【摘要】：日本三角渦蟲(Dugesia japonica)隸屬扁形動物門(Platyhelminthes),渦蟲綱(Turbellaria),在動物系統(tǒng)演化中占有重要地位,并且具有極強的再生能力。目前,雖然已克隆大量與渦蟲再生相關的基因,但其具體的再生機制及相關基因的功能仍不清楚。本文從日本三角渦蟲轉錄組數據庫中篩選出再生過程中持續(xù)上調表達但表達量較低的熱休克蛋白70和90家族的Djhsp70e、Djhsp90a、Djhsp90b和Djhsp90c基因。利用整體原位雜交、RNA干擾和Real time-PCR等分子生物學技術對四基因的時空表達模式和功能進行分析,結果如下:(1)通過Real time-PCR技術對切割損傷、離子液體暴露、冷應激和熱應激環(huán)境下日本三角渦蟲體內四基因的表達量進行分析,結果顯示:在應激條件下,四基因的表達量均明顯上調,并且隨著切割損傷時間的延長、離子液體濃度的升高,四基因的表達量逐漸上升。(2)日本三角渦蟲Djhsp70e基因的cDNA長度為1982bp,5’端有180bp非編碼區(qū),3’端有38bp的非翻譯區(qū),其中包含一個長度為1764bp的最大開放閱讀框(ORF),編碼一個由587個氨基酸構成的蛋白質,含有兩個熱休克蛋白70家族所特有的標簽序列。原位雜交結果顯示:在整體中,該基因在除腦及咽部以外的身體各部分廣泛表達,身體兩側和腸支處雜交信號較強;再生個體的芽基部位有較強的雜交信號。RNA干擾該基因后,整體渦蟲出現(xiàn)嚴重頭部溶解且不能再生,而頭再生尾片段也出現(xiàn)嚴重的溶解現(xiàn)象。Real time-PCR結果表明:干擾該基因后,Djpiwi-1和Djpiwi-b的表達量明顯下調。(3)日本三角渦蟲Djhsp90a基因cDNA長為3212bp,包括5’端1039bp的非編碼區(qū)和3’端22bp的非編碼區(qū),其中包含一個長度為2151bp的ORF,編碼一個由716個氨基酸構成的蛋白質。其中包含6個熱休克蛋白90家族所特有的序列標簽。原位雜交結果顯示:在整體渦蟲中雜交信號主要分布在除腦及咽以外的大部分實質組織中;在再生個體中的芽基部位有較強雜交信號。RNA干擾該基因后,整體渦蟲出現(xiàn)明顯的頭部溶解現(xiàn)象并伴有身體持續(xù)性皺縮;頭再生尾片段再生被抑制;尾再生頭片段與對照比沒有明顯變化。Real time-PCR結果表明:干擾該基因后,Djpiwi-1和Djpiwi-b的表達量明顯下調。(4)日本三角渦蟲Djhsp90b基因的cDNA長度為2636bp,5’端有94bp的非編碼區(qū)和3’端有132bp非編碼區(qū),含有2409bp的最大開放閱讀框,編碼一個由802個氨基酸構成的蛋白質。該基因所推導的氨基酸序列含有5個HSP90家族所特有的標簽序列。整體原位雜交結果顯示:該基因在整體渦蟲實質組織中廣泛表達且身體兩側有較強的雜交信號;再生個體的芽基部位雜交信號較強。RNA干擾該基因后,整體渦蟲身體出現(xiàn)明顯的皺縮;頭再生尾和尾再生頭片段再生速度緩慢。Real time-PCR結果表明:干擾該基因后,Djpiwi-1和Djpiwi-b的表達量明顯下調。(5)日本三角渦蟲中Djhsp90c基因的cDNA長為2573bp,其中包含一個長度為2394bp的最大開放閱讀框,編碼一個由797bp氨基酸編碼的蛋白質,5’端有114bp的非編碼區(qū),3’端有65bp的非編碼區(qū)。包含6個HSP90家族基因所特有的標簽序列。原位雜交結果顯示:該基因在整體渦蟲中雜交信號主要分布在身體兩側及尾部;再生片段芽基部位有較強的雜交信號。RNA干擾該基因后,整體渦蟲口部兩側出現(xiàn)膨脹現(xiàn)象;頭再生尾和尾再生頭片段再生受到抑制。Real time-PCR結果表明干擾該基因后,Djpiwi-1、Djpiwi-b和Djh2b基因的表達量顯著下調。以上結果表明:渦蟲體內Djhsp70e、Djhsp90a、Djhsp90b和Djhsp90c基因在應激條件均具有細胞保護作用;四基因通過調控干細胞維持渦蟲體內組織平衡,并在再生過程中起重要作用。除此之外,Djhsp90c基因通過調節(jié)Djpiwi-b和Djh2b基因調節(jié)渦蟲體內干細胞的增殖和分化,是渦蟲再生過程中重要的調節(jié)因子。
[Abstract]:Dugesia japonica belongs to the phylum Platyhelminthes and Turbellaria. It plays an important role in the evolution of animal system and has strong regeneration ability. In this paper, Djhsp70e, Djhsp90a, Djhsp90b and Djhsp90c genes, which were continuously up-regulated but low-expressed during regeneration, were screened from the transcriptome database of Trichomonas japonica. Temporal and spatial expression patterns and functions of the four genes were studied by in situ hybridization, RNA interference and Real time-PCR. The results were as follows: (1) Real time-PCR was used to analyze the expression of four genes in Trichomonas japonica under cutting injury, exposure to ionic liquids, cold and heat stress. The results showed that the expression of the four genes was significantly up-regulated