Cloning, Expression And Sequence Analysis Of Putative Zingiberene Synthase From New Plant Type Rice, Oryza Sativa

Herbivory insects select their host plants, but plants cannot choose which herbivores will feed upon them. Thus, as long-lived stationary organisms, plants must resist the onslaught of varying and multiple attackers over their lifetime. The greatest threats to plants are herbivorous insects and their associated pathogens. Plants produce a great diversity of secondary compounds, such as an enormous array of terpenoids, which may impart resistance to a variety of herbivores and microorganisms. Terpenoids are the largest family in nature, which play important roles between plants and their habitats, they are messengers. Plants produce terpenoids independently via two pathways, which are full terms MVA pathway and DXP pathway. Studies on the enzymes in the pathway of terpenoid biosynthesis are becoming hot topics.In this study, we examined the volatile compounds of four entries (varieties or breeding lines) of rice, cloned and expressed two putative terpene synthase genes from rice. Analysis of bioinformatics about the both two sequences were also performed. The major study results are as follows,(1) To study the chemical components of the volatile from rice Oryza sativa, the volatile was extracted by head space solid phase microextraction (HS-SPME) and the components separated were identified by gas chromatography-mass spectrometry technique (GC-MS). Finally, 26 compounds were identified on the basis of their mass spectral fragmentation by comparing with the NIST mass spetral data library. The relative content of each component was determinated by normalization method. There were 17 alkanes, 5 sesquiterpenes, 2 esters, 1 alcohol and 1 ketone. Sesquiterpenes in the volatile released from the Japonica breeding line TZRO were the most multiplex,β-sesquiphellandrene (3.25 %), (-)-zingiberene (3.01 %),α-bergamotene (1.7 %),α-curcumene (1.57 %) andβ-bisabolene (1.48 %), while just two sesquiterpenes from the new plant type breeding line IR68011 were found, (-)-zingiberene (6.82 %) andα-curcumene (2.4 %). No monoterpene was detected from rice volatiles in this study.(2) Using zingiberene synthase of the common basil (Ocimum basilicum) and BLAST from Knowledge-based Oryza Molecular biological Encyclopedia (KOME) database, we chose two putative sesquiterpene synthase sequences, and got two cDNAs by RT-PCR from the new plant type entry IR68011 which releases a good quantity of zingiberene, named ZIS1 and ZIS2. The largest ORFs were 1800 bp for ZIS1 and 1515 bp for ZIS2 respectively. We studied on prokaryotic expression of ZIS1 and ZIS2, cDNA sequences were inserted into pET-32a vectors and recombinant plasmids were constructed. By using IPTG inducing, expression of ZIS1 and ZIS2 fusion protein in E.coli BL21 were performed. The results showed that there were specific bands at about 90 and 80 kDa in size, which were identical with the expected molecular weight of the recombinant proteins, but most of which existed in inclusion body.(3) Constituent proportions of four base pairs in nucleotide sequences and amino acids in protein sequences were similar. The most abundant amino acid in sequences of ZIS1 and ZIS2 was Leu, 10.18 % and 10.91 % respectively.Analysis of hydrophobicity/hydrophilicity revealed that ZIS1 and ZIS2 were hydrophilic proteins. They both had no leading peptide or signal peptide, and no transmembrane region, all these results revealed that since ZIS1 and ZIS2 were synthesized in cytoplasm via MVA pathway, they were not be transferred, but stayed where they were.For ZIS1, motif analysis revealed that it contained 3 N-glycosylation sites, 7 Protein kinase C phosphorylation sites, 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 8 Casein kinase II phosphorylation sites, 4 N-myristoylation sites, 1 Tyrosine kinase phosphorylation site and 1 Amidation site. Sequence of ZIS2 contained 7 Casein kinase II phosphorylation sites, 4 Protein kinase C phosphorylation sites, 1 Tyrosine kinase phosphorylation, 2 N-myristoylation sites, 1 N-glycosylation site and 1 Cell attachment sequence. Two domains which are highly conserved in the all plant terpenoids synthases were present in ZIS1 and ZIS2.Sequences of terpenoids synthase genes of various plant species were downloaded from NCB1. The phylogenetic tree based on distances estimated with 1000 bootstrap samples using MEGA 3.1 was constructed. The result showed ZIS1 and ZIS2 were near to sesquiterpenes from rice and maize, and both had the motif of DDXXD region.