ASY （asynaptic） is a synaptonemal complex （SC） related protein in plant mei- osis. Some ASYs in plants have been cloned and functional determined. ASY in Brassica napus has not been sequenced and functional certi...ASY （asynaptic） is a synaptonemal complex （SC） related protein in plant mei- osis. Some ASYs in plants have been cloned and functional determined. ASY in Brassica napus has not been sequenced and functional certificated. Here, we first cloned ASY gene in Brassica napus （BnASY） and inserted it into vector pET-32a for expression fusion protein BnASY-6His in Escherichia coli BL21 （DE3）. Purified fusion protein was used to produce polyclonal antibody. Specificity and application of polyclonal antibody was examined by Western blot and immunofluorescence assay. Results showed that BnASY-6His recombi-nant protein and anti-BnASY antibody were successfully obtained. Polyclonal anti-BnASY antibody had high specificity. Results of immunofluorescence showed that ASY signals appeared at leptotene stage, faded away gradually at pachytene stage and almost disap-peared at diplonema stage. In this study, highly specific polyclonal antibody was success-fully acquired and used for immunofluorescence assay in plant cells. It will contribute to functional research of ASY in B. napus.We thank Zhigang Li for providing pET-32a vector and Escherichia coli BL21 pLysS strains.展开更多
基金This work was supported by National Natural Science Foundation of China （31671733,31400243 and 31201152）, Natural Science Foundation of Hubei Province （2013CFB423 and 2014CFB320）, Major Research Project of CAAS Science, Technology Innovation Program and the Cen-tral Public-interest Scientific Institution Basal Research Fund.
文摘ASY （asynaptic） is a synaptonemal complex （SC） related protein in plant mei- osis. Some ASYs in plants have been cloned and functional determined. ASY in Brassica napus has not been sequenced and functional certificated. Here, we first cloned ASY gene in Brassica napus （BnASY） and inserted it into vector pET-32a for expression fusion protein BnASY-6His in Escherichia coli BL21 （DE3）. Purified fusion protein was used to produce polyclonal antibody. Specificity and application of polyclonal antibody was examined by Western blot and immunofluorescence assay. Results showed that BnASY-6His recombi-nant protein and anti-BnASY antibody were successfully obtained. Polyclonal anti-BnASY antibody had high specificity. Results of immunofluorescence showed that ASY signals appeared at leptotene stage, faded away gradually at pachytene stage and almost disap-peared at diplonema stage. In this study, highly specific polyclonal antibody was success-fully acquired and used for immunofluorescence assay in plant cells. It will contribute to functional research of ASY in B. napus.We thank Zhigang Li for providing pET-32a vector and Escherichia coli BL21 pLysS strains.
