Identification of intron retention in a gene involved in cold hardiness in Poncirus trifoliata.

摘要

The trifoliate orange, Poncirus trifoliata (L) Raf., is a cold hardy citrus relative plant able to survive temperatures below freezing once fully acclimated. Previous studies in cold acclimation of trifoliate orange found a cold responsive gene called citrus low temperature (CLT) responsive gene induced by cold stress. Under cold stress the CLT gene is up-regulated resulting in different m-RNA transcripts. A process known as alternative splicing (AS) was suggested as having a role in regulating the expression of the CLT gene, but no confirmation has been reported.;Cold stress is reported to affect pre-mRNA processing of certain genes causing AS resulting in the exclusion or retention of certain genetic information. One of the transcripts of the CLT gene contains a 98 base pair (bp) sequence at the 3' untranslated region (UTR) that is present in acclimated trifoliate orange but absent in non-acclimated plants. Investigations on the 3' UTR of human cells have shown that the retention of introns within the 3' UTR helps improve mRNA stability. Under cold stress, the retention of intronic segments may assist in increasing mRNA stability in P. trifoliata.;In this investigation, the objective was to confirm the presence of introns in mature mRNA of the CLT responsive gene from P. trifoliata. By using semi-quantitative reverse northern blotting analysis, this study confirmed that a fragment of 98 nucleotide (nt) from intron two is retained at the 3' UTR of the transcript that is up-regulated by cold acclimation. This indicates that AS plays a role in regulation of CLT gene during acclimation.;In addition to the 98 bp fragment a small segment at the beginning of intron two seams to be retained in another transcript. This transcript does not share the same open reading frame of CLT and is also only present in acclimated plants. It was also determined that intron one did not hybridize with any of the transcripts, thus confirming the absence of intron one in mature m-RNA. By RT-PCR, the level of gene expression in acclimated and non-acclimated RNA from P. trifoliata was found to be similar for the constitutive transcripts of CLT.