| Cotton Bollworm(Helicoverpa armigera Hübner) is an important pest of cotton(Gossypium arboreum L.), which damage reduces cotton production. Polyphenol oxidases was defense enzymes,which play an importtent role of plant denfese. To elucidate the interactions between cotton bollworm and cotton, we use the method of molecular biology and biochemistry and laboratory animal science, the dynamic changes of Ghppo1 mRNA, the key gen of JA-signaling pathway(GhAOS and GhCOI1) and PPOs activity in CCRI49 and CCRI41, and the effect on H. armigera and S. exigua were measured after these cotton varieties were treated. This study is crucial to understand the interaction between cotton and cotton bollworm. The main conclusions of this study are as follows:1. The changes of Ghppo1 mRNA expression level and PPOs activity were measured after CCRI49 and CCRI41 were infested with different instars cotton bollworm. In both the conventional cotton ‘CCRI49’ and transgenic Cry1 Ac cotton ‘CCRI41’, the Ghppo1 expression levels and PPOs activity was different when induced by 1st instar and 3rd instar H. armigera larvae. The Ghppo1 expression level was significantly induced by the 1st instar H. armigera, but showed no change when exposed to 3rd instar H. armigera. The Ghppo1 expression increased from 6 h to 18 h in ‘CCRI49’ and from 6 h to 24 h in ‘CCRI41’. In both ‘CCRI49’ and ‘CCRI41’, the highest expression levels, 5.78-fold and 4.54-fold the un-induced level, respectively, appeared at 18 h. The PPO activity increased after 6 h in ‘CCRI49’ and after 12 h in ‘CCRI41’. The highest level was 5.99 U·g–1·min–1 at 18 h in ‘CCRI49’ and 4.02 U·g–1·min–1 at 12 h in ‘CCRI41’. It indicates that 1st instar larvae can induce and 3rd instar larvae may suppress Ghppo1 mRNA expression level and PPOs activity in cotton plants.2. The changes of Ghppo1 mRNA expression level and PPOs activity were measured after CCRI49 and CCRI41 were treated with Me-JA and Me-SA. 0.5 mM Me-JA could induce the expression of Ghppo1 significantly after 6 h both in ‘CCRI49’ and ‘CCRI41’, and that the highest expression levels in both ‘CCRI49’ and ‘CCRI41’, 6.80-fold and 3.17-fold that of un-induced expression, respectively, were at 18 h. The Ghppo1 expression level trend was similar in cotton treated with 1st instar H. armigera, and the fold change was not significantly different compared with 1st instar H. armigera induced at 18 h both in ‘CCRI49’ and ‘CCRI41’. In ‘CCRI49’ and ‘CCRI41’ treated with Me-SA or ethyl alcohol, the Ghppo1 expression was not appreciably changed. Spraying Me-JA on cotton plants also induced PPOs activity, and the trend was similar to that found in plants treated with 1st instar H. armigera, but the PPOs activity was lower than in cotton treated with 1st instar H. armigera.3. In ‘CCRI49’, 1st instar H. armigera infestations and Me-JA spraying significantly enhanced the expression levels of GhAOS and GhCOI1 at 12 h, 18 h and 24 h. A 3rd instar H. armigera infestation and Me-SA spraying could suppress the expression level of GhCOI1, but had no effect on GhAOS. Spraying with Me-SA could enhance the expression level of GhAOS only at 6 h and 12 h, the earliest decrease of GhCOI1 was induced by 3rd instar larvae at 6 h. The same results were found using CCRI41. The expression of key JA signaling pathway genes, GhAOS and GhCOI1, increased after an infestation with 1st instar H. armigera, indicating that the Ghppo1 expression is a JA-mediated, not an SA-mediated, defense.4.Based on the qRT-PCR and PPO activity results, cotton plants that had been treated with 1st instar H. armigera or Me-JA were infested with 3rd instar H. armigera and S. exigua. The weight gains of 3rd instar H. armigera feeding on ‘CCRI49’ that had been previously treated independently with 1st instar H. armigera and Me-JA were lower than the control. The weight gains were lower than the control, of S. exigua fed independently 1st instar H. armigera- and Me-JA-treated cotton, respectively, were similar to those of H. armigera. The same result that H. armigera and S. exigua fed on 1st instar H. armigera-,or Me-JA treated ‘CCRI41’ cotton,indicate that 1st instar H. armigera induce the cotton plant defense to H. armigera and S. exigua.5. Ghppo1 transgenic Arabidopsis were infested with 3rd instar H. armigera and S. exigua. The survival rate of 1st instar larvae and pupal weight feeding on Ghppo1 transgenic Arabidopsis was significantly lower than feeding on wild-type Arabidopsis. Weight gains and relative growth rates(RGRs) feeding on wild-type Arabidopsis are higher than those feeding on transgenic plants. When 1st instar H. armigera infested the two Arabidopsis genotypes, the weights of larvae feeding on the wild type were significantly higher than those feeding on the Ghppo1 transgenic plants between 12 d and 20 d. The result shows that Ghppo1 played a crucial role in cotton defense.6. Using the transcriptome sequencing, the CCRI49 cotton infested with 1 instar H. armigera was analyzed. 2422 Differentially Expressed Genes were obtained, including 1853 up-regulated genes, down regulated genes with 569. The result of Pathway Enrichment Analysis of DEGs shows that, DEGs mainly enriched in Biosynthesis of secondary metabolites pathway, inclouding Flavonoid biosynthesis(79 genes), and enriched in alpha-Linolenic acid metabolism pathway, which belong to Plant hormone signal transduction. |
