FUNCTIONAL ANALYSIS OF INTRONS FOR THEIR EFFECT ON TRANSGENE EXPRESSION IN PLANT

Abstract

Most plant genes contain intervening sequences known as introns that are found in nearly all plant genes and they are transcribed into pre-mRNA, the later will be removed by splicing machinery. Proteins are encoded by exons which separated by intron segments while the non-coding exons are conserved in the mature transcript. In this study, level of spliced and non-spliced introns was measured in transgenic Nicotiana tabacum lines expressing gusint-gfpint fusion gene controlled by the CaMV 35S promoter. Transcription analysis of the transgenes was performed by (RT)-PCR. All transcripts of transgenic lines generated two different length of fragments for gfp and gus genes. The results indicated that the gene expression of transcripts (without intron) in which their intron was spliced out, was higher than those of non-spliced transcript (with intron) in which the intron was maintained i.e. the both introns inside gus and gfp were partially and not completely spliced out of the transcripts. Furthermore, the relative quantifications of band intensity of gfp transcripts revealed that the percentage of non-spliced transcripts compared to the housekeeping gene, elongation factor 1-alpha (ef1α) using different amplification cycles (25, 30, 35 and 40) was 23.3 %, 28.3 %, 21.7 % and 20 % respectively and the percentage of spliced transcripts was 76.7 %, 71.7%, 78.3 % and 80 % in that order. While, the proportion of gus transcripts revealed that the non-spliced transcripts were 34.2 %, 15.7%, 12.8 % and 16.3 % and the spliced was 65.8 %, 84.3 %, 87.2 %, and 80 % at 25, 30, 35 and 40 cycles respectively. Conclusively, in the present study, the expression of gus and gfp genes might linked tothe process of splicing and not to a particular intron sequence or the partial splicing may due to splicing signals within the sequences of the both introns. In conclusion, in spite of the incomplete splicing of the IV2 second intron of the potato ST-LS-1 gene and fristintron of a castor bean gene for catalase, the expression of gus and gfp genes was not affected. Where, it was ranged from 71.7 % to 80 % and from 65.8 to 87.2 for the gus and gfp, respectively in the spliced introns. Therefore, the expression of the non-spliced gus and gfp introns was much lower than those of splicedones.