Cloning And Sequence Analyses Of CDNA Fragments Encoding Salt-responding Genes From Wheat

The object of this study is looking for the salt-responding genes from the leaves of wheat (Xinong 88) seedlings growing in normal or salt-stress conditions by Differential Display RT-PCR( DD RT-PCR) method. mRNAs was isolated and purified from leaves of wheat seedlings treated with 1% NaCl for 72 hours. The first strand of cDNA was synthesized via reverse transcription with anchor primer Oligo(dT)12GC. PCR amplification was carried out with 10bp random primers for two times, and the re-amplified products were separated in 1.5% agarose gel. Twenty four differential cDNA fragments, including nine inductive expression fragments, two intensive fragments and thirteen inhibitory expression fragments were detected. Twelve cDNA fragments with obvious difference were cloned. Sequence analyses and homologous comparisons of WSR2, WSR1, WSR3 were carried out. The result of Northern blotting exhibited that WSR2 fragment had obvious salt inhibitory expression characteristic, namely, the treated material had weaker signal, the control had stronger signal. Analysis through GenBank/BLAST database revealed that WSR2 bore 99% identity to the cDNA sequence of LyxK gene encoding L-xylulose Kinase. The high homology between WSR2 and LyxK gene showed WSR2 probably participated in the dark reaction of photosynthesis and had an restrictive effect on the dark reaction. Cloning and expression analysis of WSR2 provided new evidences to understand hexosemonophosphate pathway and the dark reaction of photosynthesis in plant. Northern blotting exhibited that WSR1 and WSR3 fragment had obvious salt inductive expression characteristic, namely, the treated material had positive signal, the control had no signal. WSR1 showed high homology with the encoding sequence of human angiogenin-inhibitor. In view of angiogenin-inhibitor possessed the activity of RNase inhibitor, the product of WSR1 gene might also work as a RNase inhibitor, it might regulate the translation process through RNA protection and cause thedefensive response of wheat to salt stress. The high homology between human angiogenin inhibitor and WSR1 indicated that they might be the earliest genes formed in the process of life evolution. It reflected that animal cells and plant cells had some common mechanisms in resistance to adversity stress. WSR3 showed high identity to the sequence at Homo sapiens BAC clone RP11-399D2 from 4, which indicated there were some common encoding sequences and some common mechanisms in the gene expression between animal genome and plant genome. WSR3 might be the conservative sequence in the process of life evolution of animal and plant. The obtainment of the cDNA fragments of salt-responding genes provided new evidences for further understanding of wheat salt-responding mechanism.