title: Strategies for cloning ion transporters in salt-resistant plants
Creators: name: al-Salameen, Fadila A.
Publisher: name: University of Glasgow
Year: 1998
Subjects: subject: Plant sciences.

description: Most crop species upon which we depend are glycophytes, which lack the mechanisms required to tolerate ion excesses present in high concentration in saline soils. Therefore, it is important to understand how salt-resistant plants respond to external salinity. What is known is that a number of physiological processes are involved in salt-tolerance and each of these processes requires the co-ordinated expression of a number of genes. One way to identify putative genes encoding Na+ and Cl- transporters from salt-resistant plants is to use an approach involving heterologous probes. Genomic Southern blots of barley and sugar beet were performed using a range of H+-ATPase, Na+ / H+ antiporter and Cl- channel sequences as probes. Some sequence homology to both p-type and v-type H+-ATPases, and to clc-0 (a voltage gated Cl- channel) was found. However, Northern analysis showed that only in barley was the clc-0 homologue expressed; the transcript was ~ 1.4 kb and probably not poly-adenylated. Attempts to isolate a voltage gated Cl- channel from barley genomic library constructed in lambda Dash II by colony hybridization using clc-0 as probe were unsuccessful. In 1996 the first plant voltage-gated Cl- channel, clc-nt1 was cloned from tobacco using an RT-PCR approach. A similar strategy was then adopted in this study. Seventeen putative CLC sequences were found in the databases and these were aligned. Three regions of consensus were identified that were suitable for designing degenerate PCR primers. Two PCR approaches were used, conventional PCR and RT-PCR, to identify clc homologues in sugar beet, barley and red beet. The conventional PCR approach used cDNA libraries and genomic DNA as template, but failed to identify any clc homologues. RT-PCR was performed on oligo dT - and random primed cDNA from root and leaf tissues of high-salt-grown plants. A 600 bp fragment, bv600, from sugar beet leaves, and two shorter fragments, bv160 and bv163, from red beet cell suspension cultures, were amplified and demonstrated to be authentic plant sequences. These fragments were then sequenced and this information used to interrogate the databases using BLAST searches. The fragment bv600 was identified by BLAST as homologous to an ATP-dependent protease. Similar comparisons using the bv160 and bv163 sequences suggested that they are almost identical, but BLAST failed to identify any homologues in the databases. The red beet fragment bv160 was used to identify a 7 kb sugar beet genomic fragment, bvFAS1.7. This fragment was sequenced and analyzed by a range of computer programs. The bvFAS1.7 fragment, containing the bv160 sequence, probably encodes a reverse transcriptase although this is by no means certain. More recent alignments of all of the clc sequences, and of the six plant sequences deposited in the databases, suggests that better (i.e. less degenerate) primers can now be designed which should improve the success of an RT-PCR approach in cloning clc homologues in salt-resistant plants.