain the transformed bacterial liquid. Each strain was transformed with 300ml, transferred to 2-3 bowls. Another transformation was performed 1 time after 7 days. The seeds of Arabidopsis thaliana (Columbia type) were inverted and immersed in the transformant bacterial liquid for 10 seconds, and both the rosette and the inflorescence were infected. And (3) drying the transformed plant bacteria liquid for 3-5 seconds after infection. The transformed plants are circled by preservative film and kept flat for 16-24 hours. And (5) uncovering the preservative film, keeping a certain humidity, and harvesting the seeds after the seeds grow for 1 month. Screening transformed plants by using 50 mu g/ml hygromycin, transplanting seedlings which normally grow on a hygromycin plate, harvesting the seedlings, and identifying positive seedlings.
Total RNA was extracted from Arabidopsis leaves grown up to 3 weeks, and the total RNA was reverse transcribed into cDNA, and PCR detection was performed on the transformed plants using R45425- (TCCGACGTCAAGATGATCGCA) and R45426- (ACCGATGACTTCATCACCAGG) as primers (see molecular cloning, A laboratory Manual, Sambrook and Russell, 2001) under the following conditions: preheating at 94 deg.C for 1 min; 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 4 min. The total of 25 cycles, with the arabidopsis thaliana Actin gene as an internal reference, the results are shown in fig. 2. From the figure, it can be seen that no target band was detected in the wild type plants, but no target band was detected in the transgenic plants, which proved that these lines were all positive seedlings, indicating that the target gene had been successfully introduced into Arabidopsis and expressed at the transcriptional level.
Example 5 rotationTmZinc-cadmium resistance test of ZRC1S gene Arabidopsis thaliana
The arabidopsis transgenic plant successfully transferred with the target gene is subjected to selfing homozygosis for 2 generations to obtain a homozygosis transformation plant, seeds are collected and sowed in soil, and meanwhile, a wild plant is cultured to serve as a control experiment. Seedlings grown at 22 ℃ for about 3 weeks were irrigated every three days with 50ml of 25mM ZnSO4Solution or 5mM CdCl2Solution, growth of Arabidopsis thaliana was observed 10 days after treatment. The specific experimental results are as follows: after passing through 25mM ZnSO4Solution or 5mM CdCl2After the solution treatment, the leaves of the control wild type arabidopsis thaliana are almost yellow, while the arabidopsis thaliana transgenic plant containing the target gene of the invention grows well, and the leaves are mostly green, as shown in fig. 3. Illustrating the inventionTmThe ZRC1S gene can improve the zinc-cadmium resistance of plants.
Example 6 transferTmZinc and cadmium enrichment of ZRC1S gene Arabidopsis thaliana
Sterilizing harvested homozygous 2-generation seeds and wild Arabidopsis seeds, sowing the seeds on an MS solid culture medium, removing water culture after 10-14 days, culturing the seeds in 1/2 Hoagland nutrient solution (replacing the nutrient solution every 3 days), and adding heavy metals (250 mu M ZnSO) with different concentrations4Or 50. mu.M CdCl2) Culturing for 3 days, harvesting the plants, washing impurities with deionized water, and then washing with 5mM CaCl2Soaking for 15min, washing with water for 3 times, drying surface water with absorbent paper, placing into envelope respectively for aerial part and root part, placing into oven, deactivating enzyme at 105 deg.C for 15min, and oven drying at 65 deg.C to constant weight. Each treatment was repeated 3 times without any heavy metal added as a control. Determining the zinc and cadmium content of aerial parts and roots of wild type and transgenic plants by ICP-MS (Agilent 7700 ICP-MS, USA), e.g.Shown in table 1.
TABLE 1 treatment of wild type and transgenic plants with heavy metal ionsTmZinc-cadmium content of ZRC1S gene Arabidopsis thaliana
The experimental results show thatTmThe zinc content of the overground part of the ZRC1S gene Arabidopsis plant is obviously higher than that of a wild plant and is 3.74 times of that of the wild plant; the zinc content of roots has the same trend, and the zinc content of the roots of the transformed strains is obviously increased by 80 percent compared with that of wild plants. Rotating shaftTmThe cadmium content of the overground part of the ZRC1S gene Arabidopsis plant is 3.36 times of that of a wild plant, and the cadmium content of the root of a transformation strain is increased by 25 percent relative to the WT. Illustrating the inventionTmThe ZRC1S gene can improve the zinc and cadmium enriching ability of plants.
