| Summary: | 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.
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