Molecular cloning and characterization of the actin and calmodulin genes of Pythium splendens Braun and their expression in the process of sporangia germination

• Main Authors: Chang, Tsz-Yin, 張慈映
• Other Authors: Ruey-Fen Liou
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/16239749943376273568

碩士 === 國立臺灣大學 === 植物病蟲害學系研究所 === 86 === Sporangia of Pythium splendens Braun infect host plants by directly producing germ tubes, rather than producing zoospores. In order to understand how expre ssion of genes are regulated upon induction of sporangia germination, effort i s made in three aspects in the present study: (1) setup of methods for inducti on of germination, (2) cloning of the actin and calmodulin gene, as well as th e partial sequences of the rRNA gene of P. splendens, and (3) analysis of the mRNA and protein profiles of sporangia at different time intervals after induc tion of sporangia germination. In the first part, it is found that germinatio n of sporangia occurs only after addition of 10% CV8 broth. Formation of germ tube begins 60 minutes after addition of CV8 broth. It then elongates at a me asurable speed and attains a length 3-5 fold the diameter of sporangia by 120 min. Germination of sporangia proceeds in CV8 broth in a way similar to what is observed in planta, indicating that CV8 broth is an appropriate inducer for sporangia germination. In the second part, the full-length cDNA sequences of actin and calmodulin genes of P. splendens are obtained by 5'- and 3'-RACE, a nd partial sequences of the 18S rRNA gene by PCR. Analysis of these sequences indicate that they are closely related to the corresponding genes of other or ganisms. Besides, Southern hybridization analysis is performed, and it is fou nd that there exists one copy of calmodulin gene and at least 2 copies of acti n genes in P. splendens. In the third part, as a first step to uncover the me chanism of gene regulation in the process of sporangia germination, expression of the actin and calmodulin gene are analyzed. Results from Northern hybridi zation analysis indicated that, while the amount of 18S rRNA and actin message remains unaltered throughout the process of germination, it is obvious that t he amount of calmodulin mRNA changes as germination of sporangia proceeds. Sp orangia obtains immediately after the preparation process carry the highest am ount of the calmodulin message. As germination of sporangia induced and the g erm tube elongating, it decreases gradually. This observation suggests that c almodulin may play some role in induction of sporangia germination. In order to know whether the amount of protein changes during germination, protein prof ile is also analyzed by SDS-PAGE. The result indicates that protein pattern c hanges overtime as germination of sporangia proceeds.