Team:NEAU-Harbin/Project

1.The high-efficiency expression host—Aspergillus niger is coming!

1. Aspergillus Niger is an important fermentation microbial ,which is widely used in industrial enzymes'production and organic acid fermentation.It can produce more than 30 kinds of enzyme product such as amylase, acid protease, cellulase, pectinase and glucose oxidase. In fact, Aspergillus Niger has been applied in the field of food production for a long time.Because of its excellent ability of secreting proteins, Aspergillus Niger is developed for universal heterologous protein expression host.

　　As receptor bacteria of? genetic engineering ,Aspergillus Niger has? unique advantages upon bacteria and yeast .It's able to do majority? kinds of processing after translation , and the glycosylation system is similar to that of higher eukaryotes.It's well known for high protein secretion capacity and recognized as the safe strain for heterologous protein production,in that the fermentation and post-processing technologies are mature.In addition,its genome sequences has already been published. ?

2.Why AMT？
　　Agrobacterium-mediated fungal transformation(AMT)is a potential tool for performing? targeted and random mutagenesis .This method is commonly used for plant-cell transformations and recently widely applied to various fungus. In the AMT system,A.Tumefaciens is able to transfer T-DNA to a wide variety of fungi ,and it has especial high efficiency.

1. The gram-negative bacterium A. tumefaciens is a plant pathogen, which causes crown gall tumors.A. tumefaciens induces this tumorous growth by transferring a part of its DNA (T-DNA) ,which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of metabolites and regulators for the plant growth. But another segment,the virulence region, which is composed of a large number of vir genes,is necessary for the tumorigenicity .So the binary vector system is used,in which the T-DNA and the virulence region are placed on two separate plasmids.

3. Targeted gene replacement system

• (1)The targeted gene replacement system mainly adopts the principle of homologous gene recombination. ?We import homologous gene sequences to the nuclear genome specific receptor gene loci and replace the original genes ,so that we can achieve our purpose of modifying the original genome. In the? process of target gene replacement , the construction of vectors ?is of great importance, which typically includes 2 arms ,respectively in homology with the target gene’s homologous sequences on both sides ,and 1 selection marker gene which is used to eliminate noise bacteria. In our universal system, we choose GlaA5 and GlaA3 as the homologous arms on either side of the target gene site and HPH (hygromycin) as selective marker.

　　(2)In our previous detection methods in target gene replacement, we usually extract the genome and ?conduct the PCR detection, using the homology arms as primiers, which is somewhat complex and time-consuming.
(进行PCR检测的路线图)

　　(3)The noise from NHEJ
　　The insertion of exogenous gene may include another circumstance that the nonhomologous end,? not dependent on DNA homology, force the two DNA end to connect together,which is called  non-homologous end joining.  This mechanism allows the possibility that displacement vector is randomly inserted into chromosomes, greatly reducing the efficiency of homologous recombination.