Cloning And Characterization Of The Nitric Oxide Synthase From Locusta Migratoria Manilensis

The recent discovery of nitric oxide as a metabolic intermediate has exploded into a swiftly developing area of research. The intense interest and rapid progress have developed primarily because (i) NO synthesis has been found in a variety of different cell types; (ii) NO controls and influences a number of critical physiological processes; (iii) NO synthesis via the oxidation of L-arginine has been shown to involve unusual oxidative chemistry and has uncovered novel aspects of eukaryotic enzymology. The formation of NO from L-arginine is catalyzed by the enzyme NO synthase. The best characterized examples of biological reactions controlled by NO include vasodilation and regulation of normal vascular tone, inhibition of platelet aggregation, neuronal transmission, and cytostasis. The roles of nitric oxide in invertebrates are closely related to the physiological actions described in vertebrates, namely, neurotransmission, defence, and salt and water balance. All the identified functional domains of NO synthase enzymes: heme-, calmodulin-, FMN-, FAD-, and NADPH-binding sites. There have been many reports about nitric oxide synthase in insects, the cDNA sequences encoding NOS have also been cloned. The NOS genes in locusts, a global agricultural pest, have not been studied. The complete cDNA of a NOS gene of Locusta migratoria manilensis (Meyen) was cloned by means of RT-PCR,3'-RACE and 5'-RACE. The result showed that the cDNA was 3508bp in length and contained an open reading frame (ORF) of 3006bp, which encoded 1002 amino acids. The semi-quantitative RT-PCR was used to detect the organizational expression characteristics of nitric oxide synthasemale from male locust adult emergence 3 days and then discovered that: LmNOS was expressed in all the locust's head, legs, fat body, midgut, and blood cells, and highly expressed In the fat body, head, followed by blood cells, but lowly expressed in the legs. It can be inferred that nitric oxide synthase gene play a vital functionin insects through the tissue localization of Nitric oxide synthase gene from the locust, its function that can be not only a signal molecule but also a responsive molecule is similar to the function studies of nitric oxide synthase gene from mammalian. The Metarhizium CQM102 strains have been used to xenogenetic inoculation infect the locusts gregarious phase, the expression characteristics of LmNOS in different periods and different organizations were studied after infection, and found that it may play an extremely important immunologic function in the period from 36h to 48h.