家蚕山梨醇脱氢酶基因启动子的特性分析
发布时间：2018-09-12 06:05
【摘要】：山梨醇脱氢酶(sorbitol dehydrogenase,SDH)是山梨醇转化为糖原的关键酶,家蚕卵中山梨醇含量的变化与卵期滞育的发动、持续和停止有着密切的平行关系。家蚕有3个SDH基因,分别为BmSDH-1、BmSDH-2a和BmSDH-2b。虽然有不少有关家蚕山梨醇脱氢酶的研究报道,但他们研究重点主要集中在蛋白酶的表达上,并未发现有对这3个SDH基因启动子特性进行研究的相关文献。为了探究BmSDH是如何参与调控二化性家蚕滞育的分子机制,本文对家蚕二化性品种“秋丰”3个SDH基因相同长度启动子,BmSDH-2a不同长度启动子以及激素对BmSDH-2a启动子活性影响进行了研究分析,取得如下主要结果。1.家蚕体内山梨醇脱氢酶活性主要来自BmSDH-2a的表达根据家蚕3个山梨醇脱氢酶基因所在的核苷酸序列,设计特异性引物,以二化性(bivoltine,bvt)品种秋丰基因组DNA为模板,PCR扩增得到3个BmSDH约1.0Kb的启动子片段;以pGL3.0 basic质粒为载体,分别构建由BmSDH启动子片段驱动荧光素酶报告基因(luc)表达载体pGL3-BmSDH-1-bvt-1074,pGL3-BmSDH-2a-bvt-1082和pGL3-BmSDH-2b-bvt-1175;分别转染家蚕卵巢来源的BmN细胞,以转染空载的BmN细胞为对照,分析luc表达水平,并以此衡量BmSDH启动子片段的活性,结果显示:同样长度的BmSDH-2a启动子活性明显高于BmSDH-2b和BmSDH-1的启动子活性,分别是后者的9.7倍和21.0倍,暗示家蚕体内山梨醇脱氢酶活性主要来自BmSDH-2a的表达。2.BmSDH-2a基因启动子-355-1082bp之间存在负调控元件为了进一步研究BmSDH-2a基因启动子的特性,从BmSDH-2a基因启动子的5’端截短处理,分别克隆二化性品种BmSDH-2a 674 bp和355 bp的启动子片段,构建不同长度的BmSDH-2a启动子片段控制的报告质粒pGL3-BmSDH-2a-bvt-674和pGL3-BmSDH-2a-bvt-355,并与pGL3-BmSDH-2a-bvt-1082分别转染BmN细胞,结果显示:BmSDH-2a的355bp启动子活性明显高于674 bp和1082bp的启动子,分别是它们的1.3倍和3.3倍,提示在BmSDH-2a启动子-355 bp~-674 bp和-674 bp~-1082bp之间存在负调控元件。3.不同的昆虫激素对BmSDH-2a基因启动子活性的影响存在差异以长度为1082bp的BmSDH-2a基因启动子驱动的荧光素酶报告质粒转染BmN细胞,并分别在培养基中添加滞育激素、保幼激素和蜕皮激素,结果发现:当使用不同浓度滞育激素进行处理的时,BmSDH-2a基因启动子的活性与滞育激素浓度为0时的活性并没有显著变化,说明滞育激素不能直接调节BmSDH的表达;当保幼激素浓度为2,4,6μg/mL时,启动子的活性显著降低,分别是浓度为0时的0.67,0.64和0.67倍;当保幼激素浓度为1μg/mL时,启动子的活性变化不显著,说明高浓度的JH抑制了BmSDH的表达;蜕皮激素浓度为1μg/mL时,启动子的活性显著降低,是浓度为0时的0.63倍;而浓度为2,4,6μg/mL的条件下,启动子活性极显著减弱,是浓度为0时的0.22,0.17,0.16倍,说明高浓度蜕皮激素也抑制了BmSDH的表达。上述研究结果为研究BmSDH的功能积累了实验数据,也有助于阐明家蚕滞于分子机制。
[Abstract]:Sorbitol dehydrogenase (SDH) is the key enzyme in sorbitol-to-glycogen conversion. The change of sorbitol content in silkworm eggs is closely related to the initiation, persistence and cessation of egg diapause. There are three SDH genes in silkworm, namely BmSDH-1, BmSDH-2a and BmSDH-2b. Although there are many related sorbitol dehydrogenase genes in silkworm eggs. In order to explore how BmSDH participates in regulating the molecular mechanism of diapause in dimorphic silkworm, three SDH gene promoters of the same length of Qiufeng were studied. The main results were as follows. 1. The activity of sorbitol dehydrogenase in silkworm was mainly derived from the expression of BmSDH-2a. Specific primers were designed according to the nucleotide sequence of three sorbitol dehydrogenase genes in silkworm, Bombyx mori. Three promoter fragments of BmSDH about 1.0Kb were amplified by PCR using the genomic DNA of Qiufeng cultivar as template, and the Luc expression vectors pGL3-BmSDH-1-bvt-1074, pGL3-BmSDH-2a-bvt-1082 and pGL3-BmSDH-2b-bvt-1175 were constructed using pGL3.0 basic plasmid as vector respectively. The activity of BmSDH-2a promoter with the same length was significantly higher than that of BmSDH-2b and BmSDH-1 promoter, which were 9.7 and 21.0 times higher than that of BmSDH-1respectively, suggesting that the activity of sorbitol dehydrogenase in silkworm. BmSDH-2a gene promoter-355-1082 BP has negative regulatory elements. In order to further study the characteristics of BmSDH-2a gene promoter, the promoter fragments of BmSDH-2a gene were cloned from 5'end truncation of BmSDH-2a promoter, and the promoter fragments of BmSDH-2a 674 BP and 355 BP were constructed. Activator-controlled reporter plasmids pGL3-BmSDH-2a-bvt-674 and pGL3-BmSDH-2a-bvt-355, and pGL3-BmSDH-2a-bvt-1082 were transfected into BmN cells respectively. The results showed that the 355bp promoter activity of BmSDH-2a was significantly higher than that of 674 BP and 1082 BP promoters, which were 1.3 and 3.3 times higher than those of them, respectively, suggesting that the promoters of BmSDH-2a-355 BP and-674 bp-674 BP were transfected into BmN cells. Different insect hormones have different effects on the promoter activity of BmSDH-2a gene. The luciferase reporter plasmid driven by the promoter of BmSDH-2a gene was transfected into BmN cells with 1082 BP in length. Diapause hormone, juvenile hormone and ecdysone were added to the medium, respectively. The results showed that: when used, BmSDH-2a gene was transfected into BmN cells with luciferase reporter plasmid. The activity of promoter of BmSDH-2a gene did not change significantly when the concentration of diapause hormone was 0, indicating that diapause hormone could not directly regulate the expression of BmSDH; when the concentration of juvenile hormone was 2,4,6 ug/mL, the activity of promoter decreased significantly, which was 