蓝光作用下黑曲霉形态发育与消减文库的分析

以持续黑暗培养为对照,采用扫描电镜观察了蓝光对黑曲霉孢子发育的影响,结果表明蓝光处理使菌丝粗壮,孢囊增大。以黑暗培养至36h再经蓝光处理3~4h的菌丝为实验组,对应时间段的黑曲霉菌丝为对照组,采用抑制性消减杂交(SSH)方法,构建了蓝光作用下黑曲霉的差异表达基因的cDNA文库。阳性克隆菌落经PCR扩增出200~500bp差异表达基因的cDNA片段,对序列进行同源性分析后发现两个差异表达的未知基因片段,为进一步研究蓝光诱导下黑曲霉差异表达的未知基因的功能提供依据。差异表达的基因片段大都与黑曲霉线粒体氧化还原酶类同源;荧光定量PCR分析结果表明所选取的部分差异基因片段在蓝光诱导下表达量均有提高。

Study on the Asexual Sporulation of Aspergillus niger under Blue Light Induction and Analysis of its Subtractive Library

The effect of blue light (BL) on the morphological development of Aspergillus niger was studied by the scanning electron microscopy (SEM) observation. Comparing with the darkness, BL was able to stimulate development of sporangiophore and conidiosphore, promote grownth of mycelium. Suppression subtractive hybridization (SSH) was conducted with tester cDNA which was from 39 to approximately 40h-old mycelium cultured under darkness and driver cDNA which was from mycelium illuminated for 3 to approximately 4h under BL after dark growth. Some cDNA bands were obtained by suppression PCR (polymerase chain reaction) with the subtractive cDNA. Positive bacterial clones were randomly picked and identified by colony PCR method. Through sequence alignments from GenBank, most of differential cDNA fragments were highly identical with some redox enzymes existing in mitochondria, and the quantitative measurement of these differential mRNA by real time RT-PCR indicated that relative expression of the identified gene fragments under BL induction was higher than that under darkness. Furthermore, the result suggested that some respiratory chain redox enzymes of mitochondria were involved in the photoresponse and consequently influence the metabolism. Among differential cDNA fragments two unkown sequences were found and their complete gene and gene function remained to be investigated.