| Summary: | 碩士 === 國立臺南大學 === 生物科技學系碩士班 === 100 === LRWD1（NCBI accession no.BC030547）gene has been highly expressed in testis but
low expressed in Sertoli cell-only syndrome victims. Therefore we suggest LRWD1might play
important role in spermatogenesis. In the compintatioal prediction, it shows that LRWD1
contains one LRR and three WD40 sequences. We explored the role of LRR and WD40
repeating sequence in LRWD1, as well as the role of LRWD1 involved in spermatogenesis.
We contstructed two vectors （p3x-FLAG-CMV-14 and pEGFP-N3） with lacking LRR,
WD1, WD2, WD3 and 3xWD regions by specific point mutation. Then transfected these two
vetors to NT2/D1（a cellular expressing protein） respectively. We found the stability of
LRWD1 protein is significantly decreased while losing the LRR domain （ half-life :
LRWD1wild, 8.9 hr; LRWD1△LRR , 5.1 hr）. Then we added the inhibitor of protein kinase,
MG132. The LRWD1wild protein was more stable than LRWD1△LRR. We applied flow
cytometry to detect cell number and fluorescence intensity of EGFP cells. Therefore we found
the cell numbers at strong-flourescence region of LRWD1△LRR are less than LRWD1wild , and
the cell numbers at weak-flourescence region of LRWD1△LRR are less than LRWD1wild .
Fluorescence intensity of strong-flourescence region was weak in LRWD1△LRR than in
LRWD1wild . In immunofluorescence study, the weaker fluorescence intensity of EGFP was
observed in LRWD1△LRRcompared to LRWD1wild on the same exposure time. The LRWD1△
LRR protein and bring out the low level of fluorescence intensity. LRWD1wildprotein could
bind to DNAAF2 protein ,We suggested that LRWD1 protein may involve in forming the cilla;
LRWD1△LRR, LRWD1△WD1, LRWD1△WD2, LRWD1△WD3 and LRWD1△3xWD protein could
bind to TNFRSF1B protein.In addition, mutated LRWD1 may be involve in apoptosis. These
binding-protein studies of LRWD1 provides us a further understanding of the role of LRWD1
in spermatogenesis.
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