| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 94 === Using the functional genetics approach, we have isolated an ethyl methanesulfonic acid (EMS)-mutagenized Arabidopsis heat hypersensitivity mutant named hit1-1 (heat intolerance). The HIT1 locus was located on chromosome I. Amino acid sequence analysis showed that HIT1 is homologous to yeast Vps53p. It has been shown that yeast Vps52p Vps53p and Vps54p can form a tethering complex to mediate vesicle trafficking. The growth of yeast vps53∆ mutant is inhibited at 37℃ compared to that of wild-type. Transformation of a plasmid DNA carrying the HIT1 gene into yeast vps53∆ mutant was able to improve the retarded growth at 37℃. In order to further investigate the physiological function of HIT1, we also used HIT1 promoter driven β-glucuromidase (GUS) reporter gene to observe the expression pattern of HIT1 in plant. Results showed that HIT1 is expressed specifically at the elongation zone of root, stigma, mature anther and pollen tube. On the other hand, Arabidopsis POK (poky pollen tube) gene whose product is homologous to yeast Vps52p has been shown to be required for effective pollen tube growth. T-DNA insertional mutants of both pok and hit1-2 can not be isolate homozygously. Development of hit pollen tube was therefore examined. Results showed that only 50% of the pollens from hit1-2 mutant can develop pollen tube. Moreover, while all the pollens from hit1-1 can develop pollen tube, the average length of the tube is remarkably shorter than that from wild type. Since the elongations of pollen tube and root hair all belong to tip growth, the development of root hairs of hit mutants was also observed. Again, result showed that the length of root hairs from hit1-1 is shorter than that from wild type. Collectively, it is suggested that the HIT1 gene product is a vesicle tethering factor and has crucial roles in plant tip growth and stress tolerance. In the meantime, a second EMS-mutagenized heat intolerant mutant, hit2, was isolated from Arabidopsis. The locus of the hit2 was currently mapped to be at the chromosome IV between 4700kbp and 5700kbp on the AGI map.
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