Construction Of Ethephon-induced Latex SSH CDNA Library And Clone And Expression Analysis Of EtIe1 Gene From Hevea Brasiliensis

A SSH library of differently expressed cDNA was constructed for the purpose of analyzing the molecular mechanism underlying higher latex yield stimulated by Ethephon in Hevea brasiliensis, employing two kinds of latex which produced under ethephon stimulation for 12 hr, and the control without any treatment. 118 positive clones were obtained using the technique of screening the blue-white colony and PCR, in which the 25 selected positive clones were sequenced, indicating 5 ESTs fall into no-clued unknown sequences, 6 for the deduced proteins with unknown functions, 2 for the unknown function proteins, and 3 have high similarity with the known corresponding gene respectively, through blastx analysis in Genbank. Other clones fall into deduced protein genes with unknown functions or unknown function protein genes mostly.Subsequently the related researches on EST203 sharing high similiarity with SAG102(senescence associated gene102 )in Arabidopsis thaliana were carried out and a full–length 521bp cDNA was cloned using SMART RACE technology(rapid amplification of cDNA ends technology). The results indicated it has two non-coding sequence region which has 144bp UTR on the 3'terminal and 107bp UTR on the 5'terminal of cDNA respectively. The Open Reading Frame has 270 bp encoding a peptide comprising 89 amino acids, The analysis of EtIe1 gene structure indicated it has two extron and one intron which comprises 158bp,and the analysis of its structural domain showed it has an unkown functional domain DUF581. The results of BLASTN and BLASTX showed the nucleotide identity to SAG102 is 45% and the amino acid identity to SAG102 is 48.9% respectively.RT-PCR analysis showed that the EtIe1 gene expression can be regulated by ethephon.Under the condition of application of ethephon, the EtIe1 gene expression can gradually increase within 24 hours, but no transcripts were detected in normal latex. Ethephon can improve the process of latecifer senescence and SAG102 is identified to be involved in the regulation of senescence of Arabidopsis plant. It is speculated that the gene EtIe1 may be involved in the regulation of Eth-induced senescence of latecifers in Hevea brasiliensis. The dissection of EtIe1 gene functions will be helpful to understand the molecular regulation of ethyele signal transduction in Hevea brasiliensis...