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Sequence And Function Analysis Of Peroxidase In Tamarix Hispida

Posted on:2010-10-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:X H GuoFull Text:PDF
GTID:1103360275466126Subject:Tree genetics and breeding
Abstract/Summary:PDF Full Text Request
After suffering from abiotic stresses,the active oxygen would arise in the cells of plants, posing damaging poison to plants.In order to scavenge the active oxygen,plants have engendered some protective mechanics,which mainly included enzymatic detoxication mechanics and non-enzymatic scavenging mechanics.Peroxidases play a key role in the detoxification of reactive oxygen species because they have an almost 1000-fold higher affinity for H2O2 when compared to catalases.A gene encoding peroxidase(named ThPOD) was isolated from a T.hispida NaCl-stress root cDNA library.The preliminary research on transcriptional regulation and genetic function of ThPOD gene were studied.The primary conclusions are as follows:1 Bioinformaties analysis of ThPOD showed that this gene belongs to the classⅢperoxidase multigenic family and is typically secretory peroxidase.2 The expression of ThPOD could be induced by abiotic stresses such as cold,salt, drought and exogenous abscisic acid.These findings suggested that ThPOD might be involved in the plant response to environmental stresses and ABA treatment.Moreover,mRNA transcript level of this gene was higher in roots than leaves.3 The ThPOD performed efficient expression in E coli and the fusion protein was purified used as antigen to immune rabbit for subsequent preparing for polyclonal antibody to ThPOD. This antibody specifically responsed to ThPOD.This antibody was applied to analyze the expression of peroxidase in Tamarix hispida under different stress conditions such as 20% PEG and 100/amol/L ABA.4 To elucidate the function of this gene,recombinant plasmids expressing full-length ThPOD as well as ThPOD(aa 41-337)(encoding of the plant peroxidase functional region),and ThPOD(aa 73-337)(encoding of the ascorbate peroxidase functional region) truncated polypeptides were constructed.SDS-PAGE and Western blot analyses of the fusion proteins revealed that the molecular weights of ThPOD,YhPOD(aa41-337) and ThPOD(aa 73-337) were~57,~50 and~47 kDa,respectively.The survival rate and growth curve assay showed that expression of these polypeptides in host cells could confer protective function against abiotic stresses.Especially,ThPOD(aa 73-337) could confer higher salt and drought tolerance in Escherichia coli than others.The survival ratio of E.coli harboring ThPOD(aa 73-337) Was over 2-and 10-fold higher than that of control E.coli grown on LB plates supplemented with 0.6% NaCl and 20%PEG,respectively.This finding could potentially be used to improve plant tolerance to drought stress via gene transfer.5 To further study the function of the ThPOD gene in high plant,the nucleotide sequence was cloned into pROKⅡvector which could express aimed protein by the control of CaMV 35S promoter.The gene was introduced into popular by Agrobacterium tumefaciens.It was totally obtained 17 independent lines with tolerance to kanamycin.Among the seventeen,the expected 1086 bp amplification product was detected in 14 lines.These five transgenic lines (TL1,TL3,TL5,TL8,TL12) were selected from the fourteen putative transformants for further analysis.The results of Southern and northern blot analysis indicated that ThPOD was expressed in the five transgenic lines,and had integrated into the plant genome and could successfully transcript.The transgenic and thenon-transgenic plants were cultured on the medium with 150 mmol/L NaCl or 200 mmol/L NaCl for 15 days.Growth of transgenic plants was better than non-transgenic plants under Salt-stress conditions.Assay of oxidation stress of transgenic and non-transgenic plant showed that the chlorophyll contents of non-transgenic plants were significantly lower than those of transgenic plants(except for TL3).The relative electrical conductivity of each transgenic line except for TL3 was significantly lower than that of non-transgenic plants.The experiments reflected that,under oxidative stress,the transgenic plans suffered far less damage than control.The results also reflected that the over-expression of the gene in transgenic plants was beneficial for protecting photo system as well as alleviating oxidative membrane lipid injury which boosted the anti-oxidative ability.
Keywords/Search Tags:Tamarix hispida, Peroxidase, Real time quantitative PCR, Prokaryotic expression, Transgenic popular
PDF Full Text Request
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