under stress conditions, and ionic liquids increased with the prolongation of cutting injury time. (2) The length of the Djhsp70e gene was 1982 bp, the non-coding region was 180 BP at the 5'end and the untranslated region was 38 BP at the 3'end, which contained the largest open reading frame (ORF) with a length of 1764 BP encoding a protein of 587 amino acids and two heat breaks. The results of in situ hybridization showed that the gene was widely expressed in all parts of the body except the brain and pharynx, and the hybridization signals were strong in both sides of the body and in the intestinal branches; the hybridization signals were strong in the bud base of the regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene. (3) The length of the Djpiwi-1 and Djpiwi-b gene was 3212 bp, including the non-coding region of 5'end 1039 BP and the non-coding region of 3'end 22bp. In situ hybridization showed that hybridization signals were mainly distributed in most parenchymal tissues except brain and pharynx, and strong hybridization signals were found in the bud base of regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b in T. japonicum was significantly down-regulated after RNA interference with the gene. The length of the B gene cDNA is 2636 bp, the 5'end has 94 BP non-coding region and the 3'end has 132 BP non-coding region. It contains the largest open reading frame of 2409 bp, encoding a protein composed of 802 amino acids. The amino acid sequence derived from the gene contains five tag sequences unique to the HSP90 family. The results of Real-time-PCR showed that Djpiwi-1 and Djpiwi-1 were interfered with the gene. The expression of jpiwi-b was significantly down-regulated. (5) The length of the Djhsp90c gene was 2573 bp, which contained a 2,394 BP open reading frame encoding a protein encoded by 797 BP amino acids, 114 BP non-coding region at the 5'end and 65 BP non-coding region at the 3'end. It contained six HSP90 family genes. The results of in situ hybridization showed that the hybridization signals of the gene were mainly distributed on both sides of the body and tail of the whole worm, and strong hybridization signals were found in the bud base of regenerated fragment. The results showed that Djpiwi-1, Djpiwi-b and Djh2b genes were significantly down-regulated after the gene was interfered with. In addition, Djhsp90c gene regulates the proliferation and differentiation of stem cells in vivo by regulating Djpiwi-b and Djh2b genes, and is an important regulator in the process of regeneration.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78

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本文編號：2216449