Screening And Cloning The Heavy Metal Tolerance-Associated Genes In Avicennia Marina(Forsk.) Under Cadmium Stress By Suppression Subtractive Hybridization

The halophytic Avicennia marina(Forsk.)is useful for the study of molecular mechanisms behind heavy metal pollutant tolerance in mangrove trees.Although mangrove ecosystems receive a number of pollutants from their related drainage and rivers,they possesses a great tolerance for heavy metal pollution.Avicennia species is thought to exhibit greater metal accumulative and tolerance properties than other mangrove species.To isolate these tolerance-associated genes from mangrove plants,a cDNA library of the Avicennia marina(Forsk.)leaf was constructed and screened for stress-relatedgenes by polymerase chain reaction(PCR)-based suppressive subtractive hybridization(SSH).After reverse northern blotting analysis and sequencing,a total of 46 and 53 non-redundant differentially expressed sequence tags(ESTs)were obtained from the forward and reverse libraries,respectively.Among these up-regulated ESTs,99 unique clones were considered,identified and classified.In the forward subtracted cDNA library,within the biological process category,the 40 unigenes were classified into ten primary categories,including single-organism process,cellular process,metabolic process,developmental process,multicellular organismal process,immune system process,biological regulation,localization,cellular component organization or biogenesis,and response to stimulus.Within the molecular function category,they were classified into fourteen categories,including transferase activity,hydrolase activity,oxidoreductase activity,substrate-specific transporter activity,transmembrane transporter activity,isomerase activity,organic cyclic compound binding,small molecule binding,carbohydrate derivative binding,protein binding,sulphur compound binding,heterocyclic compound binding,ion binding,and amide binding.In cellular components,the most representative categories were cell,organelle and macromolecular complex.Membrane and extracellular region were also represented.Extracellular matrix annotations were fewer.In the reverse subtracted cDNA library,49 of the 53 contigs matched homologous sequences with identities of 51-98%.And also within the biological process category,the 49 unigenes were classified into eleven primary categories,including single-organism process,metabolic process,cellular process,developmental process,multicellular organismal process,response to stimulus,cellular component organization or biogenesis,biological regulation,localization,signalling,and growth.Regarding the molecular function,they were classified into thirteen categories,including oxidoreductase activity,hydrolase activity,transferase activity,lyase activity,enzyme inhibitor activity,peptidase regulator activity,ion binding,organic cyclic compound binding,heterocyclic compound binding,small molecule binding,carbohydrate derivative binding,lipid binding,and structural constituent of ribosome.In cellular components,the most representative categories were cell,extracellular region,organelle and membrane.Symplast and cell junction were also represented.Macromolecular complex annotations were fewer.Eight genes from different categories were selected and their expression was studied by real-time RT-PCR.Expression levels were confirmed for all eight transcripts,which suggests that these genes contribute to the heavy metal pollutant tolerance of this plant.The expression levels of Fc01 and Fs12 up-regulated in the leaf,stem and root of the Cd2+ treated plants.Expression levels of Fs01 up-regulated in the leaf,stem and especially so in the root.Expression levels of Fs07 up-regulated in the leaf,but not in the stems and roots(Figure 6).Expression levels of Rs08 down-regulated in stems and roots.Expression levels of Rs18 up-regulated in the leaf,but up-regulated in the root and stem.Expression levels of Rc24 also down-regulated in the roots.On the other hand,expression levels of Rc15 in the root up-regulated in the first day and then down-regulated,firstly in the stem,with the leaf down-regulating in the first day then upregulating afterwards.The diversity of these genes indicated that heavy metal pollutant stress resulted in a complex response by mangrove plants.Moreover,most of the up-regulated ESTs were identified so as to obtain a comprehensive picture of the heavy metal pollutants tolerance response in the plant at the level of gene.This could prove a useful approach for further elucidating the molecular mechanism underlying salt tolerance.