| Developing citrus fruit will absciss under severe stresses, resulting in yield loses. Low winter temperature frequently causes pre-harvest fruit dropping and heavy loses. Citrus fruit are harvested by hands, which is very costly. For example, the harvesting cost has even surpassed the total production cost recently in USA. Therefore, achieving controllable fruit abscission is one of the main objectives being pursued by citrus growers and researchers.This study used citrus genome array to probe ethylene-induced fruit abscission and and the underlying molecular mechanism. Ethylene at the concentration of 20μL/L was used to treat citrus mature fruit (Citrus sinensis cv. Valencia) for 4 h,12h and 24 h. Fruit abscission zones were collected and extracted for total RNA that was later analyzed by microarrays. Clustering analysis demonstrated that the replicability of the chip data were acceptable. qRT-PCR studies on 12 genes showed that their expression trends were similar to those showed by chip data. Results from analysis of chip data showed that there were 510 genes that were co-induced by ethylene at all the three sampling times (P＜0.01). Gene function assignment showed that the genes up-regulated by ethylene encode mostly membrane proteins, energy metabolism related proteins, stress-related proteins, transcription factors, serine/threonine kinases, phosphorylation enzymes, calcium-binding proteins, hydrolases, ethylene metabolic pathway proteins, transfer enzymes, and receptor proteins while those down-regulated encode ribosomal protein, hydrolytic enzymes, aquaporins, cytochrome P450, metal ion binding proteins, ABC transporters, lipase and so on. Further clustering showed that the genes encoding proteins involved in ethylene biosynthesis and signal transduction, in hypoxia stress and wounding responses were coordinately induced by ethylene. Therefore, ETR2, hypoxia responsive gene and wounding induced genes were selected for further study. The major results are as follows:Hypoxia responsive gene CsHRP was cloned and analyzed for its expression. RACE was used to clone the full-lenth cDNA of the gene. The cloned cDNA was sequenced. Results showed that the full-length cDNA was 795 bp with an ORF encoding a peptide of 98 amino-acid residues. Sequence comparision showed that it shares 97%of similarity with a sweet orangen hypoxia responsive gene. Prediciton of the protein secondary structure suggested it might be related to plant resistance. Real-time PCR was used to quantify the gene's expression levels under exogenous ethylene treatment,and the results showed that that CsHRP was highly induced only at the early stage of ethylene treatment (4h), and treatment longer than 12h and 24h reversed its expression to the basal level.Ethylene induced the expression of wounding-induced protein genes (WIPs). In this study 4 wounding-induced protein genes were cloned and designated as CsWIP1, CsWIP2, CsWIP3 and CsWIP4. Sequence comparison showed that they were strucuraly classified into two groups:group 1 contains CsWIP1, CsWIP2, CsWIP3 and group 2 contains only CsWIP4. WIPs generally contain two a-helices, a N-terninalα-helix and a C-ternimal a-helix. Simulation of these citrus wounding induced proteins demonstrated that CsWIP1, CsWIP2 and CsWIP3 are conservative in both helices while CsWIP4 is only conserved in its C-termianl helix. Real-time PCR results showed that CsWIP1 was expressed only under ethylene induction, indicating its ethylene dependence. CsWIP 2 was expressed only in leaves under both ethylene and wounding treatments, indicating its expression was tissue-specific. CsWIP 3 was universly expressed both in leaves and abscission zones either under ethylene treatment or under wounding treatment, indicating its expression was tissue-specific. CsWIP4 was only induced by wounding but not by ethylene.ETR2 is a member of ethylene receptor gene family. To better understand the role of ETR2 in ethylene-induced fruit abscission, ETR2 was cloned and its expression was analyzed. Full-length cDNA of ETR2 was obtained by using RACE technology. The cloned cDNA was 1800bp in length. Sequence alignment analysis revealed that ETR2 of Valencia shares 98%homology with the same gene from other sweet orange(Citrus sinensis). Real-time PCR was used to detect the expression of ETR2 under exogenous ethylene treatment. The results showed that ETR2 was up-regulated by 2-folds and 4-folds after treatement with ethylene for 4h, and 12h, respectively, was reduced to its basal expression level after 24h of treatment.To further characterize the CsHRP and CsWIP1, transgenic plants over expressing CsHRP and CsWIP1 have been obtained. Their RNAi transgenic plants have been alsoe obtained. Characterization of the transgenic plants is under way. |
