Studies On Gene Differential Expressions Regulated With Ethephon And Cloning Of Ethylene Receptor Gene Sc-ERS In Sugarcane (Saccharum Officinarum L.)

The gaseous plant hormone ethylene induces diverse effects in plants throughouttheir life cycle. Many previous studies have showed that sugarcane growth could beregulated by foliar spraying of ethphon. The ethephon treatments induced variousphysiological changes at the early growth stage of sugarcane, such as earlier and fastersprouting, improved tillering, faster elongation, promoted photosynthesis and enhancedability resisce to diseases and adversity, and so on. To explore the molecular mechanismsof ethephon in regulating sugarcane growth, the differences in gene expressions betweenethephon treatment and control at early growth stage of sugarcane were investigated byusing cDNA-AFLP technique, and the ethylene receptor gene Sc-ERS in sugarcane wascloned and sequenced in the present study. The main results were summarized asfollows.1. The optimized cDNA-AFLP protocol and an efficient silver stain system weredeveloped, and the gene differential expressions in sugarcane regulated with ethephonwere analysed. The results indicated that the quality of template DNA was important incDNA-AFLP analysis. It is effective to reduce polysaccharides in the total RNA byremovinging the tissue residue of sugarcane from the extractive mixture timely. In thecDNA-AFLP protocol, 20 cycles for pre-amplification of primary template wasdetermined, followed by a 20-fold dilution of the secondary template before selectiveamplification. In the silver staining, better results could be realised by using ultra-pureNa₂CO₃ and limiting the time of rinsing and developing under controlled condition. ThecDNA-AFLP analysis results showed that the polymorphisms between the treatment andthe control were abundant, and 35 to 80 bands were obtained in each amplification. 2. Some transcript derived fragments (TDFs) were isolated, and then cloned,detected with reverse Northern blot and sequenced. The results showed that there were11 samples of 24 TDFs which displayed no difference in expression, and the ratio ofhypocrite positive TDFs was about 46%. The CHI, GST, ARP, LHC, nuclear bindingprotein gene and a set of unknown genes were differentially expressed by the treatmentwith ethephon. CHI and GST were important factors for resistance to diseases, andLHC and nuclear binding protein were related to light binding and CO₂ primary fixingin photosynthesis, respectively.3. A pair of degenerate primers and a pair of specific primers were designed basedon the conserved sequences of plant ethylene receptor genes, and a 4310 bp fragment ofSc-ERS was cloned and amplified with these primer pairs. The sequencing resultsshowed that Sc-ERS, consisted of five extrons and four introns, contained an entirecoding region of a gene. The coding region of Sc-ERS showed 91.4% and 61.6%homology with those of maize ERS1 and rice ERS1, respectively. The deduced protein ofSc-ERS encoded 636 amino acids, and shared 94.1% and 89.4 % similarity with those ofmaize ERS1 and rice ERS1, respectively. The architecture of protein analysis indicatedthat the Sc-ERS contained three transmembrane structures in the conserved N end asthe maize ERS1 and rice ERS1 did. The functional region of the deduced protein ofSc-ERS, maize ERS1 and rice ERS1 consisted of three transmembrane structures, i.e.GAF, HisKA domain and HATPase_c domain. These results indicated that the Sc-ERSin sugarcane was similar to the ERS1 in other plants.