Accumulation Mechanism Of Natural Astaxanthin In Haematococcus Pluvialis And Genetic Engineering Of Astaxanthin Production In Tobacco(Nicotiana Tabacum)

Astaxanthin(3,3’-dihydroxy-4,4’-dione-(3,(3’-carotene),a natural fat-soluble ketone carotenoid,has super antioxidant activity.It has been widely applied to the field of food,medicine,cosmetics,health products and aquaculture.At present,H.pluvialis is the best natural resources to realize the large-scale commercialized production of natural astaxanthin.However,astaxanthin accumulation of Haematococcus pluvialis has the characteristics of high production cost and low production efficiency.All of these restrict realization of the large-scale commercialized production for natural astaxanthin and the output can’t meet the need of growing market demand.Breeding of astaxanthin-concentrating crop new germplasm used as a bio-reactor to high-efficient generate astaxanthin.That is a hot point field with wide developing prospects and contributes to development functional agriculture and massive health industry of our country.For this purpose,growth of H.pluvialis and the effect of stress conditions to astaxanthin accumulation and metabolites associated with astaxanthin accumulation dynamic change in H.pluvialis were systematically analyzed in order to clear and definite critical factors for modulating astaxanthin accumulation in H.pluvialis;Autophagy involved in modulating astaxanthin accumulation and other biological processes of H.pluvialis under stress conditions,which were system tested in order to illuminate molecular mechanism of modulating astaxanthin biosynthesis as well as key genes mining;Tobacco(Nicotiana tabacum)with high biomass were genetically modified to assemble astaxanthin biosynthesis pathway in the hope of cultivating new tobacco varieties rich in astaxanthin in vegetative organs to highly efficient production of natural astaxanthin.The main results of the thesis are as follows:1.High light,high temperature and nitrogen deficiency are three major constraint factors in inducing astaxanthin biosynthesis of H.pluvialis.In order to establish an effective technical means for enriched astaxanthin,this paper measured the growth and metabolites such as astaxanthin in H.pluvialis under high light(≥7000 lx),high temperature(≥30℃),nitrogen deficiency(0 N)and triple induced.The results presented here demonstrated that the strength of inducing astaxanthin accumulation effect is triple induced>high light>nitrogen deficiency>high temperature in H.pluvialis 797.Adversity stresses activate autophagy and form autophagic vacuoles within H.pluvialis cells and autophagy synchronously increases with astaxanthin accumulation,oil biosynthesis and ROS content;Comparing with control,proline content increased 64.4 times from 12.61μg/mg to 812.65μg/mg under induced condition(P<0.01).And content of calcium(Ca),iron(Fe)and magnesium(Mg)increased remarkably(P<0.05).Cell biochemical or physiological changes make the ability of the stress resistance strengthen in H.pluvialis.2.To illustrate mechanism that autophagy-mediated astaxanthin biosynthesis in H.pluvialis under stress condition,autophagy inhibitor 3-methyladenine(3-MA,a compound that can effectively inhibit autophagy)was used to treat H.pluvialis cultured under high light stress.The effects of autophagy inhibition on cell growth,astaxanthin synthesis and lipid metabolism were analyzed.The present data show that 0.02 mM 3-methyladenine treatment effectively inhibited autophagosome formation but no impact on the cell growth and biomass of H.pluvialis(P>0.05),leading to a large increase in cellular reactive oxygen species(ROS)(P<0.01),astaxanthin and oil as well as palmitic acid high level enrichment(P<0.05).Furthermore,the key genes such as phytoene synthase(PSY),β-carotene ketolase(CRTO),and β-carotene hydroxylase(CRTR-b)in astaxanthin pathway are upregulated significantly,several times higher than control H.pluvialis(P<0.05);However the expressions of the lipid-related genes biotin carboxylase(BC)and diacylglycerol acyltransferase(HpDGAT2D)just increased slightly in the 3-MA-treated cells,compared with control cell of H.pluvialis.Astaxanthin accumulation is not perfectly linear with fatty acid biosynthesis in H.pluvialis treated by 3-MA(inhibition of autophagy)under high light.The research shows that autophagy takes part in the regulation of biosynthesis and accumulation of astaxanthin and oil.PSY,CRTO and CRTR-b are the key enzyme gene that controls astaxanthin synthesis.3.To further demonstrate whether the enzyme genes PSY,CRTO(BKT)and CRTR-b(BHY)can function and promote astaxanthin biosynthesis in heterologous hosts.Beta carotene hydroxylase of H.pluvialis(HpBHY),phytoene synthase of Zea mays L.