| The Locusta migratoria manilensis (Meyen) is a widespread agriculture pest in China with a broad geographic distribution covering both temperate and tropical climatic zones. The species also serves as a model insect for the study of morphology, physiology, developmental biology, and neurosciences and so on. In recent years, more scientists have been interesting in the research of the cell and molecular biology of the locust, and lots of genes have been cloned. The development of methods facilitating molecular manipulation of the primitive insect is thus of wide interest and urgent need. Nowadays, RNA interference (RNAi) has been quite effective for the investigation of gene function in animals, especially in insect species.The orthodenticle (otd) gene was known to be functional in segment patterning in Drosophila melanogaster at very beginning. In Drosophila, otd is activated by Bicoid(Bcd) and acts as a zygotic head gap gene that specifies the ocular and the antennal segments. In Tribolium castaneum, otd belongs to maternal gene, and otd mRNA is initially distributed uniformly in the egg, present at the beginning of blastoderm formation. During blastoderm stages, otd mRNA and protein recede first from the posterior and later also from the anterior pole, resulting in a belt-like domain in the prospective head region. In other insects, there are also some reports on the otd gene, such as in the species Apis mellifera, Nasonia vitripennis, Episyrphus balteatus, Ceratitis capitata, Acyrthosiphon pisum and Anastrepha suspensa.The present study aims to: 1) clone the otd gene fragments of the locust, and investigate the function of the gene in the locust with embryo RNAi and parental RNAi; 2) probe and compare the effectivity of these two RNAi technologies with the otd gene. The main results were as follows:â‘ Cloning and sequence analysis of the otd fragmentsThe two fragments of the gene were obtained by PCR, RT-PCR and other methods. One is 91 bp long with its predicted amino acids sequence well aligned to otd gene family. Another is 235 bp long that was aligned to the non-coding region of otd2.â‘¡RNAi technology with otd The dsRNA of otd was injected directly into eggs and adult virgin female's body cavity. The injected eggs had lower survival rate and showed the specific RNAi phenotypes, whereas injections with H2o or no-dsRNA gave no significant difference compared with non-injected eggs, which indicating that the injection of eggs cause body injury. All injected female adults survived and produced clutches of eggs which subsequently formed specific RNAi phenotypes of embryos, whereas injections with H2o or no-dsRNA gave no specific phenotypes. The results indicated that dsRNA injection did not affect the fecundity of female adults.The embryo RNAi worked well in our research of otd gene function in the process of the locust embryonic development. dsRNA concentration was found to significantlty affect the embryo RNAi reaction; however, the length of dsRNA fragments did not significantly affect the embryo RNAi phenotypes. We injected the same dose of dsRNA into the body cavity of locust nymph at 5th instar and female adults at the age of 6, 7 and 8 days after their emergence. Result showed that the female adults at 6, 7 and 8 days after their emergence had specific RNAi phenotypes, and also the most of the parental RNAi phenotypes appeared at the age of 7 day.Both embryo RNAi and parental RNAi resulted in identical embryo phenotypes. The results showed that only the abdomen and one thoracic (T3) segments developed in the embryos, and the head (including antenna, eyes, labral, intercalary, maxillary and mandibular) and two thoracic (T1 and T2) segments were totally depressed.â‘¢The otd functionAccording to embryo RNAi and parental RNAi phenotypes, most body segments in the locust RNAi embryos were deleted, including head and two thoracic (T1 and T2) segments. These results indicated that otd may function as a gap gene in a large region in the locust. otd has apparently divergent segmentation functions in different insects, and otd may control more segments in basally branching insects. |
