| At present, the heavy metal pollution of soil and water are becoming more serious.In hence, the nation established the experimental unit in order to repair to the farmlandof the heavy metal pollution. Heavy metal (Mainly is the cadmium, lead, arsenic,mercury, chromium, etc) affects not only the growth and development of plants, but alsothreatens human health through entering in to the body by their accumulation in thefood vegetables. The toxicity of cadmium and lead are the strongest, the pollution ofwhich are the most serious.Isoetes sinensis is Lobular type ferns, mire indicating Isoetes plants of Isoetaceae.It has been endangered species due to the change of the living environment and habitatloss, which has been listed as key state protected wild plants. The survey about thehabitat of Isoetes sinensis found that the content of cadmium and lead in soil exceededstandard badly. Researching on response of Isoetes sinensis we called “living fossilplant” to heavy metal stress which especially in aspect of gene expression play asignificant role on the mechanism of the molecular and original reaction of plant stress.Herein, Isoetes sinensis Palmer was acted as test material stressed by200μMcadmium chloride and2g/L lead nitrate respectively. The technique of cDNA-amplifiedfragment length polymorphism (cDNA-AFLP) was used to identify differentialexpression gene and homology alignment of Blast (The Basic Local Alignment SearchTool) was used to analyze the function of gene. The study may supply the molecularbiology foundation for searching the genes related with heavy metal stress and revealingsensitivity of Isoetes to environmental change. The results are as follows:(1) cDNA-AFLP technology was used to analyze different expression gene ofIsoetes sinensis Palmer under different times of Cd exposure and Pb exposure.According to display of cDNA-AFLP which under Cd exposure and Pb exposurerespectively, differentially expressed transcript-derived fragments (TDFs) were dividedinto four types. Type I: the fragments existed in normal condition and the whole processof heavy metal stress, amount of the expression of the genes did not change. Type II: thefragments existed in normal condition and the early time of heavy metal stress. With theincreasing heavy metal stress, the expression of genes were down-regulated, butrecovered after a period of time. Type III: amount of the expression of genes had no or less in normal condition, the expression of genes were up-regulated in the wholeprocess of heavy metal stress. Type IV: the fragments existed in normal condition, butdid not exist in the whole process of heavy metal stress.(2) According to cDNA-AFLP display, the expression of one hundred andeighty-four fragments were identified to be differential under different times of Cdexposure and Pb exposure respectively. The number of TDFs (type I) were twenty-four,which was13percent of the total. The number of TDFs (type II) were same as the typeI. The number of TDFs (typeIII) were one hundred and nine, which was59percent ofthe total. The15percent of the total is type IV, the number were twenty-seven,.(3) We cloned and sequenced Some TDFs obtained from cDNA-AFLP. Fifteen ofthem had been cloned and sequenced successfully. The cloned genes were analyzed byBLAST in NCBI, of which13genes could find homologous genes in GeneBankdatabase.(4) There were3genes related to heavy metal stress. TDF9stressed by Cd washomologous with ABC transporter C family member gene and the homologies was84percent. TDF4and TDF7stressed by Pb were homologous with zinc finger RING-typeprotein respectively and the homologies was75percent. The genes may supply themolecular biology foundation for revealing Isoetes sensitivity to environmental change.Furthermore, the homologies of TDF6stressed by Pb was100percent,whichhomologous with Isoetes engelmannii18S ribosomal RNA gene, the discover of TDF6proved the reliability of cDNA-AFLP in some extent. |
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