Construction and differential screening of a cDNA library specific to osmotic stress of Haloxylon ammodendron seedlings.
A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward, reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.
Jiang
Xiao-Cheng
XC
College of Life Science, Hunan Normal University, Changsha 410081, P. R. China. jxclc@hunnu.edu.cn
Guo
Xin-Hong
XH
Pan
Xiao-Ling
XL
Song
Song-Quan
SQ
D027703:Amaranthaceae / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009997:Osmotic Pressure; D016133:Polymerase Chain Reaction; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D036226:Seedlings / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D018749:RNA, Plant