蝴蝶蘭60Co-γ射線輻照植株組織培養(yǎng)及性狀遺傳的研究.pdf

1、安徽農(nóng)業(yè)大學(xué)碩士學(xué)位論文蝴蝶蘭60Co-γ射線輻照植株組織培養(yǎng)及性狀遺傳的研究姓名：馬麗婭申請學(xué)位級別：碩士專業(yè)：生物物理指導(dǎo)教師：章鐵2011-06II Abstract Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing

2、with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for c

3、omprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in E. coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of e

4、xpression conditions, particularly the culture temperature of PPO­transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. The results are as follows: 1. All Drosophila

5、 melanogaster PPO1/2/3 genes could be expressed in E. coli. At 37 almost all PPO proteins were expressed in conclusion body ℃ ，which were more than proteins expressed at 16 . At 16 , ℃ ℃ about half of expressed prote

6、ins were in conclusion body and all the left were soluble protein. PPO1 and PPO3 expressed in E. coli cells have the same molecular weight as PPO1 and PPO3 expressed in Drosophila melanogaster S2 cell according to Wester

7、n blot assay. The reason might be that PPO1 and PPO3 didn’t need posttranslational modification or too much modification. However, the reason why PPO2 expressed in Drosophila melanogaster S2 cell was little bigger than t

8、hat expressed in E. coli cells might be due to PPO2 posttranslational modification in S2 cells. 2. Activity assay of PPO expressed in E. coli cell at 16 . Both ℃ transformational E. coli cells and their crude protein c

9、ould be activated by 30% ethanol or 0.02% CPC. PPO1 had the strongest activity among three PPOs, and PPO3 had stronger activity than PPO2. 30% ethanol could activate much more PPOs than 0.02% CPC for PPO1 and PPO3, but 0

10、.02% CPC was better than 30% ethanol for PPO2. 3. Expression amounts of PPO1. The amouts of PPO1 expressed in E. coli cell at 16℃ gradually increased in 12 h. It reached at submit when PPO1 was induced at the 12 th h. 4

11、. The influence 30% ethanol and Cu 2+ on the PPO1 activity. We could dectect the obvious enzyme activity no matter which one, Cu 2+ or 30% ethanol, was added firstly. However, we observed that the activated PPOs had hi