Full-length cDNA Library and EST Analysis on Pesudocohnilembus persalinus
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摘要: 水滴伪康纤虫为海水养殖鱼类的重要危害性寄生虫。采用SMART技术构建营养期水滴伪康纤虫全长cDNA文库，初始文库滴度3.3×106pfu·mL-1，文库容量约为1.5×107 pfu，重组率97%。随机挑取24个克隆，经PCR检测插入的片段长度在0.3~2.0 kb，平均长度>500 bp。挑选文库中的1 200个克隆进行表达序列标签测序，得到1 032条高质量的EST，与NCBI数据库进行比对后，获得292个同源基因，其中包含190条单拷贝EST和102个重叠群，冗余度为71.7%。有215条EST在Tetrahymena thermophila，Paramecium tetraurelia，Ichthyophthirius multifiliis，Cryptocaryon irritans，Oxytricha trifallax等自由生活及寄生种类的纤毛虫的基因组中比对到同源基因，其中有134条EST具有功能注释，其余为未知功能的假定蛋白基因。水滴伪康纤虫cDNA文库的构建及EST测序的完成，为进一步发现和研究水滴伪康纤虫的功能基因奠定了基础。Abstract: Pesudocohnilembus persalinus is a harmful parasite found in marine fish. Using SMART technology, a full-length cDNA library of P. persalinus was constructed. Titer of the primary library was 3.3×106 cfu·mL-1, and the library capacity was 1.5×107 cfu with a recombination rate of 97%. A PCR amplification on 24 random clones revealed that the inserted cDNA fragments ranged from 300-2 000 bp, with an average length over 500 bp. Subsequently, 1 200 clones were selected from the library for EST sequencing. As a result, 1 032 high-quality ESTs were obtained and assembled into 292 unigenes including 102 contigs and 190 unique ESTs with a redundancy rate of 71.7%. In a blast analysis against NCBI database, 215 of the unigenes were homologous to the corresponding genes of ciliates with free-living and parasitic lifestyle, such as Tetrahymena thermophila, Paramecium tetraurelia, Ichthyophthirius multifiliis, Cryptocaryon irritans, Oxytricha trifallax, etc. But, only 134 ESTs had been annotated. The remainders were hypothetical protein genes. The results obtained from this study would substantially facilitate the molecular cloning and characterization of the functional genes in P. persalinus.
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Key words:
- Pesudocohnilembus persalinus /
- cDNA library /
- EST sequencing
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图 1 水滴伪康纤虫总RNA(A)及双链cDNA(B)的凝胶电泳
注：M为DL2000 DNA marker; a为total RNA; b为ds cDNA。
Figure 1. Agarose gel electrophoresis of total RNA (A) and double strand cDNA (B) from P. persalinus
图 2 24个随机克隆中插入片段扩增产物的凝胶电泳
注：M为DL2000 DNA marker; 1~24为positive clones。
Figure 2. Agarose gel electrophoresis of inserted fragments in 24 random clones
图 3 EST的长度分布(A)及单一序列的冗余度(B)
Figure 3. Distribution of lengths(A) and redundancy(B) of singlets of ESTs
表 1 部分EST序列的功能注释及与GenBank中同源比对
Table 1. Function annotation and homology of partial EST sequences compared with data from GenBank
编号 长度 功能注释 同源性/% E值 Pp-10-B02 1131 磷酸烯醇式丙酮酸羧激酶 94 7.00E-152 Pp-109-D12 731 NADH脱氢酶亚基7 81 7.00E-174 Pp-A3-C01 1220 3-磷酸甘油醛脱氢酶 74 1.00E-168 Pp-A82-B11 1220 多聚泛素5 93 3.00E-136 Pp-E5-E01 1068 组织蛋白酶L样半胱氨酸蛋白酶 51 2.00E-87 Pp-B58-B08 1073 组织蛋白酶B 92 0 Pp-G91-C12 669 2型甲硫氨酸胺基肽酶 65 4.00E-77 Pp-I36-D05 1204 粪卟啉原Ⅲ氧化酶 56 2.00E-111 Pp61-E08 662 磷酸吡哆醛依赖转移酶 78 9.00E-61 Pp0382 1114 肌醇磷酸合成酶 67 9.00E-125 Pp-K73-A10 781 丙氨酰-tRNA合成酶 72 1.00E-119 Pp-A1-A01 1147 热休克蛋白90 69 1.00E-106 Pp-B10-B02 436 延长因子2 71 1.00E-31 Pp-D20-D03 472 核苷结合外膜蛋白 76 5.00E-49 Pp-D34-B05 1225 醌氧化还原酶 77 0 Pp-D4-D01 976 ATP合成酶γ亚基 51 5.00E-81 Pp-D53-E07 455 肽酰-脯氨酰-顺反式异构酶 69 1.00E-32 Pp-E56-H07 811 电压门控性钾通道β2亚基 77 5.00E-125 Pp-F5-E01 547 转录因子Dp-1 62 8.00E-23 
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