Abstract
Nuf2p, a centromere-associated protein, is an indispensable component of Ndc80 complex which is important for stable microtubule-kinetochore attachment, chromosome pairing and spindle assembly in mitosis. Based upon contig sequences coding for Nuf2p from a pyrosequencing transcriptome of Saccharina japonica, primers were designed for molecular cloning of this kinetochore protein. The open reading frame (ORF) of this gene SjNuf2 was 1,452 bp long, encoding a putative protein consisting of 483 amino acids. Multi-sequence alignment showed SjNuf2p possessed several conserved domains of Nuf2, Spc7, SPT2 and MIT. To illustrate the location of SjNuf2p using immunocytochemistry, its ORF was inserted into a prokaryotic expression vector pET-28a. Then the recombinant SjNuf2p (rSjNuf2p) was induced to be expressed in Escherichia coli. The rSjNuf2p was verified by Western blotting with the commercial anti-6His-tag antibody and then purified using affinity chromatography. The polyclonal antibody of anti-SjNuf2p was raised against the purified rSjNuf2p. Western blotting with this prepared antibody showed the presence of SjNuf2p in the gametophytes of a Saccharina cultivar. SjNuf2p and alpha/beta-tubulin were subsequently co-localized to the paraffin sections of kelp chromosomes with their corresponding antibodies, thus illustrating that SjNuf2p could reside at the kelp centromeres. This study laid a solid foundation for kelp centromere and karyotype analyses.
Highlights
BLASTx searching result showed that the similarity of this cloned gene with other Nuf2 genes was interestingly low, and the highest only 58.37% of identity was found between SjNuf2 and its homolog (GenBank No CBN74724.1) from Ectocarpus siliculosus that belonged to brown algae
According to our previous explorations of kelp cytogenetics using FISH technique (Liu et al, 2012; Liu et al, 2017; Yang et al, 2017), we propose that centromere is a breakthrough point for karyotype analysis of S. japonica
A lot of publications uncover the varied centromeric DNA in different organisms, but the kinetochore proteins assembled at the centromeres are evolutionarily conserved (Richards and Dawet, 1998; Jiang et al, 2003; Przewloka and Glover, 2009; Comai et al, 2017)
Summary
W. Saunders (=Laminaria japonica Aresch.) are numerous in number and small in size (Evans, 1963; Yabu and Yasui, 1991; Lewis, 1996). Saunders (=Laminaria japonica Aresch.) are numerous in number and small in size (Evans, 1963; Yabu and Yasui, 1991; Lewis, 1996) Their features, short rod-like or even dot-like shaped, prevent the karyotype analysis using routine cytological methods only. After the enzymatic treatment for kelp chromosome preparation and the staining with 4 ,6-diamidino-2-phenylindole (DAPI), Liu et al (2012) successfully localized a female-specific marker on one of the kelp gametophyte
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