Effect Of ARM Domain Deletion On SPAG6Subcellular Localization In Eukaryotic Cell

Objective：To construct the eukaryotic plasmid containing the SPGA6(full length anddifferent ARM domains sequences), express the protein in the mammalian cell，andfurther examine the effect of armadillo (ARM) domains on Sperm-associated antigen6(SPGA6) localization in CHO cells.Methods:1. Looking for SPAG6protein in mice conservative functional areas, analyzethe full-length protein sequences, retrieving the SPAG6seven ARM domains;2. cDNAlibrary was constructed by reverse transcription with testicular tissue from healthy,matured male mice;3. The SPAG6coding sequences (C-terminal＆full length) wereamplified by PCR with the cDNA template; The sequences coding different ARMdomains were amplified by polymerase chain reaction (PCR);4. the sequences wereanalized after the TA Clone;5. The correct SPAG6and different ARM domainssequences were subcloned into the pEGFP-N2vector．These plasmids were transfectedinto CHO cells．6. The expression of the GFP-tagged SPAG6proteins was proved byWestern blot analysis, and localization of GFP-tagged full length and truncated SPAG6proteins was observed by using laser scanning confocal microscopy.Results: Truncated SPAG6proteins with series deletion of ARM domains weresuccessfully constructed into the mammalian expressing vector carrying the greenfluorescent protein gene, pEGFP-N2． The expression of the fusion protein wasconfirmed by Western blot analysis. The full length GFP-tagged SPAG6protein was1ocalized on the microtubule in the transfected CHO cells, but none of the truncatedproteins was localized on the microtubule.Conclusion: Microtubule localization of mouse SPAG6protein is dependent on fulllength sequence. Construction of the plasmids expressing full length and truncatedSPAG6proteins provides an important tool for further study SPAG6function.