蜂王受精囊腺cDNA文库的构建与分析

Construction and analysis of a cDNA library from queen honeybee spermatheca gland

从400只人工培育的处女蜂王中解剖受精囊及其腺体,利用其mRNA构建了一个cDNA文库.该文库的滴度为1.1×106,重组效率大于99%.进一步对文库克隆进行小规模测序和分析,获得了一批编码蜂王受精囊腺内溶蛋白的基因克隆,同时从中也检测到编码3种蜂毒蛋白(明肽、镇静肽和icarapin)、2种王浆蛋白(MRJP8-9)以及睾丸增强因子等克隆.

Spermatheca is a tissue in queen honeybee for storing the sperm from the drone honeybee in copulation.The long term sperm storage in spermatheca is related to the secretion of spermathecal gland.Gene expression and regulation of spermathecal gland is a basement for understanding the mechanism of long term sperm storage.In this study,four hundred queen honeybees were reared artificially.The spermathecal gland of queen honeybee was dissected under microscope for isolating the total RNA and mRNA.The double strands cDNA were synthesized using the SMART（switching mechanism at 5′ end of RNA transcript） technology and then used to construct a cDNA library of spermathecal gland.The titre of the cDNA library was about 1.1×10^6.The recombination rate of the cDNA library was over 99%.Many clones coding the spermathecal fluid protein were obtained by small scale sequencing and analyzing the cDNA library clones.Among them three clones coding the honeybee venom protein of apamin,secapinand icarapin,two major royal jelly protein clones（MRJP8 and MRJP9） and testis enhanced gene transcript clone were also detected.These works will be helpful for understanding the mechanism of long term sperm storage in spermatheca.