CN116004654B - Application of willow transporter SsIRT9 in cadmium contaminated soil restoration genetic engineering - Google Patents
Application of willow transporter SsIRT9 in cadmium contaminated soil restoration genetic engineering Download PDFInfo
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Abstract
The invention discloses application of a willow transporter SsIRT9 in cadmium contaminated soil restoration genetic engineering. The salix psammophila transporter gene SsIRT9 has cadmium transporting activity, and after the gene is overexpressed in dicotyledon tobacco by using agrobacterium-mediated genetic transformation technology, the cadmium concentration in transgenic plant tissues is obviously increased in a cadmium exposure environment, so that the absorption and enrichment of cadmium are enhanced, and the salix psammophila transporter SsIRT9 has important potential in the remediation of cadmium contaminated soil.
Description
Technical Field
The invention relates to a genetic engineering technology, in particular to application of a willow transporter gene SsIRT9 in cadmium polluted soil restoration genetic engineering.
Background
Soil cadmium pollution is a serious environmental problem facing the world, and seriously endangers soil quality and human health. The phytoremediation is a process of removing cadmium in soil by means of plant fixation extraction and the like, is environment-friendly, and can realize in-situ remediation and increase carbon sink. Willow is woody plant capable of efficiently absorbing cadmium, and capable of effectively removing 30-50% of cadmium in soil, and is widely applied to cadmium polluted soil restoration engineering at present, but key genes and functions thereof in the cadmium absorption process of the willow are not clear, which becomes a bottleneck to be broken through in order to further improve the repair efficiency of the willow. Therefore, the biological functions of the critical genes responsible for cadmium absorption of the willow are excavated and analyzed, and important gene resources can be provided for improving the repair capability of the willow by utilizing genetic engineering.
Disclosure of Invention
The invention aims to: the invention aims to provide application of a willow transporter gene SsITR9 in improving cadmium enrichment capacity of plant soil.
Application of a willow transporter gene SsIRT9 in cadmium contaminated soil restoration engineering, wherein the accession number of the willow transporter genome sequence in NCBI database is KAG5240077, the accession number of the coded protein sequence in NCBI database is KAG5240077, and the CDS sequence of a gene KAG5240077 (SsIRT 9) is SEQ ID NO 15.
And (3) carrying out expression pattern analysis on the willow SsIRT9, wherein the willow SsEAF1 is taken as an internal reference gene, and the primer sequence is as follows:
SsEF1a-F：
SsEF1a-R：
SsIRT9-F：
SsIRT9-R：
and (3) performing heterologous function verification on the salix SsIRT9 yeast, performing restriction enzyme digestion site analysis on the coding region sequence of the SsIRT9, and designing PCR amplification primers containing two enzyme digestion sites of BamHI and EcoRI as follows:
PF：
PR：
SsIRT9 subcellular localization analysis, restriction enzyme cutting site analysis is carried out on the coding region of the SsIRT9 gene, and PCR amplification primers containing EcoRI and BamHI enzyme cutting sites are designed:
pSAT6-EGFP-C1
GFP-SsIRT9-F：
GFP-SsIRT9-R：
pSAT6-EGFP-N1
SsIRT9-GFP-F：
SsIRT9-GFP-R：
designing homologous recombination primers according to pRCS2-2288 vector sequences by taking an intermediate expression vector plasmid containing SsIRT9 gene fragments as a template:
pF：
pR：
SsIRT9 over-expressed transgenic tobacco, PCR amplification primers containing two enzyme cutting sites of BamHI and PstI are designed:
PF：
PR：
SsIRT9 transgenic plants include OxIRT9-1, oxIRT9-2, oxIRT9-3 and OxIRT9-4.
The transgenic tobacco for over-expressing the willow transporter gene SsIRT9 is constructed through genetic engineering, so that the capacity of the transgenic plant for enriching the heavy metal cadmium in soil is improved.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the willow transporter gene SsIRT9 has cadmium transporting activity, and after the 35S strong promoter starts the gene to be expressed excessively, the cadmium concentration in transgenic plant tissues is increased, the enrichment of cadmium is enhanced, and the method has important significance for cadmium polluted soil restoration engineering.
