Construction Of CDNA Library And Analysis On Expressed Sequence Tags Of Genus Of Lycoris Plant

The generation of expressed sequence tags (ESTs) has been proven to be a rapid and efficient approach by which to identify the novel genes and analyze the function of genes.To this aim,we constructed two cDNA libraries from wild leaf and mutant leaf of Lycoris awrea,named LAU-CK,LAU-MU respectively.The titer of LAU-CK is 1.2 X 106pfu/mL,and the recombinants accounted for 93.7%.The titer of LAU-MU is 5.6X 105pfu/mL, and the recombinants accounted for 96.0%.The insert fragment length of the two libraries both ranged from 500bp to 3000bp.From the two libraries, 1606 ,2132 5'ESTs were sequenced respectively.Both 1606 and 2132 ESTs from Lycoris aurea, and together with 4992 5'ESTs from Lycoris longituba were analyzed. 4687 ,1312 and 1810 effective ESTs were isolated by bioinformatical analysis from Lycoris longituba, LAU-Ck and LAU-MU respectively.The average length of effective ESTs was 545bp,403bp and 437bp respectively;The average quality was 39.28, 39.54 and 40.13 respectively;The average GC content was 39.46, 48.33 and 47.51 respectively. 4624,1304 and 1800 ESTs from effective ones have been subscribed to GenBank and gotten the Acce.No..The Phrap software was used for contig analysis of all the ESTs. From the 4687 effective ESTs of Lycoris longituba bud,a total of 2310 different contigs were obtained ,1343 of the 2310 contigs were composed of only one EST,and such contig was also called singlet;735 contigs were obtained from the 1312 effective ESTs of wild leaf of Lycoris aurea,among which 535 contigs were singlets;879 contigs were obtained from the 1810 effective ESTs of mutant leaf of Lycoris aurea,among which 574 contigs were singlets.All the contigs also called unique genes,which were identified by redundancy analysis.In the Lycoris longituba bud,wild leaf and mutant leaf of Lycoris aurea 1343(58.1%),535(72.8%) and 574(63.3%) of contigs presented low expression abundance genes respectively;788(34.1%), 152(20.7%) and 234(26.6%) of them presented moderate expression abundance genes respectively;! 79(7.8%),48(6.5%) and 71(8.1%) presented high expression abundance genes respectively.The results indicated that most genes in the genus of Lycoris were low or moderate expression abundance genes.4687 effective ESTs of Lycoris longituba bud were compared with the sequences in the non-redundant protein and nucleotide databases of NCBI,31%(1458/4687 ) ESTs sharing significant similarity with sequencesregistered in these databases after being analyzed using BLASTX and BLASTN program.Out of these,3.3%(l 56/4687) ESTs were transposable elements (TEs),which was proposed to be related to various flower color and form of Lycoris longituba.At the same time,the gene of AGAMOUS which is a MADS-box gene was identified.Genes with function annotation or putative function annotation were assigned to 11 functional gene classes by the means of function classification in Arabidopsis.The most were genes related to protein synthesis ,the others were ones related to energy metabolism,signal transduetion etc.The high abundance expression genes were polyprotein genes,retrotransposons related protein genes,and so on.BLAST search showed that 74.9% (983/1312) and 78.1% (1413/1810) effecive ESTs had homologues in GenBank of wild leaf and mutant leaf of Lycoris aurea respectively. By the means of function classification in Arabidopsis,the most were genes related to protein synthesis,the others were energy metabolism,cell defense,cell structure ,ogranization and biogenesis,and signal transduetion etc.There were differences in expression of protein synthesis genes,cell defense genes and miscellaneous genes expression abundance bewteen wild leaf and mutant leaf of Lycoris aurea.ln the wild leaf of Lycoris aurea the protein synthesis genes and cell defense genes expression abundance were higher than that of the mutant leaf of Lycoris aurea,and the miscellaneous genes expression were lower than that of the mutant leaf of Lycoris aurea.This result indicated that mutation resulted in ability of protein synthesis and cell defense was declining...