Cloning And Expression Analysis Of CTR1 And EIN2 Involved Ethylene Signaling (Prunus Persica (L.) Batsch)

Ethylene is an important factor for involving maturation of fruits, as a resultant which has an intimately connection with ripening and softening of peach. Pp-CTR1 and Pp-EIN2 cDNA full sequence involved ethylene signaling were cloned from prunus persica cv'Rui pan 4'by biotechnology including RT-PCR,RACE and the bioinformation were analysed in this article. The expression of Pp-CTR1 and Pp-EIN2 in the flowers, fruits and seeds of ruipan 4 and ruipan 5 were researched. In addition, function of Pp-EIN2 was analysed through in vitro expression approach by construction the prokaryotic vector pET-32-EIN2 in this research. All of those provide a foundation for appliction and research in gene engineering in the future. The main results were as follows.1 Through 3'and 5'RACE, and RT-PCR, thePp-CTR1 obtained two fragments 2.5Kb and 1.2Kb, Pp-EIN2 amplified 2.5Kb and 2.0Kb by Touchdown PCR. The results of sequencing and splicing displayed the full length sequence of Pp-CTR1 cDNA which coding 834 amino acid is 3124 bp, including 2532bp ORF, 145bp 5'UTR and 447bp 3'UTR. Pp-EIN2cDNA which coding 1304 amino acid is 4399bp, including 3915bp ORF, 233bp 5'UTR and 251bp 3'UTR. The Genbank numbers of Pp-CTR1 and Pp-EIN2 were FJ982370 and GU120631, respectively.2 The analysis results displayed: Pp-CTR1 encoding a protein which molecular is 93.2KD. pI 5.844, the whole protein is negative charged. Pp-EIN2 encoding a 93.2KD protein, pI 5.982, the whole protein is negative charged also. The prediction of CTR1 secondary structure foundα-helix (21.00%), extend main chain (19.35%) and random coil (59.67%), respectively. The EIN2 isα-helix (21.00%), extend main chain (19.34%) and random coil (59.67%), respectively. In addition, we also found CTR1 and EIN2 protein are hydrophilcity protein. CTR1 protein has one transmembrane domain which located in 321-330 amino acids and EIN2 protein has 10.3 The expression of pp-EIN2 in flowers, fruits and seeds of ruipan 4 and ruipan 5 were analysised by the RealTime PCR. The results displayed that the expression of pp-EIN2 in the flowers that completed pollination and fertilization is higher than the alabastrum and full flowers stage. The expression of pp-EIN2 also has a significant summit corresponding, when a lot of ethylene produced, that indicted pp-EIN2 has significant relation with ripening and softening.4 The expression of pp-CTR1 in flowers, fruits and seeds of ruipan 4 and ruipan 5 were analysised by the RealTime PCR. The results showed that the expression of pp-CTR1 in the flowers that completed pollination and fertilization has not insignificant increase compared with the alabastrum and full flowers stage in ruipan 4. The expression of pp-CTR1 displayed some increase compared with the alabastrum and full flowers stage in ruipan 5. Correspondingly, when a lot of ethylene produced the pp-CTR1 has a lower expression than other period, that indicted pp-CTR1 has significant relation with ripening and softening.5 Recombined plasmid Pet-32-EIN2 was constructed, then transformed into BL21 (DE3). The result displayed a special protein which molecular maker is 161KD was produced by IPTG (1.0mmol/L) induced. The production including Trx which molecular maker is 20KD coupling fusion protein and 141 KD protein that was encoded by EIN2, and the expression of the reconstructing protein was increased with the induced time. Those indicted that the whole EIN2 was expressed in the colibacillus BL21 (DE3).