Cloning And Function Analysis Of The LeEIN2 From Tomato

The phytohormone ethylene is a very important regulator in plant growth and development. Now the researches on ethylene signal transduction pathway and the regulation mechanism have received considerable attention in plant molecular biology. As a central signal transducer in ethylene signaling, EIN2 mediated an essential step in the signal transduction between the CTR1 and EIN3. Furthermore, EIN2 may also mediate other phytohormone and the stress factors signaling. So it is significant to identify the function and the mechanism of EIN2 in signal transduction pathway. So far, there is no report about the isolation of full-length EIN2 from tomato, which is the most studied model for fruit ripening. Study on EIN2 in tomato will make us better understand the mechanism of ripening-related genes expression regulated by ethylene and further elucidate ethylene transduction pathway.A full-length cDNA, named LeEIN2, was cloned from wild-type tomato (Lycopersicon esculentum) by RT-PCR and RACE. Degenerated primers were designed based on the conserved sequences in EIN2 cDNAs. Then the genomic DNA sequence of LeEIN2 was isolated by LA (Long and Accurate) PCR and Inverse PCR. This DNA sequence was 6838 bp including six introns. The LeEIN2 cDNA was 4343 bp and contained a 3951 bp open reading frame, encoding a polypeptide of 1316 amino acids with a molecular mass of 142.63kD and dimorphic structure. The protein sequence predicts 11 transmenbrane helices. Comparison of this protein sequence with those of Arabidopsis and Petunia showed that the amino acid homology was 50.6% and 86.0%, respectively. The cDNA sequence and DNA sequence of LeEIN2 were deposited in GenBank (GenBank Accession Number AY566238, DQ409173).Northern blotting analysis indicated that the expression level of the LeEIN2 in young leaves was higher than in mature leaves and senescent leaves. During the development of fruits, the expression of the LeEIN2 was detected before mature green stage and got up to maximum at mature green and breaker stages, then reduced rapidly after breaker stage. The expression level of LeEIN2 in wild type tomato fruit at mature green stage did not distinctively change when treated with exogenous ethylene. This result demonstrated that ethylene could not induce LeEIN2 mRNA expression at this stage.In order to investigate the role of LeEIN2 in tomato fruit ripening, an...