氮肥运筹对转基因棉Bt蛋白表达与降解的调控作用
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摘要
本文以转单价基因抗虫杂交棉中棉72为实验材料,采用不同的施氮量和施氮方式处理,系统研究了氮肥运筹对转基因棉叶片和棉铃中Bt蛋白的表达与降解的调控作用及其初步机理,氮肥运筹对棉蕾中Bt含量、叶片中氮代谢生理活性及衰老的影响,主要研究结果如下:
1氮肥运筹对抗虫棉叶片中Bt蛋白表达与降解的调控
随着棉花叶片的生长发育,Bt蛋白的含量呈现先上升后下降的趋势,至功能期时达到高峰,衰老期和脱落期时含量明显下降。不同的施氮量处理和施氮方式处理对抗虫棉叶片中Bt蛋白的表达与降解均具有明显的调控作用。就施氮量处理而言,随着氮肥施入量的加大,叶片中Bt蛋白的含量整体呈现增大趋势,但当氮肥施入量过大时,叶片中Bt蛋白含量反而会下降。前期表达量较多的处理后期残留量也较多。对施氮方式处理来说,不施底肥会严重影响前期叶片中Bt蛋白的表达;施入较多的盖顶肥时,Bt蛋白的降解速度缓慢,后期叶片中残留的Bt蛋白含量较高。
2氮肥运筹对转基因棉棉铃中Bt蛋白表达与降解的调控
随着棉铃的发育,铃壳中Bt蛋白含量呈现出先增大后减小的趋势,充实期时含量最高,棉铃吐絮时迅速下降。而棉籽中Bt蛋白含量却逐渐增加,吐絮期时各处理棉籽中含量达到最高峰。不同的施氮量处理对棉铃各器官中Bt蛋白的表达和降解具有一定的调控作用。随着施氮量的加大,铃壳、棉纤维和棉籽中的Bt蛋白含量整体呈增大趋势,前期表达较多的处理后期残留也较多。棉铃各器官中Bt蛋白的表达和降解也受到不同施氮方式的影响。在膨大期和充实期,B3处理棉铃各器官中Bt蛋白的含量总体高于其他处理;在吐絮期时,盖顶肥施入最多的B1处理棉铃各器官中Bt蛋白降解的速度较慢,残留量最高,未施盖顶肥的B4处理棉铃各器官中Bt蛋白的含量最低。
3氮肥运筹对抗虫棉叶片氮代谢生理活性的影响
不同的施氮量处理显著影响了抗虫棉叶片中的氮代谢生理活性,随着氮肥施入量的加大,叶片中的游离氨基酸、可溶性蛋白和全氮的含量整体呈增加趋势,提高了谷氨酰胺合成酶(GS)的活性,叶片中的氮代谢生理活性随之增强。抗虫棉叶片中的氮代谢生理活性也受到了不同施氮方式处理的影响,叶片中的游离氨基酸、可溶性蛋白和全氮的含量以及谷氨酰胺合成酶(GS)的活性在展开期时以施入底肥最多的B4处理的含量最高,在衰老期、脱落期时均以施入盖顶肥最多的B1处理含量最高,这说明施入一定量的盖顶肥能够显著增强抗虫棉叶片在后期时的氮代谢的生理活性。
4氮肥运筹对抗虫棉叶片衰老的影响
对不同氮肥处理的叶片中的丙二醛(MDA)的含量、超氧化物歧化酶(SOD)的活性以及叶绿素的含量进行了测定,结果表明随着氮肥施入的增多,降低了各个时期的叶片中的丙二醛(MDA)的含量,增强了超氧化物歧化酶(SOD)的活性,提高了叶绿素的含量,进而延缓了叶片的衰老进程。就施氮方式处理而言,底肥和花铃肥施入较多的处理在叶片的展开期和功能期时的丙二醛(MDA)的含量较低,超氧化物歧化酶(SOD)的活性较高,叶绿素的含量也较其他处理高。在叶片的衰老期和脱落期,盖顶肥施入较多的处理丙二醛(MDA)的含量低于其他处理,超氧化物歧化酶(SOD)的活性仍较高,叶绿素的降解速度也低于其他处理,这说明对抗虫棉及时补施盖顶肥能够防止棉株因缺肥而早衰,延缓叶片的衰老。
5氮肥运筹对抗虫棉叶片中Bt蛋白表达与降解调控的初步机理
就施氮量处理而言,增施氮肥总体上促进了抗虫棉叶片在展开期、功能期、衰老期和脱落期的谷氨酰胺合成酶(GS)活性的增强,游离氨基酸、可溶性蛋白质的合成和全氮含量增加。同时也促进了棉花叶片中的Bt蛋白合成,导致叶片生育前期Bt蛋白表达量增加,在生育后期残留量也较大。对施氮方式处理来说,底肥和花铃肥施入较多的B4处理,促进了抗虫棉叶片在展开期时谷氨酰胺合成酶(GS)活性的增强,游离氨基酸、可溶性蛋白质的合成和全氮含量增加,其合成的Bt蛋白量也增加。盖顶肥施入较多的B1处理在衰老期、脱落期时谷氨酰胺合成酶(GS)活性仍较高,游离氨基酸、可溶性蛋白质和全氮含量也比其他处理高,叶片中残留的Bt蛋白含量最高。
In this paper,transfer gene unit hybrid cotton zhongmian72 was used as experimental material, using different nitrogen fertilizer rates and patterns treatments, the effects of nitrogen management practices on expression and degradation of Bt-protein in leaves and bolls and its preliminary mechanism、the effects of nitrogen management practices on the content of Bt-protein in squares、the effects of nitrogen management practices on the activities of nitrogen metabolism and senescence in the leaves were studied. The results as follows:
1 The effects of nitrogen management practices on expression and degradation of Bt-protein in leaves
