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Screening Of Arabidopsis Cesium Resistants Mutants Atbe1-5 And Its Mechanism Of Cesium Resistance

Posted on:2021-01-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:J X ZhangFull Text:PDF
GTID:1360330620976620Subject:Biochemistry and Molecular Biology
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The disaster accidents of Chernobyl Nuclear Power Plant and Fukushima Daiichi Nuclear Power Plant released a large amount of radioactive 137Cs and 134Cs,resulting in severe contamination of soil in surrounding areas.Cesium?Cs?,as the first principal group element,possesses similar chemical properties to potassium?K?and can be absorbed by plant roots easily.It is an efficient approach to abate pollution by using plants to absorb Cs from soil.Nevertheless,Cs has toxic effects on plants,and high-concentrated Cs may result in the death of plants.This experiment furnishes materials for phytoremediation of environmental Cs pollution by screening arabidopsis thaliana EMS mutant library to obtain Cs-resistant mutant and exploring its Cs-resistant mechanism.The main contents and results of this study are followed:?1?Arabidopsis thaliana Columbia ecotype?Col-0?background EMS mutagenic mutant atbe1-5 is screened out on the context of a medium containing 1mM Cs+.The mutant maintaining alive and Cs+accumulation capacity,while the wild type Col-0 shows necrosis phenotype in present of sub-millimolar Cs+.?2?atbe1-5 was conferred by a single nucleotide polymorphism?SNP?in Arabidopsis thaliana BRANCHING ENZYME 1?AtBE1,At3g20440.2?.The mutation site of atbe1-5 is initially positioned at the upper arm of Arabidopsis thaliana chromosome 3 through map-based cloning,and is linked with CER457071molecular marker for inheritance.By high-throughput sequencing,it is testified that mutations also occur in this region.Through further analysing candidate genes,cloning target gene,it is found that atbe1-5 has an SNP at 5320 of the 16thh exon of AtBE1,which changes from C?Col-0?to T?atbe1-5?,resulting in the 749thh amino acid encoded by atbe1-5 being changed from proline?P?in Col-0 to serine?S?in atbe1-5.By analyzing the homology of AtBE1 in higher plants,it is discovered that this site is highly conserved in these plants.The SNP does not affect the transcription of AtBE1and exerts little effect on the secondary structure of C-terminal,but increases the ratio of random coil in N-terminal fragment from 36.96%to 40.69%,decreases the ratio of?helix from 36.39%to 34.38%.Through genetic complementary testing,the coding sequence?CDS?of AtBE1 of wild-type Col-0 is transferred to atbe1-5,and the Cs-resistant phenotype is analyzed for atbe1-5/AtBE1 of mutually complemented plants,then it is discovered that the mutually complemented plants recover to the wild-type Cs sensitive phenotype,and finally it is determined that the Cs-resistant phenotype of atbe1-5 is indeed triggered by SNP of AtBE1.?3?Cs has markedly suppressed the photosynthesis of Col-0,but promoted the photosynthesis of atbe1-5.Under CK conditions,the atbe1-5 are blading yellowing.By measuring chlorophyll content,the experimental results reveal that the chlorophyll content of atbe1-5 leaves is markedly lower than that of Col-0,suggesting that the photosynthetic efficiency of atbe1-5 is lower than that of Col-0.Net photosynthetic efficiency?An??chlorophyll fluorescence parameters PSI?ETR1??PSI[Y?I?]?PSII?ETRII?and PSII[Y?II?]of atbe1-5 and Col-0 are measured respectively.The results turn out that under CK conditions,the[An]PSI?ETR1??PSI[Y?I?]?PSII?ETRII?and PSII[Y?II?]of atbe1-5 are markedly lower than those of Col-0.While after the treatment of 0.4 mM Cs+,the results are the[An]PSI?ETR1??PSI[Y?I?]