【作者】 董自梅；

【导师】 陈广文；

【作者基本信息】 河南师范大学 ， 动物学， 2011， 博士

【摘要】 本文利用整体原位杂交技术、实时荧光定量PCR技术(Real-time PCR).RACE技术、石蜡切片技术和生物信息学技术等,对日本三角涡虫和阳城多目涡虫再生过程中眼点的形态发生及相关基因的实时和原位表达情况进行了系统研究,结果如下：1.利用形态学和组织学方法,观察了日本三角涡虫(D.japonica)和阳城多目涡虫(P.yangchengensis)眼点的形态发生过程。结果表明：(1)在最适温度22℃恒温培养条件下,日本三角涡虫在切割后第4天能够看到眼点,第5天形成小的视杯,第15-16天左右眼点发育完成；在最适温度14℃恒温培养条件下,阳城多目涡虫在切割后第7天能够明显看到新生芽基,第14-15天隐约观察到眼点,第20-27天眼点数目增加较快。2.通过Real-time PCR技术,首次对三个内参基因——18sRNA、ACTB和DjEF2在涡虫头部再生过程中的表达水平和稳定性进行了研究,发现ACTB的原始Ct值变异系数最小；将所得数据分别用GeNorm和NormFinder软件分析后,提出在涡虫再生研究中,ACTB为首选内参基因。3.利用RACE技术对日本三角涡虫的Djopsin基因、阳城多目涡虫的Psix-1和Pβ-actin基因的全长cDNA进行了克隆、测序和序列分析,并根据Djopsin.Psix-1基因推导的氨基酸序列和Pβ-actin基因的核苷酸序列用邻接法构建了系统进化树；同时利用整体原位杂交技术和Real-time PCR技术研究了Psix-1基因在阳城多目涡虫成体及再生个体中的时空表达模式。研究发现：Djopsin基因的动态表达趋势与眼点形态发生的过程以及眼点避光反应的恢复趋势相对应,提出Ojopsin基因与眼点的避光反应等功能恢复密切相关；原位杂交结果表明：阳成多目涡虫成体Pstx-1基因的阳性信号主要出现在头部,眼点部位呈强阳性信号,而再生个体的杂交信号在新生芽基的基部表达最强；Real-time PCR结果显示Pstx-1的mRNA在切割后的第13、21天达到峰值。据此推测,Psix-1基因的主要功能是促进涡虫眼点前体细胞的分化,并对眼点的形成及维持起着重要作用。结合Djsix-1基因在日本三角涡虫中动态表达分析,指出Six-1i是涡虫眼点发育的关键性和标志性基因,在涡虫眼点整个发育过程中起着重要作用。4.利用Real-time PCR技术,首次对与日本三角涡虫(D.japonica)眼点再生相关的基因网络进行了全面分析。在同样实验条件下,对同一物种九个眼点再生相关基因(Djeya, Djsix-1, Eye53, DjotxA, Djnos, Djpax6, Djopsin, Djnetrin和1020HH)的动态表达情况进行了系统研究,根据它们在眼点再生不同时期的表达差异以及表达趋势将其分为早期表达组(Djeya, Djsix-1,Eye53,DjotxA和Djnos)、中期表达组(Djpax6)和晚期表达组(Djopsin, Djnetrin和1020HH)三组。提出涡虫眼点形态发生的过程也是基因网络协同作用的过程,同一基因在眼点再生的不同阶段可能执行着不同的功能,不同基因的功能可能是不同的,也可能是协同作用的。涡虫眼点再生相关基因的实时表达与眼点形态发生之间存在一定的对应关系。更多还原

【Abstract】 The eyespots morphogenesis and the transcripts expression of transcripts of eye-related genes in the genus Dugesia and Polycelis during eyespots regeneration were studied in this paper using whole-mount in situ hybridization, quantitative Real-time PCR, RACE technique, paraffin sections and bioinformatics. The results were as follow:1. Investigation of eyes morphogenesis in two species D.japonica and P. yangchengensis. Results showed that the eyes of D.japonica were first appeared about at 4 days after amputation, a semilunar eyecup were distinctly observed at 5 days, and the shape and size of new eyes were close to the intact planarians about 15-16 days in the dark at 22℃. then in P. yangchengensis, the regenerated tissue could be observed at 7 days after amputations in the dark at 14℃. The eyes were first appeared about 14-15 days after amputation and the regenerative rate was the fasest about 20-27 days.2. Selection of the most stable endogenous reference genes during planarian head regeneration. Three housekeeping genes,18s RNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder softwares. Results showed that ACTB exhibited the lowest variability and the most stable expressed control gene in the planarian head regeneration.3. Cloning and sequencing the full-length of three genes. Djopsin from D. japonica, Psix-1 and Pβ-actin from P. yangchengensis were cloned using RACE technology and sequenced. Three phylogenetic trees were constructed according to the deduced amino acid sequences of Djopsin and Psix-1, and nucleotide sequence of Pβ-actin by the neighbor-joining method, respectively. Results indicated that eyespots morphogenesis, recovery of photophobic response, and quantitative expression levels of Djopsin during eyes regeneration were corresponding, which implied that Djopsin were closely related to the functional recovery and maintenance of the planarian eyes. At the same time, the temporal and spatial expression of Psix-1 was studied by whole-mount in situ hybridization and quantitative real-time PCR in intact planarians and regenerative fragment, the results of whole-mount in situ hybridization showed that Psix-1 were expressed in both regenerating fragments and eyes of intact adults; the transcripts of Psix-1 was up-regulated at 13 and 21 days after amputations and in these days the eyes precursor cells were divised and the eyes were formed. So we speculated that Psix-1 played a key role in the eye differentiation (?) the functional maintenance of visual system.4. Analysis of nine eye-related genes in planarian D.japonica by quantitative real-time PCR. Nine (?) were selected, Djeya, Djsix-1, Eye53, DjotxA, Djnos, Djpax6, Djopsin, Djnetrin and 1020HH. According to their expression levels at different time, these genes could be divided into three groups:(1) early expression group, including Djeya, Djsix-1, Eye53, Djnos and DjotxA. which expression levels were significant increase at 1-3 days after amputation; (2) medium-term expression group, only including one gene, Djpax6, which expression value reached the peak on day 5; and (3) the late, including Djopsin, Djnetrin and 1020HH, which transcripts gradually increased with the eye regeneration. The data suggested that these genes consisted of the eye genetic network to control the eyes regeneration and development. In the genetic network, the single gene could play different function at different regeneration stage, the various genes could be distinctt function or the synergies. The corresponding relationship existed between eyespots morphogenesis and quantitative expression of these genes.更多还原