家蠶HSP基因的表達調(diào)控研究.pdf

1、 I家蠶 HSP 基因的表達調(diào)控研究 中文摘要 家蠶(Bombyx mori) 是重要的經(jīng)濟昆蟲和模式生物。熱激蛋白(heat shock protein，HSP) 是在生物界廣泛存在的蛋白質(zhì)，不僅在應(yīng)激條件下高表達，在正常情況下的組織中也廣泛存在。 蛻皮激素屬于類固醇激素， 能促進核酸和蛋白質(zhì)的合成， 影響糖類、脂類的代謝， 影響中樞神經(jīng)系統(tǒng)并參與免疫調(diào)節(jié)。 蕓香苷是食物中常見的類黃酮物質(zhì)，屬黃酮醇類， 有廣泛的生物學(xué)活性， 能抑制細

2、胞增殖和誘導(dǎo)細胞凋亡。 我們從NCBI 上獲得了家蠶10 種熱激蛋白基因和蛋白序列，用生物信息學(xué)的方法對BmHSP進行分析，并采用雙跟蹤標(biāo)定實時熒光定量RT-PCR (dual-spike-in qPCR) 方法，對家蠶BmHSP的基因轉(zhuǎn)錄水平進行了定量研究，主要研究結(jié)果如下： 1、熱激蛋白的生信息學(xué)分析 用生物信息學(xué)方法對熱激蛋白進行分析，結(jié)果表明：Bmhsp20.4、Bmhsp20.8 和Bmhsp19.9 的親緣關(guān)系最近， 小分子

3、熱激蛋白的同源性較高，Bmhsp21.4 在進化中處于獨立的分支。 對小分子熱激蛋白二級結(jié)構(gòu)域的分析比較認為， 家蠶的 7 種小分子熱激蛋白序列有較高的保守性， 而且都相似的 α 螺旋區(qū)域。 對啟動子區(qū)域的轉(zhuǎn)錄因子結(jié)合位點預(yù)測發(fā)現(xiàn)，結(jié)合位點存在差異，又有相似性，推測與基因的功能相關(guān)。 2、蛻皮激素對 BmHSP 的誘導(dǎo)表達定量分析。 測定了家蠶幼蟲經(jīng)蛻皮激素誘導(dǎo)后， 熱激蛋白在中腸、 脂肪體和馬氏管中的表達水平。結(jié)果表明：Bmhsp70

4、 在各個組織中檢測不到表達。在中腸中， Bmhsp21.4 的表達水平在刺激后顯著升高， 表明該基因在中腸中起重要作用； Bmhsp25.4 在刺激后的脂肪體中持續(xù)高表達， 表明該基因在脂肪體中有重要作用。 不同的基因在不同的組織中，表達水平表現(xiàn)出時序的差異。 3、蕓香苷誘導(dǎo)家蠶 HSP 基因的表達變化 用實時熒光定量 RT-PCR 的方法測定了蕓香苷刺激家蠶后熱激蛋白在中腸、 脂肪體和馬氏管中的表達水平。結(jié)果表明：Bmhsp70 在各

5、個組織中檢測不到表達；不同的組織中基因的轉(zhuǎn)錄水平差異很大， 并且不同的基因呈現(xiàn)出不同的表達模式， 且基因表IIIExpression and Regulation of HSP Genes in Silkworm, Bombyx mori Abstract Silkworm is an important economic insects and model organisms. Heat shock protein (heat sh

6、ock protein, HSP) exists in biology widely, not only in the high expression under stress, but also widespread under normal circumstances. Ecdysone can promote the synthesis of nucleic acids and proteins, affecting carboh

7、ydrate lipid metabolism, affecting the central nervous system and immune regulation. Rutin is a common flavonol of flavonoids can inhibit cell proliferation and induce apoptosis. In this paper, we analysised BmHSPs with

8、the method of bioinformatics, investigated the transcription levels of BmHSP genes in Bombyx mori with dual-spike-in qPCR method. The main results are as follows: 1 bioinformatics analysis of heat shock proteins Using b

9、ioinformatics methods analyzed heat shock proteins, the results showed that: Bmhsp20.4, Bmhsp20.8 and Bmhsp19.9 have the closest relationship, small heat shock proteins were highly homologous, and Bmhsp21.4 was an indepe

10、ndent branch in evolution. The secondary structure analysis of small heat shock proteins revealed that the sequences of 7 silkworm small heat shock proteins were quite conservative containing the similar α-helix region.

11、Predicting the binding sites of transcription fators on promoters of BmHSPs revealed that the binding sites were different but had similarities, suggesting that maybe they were associated with fuctions, 2 the expressions

12、 of BmHSPs in tisusses inducted by ecdysone Expressions were determined in the midgut, fat body and malpighian tubules after induction by ecdysone. The results showed that: we could not detect the expression of Bmhsp70.

13、 In midgut, expression of Bmhsp21.4 increased at 2h after induction implied its importance. In fat body, transcription of Bmhsp25.4 kept in a high level. The expressions of genes were different in tusseus and timing, 3 t