Cloning And Bioinformatic Analysis Of The Genes In Ethylene Synthesis And Signal Transduction Pathway From Watermelon And Cucumber

Watermelon, cucumber and melon are important Cucurbita crops, ethylene play impormant role in the fruit ripening, the floral sex determination and the yield, now the study on ethylene and Cucurbita crops becomes a hotspot. Cloning and identifying the key genes in the ethylene synthesis and signal transduction path will help to reveal the mechanism of action of ethylene in the floral sex determination of Cucurbita crops. Nowadays most research focus on melon and cucumber, there was few report in watermelon.There are some key genes in ethylene biosynthesis and signal pathway, in this experiment, we designed the primers based on the conserved sequence of those genes in cucumber and melon. Use the watermelon subgynoecious lines (XinHongBao mutant) and monoecious lines (XinHongBao)'s genomics DNA; and the cucumber subgynoecious lines (OuZhou No 8) and monoecious lines (QiuPeng)'s genomics DNA, as the template. With the help of homologous genes cloning, obtain some genomic DNA fragments of ACS,ACO,ETR1,ETR2,ERS,EIN3,ERF in ethylene biosynthesis and signal pathway from watermelon and cucumber genomics. In the mean time analysis those DNA fragments by bioinformatics knowledge, do some preparations for the further research about the molecular mechanism of the sex phenotype various in Cucurbita crops. And the results of the experiment as the follows:1 Designed the primers from the conserved sequence of ACS gene. Amplification a ACS gene about 3385bp from each of (XinHongBao mutant) and (XinHongBao)'s genomics DNA, and the nucleotide sequence of two genes were identical had a complete ORF, named as CLACS. The length of coding region was 1485bp, encoded 494 amino acid, the amino acid sequence was more than 90% homologous with those ACS amino acid sequence from melon and cucumber, which had been reported in GenBank. The protein of CLACS contained 7 conserved sequences, and the fifth conserved sequence was the active site of the protein. Predicted size of 5.57kD and a calculated PI of 6.61, predict tertiary structure of ACS protein in watermelon was very similar with the X-ray structure of ACS protein in tomato (MMDB:15810;PDB:1IAX).2 Designed the primers from the conserved sequence of ACO gene. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA, as the template, we had amplified two fragments about 1522bp from each of them, the nucleotide sequence of two genes were identical, named as CLACO, contained 4 extrons and 3 introns, the length of coding region was 912bp, encoded 303 amino acid; use the cucumber (OuZhou No 8) and (QiuPeng)'s genomics DNA, as the template, we had amplified two fragments about 1270bp from each of them, the nucleotide sequence of two genes were identical, named as CSACO, contained 4 extrons and introns, the length of coding region was 909bp, encoded 302 amino acid. The coding region of the similarity from CLACO and CSACO was 92.3% and the similarity of the two amino acid sequences was 93.4%. Predicted tertiary structure of ACO in watermelon and cucumber was very similar with the X-ray structure of ACO protein in petunia (MMDB:35451;PDB:1W9Y). The phylogenetic trees showed that CLACO and CSACO were closely related to the cucurbita.3 Designed the primers from the conserved sequence of ETR1 gene. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA, as the template, we had amplified two fragments about 1633bp from each of them, the nucleotide sequence of two genes were identical, named as CLETR1; use the cucumber (OuZhou No 8) and (QiuPeng)'s genomics DNA, as the template, we had amplified two fragments about 1491bp from each of them, the nucleotide sequence of two genes were identical, named as CSETR1. The length of the coding region was the same from CLETR1 and CSETR1, was about 795bp,encoded 264 amino acid. The similarity of the two coding regions was 96% and the similarity of the two amino acid sequences was 98%. The single nucleotide variations were found in the coding region between CLETR1 and CSETR1, there were 23 SNPs in two regions, and 5 of theSNPs resulted 4 amino acids difference.4 Designed the primers from the conserved sequence of ETR2 gene. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA, as the template, we had amplified two fragments from each of them, the nucleotide sequence of two genes were identical, named as CLETR2; use the cucumber (OuZhou No 8) and (QiuPeng)'s genomics DNA, as the template, we had amplified two fragments from each of them, the nucleotide sequence of two genes were identical, named as CSETR2. The length of CLETR2 and CSETR2 was the same, was about 417bp, they all belonged to coding region, encoded 138 amino acid. The similarity of the two fragments was 95.68% and the similarity of the two amino acid sequences was98.5%. There was a conserved REC domain predicted by NCBI, predict tertiary structure of REC domain was very similar with the X-ray structure of REC domain in Arabidopsis thaliana (PDB:1DCF_A).5 Designed the primers from the conserved sequence of ERS gene. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA, as the template, we had amplified two fragments about 837bp from each of them, the nucleotide sequence of two genes were identical, named as CLERS; use the cucumber (OuZhou No 8) and (QiuPeng)'genomics DNA, as the template, we had amplified two fragments about757bp from each of them, the nucleotide sequence of two genes were identical, named as CSERS. CLETRS and CSERS were composed of 2 extrons,1 intron and 3'UTR region; The length of the coding region was the same from CLETRS and CSERS, was about 291bp, encoded 96 amino acid. The similarity of the two coding regions was 94.85% and the similarity of the two amino acid sequences was 93.76%. There was a conserved HATPase-c domain predicted by NCBI.6 Designed the primers based on a EST sequence from fruit and development bank of watermelon. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA, as the template, we had amplified two fragments about 603bp from each of them, the nucleotide sequence of two genes were identical, named as CLEIN3; use the cucumber (OuZhou No 8) and (QiuPeng)'s genomics DNA, as the template, we had amplified two fragments about 619bp,604bp from each of them, named as CSEIN3 A,CSEIN3 B. The length of CLEIN3,CSEIN3A and CSEIN3B were 399bp,423bp,408bp respectively, and encoded 132,140,135 amino acid respectively. The similarity of the three coding regions was 92.83% and the similarity of the three amino acid sequences was 93.1%. Close to the N-terminal, CSEIN3A encoded 5 more Asn than CSEIN3B.7 Based on suppression subtractive hybridization(SSH) cDNA library about Fusarium Wilt Resiatance from watermelon, designed a pair of primers. Use the watermelon (XinHongBao mutant) and (XinHongBao)'s genomics DNA as the template, we had amplified two fragments about 627bp from each of them, the nucleotide sequence of two genes were identical, named as CLERF; use the cucumber (OuZhou No 8) and (QiuPeng)'genomics DNA, as the template, we had amplified two fragments about 625bp from each of them, the nucleotide sequence of two genes were identical, named as CSERF. CLERF and CSERF were composed of 1 extron and 3'UTR region; the length of the coding region was the same from CLERF and CSERF, was about 363bp, encoded 120 amino acid. The similarity of the two coding regions was 94.49% and the similarity of the two amino acid sequences was 95%.