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Expression Of Genes From Limonium Bicolor Under NaHCO3 Stress And Functional Identification Of The VHA-c

Posted on:2009-03-20Degree:MasterType:Thesis
Country:ChinaCandidate:H MaFull Text:PDF
GTID:2120360275466666Subject:Tree genetics and breeding
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Limonium bicolor,a halophytic species of flowering plant,thrives in saline and saline-alkali soil.It's with high capacity of salt resistance,is an excellent target for plant salt tolerance research.We employed expressed sequence tags(EST) analysis combined with cDNA microarray in order to have a better view of transcript expression in L.bicolor during saline-alkali induced stress.And we cloned a Vacuolar H+-ATPase Subunit c gene from the library,the analyses of biological information,the expression pattern,and function identification of H+-ATPase Subunit c(VHA-c) were conducted.A cDNA library was constructed from the leaf of Limonium bicolor exposed to 0.4 mol/L NaHCO3 for 48 h.Primary titer of the library was 9.6×105 pfu/mL,and the rate of recombination was 95%,with an average inserts size of 0.9kb,which were determined by both PCR and sequencing analysis.A Total of 2358 high quality sequences were generated from 2880 sequenced clones which were selected randomly from the library.The GenBank accession numbers of these ESTs are EH792855-EH795212.BlastX search results of 2358 ESTs showed that 1148 sequences had high homologous to known sequenees.The database-matched ESTs(1418) were grouped into 12 function categories.The most recurrent categories were metabolism(18.48%),photosynthesis(14.6%),unclassified(11.99%),cell rescue, defense(11%) and transport facilitation(10.01%).EST analysis showed that 245 ESTs were homologous to genes earlier implicated in stress responses,most prominently metallothionein (EH793637) and lipid transfer protein(EH794695),each of which accounted for 1.4%of total ESTs.1330 ESTs were selected for construction a cDNA microarray.We obtained transcript profiles of L.bicolor response to NaHCO3 for 6,24 and 48 h.A total of 142 transcripts were differentially regulated under the conditions studied,and 108,43,and 46 transcripts were differentially regulated by NaHCO3 stress for 6,24 and 48 h,respectively.The microarray results showed that 76%of differentially regulated transcripts were identified at the early stress period(stress for 6 h),suggesting that the response at an early stage is important for saline-alkali stress tolerance in L.bicolor.This study provides informative preliminary data and a starting point for more in-depth analyses of extreme stress tolerance in L.bicolor.The biological information analysis of the Vacuolar H+-ATPase gene(VHA-c) was analyzed.Sequence analysis showed that the VHA-c gene is 729 bp in length,including 498 bp of open reading frame(ORF),and encodes a protein of 165 amino acids with a predicted molecular mass of 16.6kD and pI 8.62.In addition,the expression patterns of VHA-c gene in L. bicolor exposed to NaCl,NaHCO3 and Na2CO3 were performed by real time RT-PCR.The result showed that the expression of VHA-c gene was induced by NaCl at leaves of L.bicolor and inhibited in root;reached to the highest level at leaves and root after NaHCO3 stress for 12 hand 6h;and reached to the highest level after Na2CO3 stress for 6 h,48 h at the leaves of the highest expression.Above all results indicated that the VHA-c gene can be responsive to abiotic stress;therefore,it may be a salt tolerance gene.The VHA-c gene was inserted into the plant expression vector pROKⅡ,and transferred into model plant,Nicotiana tabacum cv.Petit Havana SR-1,using Agrobacterium-mediated transformation method.The function of the exogenous VHA-c gene was examined by the salt tolerance test of transgenic lines.The result indicated that compared with nontrangenic line,the expression of exogenous VHA-c gene increased the activities of SOD and the POD,and reduced the content of MDA in transgenic lines,enhancing the salt tolerance of the transgenic plant.our results indicated that the expression of VHA-c gene may increase the salt tolerance of plant by protection of the plant active oxygen elimination enzyme class,such as POD,SOD and so on,and reduction of the membrane oxidation in plants.This study confirmed the VHA-c gene from L.bicolor can confer salt tolerance to plant.The process of plant bears salty is a complex system associated with multi-genetic coordinated operating.Our research obtained that the genes expression profiles through the EST analysis and cDNA microarray,and will make a solid foundation for revealing the molecular mechanism of salt-alkali tolerace in L.bicolor.This study provides informative preliminary data and a starting point for more in-depth analyses of extreme stress tolerance in L. bicolor.The experiments results indicated that the constitutively expressed VHA-c gene from L. bicolor can improve the tolerance of plant to adverse environments,and made a foundation for employing the VHA-c gene in plant genetic.
Keywords/Search Tags:Limonium bicolor, saline-alkali stress, expressed sequence tags, cDNA microarray, Vacuolar H~+-ATPase Subunit c gene, genetic transformation
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