Drawings
FIG. 1 analysis of the expression pattern of the willow SsIRT9 gene;
FIG. 2 shows that the willow SsiRT9 has cadmium transporting activity;
FIG. 3 subcellular localization analysis of the willow SsiRT 9;
FIG. 4 identification of the willow SsIRT9 transgenic line;
FIG. 5 shows that the SsIRT9 transgenic plant has enhanced cadmium uptake and enrichment capacity in a cadmium exposure environment, (a) the transgenic plant accumulates cadmium in an aqueous cadmium treatment; (b-c) accumulation of cadmium by transgenic plants exposed to earth-cultivated cadmium;
WT and Trans (-) referred in the above figures represent transgenic tobacco wild type and transgenic negative seedlings, oxIRT9-1, oxIRT9-2, oxIRT9-3, oxIRT9-4 represent over-expressed transgenic materials OxIRT9-1, oxIRT9-2, oxIRT9-3, oxIRT9-4 of which the 4 35S promoters activate OsIRT9, respectively.
Detailed Description
EXAMPLE 1 willow SsiRT9 expression Pattern analysis
Willow (dustpan willow Salix suchowensis) cuttings with the length of 12em and the diameter of 0.8em are selected, fixed type air-filled water culture is adopted, after the cuttings root, 1/4Hoagland nutrient solution is used for culturing willow cutting seedlings, fresh root systems, leaves and other parts of the willow are collected, total RNA of all parts is extracted by a Trizol method, prime script RT kit is used for reverse transcription to obtain cDNA, SYBR Premix Ex Taq TM II kit is used for quantitatively amplifying genes, real-Time PCR is used for detecting SsIRT9 gene expression, and dustpan willow SsEAF1 is used as an internal reference gene. Designing specific quantitative primers according to the gene sequence, wherein the primer sequences are as follows:
SsEF1a-F：
SsEF1a-R：
SsIRT9-F：
SsIRT9-R：
according to Ct values of internal reference genes SsEAF1 and SsIRT9 and according to 2- ΔCt And calculating the gene expression quantity. As a result, ssiRT9 was found to be expressed at a higher level in the root system (FIG. 1).
Example 2 Salix SsiRT9 Yeast heterologous functional verification
Restriction enzyme cutting site analysis is carried out on SsIRT9 coding region sequence by DNAMAN8.0, and PCR amplification primers containing two enzyme cutting sites of BamH I and EcoR I are designed as follows:
PF：
PR：
the cDNA obtained by reverse transcription of RNA of the root system of the dustpan willow is used as a template, ssIRT9 complete coding region containing enzyme cutting sites is obtained by PCR amplification of high-fidelity enzyme 2X Phanta Max Master Mix (Dye Plus) (Vazyme, china), after double enzyme cutting, the coding region is connected to a yeast expression vector pYES2 (Zhang L, wu J, tang Z, huang XY, wang X, salt DE, zhao FJ.variation in the BrHMA3 coding region controls natural variation in cadmium accumulation in Brassica rapa vegetables J Exp Bot.9 Oct 24;70 (20): 5865-20178.doi:10.1093/jxb/erz310.PMID 313675770; PMCID: PMC 687176) by T4 ligase, and the recombinant plasmid is obtained after sequencing verification and preservation for standby. Competent cells were prepared according to the instructions of Yeastmaker Yeast Transformation Kit (TaKaRa, japan) and the empty pYES2 and recombinant plasmids were transferred to the Saccharomyces cerevisiae wild-type strain BY4741 (Yue X, song J, fang B, wang L, zou J, su N, cui J.BcNRAMP1 promotes the absorption)of cadmium and manganese in Arabidopsis. Chemosphere.2021 Nov;283: 131113.). Selecting single colony of yeast, culturing in liquid SD-Ura culture medium to logarithmic phase, centrifuging to remove supernatant, cleaning with sterilized water for 3-5 times, and regulating OD 600 To 1.0, according to 1:10 proportion gradually dilute 4 concentration gradients, carry out dotting experiment in the culture medium flat plate containing different cadmium concentrations, cultivate for 3-4 days at 30 ℃, observe the growth condition of yeast, record by photo. As a result, it was revealed that the yeast strain transformed with empty pYES2 was still able to grow with an increase in the cadmium concentration in the medium, while the yeast strain expressing SsIRT9 contained 20. Mu.M, 30. Mu.M, 50. Mu.M CdCl 2 The growth in the medium of (a) was inhibited to varying degrees, indicating that SsIRT9 has cadmium transporting activity in yeast cells (fig. 2).
