| Aspergillus oryzae is an important filamentous fungus in traditional fermentation industry,and it is also an important expression system of heterologous protein which is listed as GRAS strain by US Food and Drug Administration(FDA).A.oryzae is a typical semi-cognic fungus and its conidia have various karyotypes,most of which are multinucleated and a few mononucleated,which brings challenges to strain modification and breeding industrial.In the process of strain modification,the heteronuclear transformants are more likely acquired,which will properly lead to decrease in the expression level of recombinant proteins and loss of target genes during passage.The characteristic of polykaryotic in conidia will dilute the phenotypic effects of genetic manipulation in industrial breeding.In order to solve the dilemma,it is urgent to find a way to obtain homozygous monospora.At present,the methods of single spore separation mainly include direct single spore isolation,membrane filtration and enrichment of monocyte spores,capillary electrophoresis,and passive separation based on microfluidic platform.In this paper,we have devised a group of microfludic passive sepration structions,which are consist of Inertial Focusing and Determined Lateral Displacement and can achieve high throughput and high precision separation of mononuclear conidia of A.oryzae H4.This method also provides a solution to the difficulty of obtaining homozygous transformant by genetic modification of A.oryzae.The achievements are mainly described as follows:1.Karyotype analysis of conidia of A.oryzae H4.DAPI nuclear staining of conidia showed four karyotypes of Aspergillus oryzae H4,they are respectively:mononuclear,dinuclear,trinucleate and tetranucleate.Futher analyzed the morpholology of nucleate,it is found that there are two kinds of arrangement in the trinucleate,one form is curved and close to the wall,the other is equilateral triangle.The diameter of conidia of A.oryzae was measured by the software of Nano Measure.The result showed that the diameter of conidia collected on MES medium was mainly distributed in the range of 2～6μm and there was a certain proportional relationship between the diameter and the number of nuclear of the condia.We can also know that the proportion of mononuclear spores below 3μm was 8.1%.At the same time,the improvement effect of inositol on mononuclear rate was further investigated,it was found that the proportion of mononuclear spores under 3μm collected on inositol medium increased to 34.4%.It is proved that inositol has certain effect on the increase of mononuclear rate.2.Based on the basic theories of inertial focusing and deterministic lateral displacement,a microfluidic chip was designed for single conidia separation.According to the theory and empirical formula of inertial focusing and deterministic lateral displacement and combining with the purpose of this experiment,a set of secondary microfluidic separation structures were designed for the separation of mononuclear spores below 3μm.The first separation structure is a sprial inertial focusing which has a cross section of 100×50μm(w×h),initial radius of 3 mm,channel spacing of 250μm and total spiral number of 5 turns and used for separating large impurities and conidia larger than 3.5μm in diameter.In order to further improve the purity of the target particles,the target particles collected by the primary separation structure are connected to a secondary DLD structure with high precision separation,which design parameter is D_C=3μm,ε=0.05,column spacing G=9μm,and microcolumn diameter=25μm.Conidia with diameter less than 3μm are expected to be obtained by the process of the two structure above.3.Rigid polystyrene microspheres were used to characterize the separation performance of the two structures.Before the actual separation experiment of biological particles,the separation performance of the sprial inertial focusing chip and DLD chip was characterized by rigid polystyrene microspheres which shape is regular and not easy to deform.For accurate characterization the performance of the chip,the separation performance is characterized from two main influencing aspects:the particle concentration and velocity.We will explore the optimal particle sorting conditions from the recovery rate of target particles under different conditions.Polystyrene microspheres with different diameters were used to simulate the proportion of particles with different diameters in actual biological samples,and the separation ability of secondary separation structure was characterized from three aspects of recovery,purity and enrichment factor.The optimal conditions for particle sorting are as follows:For the first inertial focusing chip,with the concentration of 10~7 particles/m L and the speed of 18 m L/h,the recovery rate of the target particles at the target outlet is 17.42%,the purity is 90%,and the enrichment factor is 17.5;Similiarly,for the secondary separation structure,with the concentration of 10~6 particles/m L and the speed of 60μL/min,the recovery rate of target particles at the target outlet is 85.42%.the purity is 100%and the enrichment factor is 11.1.It was proved that the secondary structure designed in this paper could achieve high-throughput and high-precision separation of mononuclear conidia below3μm.4.The lipase gene was transformed into A.oryzae H4 by agrobacterium-mediated transformation,and the monocytes were obtained by the secondary microfluidic separation structure.After sorting performance characterization of inertial focusing and DLD secondary structures using rigid polystyrene microspheres,the conidia of the actual biological particle A.oryzae H4 were sorted according to optimized conditions.The sorting effect was also verified by DAPI nuclear staining.The obtained results are basically consistent with the sorting effect of the rigid polystyrene microsphere.The lipase gene carrying with the reporter gene of green fluorescence gene was transferred into A.oryzae H4 by agrobacterium-mediated transformation.We will obtain the heterokaryon after the initial screening.then collected the conidia of the heterokaryon on the inositol medium and processing it with the two stage separation structure step-by-step,finally we will obtain the single-core homozygous.In order to compare the difference between homozygous and heterozygous,the fermentation culture was carried out and the concentrated crude protein was verified by polyacrylamide gel electrophoresis.It can be roughly inferred that the expression of lipase in homozygous is higher than that in heterozygous. |
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