Effect of Phytoecdysteroids on Insect and Construction of Biological System for Analyzing of Phytoecdysteroids Biosynthesis

Abstract

Ecdysteroids were first recognized as steroidal hormones controlling and metamorphosis in insects. Today, it is realized that these steroids are present at all stages of insect development, regulating many biochemical and physiological processes: in newly-laid eggs, during embryonic and postembryonic developments and in adult insects, regulating aspects of development, metamorphosis, reproduction and diapauses. Ecdysteroids are the steroid hormones of all classes of arthropods and probably of other invertebrates too. Analogues of ecdysteroids (phytoecdysteroids) also occur in a certain proportion of plant species. In 1954, the first ecdysteroid (ecdysone, E) was isolated by Butenandt and Karlson from pupae of silkworm Bombyx mori. Since then, the terms of zooecdysteroid (ZE) and phytoecdysteroid (PE) was used on the basis of the isolation sources. Furthermore, finding of PEs promoted the screening of source plants and understanding of PEs. Over the past 50 years, PEs researches have been advanced concerning the isolation, identification, distribution, biosynthesis and functional role in plant. Until now, more than 440 structurally different PEs have been isolated. Nevertheless, biosynthesis and functional role of PEs in plant are not fully understood. In this study, the biological activity, biosynthesis and related genes of PEs were investigated. In part ▷, the biological activities of PEs for some insect pests were studied to confirm the antifeedant activity, repellent activity and insecticidal activity. In part ▶, the genes in PEs biosynthesis were isolated from Spinach oleracea and its codon was optimized and synthesized for expression in Escherichia coli or plant. In part ♤ several genes in PE metabolism were expressed in E. coli, and their genes were used to construct the transformation system in PE-positive and negative plants. And eventually, the changes of putative PE metabolites were detected in transgenic line since experimental conditions to profile metabolites by HPLC were established. In the antifeedant activity of PE, three insect pests were studied. For Anomala albopilosa, the PE showed the strong antifeedant activity. PE-treated cabbage was not fed after 48 hours, while the control cabbage was almost eaten up. For beet armyworm Spodoptera exigua, PE also had the potent antifeedant activity. The activity ratio of PE extraction from A. japonica and PE standard was 18.1% to 39.1% and 20.5% to 45.5%, respectively. The highest antifeedant ratio of PE was 45.5% at 400??g/ml. For Diamondback moth Plutella xylostella, PE also had the high antifeedant activity. Its activity ratio was 17.7% to 45.9% for PE standard, 24.3% to 45.5% for PE extracts from A. japonica, and 29.1% to 64.3% for PE extracts from S. oleracea. The highest antifeedant ratio of PE was 64.3% at 800??g/ml. In repellent activity test of PE for Cryptotympana dubia, the remarkable repellent activity was observed. In the insecticidal activity of PE for Culex pipiens pallens, there was a strong insecticidal activity. The mortality of mosquito of PE standard was 27.1% to 60.4%, and the value of LC50 was 1.9??g/ml. At the conclusion of part ▷, the PE showed a remarkable antifeedant, repellent and insecticidal activity on tested insect pests. These results showed that the PE might operate on the plant defense against non-adapted phytophagous insect. Spinach, S. oleracea, was chosen for cloning of genes related to PE biosynthesis, due to a potent biological activity of its PE extracts against herbivorous insect. Four genes related to PE biosynthesis, DHCR, CYP85, CYP90B and CYP92A were isolated from S. oleracea. All of these genes were homologous to other plant genes identified with same function and shared more than 60% identities at the amino acid level. To enhance the expression and solubility of plant P450s (CYP85, CYP90B, CYP92A) and insect CYP314A1 in E. coli, the peptide segment, MAKKTSS residues, replaced the hydrophobic region in the amino-terminal region of the enzyme. And 5 histidine-tags were attached to C-terminal of protein for Ni2+-chelate affinity column for purification. At the conclusion of part ▶, four candidate genes were isolated from spinach. And then, all codons in each gene were optimized and synthesized for over expression in E. coli or plant. Based on the above results, four candidate genes were obtained the over-expression in E. coli in part III. Transformation systems in PE-positive (spinach) and negative (tobacco and Arabidopsis) plants were constructed for stable expression and transient expression of candidate genes of PE biosynthesis, respectively. In the former case, transgenic callus of spinach obtained by transformation of Aj-CYP85 gene to get a constant expression. Transgenic plants of Arabidopsis also acquired by floral dipping method for DHCR, CYP85, CYP90B and CYP92A, respectively. In the latter case, three genes, CYP85, CYP90B and CYP92A, were infiltrated into a leave of spinach to get over-expression or knock-down, and obtained a temporarily expression in plant leaves. Metabolite profiling was established by HPLC. This condition could separate several metabolites between 7-dehydrocholesterol and 20-hydroxyecdysone in downstream of putative PE metabolic pathway. The over expressed plant of CYP85 gene and the knockdown plants of CYP90B and CYP92A genes were analyzed in metabolites changes. At the conclusion of part ♤, four genes were expressed in E. coli for the enzymatic study. Several transgenic lines against those genes were constructed through the transformation system in PE-positive and negative plants. And finally, metabolite profiling was established by HPLC, and changes of putative PE metabolites were detected in transgenic lines. Consequently, defensive roles of PE against insects were elucidated, and basic information on a molecular level was established for insight into regulation of PE pathway in this study.