EXAMPLE 3 SsIRT9 subcellular localization assay
Restriction enzyme cutting site analysis is carried out on SsIRT9 gene coding region by DNAMAN software, and PCR amplification primers containing EcoRI and BamHI enzyme cutting sites are designed:
pSAT6-EGFP-C1
GFP-SsIRT9-F：
GFP-SsIRT9-R：
pSAT6-EGFP-N1
SsIRT9-GFP-F：
SsIRT9-GFP-R：
the cDNA obtained by reverse transcription of RNA of the root system of the willow is used as a template, PCR amplification is carried out by using high-fidelity enzyme 2X Phanta Max Master Mix (Dye Plus) (Vazyme, china), PCR products are cloned to pCE2 TA/Blunt-Zero cloning Vector (in the family of the engine, china) after gel recovery and purification, plasmids with correct sequence are cut by EcoRI and BamHI, and the plasmids are connected into intermediate vectors pSAT6-EGFP-C1 and pSAT6-EGFP-N1 (commonly star. Rice phosphate transporter OsPHT1; functional research [ D ] of Nanjing agricultural university, 2019.DOI:10.27244/D. Cnki.gnjnu.2019.000134) through T4 ligase, so that the fusion GFP recombinant plasmid of the N end and the C end of the target gene is obtained. Designing homologous recombination primers according to pRCS2-2288 vector sequences by taking an intermediate expression vector plasmid containing SsIRT9 gene fragments as a template:
pF：
pR：
the product was recovered after PCR amplification and ligated with expression vector pRCS2 (Changming. Rice phosphate transporter OsPHT1; functional research [ D ]. Nanjing university of agriculture, 2019.DOI:10.27244/D. Cnki. Gnjnu. 2019.000134.) using ClonExpress II One Step Cloning Kit (Vazyme, china), while constructing marker protein fusion m-herry expression vector pm-rbCD3-1008 (Nelson BK, cai X, nebenf u hr A.A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plant J.2007Sep;51 (6): 1126-36.) positioned at the cell membrane site, and the obtained recombinant plasmid was stored after sequencing verification was correct.
The empty expression vector, to which the gene of interest was ligated, and the pm-rbCD3-1008 plasmid were transferred into Agrobacterium GV3101 (Optimaceae, china), and Agrobacterium containing the functional plasmid was selected for resistance with Spec, str and Rif. Inoculating agrobacterium monoclone into LB liquid culture medium containing antibiotics, shake culturing at 28deg.C to make bacterial liquid OD600 reach 0.8-1.0, centrifuging to collect bacterial body, suspending with 1mL of indication medium (10mM MES,10mM MgCl2 and 200 mu Macetosporine), diluting OD600 to 0.1-0.2, injecting into tobacco leaf lower epidermis, dark culturing for 2-3 days, observing fluorescent signal with laser confocal microscope, GFP excitation light wavelength is 578 nm, RFP excitation light is 561 nm. As a result, no fluorescence signal was observed after the gene N-terminal fusion GFP infects tobacco, and no fluorescence signal was detected in tobacco epidermal cells after the gene C-terminal fusion GFP infects tobacco, indicating that the signal peptide of SsIRT9 is at the N-terminal, and that the SsIRT9 green fluorescence signal is located on the cytoplasmic membrane, coinciding with the red fluorescence signal emitted by the plasma membrane Marker, indicating that SsIRT9 is located on the plasma membrane, and is a membrane transporter mediating cadmium transmembrane transport in willows (FIG. 3).