With the growth of the leaves,the content of Bt-protein showed that first increased and then decreased,it was maximum in leaves of functioning stage, it declined obviously in leaves of senescing stage and shedding stage.The nitrogen can regulate expression and degradation of Bt-protein in leaves. In terms of nitrogen fertilizer rates treatments, with the increasing amount of nitrogen fertilizer,the content of Bt-protein trend to increase,but when the nitrogen fertilizer was excessive, the content of Bt-protein will decrease. The later residues were more when Bt-protein expressed more in prophase. In terms of nitrogen fertilizer patterns treatments, if the base fertilizer is not applied, the expression of Bt-protein will be affected;when using more fertilizer of roofing,the rate of degradation of Bt-protein will be slow,the residual Bt-protein were numerous.
2 The effects of nitrogen management practices on expression and degradation of Bt-protein in bolls
With the development of boll,the content of Bt-protein in shell showed that first increased and then decreased,it was maximum in the stage of filling,it rapid declined in the stage opening. The content of Bt-protein in the seed will increase,it was maximum in the stage of opening. With the increasing amount of nitrogen fertilizer, the contents of Bt-protein in shell, fiber and seed of bolls were increasing in the mass, the later residues were more when Bt-protein expressed more in prophase. The expression and degradation of Bt-protein in the boll was affected by different nitrogen fertilizer patterns. The contents of Bt-protein in shell, fiber and seed of bolls of treatment B3 were more than other treatments in the stage of expanding and filling; in the stage of opening, the contents of Bt-protein in the organs of boll of treatment B1 were more than other treatments because of using major fertilizer of roofing, the contents of Bt-protein of treatment B4 were minimal.
3 The effects of nitrogen management practices on the activities of nitrogen metabolism in leaves
The activities of nitrogen metabolism in leaves were obviously affected by nitrogen fertilizer rates treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves increased with the nitrogen fertilizer rates. The activities of nitrogen metabolism in leaves were also obviously affected by nitrogen fertilizer patterns treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves in treatments B4 were higher than others in the stage of expanding, the activities of nitrogen metabolism in leaves in treatment B1 were stronger than others in the stage of senescing and shedding. This shows that using a certain amount of fertilizer of roofing can evidently improve the activities of nitrogen metabolism in late stages.
