JIA-2018-09

1996 ZHANG Xiang et al. Journal of Integrative Agriculture 2018, 17(9): 1991–1998 increased by 1.30 and 2.79 mg pro g –1 FW h –1 , respectively, compared to the control. On the corresponding days, those activities for SK-3 increased by 0.98 and 2.80 mg Pro g –1 FW h –1 , respectively. We also observed a greater increase of protease activity in SK-1 than that in SK-3. 4. Discussion 4.1. Delayed decrease of Cry1Ac protein content under high day/normal night temperature stress in comparison with constant high temperature stress Studies have shown that several environmental factors, such as temperature, water, and fertilizers, affect the level of resistance to pests and the expression of the Bt cotton Cry1Ac protein. High temperature is a main factor confirmed by many researchers. However, the stress regime used in previous studies was conducted at a constant temperature over 24 h for several consecutive days (Chen et al . 2005b, 2012; Jiang et al . 2012). In our study, the significant decrement of Cry1Ac protein content was found after the Bt cotton was exposed to 38°C continuously for 24 h in 2011 and 2012 (Fig. 1). However, this temperature regime does not accurately mimic nature where there are periodic (hourly) variations of temperature. In nature, the duration of high temperature stress is usually less than 12 h. Therefore, the effects of a constant high temperature on toxic protein expression of Bt cotton are likely different from the effects of a periodic variation of temperature. Moreover, Chen et al . (2013) found that the expression of the insecticide Cry1Ac protein in the leaf could recover completely after short periods (24 h) of extreme temperature. In summer, it is possible that the exposure of Bt cotton to high temperatures will terminate at night. Thus, the results of previous studies might not be truly representative of the condition of heat stress, meaning that results of these studies are not practically applicable in the field. It is more important to study the effects of periodic variations of high temperature on the level of pest resistance for Bt cotton due to its significance in guiding agricultural practices. The studies in 2011 and 2012 indicated that 38°C might be the temperature threshold for significant effects on boll shell insecticidal protein content. Thus, the temperature regime of 38°C from 6:00 a.m. to 6:00 p.m. followed by a normal temperature of 27°C during the night was imposed in 2012 and 2013 to study the effects of alternating temperature during day and night on boll shell Bt protein content. Our results showed that under the stress of the 38/27°C day/night temperature regime, Cry1Ac protein contents in the boll shell decreased with prolonged duration of the stress. Compared with their corresponding controls, the difference of Cry1Ac protein content for both cultivars was significant after 7 d and longer. The study also showed that the reduction in content was not significant in Bt cotton subjected to 4 d of stress. Therefore, the reduction in Cry1Ac protein content was influenced by the duration of the stress; the longer the stress period, the greater decline in the content. Thus, when compared to constant high temperature stress, we see a difference in the actual stress time required to make a significant reduction of the Bt protein content. This indicates that a periodic, cyclical change of temperature (38/27°C day/ night temperature regime) delayed the reduction of Cry1Ac protein content. The possible explanation for the delayed insecticidal protein reduction might be that under the day/ night temperature regime, the effects of high temperature on the plants during daytime would be counteracted at night. Hence, the weather should be closely monitored in crops at the flowering stage. When the temperature is above 38°C during the day and lasts for 4 d or more, a significant loss of Cry1Ac protein should be expected in Bt cotton with little possibility of recovery. This means that the level of insect resistance of these plants will also decrease significantly. These results will help farmers implement efficient agricultural techniques to control the bollworm and increase farmers’ profitability. This study also provides a comparison of the stress effects on the conventional SK-1 versus the hybrid SK-3. The extent and rate of decrease of Cry1Ac protein in the boll shell was higher in SK-1 than SK-3 (Table 1). These conclusions are consistent with previous research and could be explained by two possible reasons. First, tolerance for high temperature was greater for the hybrid cultivar SK-3 than for the conventional cultivar SK-1. The hybrid plants seemed more resistant to protein loss under high temperature. This supports the work of Chen et al . (2005b). Second, the recovery rate of insecticidal protein in the conventional cultivars was weaker than their hybrid counterparts. This is consistent with the observations of Chen et al . (2013). 4.2. Decreased protein synthesis and increases protein degradation under high day/normal night temperature reduced Bt protein content Nitrogen metabolism is associated with the level of insecticidal protein in Bt cotton plants. Research has shown that nitrogen is absorbed, transmitted, reduced, and assimilated throughout the expression of Cry1Ac insecticidal protein (Saini and Dhawan 2014). Therefore, enzymes taking part in nitrogen metabolism, such as GPT and GOT, can be used to determine the content of insecticidal protein (Hallikeri et al . 2011; Dadgale et al . 2014). The specific measurements showed that, under the high day temperature (38°C)/normal night temperature (27°C)