Response Of Cotton Polyphenol Oxidase GhPPO To The Feeding Of Cotton Bollworm Helicoverpa Armigera (Hubner)

By using the method of molecular biology and biochemistry and laboratory animal science, the dynamic changes of GhPPO mRNA expression level and PPO activity in CCRI41, CCRI49and Shiyuan321were measured after these cotton varieties were treated with mechanical damage or cotton bollworm larvae feeding or oral secretion mimic feeding. This research is helpful to elucidate the interaction between cotton bollworm and cotton. The main conclusions of this study are as follows:1. The dynamic changes of GhPPO mRNA expression level and PPO activity were measured after3different cotton varieties treated with mechanical damage. The fold change of GhPPO mRNA expression level in CCRI41, CCRI49and Shiyuan321rise up to a maximum at9h,6h, and6h separately after mechanical wounding, with a significant change of5.62fold,3.29fold and9.61fold compared with untreated leaves. The PPO activity in the3former cotton varieties get to maximum at36h,36h and60h separately after mechanical wounding, with48.2%,39.3%and26.5%higher than untreated leaves. It indicates that cotton GhPPO can respond to mechanical wounding.2. The expression level of GhPPO which shows an increased at first and then decreased trend, and the PPO activity shows a gradually increasing variation trend was cleared after different cotton varieties treated with1st instar larvae. The fold change of GhPPO mRNA expression level in CCRI41, CCRI49and Shiyuan321rise up to a maximum after18h,18h and12h separately after larvae feeding, with a significant change of8.68fold,15.48fold and26.92fold compared with untreated leaves. The PPO activity in the3varieties at60h after1st instar larvae feeding were71.4%,61.3%and61.3%higher than untreated leaves. It showes that cotton GhPPO can also respond to cotton bollworm feeding, but the maximum change and the speed to rise up to maximum is different between various cotton varieties.3. The dynamic changes of GhPPO mRNA expression level and PPO activity were measured after CCRI41were treated with cotton bollworm larvae feeding of different instars. There was no significant difference in GhPPO mRNA expression level at each time point in CCRI41leaves treated with3rd instar larvae. And the expression level of GhPPO was always lower in3rd instar than in1st instar larvae treated leaves. At each time point after the CCRI41leaves were treated with cotton bollworm larvae feeding, the PPO activity were lower in3rd instar larvae than in1st instar larvae. After6h feeding by3rd instar larvae, the PPO activity was significantly lower than untreated sample, which decreased by20.6%. It indicates that old stage larvae may suppress GhPPO mRNA expression level and PPO activity in cotton plants.4. By spraying cotton bollworm larvae oral secretion to the wounding area of different cotton varieties, it revealed that oral secretions could inhibit the expression level of GhPPO and the activity of PPO. Compared with the water and oral secretion treatment, the expression of GhPPO was decreased by1.24times,4.63times and2.72times respectively. On the other hand, the enzyme activity of PPO decreased by7.5%,23.3%and14.4%in CCRI41, CCRI49and Shiyuan321, respectively. It indicates that oral secretions can inhibit the production of cotton defensive materials.5. Two key genes including Allene Oxide Synthase and CORONATINE INSENSITIVEl in jasmonic acid (JA) signal pathway were cloned and there expression level were measured after1st and3rd instar larvae feeding. Compared with1st instar larvae feeding, the mRNA expression level of GhAOS was lower during the first24h after3rd instar larvae feeding. It indicates that old stage larvae can suppress the expression of GhAOS gene and the expression of GhPPO may related to JA signal pathway.6. We successfully constructed the prokaryotic expression vector of GhPPO and it laid the foundation to further study the defensive role of cotton GhPPO.