شناسایی ژن پروتئین اصلی اسپرم در جمعیت خوی نماتد سیستی چغندر

Nematode sperm uses an actin-like ameboid motility which is mediated by a dynamic cytoskeleton composed of major sperm protein (MSP). This basic protein of spermatozoa is 14.5-kDa and makes up 15–20% of the total protein in nematode sperm, but it is not found in any other cells of the body. The aim of this study was identification and molecular characterization of the major sperm protein (msp) gene in beet cyst nematode. By using the nucleotide sequence of msp gene in 15 different species of nematodes, deposited in NCBI database, a pair of degenerate primers was designed. RNA was extracted from 300 male specimens. The cDNA was synthesized and used as PCR template. The amplified 299 bp fragment was cloned into pTZ57R/T vector and then was transformed into Escherichia coli strain DH5α. The recombinant plasmids were characterized following by bi-directional sequencing using universal M13 primers. Afterwards, the obtained nucleotide sequences was used to design primers required by the RACE technique for identification and isolation of 5′ and 3′ untranslated regions (UTR). The 5ˊ- and 3ˊ-RACE, which produced two and one fragments respectively, were cloned and sequenced. The full-length cDNA of msp was obtained following sequence assembly. The larger fragment named msp1-khoy (GenBank accession number MH282870) and the smaller fragment named msp2-Khoy (GenBank accession number MH282871) contained a 129 and 93 bp of the 5ˊ-UTR respectively, while both fragments contained a similar ORF with 381 bp and 215 bp at 3ˊ-UTR followed by a poly(A) tail. In addition, the 3ˊ UTR contained a typical polyadenylation signal (AATAAA). In order to isolate the genomic fragment, new primers were designed, based on the flanking region of the ORF, and the PCR product was cloned and sequenced. Aligning of the genomic nucleotide sequence to the corresponding cDNA sequence showed that Hs-msp genomic fragment contained a single intron of 55 bp in length. This intron located between the nucleotides 96 and 97 and followed the ‘GC-AG rule’ for splicing.