EXAMPLE 4 creation and identification of SsIRT9 overexpressing transgenic plants
1. Creation of SsIRT9 transgenic plants
SsIRT9 overexpressing transgenic tobacco: PCR amplification primers containing two cleavage sites of BamHI and PstI were designed:
PF：
PR：
the cDNA obtained by reverse transcription of the RNA of the willow root system of dustpan is used as a template, ssIRT9 complete coding region containing two enzyme cutting sites of BamHI and PstI is obtained by PCR amplification of high-fidelity enzyme 2X Phanta Max Master Mix (Dye Plus) (Vazyme, china), after double enzyme cutting, the coding region is connected to an expression vector pCAMBIA1305 (commonly used star. Rice phosphate transporter OsPHT1; functional research [ D ] of Nanjing agricultural university, 2019.DOI:10.27244/D. Cnki. Gnjnu.000134.) by using T4 ligase (TransGen, china), a recombinant plasmid 2019 is obtained, and the SsIRT9 over-expression transgenic tobacco material is obtained by a transgenic technology and is transferred into agrobacterium tumefaciens EHA105 (Optimago, china).
2. Identification of SsIRT9 transgenic plants
And obtaining T0 generation transgenic tobacco positive seedlings by hygromycin screening and GUS staining. By qRT-PCR detection, 4 over-expressed lines were obtained, designated OxIRT9-1, oxIRT9-2, oxIRT9-3, oxIRT9-4 (FIG. 4).
Example 5 functional verification of uptake enrichment of cadmium by SsIRT9 transgenic plants
1. Cd absorption experiment of transgenic tobacco
To verify the cadmium uptake capacity of the overexpressing transgenic plants, the wild-type and transgenic plants were pre-cultured in 1/5 Hoagland nutrient solution for 14 days and transferred to fresh plants containing 0.2. Mu.M CdCl 2 Is treated in the treatment liquid for 7 days. The root system and the aerial parts were collected, baked to constant weight in an oven at 70 ℃, weighed and ground, and the Cd concentration in the tissue was measured by inductively coupled plasma mass spectrometry (ICP-OES). As a result, it was found that 0.2. Mu.M CdCl 2 Under treatment, there was no significant difference in the concentration of Cd in the roots of wild-type and overexpressing lines, while the concentrations of Cd in the aerial parts of the overexpressing lines OXIRT9-1, OXIRT9-2, OXIRT9-3, OXIRT9-4 were significantly higher than in the wild-type and transgenic negative seedlings, indicating that overexpressing willow IRT9 was able to significantly increase cadmium uptake by plants (FIG. 5 a).
2. Cd pollution soil culture experiment of transgenic tobacco
When wild type and transgenic expression plants were cultivated in soil to 4-5 leaves, cadmium contaminated soil (3 mg/kg CdCl) 2 ) In the pot experiments, the soil without cadmium treatment is used as a control. Watering every 2 days, keeping the water holding capacity of the soil field at about 60%, taking pictures after cadmium treatment for one month, recording plant phenotype, and using 20mmol/L EDTA-Na 2 Rinsing root system for 10 min, washing with deionized water for 3 times, collecting tobacco root and aerial parts, baking to constant weight in oven at 70deg.C, weighing, grinding, and measuring tissue Cd concentration with inductively coupled plasma mass spectrometer (ICP-OES). The results found that the root system and overground cadmium concentration of the over-expressed strain were significantly higher than that of the wild-type and negative seedlings in the cadmium-containing soil, further demonstrating that over-expressed willow IRT9 was able to significantly increase the uptake and accumulation of cadmium by plants (fig. 5b, c).