0.67,0.64 and 0.67 times higher than that at the concentration of 0, respectively. When juvenile hormone concentration was 1 ug/mL, the activity of promoter was not significantly changed, indicating that high concentration of JH inhibited the expression of BmSDH; when ecdysone concentration was 1 ug/mL, the activity of promoter was significantly decreased, which was 0.63 times as high as 0; and when the concentration was 2,4,6 ug/mL, the activity of promoter was significantly decreased, which was 0.22,0.17 at 0 ug/mL. These results accumulated experimental data for studying the function of BmSDH, and also helped to elucidate the molecular mechanism of silkworm stagnation.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;S881.2
[Abstract]:Sorbitol dehydrogenase (SDH) is the key enzyme in sorbitol-to-glycogen conversion. The change of sorbitol content in silkworm eggs is closely related to the initiation, persistence and cessation of egg diapause. There are three SDH genes in silkworm, namely BmSDH-1, BmSDH-2a and BmSDH-2b. Although there are many related sorbitol dehydrogenase genes in silkworm eggs. In order to explore how BmSDH participates in regulating the molecular mechanism of diapause in dimorphic silkworm, three SDH gene promoters of the same length of Qiufeng were studied. The main results were as follows. 1. The activity of sorbitol dehydrogenase in silkworm was mainly derived from the expression of BmSDH-2a. Specific primers were designed according to the nucleotide sequence of three sorbitol dehydrogenase genes in silkworm, Bombyx mori. Three promoter fragments of BmSDH about 1.0Kb were amplified by PCR using the genomic DNA of Qiufeng cultivar as template, and the Luc expression vectors pGL3-BmSDH-1-bvt-1074, pGL3-BmSDH-2a-bvt-1082 and pGL3-BmSDH-2b-bvt-1175 were constructed using pGL3.0 basic plasmid as vector respectively. The activity of BmSDH-2a promoter with the same length was significantly higher than that of BmSDH-2b and BmSDH-1 promoter, which were 9.7 and 21.0 times higher than that of BmSDH-1respectively, suggesting that the activity of sorbitol dehydrogenase in silkworm. BmSDH-2a gene promoter-355-1082 BP has negative regulatory elements. In order to further study the characteristics of BmSDH-2a gene promoter, the promoter fragments of BmSDH-2a gene were cloned from 5'end truncation of BmSDH-2a promoter, and the promoter fragments of BmSDH-2a 674 BP and 355 BP were constructed. Activator-controlled reporter plasmids pGL3-BmSDH-2a-bvt-674 and pGL3-BmSDH-2a-bvt-355, and pGL3-BmSDH-2a-bvt-1082 were transfected into BmN cells respectively. The results showed that the 355bp promoter activity of BmSDH-2a was significantly higher than that of 674 BP and 1082 BP promoters, which were 1.3 and 3.3 times higher than those of them, respectively, suggesting that the promoters of BmSDH-2a-355 BP and-674 bp-674 BP were transfected into BmN cells. Different insect hormones have different effects on the promoter activity of BmSDH-2a gene. The luciferase reporter plasmid driven by the promoter of BmSDH-2a gene was transfected into BmN cells with 1082 BP in length. Diapause hormone, juvenile hormone and ecdysone were added to the medium, respectively. The results showed that: when used, BmSDH-2a gene was transfected into BmN cells with luciferase reporter plasmid. The activity of promoter of BmSDH-2a gene did not change significantly when the concentration of diapause hormone was 0, indicating that diapause hormone could not directly regulate the expression of BmSDH; when the concentration of juvenile hormone was 2,4,6 ug/mL, the activity of promoter decreased significantly, which was 0.67,0.64 and 0.67 times higher than that at the concentration of 0, respectively. When juvenile hormone concentration was 1 ug/mL, the activity of promoter was not significantly changed, indicating that high concentration of JH inhibited the expression of BmSDH; when ecdysone concentration was 1 ug/mL, the activity of promoter was significantly decreased, which was 0.63 times as high as 0; and when the concentration was 2,4,6 ug/mL, the activity of promoter was significantly decreased, which was 0.22,0.17 at 0 ug/mL. These results accumulated experimental data for studying the function of BmSDH, and also helped to elucidate the molecular mechanism of silkworm stagnation.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;S881.2
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