4 The effects of nitrogen management practices on the characteristic of senescence in leaves
The content of the malondialdehyde(MDA) and chlorophyll、the activity of superoxide dismutase (SOD) in leaves under different nitrogenous fertilizer treatments were assayed, the results showed that the content of the malondialdehyde(MDA) reduced、the activity of superoxide dismutase (SOD) enhanced、the content of chlorophyll increased in leaves with the increasing of nitrogenous fertilizer,the process of the senescence of leaves was delayed.In terms of nitrogen fertilizer patterns treatments, the content of the malondialdehyde(MDA)was lower、the activity of superoxide dismutase (SOD) and the content of chlorophyll were hinger than other treatments in the stages of expanding and functioning inleaves withmore base and boll fertilizer. the content of the malondialdehyde(MDA) was lower、the activity of superoxide dismutase (SOD) and the content of chlorophyll were hinger than other treatments in the stages of senescing and shedding in leaves with more roofing fertilizer.These show that applying fertilizer in time for transgenic cotton can prevent the premature senescence because of lack of fertilizer,and then the process of the senescence of leaves was delayed.
5 The preliminary mechanism of the effects of nitrogen management practices on expression and degradation of Bt-protein in leaves
In terms of nitrogen fertilizer rates treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves increased with the nitrogen fertilizer rates in expanding stage﹑functioning stage、senescing stage and shedding stage.At the same time, the synthesis of Bt-protein was promoted, led to the expression of Bt-protein increased in the early growth stage,the residues in the late growth stage were large. For nitrogen fertilizer patterns treatments, the treatment B4 because of base fertilizer and boll fertilizer applied more, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity were promoted in the stage of expanding, the expression of Bt-protein also increased. The activities of nitrogen metabolism in leaves of treatment B1 were stronger than others in senescing stage and shedding stage, the residues were more than others.
1氮肥运筹对抗虫棉叶片中Bt蛋白表达与降解的调控
随着棉花叶片的生长发育,Bt蛋白的含量呈现先上升后下降的趋势,至功能期时达到高峰,衰老期和脱落期时含量明显下降。不同的施氮量处理和施氮方式处理对抗虫棉叶片中Bt蛋白的表达与降解均具有明显的调控作用。就施氮量处理而言,随着氮肥施入量的加大,叶片中Bt蛋白的含量整体呈现增大趋势,但当氮肥施入量过大时,叶片中Bt蛋白含量反而会下降。前期表达量较多的处理后期残留量也较多。对施氮方式处理来说,不施底肥会严重影响前期叶片中Bt蛋白的表达;施入较多的盖顶肥时,Bt蛋白的降解速度缓慢,后期叶片中残留的Bt蛋白含量较高。
2氮肥运筹对转基因棉棉铃中Bt蛋白表达与降解的调控