?PSII?ETRII?and PSII[Y?II?]of atbe1-5 are markedly higher than those of Col-0.Revealing that the treatment of 0.4 mM Cs+has markedly suppressed the photosynthesis of Col-0,but promoted atbe1-5,which is consistent with the Cs-resistant phenotype of the mutant.?4?The Cs+-resistant mutant atbe1-5 has normal Cs+but altered K+ accumulation capacity.By measuring the contents of Cs+and K+in roots and leaves of atbe1-5 and Col-0,under the treatment of CK or 0.4 mM Cs+,it is discovered that there is no obvious difference between Col-0 and atbe1-5.It demonstrates that the Cs-resistant mutant atbe1-5 has normal Cs+accumulation capability of Col-0 plants.Nevertheless,the accumulation mode of K+has changed compared with that of Col-0:the content of K+in roots of atbe1-5 is markedly lower than that of Col-0,while the content of K+in leaves is markedly higher than that of Col-0,suggesting that K+in atbe1-5 is more accumulated in leaves,the accumulation of K+of Col-0 was more in roots,indicating that Col-0 plants roots by accumulating as much K+as possible in order to survive resist Cs+,which also indicated that Col-0 was more sensitive to Cs+.?5?The 749thh amino acid residue of AtBE1 is the binding site of Cs+and K+,and the binding capacity of this residue to Cs+is stronger than that of K+.By Isothermal Titration Calorimetry?ITC?analysis thermal dynamic between Cs+?K+and synthetic peptide,synthetic peptides corresponding to the 707-802thh residues?contains mutant residues?.Col-0 peptide(AtBE1?707-802749P)shows strong thermokinetics during the titration of Cs+or K+,while the thermokinetics of atbe1-5 peptide(atbe1-5?707-802749S)is eliminated.The thermal amplitude of variation triggered by interaction between Cs+and Col-0 peptide is markedly higher than that of buffer solution and K+,which suggests that the binding capacity of Cs+to this residue is stronger than that of K+.It is speculated that Cs+competes to replace K+to bind to the749thh residue of AtBE1,resulting in loss of the activity of AtBE1 enzyme.In atbe1-5,the residue of AtBE1 mutated from P?Col-0?to S?atbe1-5?and did not bind to Cs+.Therefore,the mutation of AtBE1 made atbe1-5 insensitive to Cs+and showed an Cs-resistant phenotype.In addition,Cs+competitive substitution K+binds to the749thh residue of AtBE1,resulting in the loss activity of AtBE1 enzyme.?6?atbe1-5 is defective in sucrose and starch biosynthesis.The contents of different monosaccharides and disaccharides in Col-0 and atbe1-5 are compared in this experiment.As a result,maltose level in atbe1-5 leaves is markedly higher than that of Col-0,but the sucrose level is markedly lower than that of Col-0,and there is no obvious difference between other saccharides.Besides,starch contents of Col-0and atbe1-5 at the end of photoperiod and dark period are measured respectively,displaying that amylose and amylopectin contents in atbe1-5 are merely 60%and 1%of Col-0,suggesting that AtBE1 plays an important role in the biosynthesis of sucrose,amylose,especially amylopectin.In conclusion,Arabidopsis thaliana Cs-resistant mutant atbe1-5 is acquired in the current research,which boasts the capability of Cs+survival and normal accumulation,providing ideal materials for phytoremediation of environmental Cs pollution.The mutant gene is AtBE1,which testifies that the gene plays a vital role in the biosynthesis of sucrose,amylose,especially amylopectin.The 749thh amino acid residue P of AtBE1 is found to be the key binding site for the enzyme to Cs+and K+.The binding capacity of this site to Cs+is stronger than that to K+,while the 749thh amino acid residue of atbe1-5 is mutated to S,which caused the enzyme not to bind to Cs+and K+,thus rendering atbe1-5 had an Cs-resistant phenotype.
Keywords/Search Tags:cesium, phytoremediation, BRANCHING ENZYME 1, sucrose, amylopectin
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