| Zostera marina L.,as one of the common seagrass, belongings to Helobieae, Potamogetonaceae, Zostera and is an important submerged saline plant. To investigate stress-tolerant mechanism and discover stress-tolerant genes of Z. marina, in this paper we contruct a cDNA library which were teated with different concentration of salinity, using SMART (Switching mechanism at 5'end of RNA transcript method) cDNA library construction method. In addition, 5'-sequencing and the bioinformatics analysis of single clones were performed.Using mixed mRNA isolated from Z. marina samples which were teated with three concentration of salinity, we construct a full-length cDNA library from leaves tissues. The titer of the primary cDNA library was 8.675×10~6 pfu/mL, and that of the amplified library was 1.03×10~9pfu/mL. The percentage of recombinant clones was 97.19% and the length of inserted cDNA fragments was about 750～2000bp with averaged length of above 800bp.The 4081 single clones were randomly selected to be sequenced from 5'-end, and yielded 3986 high-quality expressed sequence tags. These ESTs were assembled into 145 contigs and 1790 singletons, representing 1935 unigenes. The accession numbers in NCBI dbEST dataase are from HS089944–HS091857 and JG698933- JG699246. Accroding to the analysis of abundantly expressed ESTs, metallothionein (MT) and chlorophyll a/b-binding protein (Lhc) were the two most abundant genes, which accounted for 10.2% and 1.9%. Bioinformatics analysis shows that 1427 unigenes had significant homology to known genes against the NCBI nr database by BlastX. 37.5% of 1935 uinigenes was not annotated, which may be the novel genes. Compared to the Clusters of Orthologous Groups (COG) categories, 452 unigenes were annotated. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG), 525 unigenes were assigned into 241 pathways (E value≤10-5).,and a carbon fixing pathway invovlved C3/C4/CAM pathway was profiled. For Gene Ontology (GO) hierarchy analysis, a total of 574 unigenes were assigned into 740 GO terms. For InterProScan searches, 982 InterPro familes were recognized. The successfully construction of Z. marina leaf tissues cDNA library is an initial key work for screening and cloning of new genes in the future.MT and LHC gene family were chosen to be done the bioinformatics analysis. Twelve LHC encoded genes were discovered in this library, named by Lhca1,Lhca2,Lhca3,Lhca5,Lhca6,Lhcb1.1,Lhcb1.2,Lhcb2,Lhcb3,Lhcb4,Lhcb5,Lhcb6, belonging to Lhca1,Lhca2,Lhca3,Lhca5,Lhca6 of PSⅠand Lhcb1,Lhcb1,Lhcb2,Lhcb3,Lhcb4,Lhcb5,Lhcb6 of PSⅡgene family, repectively. The motifscan analysis showed that all of these twelve proteins owned the chlorophll a/b binding domain. Lhcb1.1 and Lhcb2 shared the highest similarity about 75%. According to the phylogenic tree contruction analysis indicates that taxonomic distribution of most Z. marina LHC showed more similarity with dicotyledons than monocotyledons.Three metallothionein genes were found in the library, named by Mtlp-Zm2.1, Mtlp-Zm2.2 and Mtlp-Zm3。The fomer belong to type2 metallothionein, and Mtlp-Zm3 belongs to type3 metallothionein. Based on the results of sequenced analysis, Mtlp-Zm2.1 encodes a protein of 62 amino acids, which owned 8 cysteines, accounting for 12.9% of total amino acids. The protein molecular weight was 6.3KD and theoretical isoelectric point was 4.65. Mtlp-Zm2.2 encodes a protein of 82 amino acids, which has 14 cysteines accounting for 17.1% of total amino acids. The protein molecular weight was 8.2KD and theoretical isoelectric point was 5.18. The phylogenic tree analysis indicated that Mtlp-Zm2.2 f had high level similarity with submerged saline plants., which has ealier evolutionary position than land plants and aquatic plants. Key words: Zostera marina; full-length cDNA library; expressed sequence tags;... |
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