随着棉铃的发育,铃壳中Bt蛋白含量呈现出先增大后减小的趋势,充实期时含量最高,棉铃吐絮时迅速下降。而棉籽中Bt蛋白含量却逐渐增加,吐絮期时各处理棉籽中含量达到最高峰。不同的施氮量处理对棉铃各器官中Bt蛋白的表达和降解具有一定的调控作用。随着施氮量的加大,铃壳、棉纤维和棉籽中的Bt蛋白含量整体呈增大趋势,前期表达较多的处理后期残留也较多。棉铃各器官中Bt蛋白的表达和降解也受到不同施氮方式的影响。在膨大期和充实期,B3处理棉铃各器官中Bt蛋白的含量总体高于其他处理;在吐絮期时,盖顶肥施入最多的B1处理棉铃各器官中Bt蛋白降解的速度较慢,残留量最高,未施盖顶肥的B4处理棉铃各器官中Bt蛋白的含量最低。
3氮肥运筹对抗虫棉叶片氮代谢生理活性的影响
不同的施氮量处理显著影响了抗虫棉叶片中的氮代谢生理活性,随着氮肥施入量的加大,叶片中的游离氨基酸、可溶性蛋白和全氮的含量整体呈增加趋势,提高了谷氨酰胺合成酶(GS)的活性,叶片中的氮代谢生理活性随之增强。抗虫棉叶片中的氮代谢生理活性也受到了不同施氮方式处理的影响,叶片中的游离氨基酸、可溶性蛋白和全氮的含量以及谷氨酰胺合成酶(GS)的活性在展开期时以施入底肥最多的B4处理的含量最高,在衰老期、脱落期时均以施入盖顶肥最多的B1处理含量最高,这说明施入一定量的盖顶肥能够显著增强抗虫棉叶片在后期时的氮代谢的生理活性。
4氮肥运筹对抗虫棉叶片衰老的影响
对不同氮肥处理的叶片中的丙二醛(MDA)的含量、超氧化物歧化酶(SOD)的活性以及叶绿素的含量进行了测定,结果表明随着氮肥施入的增多,降低了各个时期的叶片中的丙二醛(MDA)的含量,增强了超氧化物歧化酶(SOD)的活性,提高了叶绿素的含量,进而延缓了叶片的衰老进程。就施氮方式处理而言,底肥和花铃肥施入较多的处理在叶片的展开期和功能期时的丙二醛(MDA)的含量较低,超氧化物歧化酶(SOD)的活性较高,叶绿素的含量也较其他处理高。在叶片的衰老期和脱落期,盖顶肥施入较多的处理丙二醛(MDA)的含量低于其他处理,超氧化物歧化酶(SOD)的活性仍较高,叶绿素的降解速度也低于其他处理,这说明对抗虫棉及时补施盖顶肥能够防止棉株因缺肥而早衰,延缓叶片的衰老。
5氮肥运筹对抗虫棉叶片中Bt蛋白表达与降解调控的初步机理
就施氮量处理而言,增施氮肥总体上促进了抗虫棉叶片在展开期、功能期、衰老期和脱落期的谷氨酰胺合成酶(GS)活性的增强,游离氨基酸、可溶性蛋白质的合成和全氮含量增加。同时也促进了棉花叶片中的Bt蛋白合成,导致叶片生育前期Bt蛋白表达量增加,在生育后期残留量也较大。对施氮方式处理来说,底肥和花铃肥施入较多的B4处理,促进了抗虫棉叶片在展开期时谷氨酰胺合成酶(GS)活性的增强,游离氨基酸、可溶性蛋白质的合成和全氮含量增加,其合成的Bt蛋白量也增加。盖顶肥施入较多的B1处理在衰老期、脱落期时谷氨酰胺合成酶(GS)活性仍较高,游离氨基酸、可溶性蛋白质和全氮含量也比其他处理高,叶片中残留的Bt蛋白含量最高。
In this paper,transfer gene unit hybrid cotton zhongmian72 was used as experimental material, using different nitrogen fertilizer rates and patterns treatments, the effects of nitrogen management practices on expression and degradation of Bt-protein in leaves and bolls and its preliminary mechanism、the effects of nitrogen management practices on the content of Bt-protein in squares、the effects of nitrogen management practices on the activities of nitrogen metabolism and senescence in the leaves were studied. The results as follows:
1 The effects of nitrogen management practices on expression and degradation of Bt-protein in leaves
With the growth of the leaves,the content of Bt-protein showed that first increased and then decreased,it was maximum in leaves of functioning stage, it declined obviously in leaves of senescing stage and shedding stage.The nitrogen can regulate expression and degradation of Bt-protein in leaves. In terms of nitrogen fertilizer rates treatments, with the increasing amount of nitrogen fertilizer,the content of Bt-protein trend to increase,but when the nitrogen fertilizer was excessive, the content of Bt-protein will decrease. The later residues were more when Bt-protein expressed more in prophase. In terms of nitrogen fertilizer patterns treatments, if the base fertilizer is not applied, the expression of Bt-protein will be affected;when using more fertilizer of roofing,the rate of degradation of Bt-protein will be slow,the residual Bt-protein were numerous.
