Isolation Of OsRwp34, A New Rice Gene And Its Protein Toxic Effect On The Growth Of E.Coli

Rice (Oryza sativa L.) is one of the most important cereal crops in the world. It is obvious that the stable high yield of rice has very important assurance for national food security. However, rice often suffer from various unfavourable conditions during its growth development. Every year, the natural disasters result in significant decrease of rice production and its quality in the world. Rice grows extensively in different ecological situations and its planting area is large in China.Temperature is an important circumstance factor for rice growth, the lower temperature and too hot weather will cause a obvious decrease of rice output in a large area.Rice often meets with heat stress during its growth in the valley of Changjiang River. Rice will drop significantly its seed-setting rate and decrease heavily its yield under heat stress at the heading and flower stages. In order to study the phenomena, a cDNA library was constructed and used to identify and isolate differential expression genes under heat stress at rice seedlings, which is considered as a new technology platform to study further mechanism of pollen fertility change at molecular level. The major results were as follows:1) RNA was extracted from a rice variety 9311 seedlings treated at temperature of 45℃ for 2 hr, and cDNA library was constructed by CLONTECH SMART? cDNA Library Construction Kit. The titer of cDNA library is 115000000 pfu/mL, the average length of the cDNA library is 800 bp.2) A new rice gene,OsRwp34, was isolated in the cDNA library by sequencing. The length of the cDNA of OsRwp34 is 659bp,including an ORF of 378 bp which encodes a 125 aa peptide. As an orphan gene, OsRwp34 is not found the conservative function domain by BLASTP and does not belong to any gene family and gene superfamily. Thereis a transmembrane hydrophobic helics at the OsRwp34 C terminate and its N terminate locates at cytoplasm by HMMTOP analysis. The Probability of OsRwp34 in endoplasm is 66.7% by PSORT II analysis. There is a single OsRwp34 copy from Oryza sativa nipponbare(G3), OsRwp34 is mapped at the 9 chromesome 68.2 cM. However, there are three OsRwp34 copies in the rice variety 9311 by Southern blot analysis. The expression analysis of OsRwp34 emerges very differently in various rice varieties by Northern blot, it expresses poorly in the rice variety 9311 and plentifully in another rice variety JXNL, but there is a single transcript in both 9311 and JXNL3) In order to study the function of OsRwp34, a vector pET-28(a)OsRwp34 which included OsRwp34 ORF was constructed and transformed into the E.coli strain BL21(DE3). The bacteria was grown in LB medium and induced by IPTG, and the host bacteria was found to be dying rapidly in 30min through the ODgoo monitor and quantitative plating. The result suggests that the expression product of OsRwp34 causes death of the bacteria. Continuously, three deletion mutants:one of them deleted 15 aa of N terminate, the another two deleted 12 aa and 47 aa of C terminate respectively, were constructed, and the toxic effect to the bacteria growth was not found after the induction of IPTG. It is guessed that the exist of both N and C terminates is necessary for the toxic effect of OsRwp34. A further study will be required on the toxic mechanism of OsRwp34.