Construction And Analysis Of Suppression Subtractive Hybridization CDNA Library About The Diapause And Non-Diapause Larvae Of Grapholita Molesta Busck

Grapholita molesta Busck (Lepidoptera: Olethreutidae) is a key pest seriously damaging fruit trees such as peach and pear. In this research, we constructed suppression subtractive hybridization cDNA library about diapause and non-diapause larvae of G. molesta to obtain the diapause-related genes which were then sequenced and analyzed, and we also cloned the housekeeping genes of G. molesta by RT-PCR. The paper aims to reveal the molecular mechanism of diapause and provide a new theory preventing and controlling the pest by methods of ecological regulation. The main results are as follows:1. Two suppression subtractive hybridization cDNA libraries were constructed with diapause and non-diapause G. molesta as positive and reverse library. After identifying the quality of the libraries, the differential fragments of the two libraries were respectively selected for sequencing, of which 128 fragments were from the positive library, and 132 fragments were from the reverse library. After cluster analysis and ordering repeat sequences, finally 42 ESTs sequences with diapause difference and 46 ESTs sequences with non-diapause difference were obtained. After BLASTN, we found most of genes screened from two libraries are related to diapause and basically reflect gene expression profiles of G. molesta in duration of diapause and non-diapause. The results also established a foundation for further research of the molecular mechanism of diapauses of G. molesta.2. Some high homology informative genes related to diapause or pupation were screened in this two SSH libraries. In addition to transcription related genes we also found metabolic enzyme and diapause related genes with higher content in positive library and storage protein and signal conduction genes are high expressed in reverse library. Malate dehydrogenase (MDH) gene, diapause associated protein 1 gene, diapause associated protein 2 gene, basic juvenile hormone sensitive hemolymph protein gene and pheromone biosynthesis activating neuropeptide (PBAN) gene etc. were found in positive library. And arylphorin-type storage protein gene, 14-3-3-zeta-protein gene, methionine-rich storage protein (SP2) gene, DEAD box RNA helicase gene, heat shock protein 90 gene which related to entering the pupal stage in reverse library. 3. Actin and tubulin gene of G. molesta were cloned by referring to the house-keeping genes of other Lepidoptera insects. It was found that the cloned actin and tubulin gene were both well expressed in different growth stages of G. molesta and one actin gene was not affected by RsaI digestion, thus it can be used to detect the efficiency of tester's adaptor ligation in SSH.The results of this research provide rich files of the molecular mechanism of insect diapause and found the bases for further molecular research in G. molesta as well.