探討Spo11在梨形鞭毛蟲囊體化過程中所扮演的角色

Identification of a role of Spo11 Protein in Giardia lamblia Encystation

陳宇芊(Yu-Chien Chen)

指導教授 ： 孫錦虹

國立臺灣大學/醫學院/微生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610352

摘要

梨形鞭毛蟲是一種常造成腸道疾病的原蟲類寄生蟲，其主要的症狀有腹瀉、噁心嘔吐、脫水、體重減輕、營養不良等。梨形鞭毛蟲在其生活史上主要有兩種型態：滋養體與囊體，在宿主體內時主要是以滋養體寄生，而當宿主由腸道排出體外後，滋養體會囊體化形成囊體以適應宿主體外惡劣的環境。梨形鞭毛蟲主要行二元分裂，在囊體化過程中會進行核複製，使得染色體套數由4N變為16N，這時可能透過大量表現meiotic gene homologs Spo11, Dmc1a 和 Hop1等，使雙核中的染色體進行類似減數分裂中才會進行的homologous recombination，以交換遺傳物質。
本研究想了解，梨形鞭毛蟲的gSpo11在囊體化過程中所扮演的角色。首先我們利用RT-PCR和Q-PCR來偵測gSpo11在滋養體和囊體化時期的RNA表現量，結果顯示不管是RT-PCR或是Q-PCR，在囊體化時期gSpo11的RNA表現量都明顯增加。接著建構gSpo11-HA重組蛋白質的質體並轉染到梨形鞭毛蟲內，以螢光免疫染色法偵測出gSpo11蛋白質多數分布於梨形鞭毛蟲的細胞核中，少數則分布於細胞質內。而從DNA切割活性實驗中得知gSpo11蛋白質具有切割雙股DNA的能力，並且過程中需要Mg2+離子或Ca2+離子的參與。藉由電泳位移實驗，發現gSpo11蛋白質具有結合雙股DNA與單股DNA的能力。然後由Q-PCR、西方點墨法與囊體計數得知，大量表現gSpo11蛋白質會增加囊體化相關基因cwp1-3、myb2、wrky以及topoII基因的表現與囊體形成的能力。然而當我們將gSpo11蛋白質在Toprim domain上兩個高保留性的胺基酸進行突變後，可發現gSpo11突變蛋白質其多數分布位置由梨形鞭毛蟲的細胞核改為細胞質，並且會使降低囊體化相關基因的表現與囊體形成的能力。而由染色質免疫沉澱的結果可得知gSpo11蛋白質能與囊體壁蛋白質CWP1-3的基因啟動子結合，並且由免疫沉澱法的結果顯示，gSpo11蛋白質具有與調控梨形鞭毛蟲囊體化相關轉錄因子WRKY和MYB2結合的現象。
我們的研究結果顯示，梨形鞭毛蟲的gSpo11會在囊體化時期大量表現，並且能活化囊體化相關基因的表現，我們認為這可能與gSpo11蛋白質能與囊體壁蛋白質的基因啟動子結合，並能與調控囊體化相關轉錄因子產生交互作用有關。

關鍵字

梨形鞭毛蟲 ； 囊體化 ； type IIB topoisomerase ； topoisomerase VI A subunit ； gSpo11 ； double-strand breaks (DSB)

並列摘要

Giardia lamblia is a kind of intestinal protozoan parasite which can cause disease called giardiasis. Symptoms include diarrhea, dehydration, nausea, vomiting and malnutrition. The life cycle of G. lamblia has two stages: a binucleate trophozoite and a quadrinucleate infectious cyst. G. lamblia divides through asexual, binary fission to reproduce. Evidence shows that Giardia has low levels of allelic heterozygosity in two nuclei. Some homologs of meiosis genes (HMGs) have been found in Giardia, including Spo11, Dmc1, Hop1, which only express in the cyst. It might indicate that the genetic materials of two nuclei exchange in encystation.
To identify the role of Spo11 protein in G. lamblia encystation, we used quantitative RT-PCR and Western blots to detect the gSpo11 RNA and proteins in
G. lamblia. Result shows that gSpo11 gene was up-regulated expression during the encystation stage. Immunofluorescence assay revealed that gSpo11 was major localized to nucleus. DNA cleavage assay indicated that gSpo11 can cleavage dsDNA, with the cofactors Mg2+ or Ca2+. By electrophoretic mobility shift assay, we showed that gSpo11 is able to bind ssDNA and dsDNA. Also we found the overexpression of gSpo11 can induce the expression of cwp1-3、myb2、wrky and topoII genes, and cyst formation. But when we introduced G202A and R209A mutations in the toprim domain of gSpo11, we found that mutant protein changed localization to cytoplasm, also the function of induction the expression of genes and cyst formation was inhibited comparing with wild-type protein. Use chromatin immunoprecipitation, we found that gSpo11 can bind the promotors of cwp1-3 gene. By co-immunoprecipitation assay, it indicated that gSpo11 can interact with encystation relating transcription factors, Myb2 and WRKY.
To sum up, this study suggests that gSpo11 plays an important role in induction of the cwp genes and encystation relating transcription factors. It might be related to gSpo11 can interact with Myb2 and WRKY, and bind the promotors of cwp genes.

並列關鍵字

Giardia lamblia ； Encystation ； type IIB topoisomerase ； topoisomerase VI A subunit ； gSpo11 ； double-strand breaks (DSB)

參考文獻

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