Cloning, prokaryotic expression, and bioactivity of the calmodulin gene of Magnaporthe grisea.
The cDNA encoding the calmodulin (CaM) protein was isolated from the mycelia of Magnaporthe grisea PO-041 strain by reverse transcriptase (RT)-PCR and named MgCaM. High similarities in sequence to other reported CaMs suggested that CaM is highly conserved. The MgCaM gene was expressed in Escherichia coli BL21 using a pET32a(+) plasmid. MgCaM with or without a Trx tag exhibited a Ca(2+)-dependent electrophoretic migration shift, suggesting that the activity of MgCaM depends on the presence of Ca(2+). Western blot analysis showed that Trx-MgCaM possessed specific immunoreactivities and could be specifically recognized by an anti-AtCaM2-Ig polyclonal antibody raised against AtCaM2 of Arabidopsis. Immunocytolocalization of MgCaM indicated that the number of gold particles was the most in germ tubes, next to conidia, followed by appressoria and the mycelia from liquid media, and the least in the aerial hypha from solid media. Also, in the germ tube, mycelium from liquid media and appressorium, the density of gold particles in the cell wall was markedly higher than that in the cytoplasm. In conidia, the number of MgCaM particles in the cytoplasm was obviously higher than that in the cell wall. However, in the aerial hypha, MgCaM only distributed at the cytoplasm and no gold particle was detected in the cell wall.
Ma
Zhi-bin
ZB
The Life Science College, Hebei Normal University, Shijiazhuang, China.
Zhao
Jun-xia
JX
Wang
Li-an
LA
Zheng
Xiao-bo
XB
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D020082:Magnaporthe / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
D002147:Calmodulin; D005656:Fungal Proteins