Compositions for transporting phytochelatins
一种植物螯合肽、组合物的技术,应用在转运植物螯合肽的组合物领域,能够解决有待确定等问题
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Embodiment 1
[0073] Example 1: Sensitivity test of atabcc1 and atabcc2 double gene knockout mutants to arsenic, arsenic-containing herbicides or cadmium
[0074] The Arabidopsis ABCC gene was isolated from a total of 15 Arabidopsis homozygous knockout lines, and the mutants were grown in the presence of arsenic, arsenic-containing herbicides, and cadmium. Except for abcc2-2, abcc11-2 and abcc1 knockout genes kindly provided by Dr. Markus Klein (Frelet-Barrand A et al. (2008), Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2contributestodetoxification, vacuolarorganicaniontransportandchlorophylldegradation. PlantCellPhysiol49:557-569), Mutants were all obtained from the Salk Genome Analysis Laboratory (http: / / signal.salk.edu / ). The accession and knockout numbers of all 15 abcc mutants are listed in Table 1. Among other things, a new mutant allele, Salk_017431 of ABCC1, was used in this experiment, and its nomenclature was named abcc1-3 following the previously ...
Embodiment 2
[0079] Example 2: Examination of whether AtABCC1 and AtABCC2 improve arsenic resistance and accumulation in budding yeast in the presence of PCS1 (phytochelatin synthase 1)
[0080] To determine whether AtABCC1 and AtABCC2 are resistant to arsenic (As), the PCR products of yhl035c::HIS3-MX6, yll015w::Kan-MX6 and yll048c::TRP1-MX6 were inserted by homologous recombination into the Cd-sensitive mutant, DTY167 Homologous loci (YHL035c, YLL015w and YLL048c) of yeast gDNA and preparation of budding yeast SM4 mutants lacking YCF1 and three other vacuolar ABCC-type ABC transporters, YHL035c, YLL015w and YLL048c, in the preparation of SM4 Expression of the transporters AtABCC1 and AtABCC2.
[0081] To amplify DNA fragments for homologous recombination, the following primers were used with genomic DNA obtained from a single gene null mutant: For yhl035c::HIS3-MX6, the primers were His3MX6-YHL035F (SEQ ID NO: 7) and His3MX6- YHL035R (sequence number: 8), genomic DNA is obtained from YM...
Embodiment 3
[0092] Example 3: Whether AtABCC1 and AtABCC2 can be tested as phytochelatin transporters
[0093] 用空载体(pNEV-Ura)(SauerN&StolzJ(1994)SUC1andSUC2:twosucrosetransportersfromA.thaliana;expressionandcharacterizationinbaker'syeastandidentificationofthehistidine-taggedprotein.PlantJ6:67-77)、pNEV-AtABCC1或pYES3-AtABCC2(LuYP等(1998)AtMRP2,anArabidopsisATP-bindingcassettetransporterabletotransportglutathioneS -conjugatesandchlorophyllcatabolites:functionalcomparisonswithAtMRP1.PlantCell10:267-282) Transformed SM7 yeast cells were grown in SD-uracil liquid medium (Qbiogene) at 30°C for 4 hours, and then centrifuged at 2,000g for 15 minutes to collect cell microparticles. group. All cell lines were cultured in YPD medium (USB, Duchefa) at 30°C for 30 minutes, centrifuged at 2,000 g for 15 minutes to collect cell micelles, which were then lysed with cytolytic enzyme (1,000 units / g cell fresh weight, Sigma). Microsomal vesicles were then isolated from the cell extracts using the method di...
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