| Abiotic stresses, including drought, high salinity and extreme temperatures, greatly impair the growth and development of plants. Among these abiotic stresses, water deficit is the most severe environmental factor responsible for the reduction of crop yield in many parts of the world. A number of genetic and cellular events that occur under such stress have been widely documented. Although a large and increasing number of genes induced by salt stress have been recently identified with the aid of combination of molecular and genetic approaches, their physiological roles in relation to either tolerance or sensitivity are largely unknown in higher plants and many salt-tolerant genes associated have still not been found. Thus, it is critical to study the functions of stress-inducible genes to understand the molecular mechanisms of stress tolerance of plants.Several techniques have been employed to identify the gene whose expression is differentially regulated in response to various environmental stresses in higher plants. The methods include differential screening, differential display polymerase chain reaction (DDPCR), suppression subtractive hybridization (SSH) , serial analysis of gene expression (SAGE) , DNA-chip and microarray, and cDNA-AFLP and so on. Among these techniques, the suppression subtractive hybridization (SSH) method has been shown to be quick and highly efficient, and it generates less false-positive clones. By using SSH, a cDNA subtractivelibrary of aloe under salt stress had been done. After the screening, the Expression and identity of the gene for NADP-Malic enzyme in Leaves of aloe vera L. under salt stress had been done.Methods:Construction and screening of the subtractive library: To construct a cDNA subtractive library of aloe vera L. under salt stress, the leaves of aloe vera L were removed from the seedlings which were treated with 300 mM NaCl. Suppression subtractive hybridization (SSH) was carried out and a subtractive library was constructed.Poly (A)+RNA was isolated from the salt-induced aloe vera L. leaves as tester and the normal aloe vera L. leaves as driver, and single-strand cDNA and double-strand cDNA were sythesized in turn. Tester cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R, and then hybridized with normal aloe vera L cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library.To screen the library, defferential screening had been employed. Sixty-four clones were randomly picked to perform dot blot. After identifying candidate clones which are differentially expressed at salt-treated leaves of aloe vera L We have had three cDNA clones partially sequenced and searched for homology in the GenBank database.Expression and identity of the gene for NADP-Malic enzyme in Leaves of aloe vera L under salt stress: To investigate whether the expression of NADP-ME gene and the accumulation of NADP-ME protein induced by salt treatment are related to salt tolerance in the aloe plant, Northern blot analysis was performed and the activity of NADP-ME protein was measured in a tolerant aloe, Aloe vera L , and a sensitive aloe, Aloe saponarea Haw under salt stress. To investigate the expression of AvME under several types of stress, Northern blot analysis was performed. To further confirmwhether the synthesis of NADP-ME protein was induced with hours of treatment, western blot analysis of the samples was conducted.Results:1. A salt-treated aloe vera L. subtractive libarary with high subtractive efficiency was successfully set up. Random analysis of 13 clones with enzyme restriction showed that 10 plasmids in the clones contained 300-600 bp inserts. After screening, we had identified ten candidate clones which are differentially expressed at salt-treated leaves of aloe vera L. Sequence analysis was performed for three clones. One of them scored perfect to already known NADP-malic enzyme gene. We had it registered in GenBank. The accessio...
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