2 The effects of nitrogen management practices on expression and degradation of Bt-protein in bolls
With the development of boll,the content of Bt-protein in shell showed that first increased and then decreased,it was maximum in the stage of filling,it rapid declined in the stage opening. The content of Bt-protein in the seed will increase,it was maximum in the stage of opening. With the increasing amount of nitrogen fertilizer, the contents of Bt-protein in shell, fiber and seed of bolls were increasing in the mass, the later residues were more when Bt-protein expressed more in prophase. The expression and degradation of Bt-protein in the boll was affected by different nitrogen fertilizer patterns. The contents of Bt-protein in shell, fiber and seed of bolls of treatment B3 were more than other treatments in the stage of expanding and filling; in the stage of opening, the contents of Bt-protein in the organs of boll of treatment B1 were more than other treatments because of using major fertilizer of roofing, the contents of Bt-protein of treatment B4 were minimal.
3 The effects of nitrogen management practices on the activities of nitrogen metabolism in leaves
The activities of nitrogen metabolism in leaves were obviously affected by nitrogen fertilizer rates treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves increased with the nitrogen fertilizer rates. The activities of nitrogen metabolism in leaves were also obviously affected by nitrogen fertilizer patterns treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves in treatments B4 were higher than others in the stage of expanding, the activities of nitrogen metabolism in leaves in treatment B1 were stronger than others in the stage of senescing and shedding. This shows that using a certain amount of fertilizer of roofing can evidently improve the activities of nitrogen metabolism in late stages.
4 The effects of nitrogen management practices on the characteristic of senescence in leaves
The content of the malondialdehyde(MDA) and chlorophyll、the activity of superoxide dismutase (SOD) in leaves under different nitrogenous fertilizer treatments were assayed, the results showed that the content of the malondialdehyde(MDA) reduced、the activity of superoxide dismutase (SOD) enhanced、the content of chlorophyll increased in leaves with the increasing of nitrogenous fertilizer,the process of the senescence of leaves was delayed.In terms of nitrogen fertilizer patterns treatments, the content of the malondialdehyde(MDA)was lower、the activity of superoxide dismutase (SOD) and the content of chlorophyll were hinger than other treatments in the stages of expanding and functioning inleaves withmore base and boll fertilizer. the content of the malondialdehyde(MDA) was lower、the activity of superoxide dismutase (SOD) and the content of chlorophyll were hinger than other treatments in the stages of senescing and shedding in leaves with more roofing fertilizer.These show that applying fertilizer in time for transgenic cotton can prevent the premature senescence because of lack of fertilizer,and then the process of the senescence of leaves was delayed.
5 The preliminary mechanism of the effects of nitrogen management practices on expression and degradation of Bt-protein in leaves
In terms of nitrogen fertilizer rates treatments, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity in leaves increased with the nitrogen fertilizer rates in expanding stage﹑functioning stage、senescing stage and shedding stage.At the same time, the synthesis of Bt-protein was promoted, led to the expression of Bt-protein increased in the early growth stage,the residues in the late growth stage were large. For nitrogen fertilizer patterns treatments, the treatment B4 because of base fertilizer and boll fertilizer applied more, the content of amino acids、soluble protein、total nitrogen and Glutamine synthase (GS) activity were promoted in the stage of expanding, the expression of Bt-protein also increased. The activities of nitrogen metabolism in leaves of treatment B1 were stronger than others in senescing stage and shedding stage, the residues were more than others.
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