【作者】 高雅君；

【导师】 杨玉荣；

【作者基本信息】 厦门大学 ， 动物学， 2007， 硕士

【摘要】 细胞极性(cell polarity)是多种不同类型细胞的共同特征,对细胞的分化和功能有重要作用。在C. elegans中par基因家族包括par-1到par-6六种基因,除par-2以外,其余几种在C. elegans、果蝇、哺乳动物中都有同源物存在。par基因的突变会导致受精卵极性以及很多早期不对称现象消失。除此之外PAR在细胞连接、代谢和癌症方面也有重要意义。细胞命运决定因子cdc-42在该过程中也有重要作用,但这几种基因之间的相互作用还不太清楚。本文通过原位杂交、RNAi等方法在mRNA和蛋白水平对几种基因间的相互作用进行了研究。通过构建发夹结构形成双链RNA(dsRNA)的方法和双T7启动子L4440为载体形成dsRNA的方法进行RNAi载体的构建,通过细菌喂食法进行RNAi分析,发现两种方法都可以取得较高的干扰率。保证了以后转基因虫体的RNAi分析结果。本文对par基因在mRNA水平和蛋白水平的表达情况进行了分析。通过原位杂交检测了par基因在mRNA水平基因之间的相互作用,发现:par-1、par-2、par-3、par-5、par-6的mRNA都在卵母细胞、早期胚胎和雌雄同体成虫生殖腺中分布。其中par-2和par-4对其它几种par基因的mRNA表达起到重要的作用,par-2突变导致par-1、par-3、par-6的表达量减少;par-4突变时,par-1、par-5表达量都减少了,但par-2和par-4之间没有明显的作用。另外par-5突变的情况下,导致par-1和par-3 mRNA表达量增加。par-3突变也会导致par-2和par-5的表达量改变。par-6突变的情况下,其它几种par基因的mRNA在约256细胞的胚胎开始,分布发生改变,由原来的细胞质均匀分布变为后端的局域分布,集中在胚胎后端。暗示:par-4对par-1、par-3和par-5 mRNA的表达有促进作用;par-1、par-3与par-5相互抑制,par-1对par-2有抑制作用,par-2是par-1表达所需。par-6抑制par-3和par-5 mRNA的表达,同时par-2对par-6又有明显的促进作用。分析PAR-2、PAR-5和PAR-6的GFP在C. elegans和par突变体中的分布情况,发现par-1突变的情况下PAR-2::GFP的后端皮层分布前移,而PAR-6::GFP的皮层分布消失。在par-2或者par-5突变的情况下,PAR-6的前端皮层不对称分布消失,在整个皮层中都有分布。同样par-5、par-6突变的情况下,PAR-2::GFP在皮层的不对称分布也消失了。cdc-42原位杂交结果显示,其mRNA的分布情况与par基因的分布情况类似,也是在卵母细胞、早期胚胎和成虫生殖腺中分布。par-4对cdc-42 mRNA的表达也起到重要的作用,par-4突变的情况下,cdc-42由原来的均匀分布变为细胞外周分布。par-1和par-5突变会导致cdc-42的表达量降低,par-2突变则会导致cdc-42的表达量增加。更多还原

【Abstract】 Cell polarity is a crucial factor for a variety of cellular and developmental processes. par family contains six par genes, from par-1 to par-6. In Caenorhabditis elegans, mutations in six par genes, par-1 through par-6, cause the earliest and most extensive polarity defects in the zygote, eliminate many early asymmetries. Except par-2, the other PARs have orthologs in mammals and Drosophila. PAR proteins play important roles in cell junction, metabolism and cancer. Furthermore, the cell polarity regulator cdc-42 also plays an important role in this process. But the interaction of these genes is not clear. Thus, using RNA interference and in situ hybridization, we have done some work on the interaction of these genes in C. elegans.RNAi was performed using the bacteria feeding method to produce RNA interference. Double-Stranded RNA expressed by either hair-pin structures in pBluescript II SK(+) vector or L4440 with two T7 promoters, both methods proved via experiments have produced effective and specific RNA interference. To explore the interaction of par gene, we examined their expression of mRNA in C. elegans wild type and par mutants by in situ hybridization. The results show that par-1, par-2, par-3, par-5 and par-6 mRNA are all distributed extensively in oocyte, early embryos and the gonad of hermaphrodits. par-2 and par-4 are essential for other par mRNA expression. par-1, par-3 and par-6 mRNA decreased in par-2 mutant, the expression of par-1 and par-5 mRNA decreased in par-4 mutant. But there is no obvious direct interaction betweem par-2 and par-4. In addition, the absence of par-5 gene results in the increase of par-1 and par-3 mRNA expression. And the absence of par-3 gene also results in the increase of par-2 and par-5 mRNA expression. In par-6 mutant, from about 256 cells of embryo, par mRNA distribution are different, mRNA distributed in the posterior part of the embryo but not even in the embryo. We have conclude that par-4 promotes the expression of par-1, par-3 and par-5; par-1 and par-3 repress par-5, and par-1 represses par-2, but par-2 is required in par-1 expression. par-6 represses the expression of par-3 and par-5 mRNA, while par-2 has obvious stimulation to par-6.To test the distribution of PAR-2, PAR-5 and PAR-6 GFP in C. elegans wild type and par mutant via RNAi, we found that the posterier cortical PAR-2::GFP extend toward the anterior, while the cotical PAR-6::GFP disappeared in par-1 mutant via par-1 RNAi. In par-2 or par-5 mutant via RNAi, the anterior cortical PAR-6::GFP extend to the entire cell cortical. PAR-2::GFP can accumulate in the cortex, but can’t get enriched in the posterior in the absence of par-5 or par-6.The distribution of cdc-42 mRNA in C. elegans wild type and par mutants via in situ hybridization shows similar results to par genes’in situ hybridization. cdc-42 mRNA is also distributed extensively in oocyte, early embryos and gonad of hermaphroditism. par activity is required for the expression of cdc-42 mRNA, especially par-4. In par-4 mutant, cdc-42 mRNA distributed in the periphery of zygote while which uniformly distributed in wild type. The expression of cdc-42 mRNA decreased in par-1 or par-5 mutant, but increased in par-2 mutant. Key Words: Caenorhabditis elegans; cell polarity; par; cdc-42更多还原