杂色鲍Fosmid基因组文库构建与血蓝蛋白基因筛选

以杂色鲍Haliotis diversicolor肌肉组织为材料,按照CopyControlTM Fosmid Library Production Kit中方法构建了杂色鲍Fosmid全基因组文库.从文库中随机挑取50个克隆进行NotⅠ酶切鉴定发现,阳性率达到100%,插入片段大小分布在32～48 kb之间,平均长度约37.6 kb.随机挑取8个克隆进行正、反向末端测序,获得了全部16个测序结果,其中8条序列确定为鲍来源基因序列.进一步根据杂色鲍血蓝蛋白(Hemocyanin)基因3’端序列设计特异引物,用PCR方法对基因组文库进行筛选,获得一个阳性克隆,经Primer-Walking测序、Blast比对和进化分析证实,该序列为杂色鲍血蓝蛋白Ⅱ型基因克隆.

Construction of Fosmid Genomic Library of Haliotis diversicolor Reeve and Screening of Hemocyanin Gene

The foot muscle of small abalone,Haliotis diversicolor was used to construct the Fosmid genomic library according to the CopyControlTM Fosmid Library Production Kit.Fifty clones were randomly picked from library and identified by NotⅠ digestion.The positive rate was 100%.Lengths of these inserts were between 32 to 48 kb and with the average length 37.6 kb.Another 8 clones were randomly picked to do the end sequencing.With total 16 sequence results,8 of them were sure from abalone genomes.Further more,specific primers were synthesized according to the 3′ end sequence of hemocyanin gene of small abalone.With the PCR(Polymerase Chain Reaction) based screen method,a positive clone was obtained and identified as the hemocyanin type Ⅱ gene clone after Primer-Walking sequencings,Blast and phylogenetic tree analysis.