本實驗室之前應用功能性遺傳學的方法 (functional genetics approach)，以ethyl methanesulfonic acid (EMS)為突變劑，篩選出一對高溫逆境過度敏感之阿拉伯芥突變植株，命名為hit1-1 (heat intolerance)。此基因座落於阿拉伯芥第一條染色體上，為一未曾被研究過的基因。從HIT1 cDNA對應之氨基酸所作的序列比對得知，其N端與酵母菌之Vps53p相似。酵母菌之Vps53p 為囊胞繫鍊蛋白，可與Vps52p及Vps54p結合成複合體以調節囊胞的運輸。缺乏Vps53p的酵母菌突變株，對熱逆境亦會失去耐受性。本研究先利用互補實驗，顯示出阿拉伯芥HIT1能改善酵母菌Δvps53突變株，對37℃高溫的耐受性。為進一步探究HIT1基因的功能及生理意義，本研究接著利用HIT1之啟動子(promoter)與β-glucuronidase (GUS) 報導基因相連，觀察HIT1在植物組織中表現情形。結果顯示HIT1主要在根尖伸長區、成熟的花葯及花粉管中表現。此外，已知阿拉伯芥中的POK (poky pollen tube) 蛋白質與酵母菌中的Vps52p為同源蛋白質，同時也控制花粉管的伸長。由於POK基因被T-DNA 插入進行篩選pok 突變株的時候，無法找到純合子 (homozygous) (Lobstein et al., 2004) ，而hit1 基因在進行T-DNA插入篩選突變株時，也一樣找不到純合子。本研究於是觀察花粉管在in vitro中之生長情形，發現當HIT1被T-DNA插入而失去功能時，其花粉管的生長會受到限制。由於花粉管與根毛的生長方式均屬尖端生長的一種，本研究也觀察hit1-1花粉管與根毛的生長。結果顯示hit1-1花粉管與根毛的長度，都較野生型短。綜合以上結果，HIT1在阿拉伯芥有可能是調節囊胞運輸之分子。而其所調節之囊胞運輸，對植物熱逆境之耐受性及尖端生長有決定性的角色。此外，為能更了解植物耐受高溫的生理機制及其他遺傳控制因子，繼hit1突變種的研究之後，本研究另進行hit2突變株之基因定位，目前已知其坐落在第5條染色體，AGI map 4700-5700 kb之範圍內。 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.
