| With the varieties of Bt cotton in different gene sources and different types as the tested materials, we did the research on the expression of insecticidal protein and its relationship with the nitrogen metabolism on the reproductive organs of cotton under the field conditions, while with the different types of cotton as experimental materials to study the protein expression and nitrogen metabolism under the high temperature on the reproductive organs of Bt cotton. The varieties for tested were the domestic cotton:sikang1(conventional varieties), sikang3(Hybrid) and the others were from USA:99B (conventional varieties), daiza1(hybrid), labeled as SK-1, SK-3,99B, and DZ-1. The field experiment was conducted at the Experiment Station of Yangzhou University during2009to2010, and the Laboratory determination was conducted at the Key Physiology Laboratory of Crops Genetics of the Jiangsu Province, with the pot and field experiments combined. In the field experiments, we determined the content of insecticidal protein in the reproductive organs and the substances which it related to the nitrogen metabolism during the squaring stage and the boll stage. In the high temperature tests, we designed the different temperature levels (34℃,36℃,38℃,40℃) for24hours with the artificial climate chamber, and we determined the content of insecticidal protein in the reproductive organs and the relationship with nitrogen metabolism during the squaring stage and the boll stage. The main results were as follows:1.Selecting the materials of the15days after squaring and boll of10days after flowering,the study was underteken on the content of the expression of insecticidal proteins of the four boll worm resistant transgenic cotton cultivars.The results show that the higher content of insecticidal protein expression were the materials of daizal and99B which were from the USA, among them the materials of daizal was the highest(the content of the bud was196.73ng/gFw, boll shell and fiber were163.16ng/gFw and175.24ng/gFw).The varieties which is in domestic sikang3and sikang1were lower, among them the materials of sikang1was the lowest(the content of the bud was176.57ng/gFw, boll shell and fiber were138.67ng/gFw and144.03ng/gFw). In addition the insecticidal protein in the bud and the boll was significantly lower than that in the leaves.it reached the25.76%-32.88%of the leaves. Doing further research on the nitrogen metabolism for the related substances and the key enzyme,the results show that the higher levels of Insecticidal protein in the genital organs, with higher amino acids and higher protein content, with higher glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), glutamine synthetase (GS) and glutamate synthase (GOGAT) activity,also the activity of the protease and peptidase were higher. Among the different varieties showed the same characteristics that the cotton from U.S. were higher than the cotton in domestic.and the conventional species were higher than the hybrid species. The results of the correlation analysis showed that the insecticidal protein expression, with the content of the free amino acids and the soluble protein content, the activity of the glutamate pyruvate transaminase (GPT) and the glutamate synthase (GOGAT),showed significant positive correlation (the correlation coefficients were r1=0.543**,r2=0.692**,r3=0.671**,r6=0.704**);and the insecticidal protein expression, with the activity of the glutamate oxaloacetate transaminase (GOT) and the glutamine synthetase(GS),showed not significant positively related(the correlation coefficients were r4=0.191,r5=0.191); and the activity of the protease and peptidase showed significant negative correlation (the correlation coefficients were r7=-0.291,r8=-0.095). Therefore, the intensity of nitrogen metabolism in the bud and the boll may affect the insect resistance of the reproductive organs in Bt cotton.2. The study was undertaken on the expression of the insecticidal protein with two types of Bt cotton varieties of the15days after squaring and10days after flowering at four temperature levels of stress24hours.The results showed that the expression of the insecticidal proteins have been significantly decreased, and in pace with the temperature level increased, the decrease was greater, and the exacerbated decline was above36℃. The expression of the insecticidal protein in Bt cotton with the sikangl of the conventional varieties decreased greatly.At the squaring stage, the expression of the insecticidal protein of the bud and the leaf at the high temperature over36℃, with the corresponding control were decreased by20.88%and29.43%. At the boll stage, the expression of the insecticidal protein of the boll shell, fiber and leaf,with the corresponding insecticidal control were decreased by33.43%,38.49%and34.58%.The insecticidal protein in the bud and the boll was significantly lower than that in the leaves, it reached the28.34%-35.26%of the leaves. In addition, among the squaring and boll stage, the decrease rate of the Bt protein expression of cotton was more significant at the boll stage.The results indicated that the high temperature stress at the boll stage would significantly reduce the amount of Bt protein expression, thereby affected the insect resistance of cotton.Doing further research on the nitrogen metabolism for the related substances and the key enzyme under the high temperature stress,the results showed that the higher levels decrease of the insecticidal protein in the reproductive organs, with the higher decreased content of the soluble protein, glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), glutamine synthetase (GS) and glutamate synthase (GOGAT) were also a larger decrease in the activity, but the free amino acid content and the activity of the protease and peptidase were a larger increase.Among the two different varieties showed the same characteristics that the general conventional species were higher than the hybrid species. This may closely related to that the high temperature would reduce the protein synthesis, and promote the decline of proteins.The results of the correlation analysis showed that:the Insecticidal protein expression, with the content of the soluble protein content, the activity of the glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT) and the glutamate synthase (GOGAT), showed significant positive correlation (the correlation coefficients were r2=0.884**,r3=0.688**,r4=0.468**, r6=0.453**), and the insecticidal protein expression,with the activity of the glutamine synthetase (GS), showed not significant positively related(the correlation coefficients was r5=0.273),but the content of the drift amino acids and the activity of the protease and peptidase showed significant negative correlation (the correlation coefficients were ri=-0.608**,r7=-0.558**,r8=-0.460**). Therefore, the high temperature conditions can significantly reduce the Bt protein expression of the bud and the boll. This may be closely related to the reduce protein synthesis, and promote the decline of proteins at the high temperature. |
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