| Phaeodactylum tricornutum,as diatom,is a photosynthetic eukaryotic microalgae with diverse forms,which is valuble not only in economy but also on ecology.The alga grows in a marine habitat and is able to accumulate high-value compounds such as fucoxanthin,chrysolaminaran,polyunsaturated fatty acids and so on.In addition,in virtue of its fast growth rate,no occupying fresh water resources,and accumulating lipid up to 20%-30% of its dry weight,P.tricornutum has become a potential biodiesel resource alga.In recent decades,due to the progress and build-up of the research data on its nuclear genome,chloroplast genome,mitochondrial genome,proteome,P.tricornutum has proved to be a "model organism" for study of the physiological and biochemical processes of diatom.In this dissertation,by establishing axenic culture of algal cells and optimizing the protocol of gene gun transformation and the method of transformant screening,The endogenous Pt PEPC1 and PTPEPC2 genes were knockdown and the exogenous transcription factors,At LEC1 and At LEC1-LIKE(At L1L),were transformed and heterologous overexpressed in P.tricornutum after codon optimization.The transformants with significantly increased lipid content was screened out and analized,and a complete set of genetic transformation system of P.tricornutum was formed.The primary research results are as follows:1.The axenic protocol of P.tricornutum was established.Four antibiotics with different concentrations were used to treat the algae with associated bacteria.The Ampicillin(Amp)group had no significant effect on the growth and related physiological and biochemical parameters of the algal cells.In the aspect of sterilization,the titer of Amp was relatively low,and there were still a large number of associated bacteria alive even at the concentration of 400mg/L.Chloramphenicol(Cmp)could promote the growth of P.tricornutum at low concentration(50mg/L)and inhibit the growth of the algal cells at high concentration(100-400mg/L).The analysis of chlorophyll fluorescence parameters showed that all concentration of Cmp groups were toxic to the algal cells.HPLC analysis showed that Cmp can increase the proportion of chlorophyll a in photosynthetic pigment of algal cells.The tolerance of algal cells and associated bacteria to Tetracycline(Tet)was almost the same,that is,the bacteria and alga could grow at low concentration(50-100mg/L)of Tet,and the growth was completely inhibited at high concentration(200-400mg/L)of Tet.Kanamycin(Kan)did not inhibit the growth of algal cells,and even promoted the growth of algal cells in high concentration(400mg/L).The Fv/FM value of 400mg/L Kan group was constantly higher than that of the control group.In terms of sterilization,100mg/L Kan could completely remove the associated bacteria in the culture medium.SYBR Green I staining verified the sterilization result of Kan.Therefore,Kan with the concentration of above 100mg/L is the best sterilizing agent for P.tricornutum,which has no undesirable effect on the growth of the algal cells.2.Establishment of highly efficient transformation method for microalgae by Gene Gun.A systematical study was performed on improvement of antibiotic selection medium,selection,preparation,coating with DNA and loading onto macrocarrier of microcarriers,optimization of bombardment parameters,and processing of recipient cells.The results showed that 50% salinity f/2 medium can increased the potency of zeocin,and the addition of 2216 E nutrients in the f/2 solid medium can shorten the plate selection time by 1/3.The preparation of microcarriers should be performed on centrifuge tubes with no adsorption of gold(tungsten)powder and the amount of per tube should be less than 3.5 mg.The amount of microcarriers bombardment per shot is about 0.75 mg,too much will cause a bombardment death circle,while too little will cause the bombardment cost to increase.When the bombardment spacing A is 1/4 inch,the spacing B is 11 mm,and the spacing C is 6 cm,the largest number of transformed cells can be obtained.In addition,a thicker multicellular layer of 109 receptor cells can significantly increase transformation efficiency.After the above optimization and improvement,the transformation efficiency was increased by 4.7 to 30 times than that reported in the previous literature,reaching 295±60 transformants/?g DNA.Besides,the optimized method could also be widely used for other microalgae(Dunaliella Salina and Chlorella sorokiniana)transformation.3.Optimization for microalgae direct PCR.To explore the reasons for the instability of microalgae direct PCR methods