JIA-2018-09

1992 ZHANG Xiang et al. Journal of Integrative Agriculture 2018, 17(9): 1991–1998 and protected the environment without additional pollution (Zhang 2013). By 2012, the planting area for Bt cotton had reached 18.8 million hectares, which accounted for 62.7% of the total cotton planting area in the world. Previous researchers have found that there are not only temporal and spatial variations in the expression of the Bt cotton insecticidal Cry1Ac protein (Rui et al . 2002; Bakhsh et al . 2011), but also environmental condition has an effect on its expression (Chen et al . 2005b, 2012b; Jiang et al . 2006, 2012; Luo et al . 2008; Martins et al . 2008; Blaise and Kranthi 2011; Hallikeri et al . 2011). Among the environmental factors, high temperature stress is one of the most important. Many studies have shown that the temperature of the earth has been increasing, and consequently cotton-growing regions have regularly recorded extremely high temperatures (Wang et al . 2008), which directly affects the expression of the insecticidal Cry1Ac protein and the level of Bt cotton plants’ resistance to bollworm (Chen et al . 2012a, 2014). Chen et al . (2015b) showed that the content of Cry1Ac protein in bolls decreases by 63–73% when exposed to the stress of 37°C for 48 h. Aprevious study also showed that a high threshold temperature from 38 to 40°C affects the expression of Cry1Ac protein in Bt cotton (Lü 2013). In previous studies, researchers have kept the same temperature for the entire day. However, in reality, the temperature in natural environments varies during day and night, with temperatures at night usually lower than that during the day. Some researchers believe that when the external stresses on plants terminate, their physiological metabolism can recover from the effects of those stresses. Ian (2006) found that short-term abiotic stresses had no significant effects on the expression of Cry1Ac protein. In addition, the experiments reported byAdamczyk et al . (2001) also showed that there were no significant correlations between the environment and the level of resistance to pests. However, both of these studies were conducted in a natural environment with changing environmental conditions, and the stress time during the day was less than 12 h. Therefore, we believe that the level of Cry1Ac expression might recover during the night after the environmental stress during the day terminate. This hypothesis is supported by the observation of Chen et al . (2013), who detected the recovery of Cry1Ac protein content after temperature stress ended in transgenic Bt cotton. Therefore, investigating the effects of periodic variation of temperature on Bt cotton plants is very important to guide future Bt cotton planting practices. The synthesis of the Bt protein and its cycle in cotton plants are also the physiological process of nitrogen metabolism. Several key enzymes affecting nitrogen metabolism such as soluble protein, glutamic-pyruvic transaminase (GPT), protease, and peptidase may also affect Bt protein content (Steward et al . 1965; Dong et al . 2007). Therefore, it is important to investigate the relationships between boll toxin levels and nitrogen metabolism with the stress of periodic variation of temperature. The primary aim of this study was to determine the effects of alternating temperature, 38°C (threshold high temperature for Bt cotton) during the day and 27°C (normal temperature) at night, on the expression of Cry1Ac protein. A secondary objective was to examine the relationship between the Cry1Ac protein content under alternating temperature stress and the activities of several key enzymes affecting nitrogen metabolism. 2. Materials and methods 2.1. Plant materials and experimental design In the summers of 2011, 2012, and 2013, experiments were conducted in the greenhouses at Yangzhou University, Yangzhou, China (32°30´N, 119°25´E). Two transgenic Bt cotton cultivars (medium in maturity, Gossypium hirsutum L.) Sikang 1 (conventional cultivar, SK-1) and Sikang 3 (hybrid cultivar, SK-3) were studied. InApril, seeds of the two cultivars were planted in a warm room and covered with a plastic film. After 43 d, the seedlings were transplanted to pots. Each pot (50 cm height and 40 cm diameter) was filled with sandy loam soil (Typic Fluvaquents, Entisols (U.S. taxonomy)) up to 2 cm from the upper edge and watered as required. One seedling was transplanted to each pot. In 2011 and 2012, temperatures of 32, 34, 36, 38, and 40°C were imposed for 24 h at peak boll stage (104 d after transplanting). The same air humidity (65–70%) was maintained for five temperature treatments. The study was conducted using a completely randomized design with three plants per temperature treatment for each cultivar. The temperature treatment of 32°C was treated as the control. Based on the results in 2011 and 2012, the experiment was continued during the 2012 and 2013 cotton-growing season. At the peak boll developing stage (104 d after transplanting), potted plants of the two cultivars were treated with the threshold temperature of 38°C from 6:00 a.m. to 6:00 p.m. followed by a normal temperature of 27°C during the remaining night hours (DH/NN) for 0, 4, 7, and 10 d. The temperature was regulated by air conditioning, and the air humidity (65–70%) of the greenhouse was maintained. Three replicates were used for both cultivars. Control pots were grown in another greenhouse under a 32/27°C day/ night temperature regime. 2.2. Sampling and measurement Preparation of samples Boll samples were collected from