Sequence listing
<110> Shanghai city academy of agricultural sciences
<120> zinc-cadmium-resistant gene TmZRC1S derived from tuber melanosporum and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1278
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggcattct ctcgttctgc acgtatcatc actctgctgg tcattgactc tctgttcttc 60
ctgttggaga tcatcgttgg ttactctgtc cactctcttg cacttgttgc tgactccttc 120
cacatgctga acgacgtctt ctccctgctg gttgcattgt gggcaatcaa gctggcacgt 180
cagaagtcca cctcctccta cacctacggt tggcaacgtg ctgaagtcct tggtgcactg 240
atcaacggtg tcttcttgct tgcactgtgt ctgtccatct tcctggaagc aatccagcgt 300
ttcttcgaac cacaggaaat ctctactcct tggttggtcc ttggtgttgg ttctgctggt 360
cttgcatcca acatcctggg tttgttcttg ttccatgatc atggtcactc tcatggtggt 420
aactcccatg aacacgatct tgagtcctct cttgttggtg aggagtctgc tgctgctggt 480
catgagcacc acaaacacac tcgtggtctg cgtggtcaca tccatgatcc tgctgaagat 540
gatcgtggtg acatcgacga catcctgcct gacatcgttg gtcgtaaacg tgcatactct 600
cgttcctgcc atcagaacca caaccatgca aagccaaagg agaacaagtc ctctcactcc 660
catcagaacc tgaacatgcg tggtgtcttc ctgcatgttc ttggtgatgc actgggtaac 720
gttggtgtca tgtccgttgc tggtgctctg cttcttctgc ctgagaccat ctggtggcgt 780
catctgctgg acccatccat ctctctgctg atcaccatga tcatcttctc ctctgcactg 840
ccactgtgca agtctgcatc caaaatcttg cttcaaggtg ttcctaaggg tatctctctg 900
gaggaagtca aagaagacat cgcatccatc caaggtgtcg aatccgtcca cgaactgcac 960
atctggcaac tgtccgacgt caagatgatc gcatctctgc acatccagat cgcattcgat 1020
cctgagtgcg aaggtggtgg tcgttacatg caacttgcaa acgcagtccg tacctgcctt 1080
catgcatacg gtatccactc ctccaccatt cagcctgagt acaccaaaga agaggaagct 1140
gctgcatctc gtactggctc tactcgtggt ggcactgaag aagaggctgc atgtctgttg 1200
gaatgtggtg atggttgcgt tgacggtaag tgctgtgcac ctggttctgg tgctcctggt 1260
gatgaagtca tcggttaa 1278
<210> 2
<211> 425
<212> PRT
<213> Tuber melanosporum (Tuber melanosporum)
<400> 2
Met Ala Phe Ser Arg Ser Ala Arg Ile Ile Thr Leu Leu Val Ile Asp
1 5 10 15
Ser Leu Phe Phe Leu Leu Glu Ile Ile Val Gly Tyr Ser Val His Ser
20 25 30
Leu Ala Leu Val Ala Asp Ser Phe His Met Leu Asn Asp Val Phe Ser
35 40 45
Leu Leu Val Ala Leu Trp Ala Ile Lys Leu Ala Arg Gln Lys Ser Thr
50 55 60
Ser Ser Tyr Thr Tyr Gly Trp Gln Arg Ala Glu Val Leu Gly Ala Leu
65 70 75 80
Ile Asn Gly Val Phe Leu Leu Ala Leu Cys Leu Ser Ile Phe Leu Glu
85 90 95
Ala Ile Gln Arg Phe Phe Glu Pro Gln Glu Ile Ser Thr Pro Trp Leu
100 105 110
Val Leu Gly Val Gly Ser Ala Gly Leu Ala Ser Asn Ile Leu Gly Leu
115 120 125
Phe Leu Phe His Asp His Gly His Ser His Gly Gly Asn Ser His Glu
130 135 140
His Asp Leu Glu Ser Ser Leu Val Gly Glu Glu Ser Ala Ala Ala Gly
145 150 155 160
His Glu His His Lys His Thr Arg Gly Leu Arg Gly His Ile His Asp
165 170 175
Pro Ala Glu Asp Asp Arg Gly Asp Ile Asp Asp Ile Leu Pro Asp Ile
180 185 190
Val Gly Arg Lys Arg Ala Tyr Ser Arg Ser Cys His Gln Asn His Asn
195 200 205
His Ala Lys Pro Lys Glu Asn Lys Ser Ser His Ser His Gln Asn Leu
210 215 220
Asn Met Arg Gly Val Phe Leu His Val Leu Gly Asp Ala Leu Gly Asn
225 230 235 240
Val Gly Val Met Ser Val Ala Gly Ala Leu Leu Leu Leu Pro Glu Thr
245 250 255
Ile Trp Trp Arg His Leu Leu Asp Pro Ser Ile Ser Leu Leu Ile Thr
260 265 270
Met Ile Ile Phe Ser Ser Ala Leu Pro Leu Cys Lys Ser Ala Ser Lys
275 280 285
Ile Leu Leu Gln Gly Val Pro Lys Gly Ile Ser Leu Glu Glu Val Lys
290 295 300
Glu Asp Ile Ala Ser Ile Gln Gly Val Glu Ser Val His Glu Leu His
305 310 315 320
Ile Trp Gln Leu Ser Asp Val Lys Met Ile Ala Ser Leu His Ile Gln
325 330 335
Ile Ala Phe Asp Pro Glu Cys Glu Gly Gly Gly Arg Tyr Met Gln Leu
340 345 350
Ala Asn Ala Val Arg Thr Cys Leu His Ala Tyr Gly Ile His Ser Ser
355 360 365
Thr Ile Gln Pro Glu Tyr Thr Lys Glu Glu Glu Ala Ala Ala Ser Arg
370 375 380
Thr Gly Ser Thr Arg Gly Gly Thr Glu Glu Glu Ala Ala Cys Leu Leu
385 390 395 400
Glu Cys Gly Asp Gly Cys Val Asp Gly Lys Cys Cys Ala Pro Gly Ser
405 410 415
Gly Ala Pro Gly Asp Glu Val Ile Gly
420 425