previous,one-way analysis of variance was used to analyze the effects of lysis buffer type,cell number,growth stage,and lysis boiling time on the crude DNA content in the lysate.The results showed that the highest content of crude DNA(344 ng ?L-1)was obtained with lysis buffer 1% NP-40(v/v)by using stationary phase cells,which was nearly twice of the content of crude DNA with buffer 0.5% Triton X-100(207.5 ng ?L-1)and three times of those with buffers TE(107.5 ng ?L-1)and 1% SDS(108 ng ?L-1).It was also found that increase of the number of microalgal cells led to non-linear elevation of the crude DNA content,and the amount of crude DNA from the cells within logarithmic phase was significantly higher than those within stationary phase or decline phase.The effect of lysis boiling time was insignificant when boiling time ranged from 5 to 15 min.The results of PCR amplification with the crude DNAs isolated with different buffers indicated that efficient and repeatable amplification could be realized only when the amount of crude DNA added in 20 ?L PCR reaction ranged from 50 to 200 ng,while impacts of lysis buffers themselves on the PCR reactions were negligible.Hence,it was found that the amount of crude DNA in the lysate should be measured and this was a key factor to ensure the positive PCR amplification regardless of which lysis buffer,cell number,growth stage,and lysis boiling time were used.4.Silencing endogenous Pt PEPC1 and Pt PEPC2 genes increased the lipid content of P.tricornutum.Two phosphoenolpyruvate carboxylase genes,Pt PEPC1 and PTPEPC2,were cloned from P.tricornutum.The inverted-repeat RNA interference structures were designed according to the conserved sequence region of the two genes and plasmids were constructed.After transformation,the transformants were screen out.The transformants showed significant decrease not only in m RNA transcription level but also in PEPC enzyme activity.The analysis of physiological and biochemical parameters showed that the maximum cell density of the transformants were 8% ? 12% lower than that of the wild type.The Fv/FM was about 5% lower and 20% lower than those of the wild type in the logarithmic growth period(day 8)and in the stable period(day 14)respectively.There was no significant difference in r ETR values between the transformants and the wild type.NPQ of the transformants decreased by 16% ? 31% compared with the wild type.This study showed that there might be "C4 metabolism" instaed of C4 pathway in P.tricornutum,to helping algal cells to consume excess light energy.In addition,the neutral lipid content in the transformants increased by 18-22% compared with wild type,which indirectly proved hypothesis of "substrate competition".5.Overexpression of exogenous At LEC1 and ATL1 L transcription factors increased the lipid content of P.tricornutum.In this study,the codon optimized ATLEC1 and ATL1 L transcription factors were transferred into P.tricornutum cells,and subcellular localization of the transformed genes in the selected transformants was in the nucleus.The results of Southern and Western hybridization showed that the foreign gene had been integrated into the nuclear genome of P.tricornutum and could be stably expressed in the algal cells.There was no significant difference in the maximum cell density between the transformants and wild-type algae.The neutral lipid content of At LEC1 transformants increased by 15% ? 24%,and that of ATL1 L transformants increased by 42% ? 64%.The results of transcriptome sequencing showed that there were significant changes in the expression of 1876 genes,of which 1512 genes were up-regulated and 364 genes were down-regulated.Up-regulated genes are mainly involved in fatty acid synthesis,glycerol metabolism,glycolysis,pyruvate metabolism and other pathways.Metabonomics analysis showed that the contents of glucose,fructose,mannose and other monosaccharides in At L1 L transformants decreased significantly,and the contents of organic acids such as pyruvate,malic acid,succinic acid and fumaric acid,as well as acetyl coenzyme A,malonyl coenzyme A and glycerin-3-phosphate,which were the raw materials for lipid synthesis,increased significantly.The results of this study showed that At L1 L transcription factor restricted the flow of photosynthetic fixed carbon into carbohydrate synthesis by up-regulating genes related to glycolysis and lipid synthesis pathway.Meanwhile,these fixed carbon were siphoned into lipid synthesis,which resulted in a significant increase of lipid accumulation in the transformed algae. |
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