Claims (6)
1. Willow transporter geneSsIRT9Application in cadmium contaminated soil restoration genetic engineering, the accession number of the willow transporter genome sequence in NCBI database is KAG5240077, the accession number of the coded protein sequence in NCBI database is KAG5240077,SsIRT9the gene CDS sequence is SEQ ID NO 15.
2. The use according to claim 1, characterized in that: willow treeSsIRT9Expression pattern analysis with willowSsEF1aThe primer sequences of the reference genes are as follows:
SsEF1a-F ：GGTGGTATCGGAACTGT（SEQ ID NO1）
SsEF1a-R ：GTGCATCTCAACGGACT（SEQ ID NO2）
SsIRT9-F ：CTGAATTGGTTCGCCGAAGA（SEQ ID NO 3）
SsIRT9-R ：ATTGTGCCTGTGTGATGCAG（SEQ ID NO 4）。
3. the use according to claim 1, characterized in that: willow SsIRT9 yeast heterologous function verification onSsIRT9The coding region sequence was subjected to restriction enzyme site analysis and designed to containBamH I and is provided withEcoThe PCR amplification primers of the R I two enzyme cutting sites are as follows:
PF：GGATCCATGACTCATCTTCAAGCTTGC（SEQ ID NO 5）
PR：GAATTCTCAAGCCCATTTGACCAT（SEQ ID NO 6）。
4. the use according to claim 1, characterized in that: ssIRT9 subcellular localization analysis ofSsIRT9Restriction enzyme site analysis is carried out on a gene coding region, and the gene coding region is designed to containEcoRI, and RI systemBamPCR amplification primers for HI two cleavage sites:
pSAT6-EGFP-C1
GFP-SsIRT9-F ：GAATTCCATGACTCATCTTCAAGCTTGCT（SEQ ID NO 7）
GFP-SsIRT9-R ：GGATCCTCAAGCCCATTTGACCATG（SEQ ID NO 8）
pSAT6-EGFP-N1
SsIRT9-GFP-F ：GAATTCATGACTCATCTTCAAGCTTGCT（SEQ ID NO 9）
SsIRT9-GFP-R ：GGATCCGAGCCCATTTGACCATGAAA（SEQ ID NO 10）
to containSsIRT9Designing homologous recombination primers by taking intermediate expression vector plasmids of gene fragments as templates:
pF：5’-ACCACCCATAATACCCATAATAGCTGTTTGCCAACC-3’（SEQ ID NO 11）
pR：5’-CGATGGCAAACAGCTATTATGGGTATTATGGGTGCG-3’（SEQ ID NO 12）。
5. the use according to claim 1, characterized in that: ssIRT9 over-expressed transgenic tobacco designed to containBamHI andPsti PCR amplification primers of two enzyme cutting sites:
PF：GGATCCATGACTCATCTTCAAGCTTGC（SEQ ID NO 13）
PR：CTGCAGTCAAGCCCATTTGACCAT（SEQ ID NO 14）。
6. the use according to claim 1, characterized in that: over-expressed willow transporter genesSsIRT9Can improve the enrichment capability of plants to soil cadmium, wherein the plants are tobacco or willow.
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| CN106543275A (en) * | 2015-09-23 | 2017-03-29 | 中国科学院植物研究所 | A kind of albumen with cadmium binding ability and its encoding gene and application |
| CN109536510A (en) * | 2018-12-11 | 2019-03-29 | 天津师范大学 | A kind of dry land willow zinc-iron transporter gene SmZIP and the method that its expression is detected using fluorescence RT-PCR |
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| CN106543275A (en) * | 2015-09-23 | 2017-03-29 | 中国科学院植物研究所 | A kind of albumen with cadmium binding ability and its encoding gene and application |
| CN109536510A (en) * | 2018-12-11 | 2019-03-29 | 天津师范大学 | A kind of dry land willow zinc-iron transporter gene SmZIP and the method that its expression is detected using fluorescence RT-PCR |
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