Studies On Difference Of Gene Expression Treated By Salt And Construction Of A CDNA Library In Wild Barley

Total RNAs from the leaves of wild ba.r\ey(Hordeum brevisubulatum(Trink.) Link.) treated 24hrs with 430mM NaCl(T) and unstressed control(CK) were extracted. The difference in their mRNA transcriptions was analyzed by using mRNA differential display. Differentially expressed cDNA fragments were cloned and sequenced. Forty-one of the 54 primer sets displayed differentially expressed cDNA fragments. The anchored primers with G or A residues at the 3 ' end were most efficient.Forty-one cDNA fragments from 200 to lOOObp expressed differentially were isolated by polyacrylamide gel and cloned in pGEM-T easy vector.After identifying by reverse Northern blot analysis, fragments of 14 and 31 were expressed only in wild barley treated with 430mM NaCl, and fragments of 1, 2, 13, 15 and 19 achieved expression enhanced in wild barley treated with 430mM NaCl compared to that in control plants.These seven cDNA fragments were sequenced. Results of BLAST analysis in GenBank showed that fragment 14, one of two cDNA fragments which expressed only in wild barley treated with 430mM NaCl had 95% identity with cDNA clone of unknown function isolated from barley, and over 83% identity with cDNA clones isolated from cold, heat and salt-stressed plants. The fragment may be related to plant stress responding genes. Fragment 31 had over 91% identity with cDNA clones of unknown function isolated from barley and wheat. Fragments of 1 and 13, two of the five cDNA fragments showed over 91% homology to barley and wheat cDNA clones of unknown function. Fragments of 13, 15 and 19, exhibited over 85% identity with plant stress responding cDNA clones. These fragments may be related to plant stress responding genes. Fragment 19 which expressed at higher level in wild barley treated with 430mM NaCl than unstressed control showed over 88% homology to chloroplast ATPase subunit gene. It represented encoding chloroplast ATPase subunit gene.cDNA library from wild barley leaves treated with 430mM NaCl was successfully-constructed by using pTriplEx2 vector. It contained 2.16X106 pfu/ml. The size of insert fragments ranged from 0.5~4kb, with an average length of 1.5kb.