扇贝防御素基因改良5′cDNA末端快速扩增聚合酶链反应筛选方法的研究

目的应用改良5′cDNA末端快速扩增聚合酶链反应(RACE)筛选扇贝抗菌肽基因,寻找基于基因序列同源性的双壳类抗菌肽(AMP)的筛选方法。方法 TRIzol法提取扇贝血淋巴中总RNA。根据贻贝抗菌肽的保守序列设计基因特异性简并引物,进行改良5′RACE钓取可能防御素基因cDNA的5′端序列、AT克隆、DNA测序和同源性分析。结果采用改良5′RACE从扇贝血淋巴RNA中得到多个未知基因的cDNA 5′端序列,挑选600bp以下的基因片段进行AT克隆和DNA测序后得到3个插入片段序列,其长度分别为257、275和511bp。通过BLAST程序搜索GenBank核酸数据库,未发现与之高度同源的基因。结论从扇贝血淋巴中获得的3个5′端cDNA序列可能属于新的基因。应用改良5′RACE能钓取含防御素保守序列的新基因片段,由此表明此方案具有可行性。

Study of screening for defensin genes in scallop by improved rapid amplification of cDNA ends

Objective To screen AMP genes with improved 5＇ RACE,to find out a feasible method for screening AMP genes from scallop based on gene homology. Methods Total RNA was obtained from scallop hemolymph by TRIzol method. Gene specially degenerated primer was designed according to the conserved sequence of MGDs. The 5r end sequences of possible defensin genes could be fished out with improved 5＇ RACE and their positive clones were sequenced. The homology of sequences at 5r end was blasted in GenBank. Results Several unknown cDNA 5＇ end sequences were obtained from scallop hemolymph RNA with improved 5＇ RACE. The eDNA fragments under 600 bp were AT cloned and three cDNA 5＇ end sequences were obtained, with the lengths of 257,275 and 511 bp, respectively. There was no similar genes in homology with these eDNA 5＇ end sequences in GenBank. Conclusion Three eDNAs 5＇ end sequences from scallop hemolymph could be new genes. It is available to use improved 5＇ RACE to screen gene fragments containing defensin conserved sequence.