Cloning And Expression Of Two Key-Enzyme Genes Involved In Glycinebetaine Synthesis Pathway From Rice (Oryza Sativa. L)

Most organisms accumulate highly soluble compounds termed osmoprotectants orosmolytes when they live in various abiotic stresses such as salinity, drought and cold. Oneof the most-important osmoprotectants is glycinebetaine, which is a quaternary ammoniumcompound accumulated in many organisms from archaebacteria to higher plants andanimals. Previous reports show that glybetaine can provide tolerance to the cells understress by stabilizing the quaternary structure of the complex proteins and adjusting theosmotic potential in their cytoplasm to maintain water content. Higher plants synthesizeglybetaine via a two-step oxidation of choline. In the first step, choline monooxygenase(CMO) catalyzes choline to betaine aldehyde; in the second step, betaine aldehydedehydrogenase (BADH) catalyzes betaine aldehyde to glybetaine.In order to isolate CMO cDNAs from rice, the amino acid sequence of Beta vulgarischoline monooxygenase gene (GenBank accession number: AF023132) was used as a queryprobe to search rice genome database in GenBank through BLAST algorithm program. Itwas found that there was a genomic sequence to be highly homologous with the relativeprobe. Through splicing of extrons using FGENESH program it was obtained thefull-length cDNA sequence. By RT-PCR and the specific primers of assembled sequence,the cDNA fragment was isolated from mRNAs from rice seedlings, and designated asOsCMO1, with accession number AJ578494 in GenBank. This should be the first report inrice. We also isolated BADH2 cDNAs from rice by RT-PCR, and designated as OsBADH2in order to study the mechanism of rice non-accumulating glycinebetaine.The expression profiles of OsCMO1 and OsBADH2 in various rice tissues wereinvestigated using semi-quantitative RT-PCR approach. The RT-PCR results suggested thatOsCMO1 and OsBADH2 were constitutively expressed in various rice tissues under normalgrowth conditions. To understand the functions of betaine synthesis pathway in plants undervarious environmental stresses, the expressions of two genes were investigated in the riceseedlings under various stresses including salinity, drought and cold by semi-quantitativereverse transcription (RT)-PCR approach. The results showed that the transcript level of OsCMO1 was up-regulated to different extents by drought, cold, high salinity. And theexpression of OsCMO1 was significantly induced by high salinity. Under salt stress,OsCMO mRNA levels increased more greatly in Jiucaiqing (japonica variety, salt-tolerant)than in IR28 (indica variety, salt-sensitive). The OsBADH2 was significantly induced bysalt treatment in Jiucaiqing with the salt-tolerant. However, OsBADH2 was constitutivelyexpressed in IR28 with the salt-sensitive under salt treatment. The transcript level ofOsBADH2 was slightly up-regulated by drought and cold.To study the functions of choline monooxygenase (CMO) and betaine aldehydedehydrogenase (BADH) from rice, the expression vector pET-30a-OsCMO1 andpET-30a-OsBADH2 were constructed. The plasmids (pET-30a-OsCMO1 andpET-30a-OsBADH2) were transformed into E.coli BL21 (DE3), then the fusion-proteins ofCMO or BADH were over-expressed in BL21 (DE3) induced by IPTG. The crude extractsof OsBADH2 showed the enzymatic activities of typical BADH.