家蠶山梨醇脫氫酶基因啟動子的特性分析
發(fā)布時間：2018-09-12 06:05
【摘要】：山梨醇脫氫酶(sorbitol dehydrogenase,SDH)是山梨醇轉(zhuǎn)化為糖原的關(guān)鍵酶,家蠶卵中山梨醇含量的變化與卵期滯育的發(fā)動、持續(xù)和停止有著密切的平行關(guān)系。家蠶有3個SDH基因,分別為BmSDH-1、BmSDH-2a和BmSDH-2b。雖然有不少有關(guān)家蠶山梨醇脫氫酶的研究報道,但他們研究重點主要集中在蛋白酶的表達(dá)上,并未發(fā)現(xiàn)有對這3個SDH基因啟動子特性進(jìn)行研究的相關(guān)文獻(xiàn)。為了探究BmSDH是如何參與調(diào)控二化性家蠶滯育的分子機制,本文對家蠶二化性品種“秋豐”3個SDH基因相同長度啟動子,BmSDH-2a不同長度啟動子以及激素對BmSDH-2a啟動子活性影響進(jìn)行了研究分析,取得如下主要結(jié)果。1.家蠶體內(nèi)山梨醇脫氫酶活性主要來自BmSDH-2a的表達(dá)根據(jù)家蠶3個山梨醇脫氫酶基因所在的核苷酸序列,設(shè)計特異性引物,以二化性(bivoltine,bvt)品種秋豐基因組DNA為模板,PCR擴增得到3個BmSDH約1.0Kb的啟動子片段;以pGL3.0 basic質(zhì)粒為載體,分別構(gòu)建由BmSDH啟動子片段驅(qū)動熒光素酶報告基因(luc)表達(dá)載體pGL3-BmSDH-1-bvt-1074,pGL3-BmSDH-2a-bvt-1082和pGL3-BmSDH-2b-bvt-1175;分別轉(zhuǎn)染家蠶卵巢來源的BmN細(xì)胞,以轉(zhuǎn)染空載的BmN細(xì)胞為對照,分析luc表達(dá)水平,并以此衡量BmSDH啟動子片段的活性,結(jié)果顯示:同樣長度的BmSDH-2a啟動子活性明顯高于BmSDH-2b和BmSDH-1的啟動子活性,分別是后者的9.7倍和21.0倍,暗示家蠶體內(nèi)山梨醇脫氫酶活性主要來自BmSDH-2a的表達(dá)。2.BmSDH-2a基因啟動子-355-1082bp之間存在負(fù)調(diào)控元件為了進(jìn)一步研究BmSDH-2a基因啟動子的特性,從BmSDH-2a基因啟動子的5’端截短處理,分別克隆二化性品種BmSDH-2a 674 bp和355 bp的啟動子片段,構(gòu)建不同長度的BmSDH-2a啟動子片段控制的報告質(zhì)粒pGL3-BmSDH-2a-bvt-674和pGL3-BmSDH-2a-bvt-355,并與pGL3-BmSDH-2a-bvt-1082分別轉(zhuǎn)染BmN細(xì)胞,結(jié)果顯示:BmSDH-2a的355bp啟動子活性明顯高于674 bp和1082bp的啟動子,分別是它們的1.3倍和3.3倍,提示在BmSDH-2a啟動子-355 bp~-674 bp和-674 bp~-1082bp之間存在負(fù)調(diào)控元件。3.不同的昆蟲激素對BmSDH-2a基因啟動子活性的影響存在差異以長度為1082bp的BmSDH-2a基因啟動子驅(qū)動的熒光素酶報告質(zhì)粒轉(zhuǎn)染BmN細(xì)胞,并分別在培養(yǎng)基中添加滯育激素、保幼激素和蛻皮激素,結(jié)果發(fā)現(xiàn):當(dāng)使用不同濃度滯育激素進(jìn)行處理的時,BmSDH-2a基因啟動子的活性與滯育激素濃度為0時的活性并沒有顯著變化,說明滯育激素不能直接調(diào)節(jié)BmSDH的表達(dá);當(dāng)保幼激素濃度為2,4,6μg/mL時,啟動子的活性顯著降低,分別是濃度為0時的0.67,0.64和0.67倍;當(dāng)保幼激素濃度為1μg/mL時,啟動子的活性變化不顯著,說明高濃度的JH抑制了BmSDH的表達(dá);蛻皮激素濃度為1μg/mL時,啟動子的活性顯著降低,是濃度為0時的0.63倍;而濃度為2,4,6μg/mL的條件下,啟動子活性極顯著減弱,是濃度為0時的0.22,0.17,0.16倍,說明高濃度蛻皮激素也抑制了BmSDH的表達(dá)。上述研究結(jié)果為研究BmSDH的功能積累了實驗數(shù)據(jù),也有助于闡明家蠶滯于分子機制。
[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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78;S881.2
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