【摘要】 采用改进的一步法提取坛紫菜丝状孢子体的总RNA,构建了坛紫菜丝状孢子体的cDNA文库,对测序得到的EST进行分析并与条斑紫菜的EST相比较。结果表明,cDNA文库共包含2.5×105个克隆,测序得到490个EST,其中275个与已知功能基因同源,占56.1%,260个EST与已知的条斑紫菜EST同源,占53.0%。使用GO(Gene Ontology)分类方法对275个EST进行分类,其中226个可归属于细胞组分(Cellular component)、分子功能(Molecular function)和生物学过程(Biological process)三个大类,而其余49个则为未知功能的EST。挑选出与条斑紫菜同源性高的28个EST,这些EST所包括的密码子数为3221个,发现密码子第三位的平均GC含量远高于第一位的平均GC含量和第二位的平均GC含量。统计坛紫菜丝状孢子体3221个密码子的使用频率,发现同义密码子第三位碱基偏好使用G或C。更多还原

【Abstract】 Porphyra haitanensis is only cultured in south China. The yield of P. haitanensis is much larger than that of P. yezoensis, but the economic value of P. haitanensis is much less than that of P. yezoensis. Therefore, it is important and helpful to study on P. haitanensis in order to improve the quality and economic value of P. haitanensis.Polysaccharide is abundantly contained in P. haitanensis. Because physical and chemical characters of polysaccharide are similar to those of RNA, it is difficult to extract RNA from P. haitanensis. In this paper, RNA was extracted by improved one-step method. Ethanol was slowly added to the aqueous phase until the concentration of ethanol reached 10%—20%. After centrifugation, polysaccharide was precipitated and RNA remained in the solution. In addition, high concentration of LiCl solution could also remove some polysaccharide. mRNA was separated by Oligotex mRNA Kits (QIAGEN).cDNA library of filamentous sporophyte of P. haitanensis was constructed. Recons from cDNA library were taken out on Petri dish which contained penicillin, X-gal and IPTG. White colonies were taken to 96-well plates and cultured over night until OD_ 600 reached 0.6—0.8. Plasmids were extracted and examined by electrophoresis. ET kits (Pharmacia) were used in the reactions for sequencing and the products of reactions were precipitated and purified. Dissolved with 8μl loading buffer (Pharmacia), the products were sequenced on MegaBace1000.Low quality sequences, vector sequences and repeated sequences of ESTs were removed by Cross match and ESTs were clustered into contigs by Phrap. 91 contigs and 399 singlets were obtained.Each sequence from the 490 contigs and singlets was subjected to similarity search against the NCBI-provided non-redundant protein database, nr, using the BlastX program. 275(56.1%) of the 490 clustered ESTs showed sequence similarity to genes registered in the public databases (e-value<1×10 -5) and new sequences occupied 43.9%. 275 contigs and singlets were classified via GO (Gene Ontology) method, among which 226 contigs and singlets could be classified into three sorts: cellular component, molecular function and biological process. The function of the other 49 contigs and singlets were unknown.Similarity search against over 20 000 ESTs of P. yezoensis showed that 260 (53.0%) of the 490 clustered ESTs showed sequence similarity to ESTs registered in the GenBank. 28 ESTs showed sequence similarity with the ESTs of P. yezoensis were selected and 3221 codons were contained in these ESTs. The average GC content of the third letter in the codons (73.1%) is much higher than that of the first letter (42.0%) and the second letter (58.5%). Frequency of usage of 3221 codons was analyzed. Research results shows that the GC content and usage frequency of ESTs from P. haitanensis and P. yezoensis are quite homological though they live in different environment and belong to different genus.更多还原