Plant Expression Vector Containing Antifreeze Peptide Gene And Its Applications In Transgenic Plants, Secretory Expression In Trichoderma Viride And A Study On Intron Function Of AFP Gene

This dissertation consists of three parts:1. Two primers were designed according to the published pseudo-pleuronectes americanus (a winter flounder) AFZP (antifreeze peptide) gene sequences, and by PCR, the open reading frame of AFZP cDNA was amplified from a bacterial expression plasmid containing the sequences. The amplified fragment was inserted into pUC-19, sequence analysis of the cloned fragment was then carried out. The results of sequences analysis were identical with the published pseudo-pleuronectes americanus (a winter flounder) AFZP cDNA sequences. Finally the AFZP cDNA was subcloned into the plant expression vector PBI-121 under regulation of CaMV35S promoter and NOS terminator. Through pollen tube pathway, several kinds of plants were transformed with the expression vector containing AFZP cDNA, elementary results show that, compared with the wild-type plants the transformants were characteristic of cold-resistance to some extent.2. Trichoderma viride, a species of filamentous fungi, is one ideal host for heterologous secretory expression. To investigate the secretory expression in T. viride, a fungal expression plasmid with the open reading frame of AFP gene (430 bp) fused between the promoter and terminator of trpC gene was constructed and transformed into T. viride. PEG-mediated transformation of protoplast was carried out utilizing the hygromycin B resistant gene as a selection marker. SDS-PAGE and Western-blot analysis showed that AFP was expressed and secreted into the culture supernatant of the transformant of T. viride. This study provides a basis for secretory expression of heterologous eukaryotic genes of important value in the system of T. viride.3. Mechanism of intron splicing in filamentous fungi might be similar to that in other eukaryotes. But little is known of whether it affects expression. The 430-bp open reading frame of antifungal protein (AFP) gene from Aspergillus giganteus is interrupted by two introns (89-bp and 56-bp), which have conserved splice sites and some typical features of filamentous fungal gene. To test whether the introns play roles in expression, two vectors containing either the AFP gene or its intronless derivative (i.e. AFP cDNA) are constructed and transformed into Trichoderma viride respectively. Expression analysis of transformants show that the introns are required for AFP gene expression in T. viride, suggesting that the small introns in filamentous fungal genes may not only act as intervening elements but also play critical roles in expression. Furthermore, a recent study shows that AFP has the characteristic features of the oligonucleotide binding domain. By comparison of the intron/exon structures in various ADH genes and related genes, it is shown that the evolution of the oligonucleotide binding domain is intron-dependent. Thus, in view of our current study and the evidence summarized above, it is reasonable to speculate that the evolution of AFP gene may be intorn-dependent. In addition, a previous study reported that a silent intronless AFP gene exists in the genome of T. viride. But, in my study, the data obtained by both PCR and Southern blot analysis of the wild-type recipient and transformants clearly showed that no such a sequence exists in the genome of T. viride.