【作者】 孙一萍；

【导师】 喻子牛； 张承才；

【作者基本信息】 华中农业大学 ， 生化与分子生物学， 2003， 硕士

【摘要】 本文利用生物信息学手段找出了鱼腥蓝细菌PCC7120基因组中所有的丝氨酸／苏氨酸／酪氨酸激酶和磷酸酶基因，对其结构和染色体上的遗传排列作了一定的分析。并对该细菌中部分与信号转导相关的基因进行中断，筛选得到突变体，并进行验证及初步表型分析。 1．鱼腥蓝细菌PCC7120基因组中丝氨酸／苏氨酸／酪氨酸激酶和磷酸酶基因的查找分析 经鱼腥蓝细菌PCC7120基因组的查找分析发现鱼腥蓝细菌PCC7120中共有52个丝氨酸／苏氨酸／酪氨酸激酶基因(STPK)和14个磷酸酶基因(PP)。按照STPKs结构的不同可划分为五个亚类：STK-Ⅰ亚类只含有一个催化区域；STK-Ⅱ亚类的C端含有WD40重复序列；STK-Ⅲ亚类的C端含有不同的调节区域；STK-Ⅳ亚类的催化区域位于C端，HSTK-Ⅰ亚类则既含有丝氨酸／苏氨酸激酶的催化区域，又含有组氨酸激酶催化区域。14个PPs共分为3族：属PP2C族的有8个，属PTP族的有3个，属PP1／2A／2B族的有3个。与单细胞集胞藻蓝细菌Synechocystis PCC6803基因组比较，只有11个基因可以在该基因组中找到相似的基因。经遗传排列分析发现，有12个基因在染色体上组成了6对基因簇(gene cluster)。 2．信号转导相关基因突变体的构建及表型分析 本研究全部通过插入失活获得突变体。首先将PCR扩增片段(～1.5kb)利用PstⅠ和XhoⅠ插入常规克隆载体pBluescriptSK，利用基因内部合适的酶切位点插入Sp／Sm抗性片段，再利用PstⅠ和XhoⅠ位点将已插入抗性基因的PCR片段，转入整合载体pRL271中获得体外基因中断，最后利用三亲本杂交获得体内突变。本研究共构建了91个体外基因中断结构，获得23个可能的单交换突变体，53个可能的双交换突变体，对所得的双交换突变体进行PCR验证发现有6个为野生型，4个为单交换突变体，其余43个均为真正的双交换突变体。将验证的真突变体在缺氮条件下观察其异型胞的发育以及生长状况，发现有2种突变体All2898~-，All1731~-在缺氮条件下不能生长也不产异型胞，另有3种突变体All4141~-，All1171~-，All4761~-在缺氮条件下可以产生异型胞，但仍不能维持正常生长，这可能是异型胞的结构被破坏或是异形胞中的固氮酶失活。所有这些基因在异形胞的发育过程中起重要作用。更多还原

【Abstract】 This dissertation searched for Ser/Thr and Tyr kinases and phosphatases genes by bioinformation tools in Anabaena PCC7120, inactivated the genes involved in protein phosphorylation/dephosphorylation, and analyze the phenotypes of mutants. 1. Search for Ser/Thr and Tyr kinases and phosphateses in Anabaena PCC7120We found 52 genes encoding Ser/Thr and Tyr kinases (STPK) and 14 genes encoding ser/thr and Tyr phosphateses (PP). We divided all STPKs into 5 subfamilies according to their own structure characterizes. STK-I only possess regions similar to the typical catalytic domain of STPKs, STK-II contain WD40 repeats, STK-III contain all kinds of regulator domain in their C-terminal region, STK-IV have their catalytic domains located in the N-terminal region, While HSTK-I also have a his kinase domain of two-component system. Among 14 PPs genes, 8 of them belong to PP2C, 3 belong to PTPs and only 3 genes encoding PP1/PP2A/PP2B could be found. Only 11 genes can be found similar genes in the Synechocystis PCC6803 genome by genomic analysis. And via the analysis of genetic array, 12 genes can be classified into 6 gene clusters in the chromosome.2. Inactivation of genes which involved in signal transduction and analyze the phenotypes of mutantsAll the mutants in this study were inactivated by inserting a resistance gene. PCR products were inserted into pBluescriptSK using PstI and XhoI. The element bearing resistance to streptomycin(Sm) and spectinomycin(Sp) was excised from pHP45 and inserted into gene coding region. The disrupted gene was isolated as a Pstl and Xhol fragment and cloned into pRL271. Triparental mating was then performed. As a result, we obtained 91 recombined plasmids, 23 possible single-cross mutants and 53 possible double-cross mutants. PCR analysis of the genomic DNA from the resulting possible double-cross mutants indicated that 6 of them were wild-type, 4 of them were single-cross mutants and the remainder ones were true double-cross mutants. Observe the heterocysts and growing states of these mutants after nitrogen depletion and found that A112898-, All 1731- can not different heterocyst, A114141-, A111171- and A114761- can different heterocysts but still can not maintain normal growth. The results indicated that all these genes play roles in hetetocyst differention.更多还原