(ZmPSY)and beta carotene ketonase of Chlamydomonas reinhardtii(CrBKT)ligated respectively into the expression vector.Transient expression these genes within tobacco leaves via agrobacterium-mediated.The biochemical indices of the tobacco leaves reveal that overexpression these target genes can promote astaxanthin or carotenoid enrichment.Especially,on the 5 days co-expression of HpBHY+CrBKT not only made astaxanthin content up to 0.518%(DW),but also made contents of chlorophyll a,chlorophyll b and carotenoid increase significantly to 10.33 μg/mg,3.012 μg/mg and 2.271 μg/mg in tobacco leaves(P<0.01).These results indicate that photosynthesis don’t decrease in astaxanthin-concentrating tobacco leaves at least.4.The transient expression results showed that promoting astaxanthin enrichment in tobacco leaves,enzyme genes HpBHY+CrBKT have the most effects.The two genes were introduced into tobacco by Agrobacturium-mediated leaf disk transformation method to assembly of astaxanthin biosynthesis passway in tobacco.The transit peptide sequence of tobacco ribulose-1,5-bisphosphate carboxylase small subunit(NtTp)integrated into front of the gene of HpBHY and CrBKT,respectively.Transit peptide leads target gene to locate plastid.The expression of NtTpCrBKT and NtTpHpBHY was driven by 35S promoter of cauliflower mosaic virus(CaMV)and ubq10 promoter of Arabidopsis,respectively.Molecular detection demonstrated that 19 transgenic tobacco lines could obtain co-transformation targeted fragments of HpBHY and CrBKT;Including 13 transgenic tobacco lines could transcript accumulation of HpBHY and CrBKT;Phenotype identification of transgenic tobacco lines reveal that T0 plants of floral nectary change from bright yellow of wild type to orange or red,and leaves change from green of wild type to purple-red;Co-expression of HpBHY+CrBKT made astaxanthin content up to 7.82 mg/g in T1 generation tobacco leaves at 45 day-old.The content of carotenoids in transgenic HpBHY+CrBKT tobacco leaves was up to 8.47 mg/g and 4.23 mg/g in wild tobacco.The content of chlorophyll in transgenic HpBHY+CrBKT tobacco leaves decreased to 16.45 mg/g comparing to 22.76 mg/g in wild tobacco,but no significant effect on the photosynthetic physiological indexes;Transgenic tobacco lines of co-expression of HpBHY+CrBKT enhance ability to protect themself from ultraviolet light and drought and there are no negative effects on the plant growth and other agronomic traits.5.To further improve the enrichment of astaxanthin in heterologous host tobacco,transcription factor CsMADS6 was used to construct co-expression vector with the above two enzyme genes HpBHY and CrBKT.Transcription factor CsMADS6 from citrus raises the activity of carotenoid metabolic pathways upstream genes,such as PSY,Phytoene desaturase(PDS)and Lycopene β-cyclases(LCYb),and carotenoid metabolic pathways downstream to degrade carotenoids gene,Carotenoid cleavage dioxygenase 1(CCD1).The expression of CsMADS6 was driven by mas promoter that is mannopine synthase promoter from Ti plasmid of Agrobacterium tumefaciens owing the features of plant promoter.CsMADS6+HpBHY+CrBKT co-transformation expression vector was transformed into tobacco by Agrobacterium through leaf disc transformation.Molecular detection demonstrated that 19 transgenic tobacco lines could obtain co-transformation targeted fragments of HpBHY,CrBKT and CsMADS6;Including 16 transgenic tobacco lines could transcript accumulation of HpBHY,CrBKT and CsMADS6;Phenotype identification of transgenic tobacco lines reveal that T0 plants of floral nectary change from bright yellow of wild type to orange or red,and leaves change from green of wild type to purple-red,and sepals of bud change from green of wild type to orange red;Co-expression of HpBHY+CrBKT+CsMADS6 made astaxanthin content up to 10.32 mg/g in T1 generation tobacco leaves at 45 day-old.The content of carotenoids in transgenic HpBHY+CrBKT+CsMADS6 tobacco leaves was up to 12.81 mg/g and 4.23 mg/g in wild tobacco.Corresponding,the content of chlorophyll in transgenic HpBHY+CrBKT+CsMADS6 tobacco leaves decreased to 14.94 mg/g comparing to 22.76 mg/g in wild tobacco;There are no negative effects on the plant growth and the photosynthetic physiological indexes of tobacco leaves compared with wild tobacco(P>0.05).Moreover,transgenic tobacco lines of co-expression of HpBHY+CrBKT+CsMADS6 enhance ability to protect themself from ultraviolet light and drought.6.For further analysis the global effect of co-expression three target genes on genome-wide expression of related genes in heterogenous host tobacco,especially that of carotenoid biosynthetic metabolism-related genes and carotenoid biosynthetic-related biochemical and physiological processes.Transgenic tobacco plants(BBC)with high expression of three target genes and high-yield production for astaxanthin and the wild-type tobacco(CK)as control were used as material.They were used to combine analysis of carotenoid metabolites and the transcriptome.A total of 33 carotenoids were tested,including 6 carotenes,15 carotenoid esters and 12 luteins.The main carotenoids are astaxanthin,lutein and β-carotene in transgenic tobacco BBC’s leaves and which represented 94.15%of total carotenoids.The main carotenoids are lutein,β-carotene and neoxanthin in wild tobacco CK’s leaves and which represented 87.91%of total carotenoids.High efficiency expression of exogenous gene,β-carotene hydroxylase(HpBHY),lead to down-regulate the expression level of endogenous gene,beta-carotene 3-hydroxylase 2(BCH2),in transgenic tobacco BBC.The content of β-cryptoxanthin and zeaxanthin increased and catalyzed by HpBHY.Canthaxanthin,echinenone and the goal astaxanthin catalyzed by exogenous gene,HpBHY and CrBKT,so canthaxanthin,echinenone and astaxanthin can biosynthesis in transgenic tobacco BBC’s leaves but missing in wild tobacco CK’s leaves.Gene co-expression analysis suggests that the goal astaxanthin accumulation had a positive relation with 9 key genes(geranylgeranyl pyrophosphate synthase(GGPPS),lycopene epsilon cyclase(LCYE)and zeta-carotene desaturase(ZDS),et al.)of tobacco endogenous carotenoid metabolic pathway in transgenic tobacco BBC of co-expression three target genes.The goal astaxanthin accumulation had a negative relation with beta-carotene 3-hydroxylase 2(BCH2),neoxanthin synthase(NXS)and 9-cis-epoxycarotenoid dioxygenase(NCED1)in transgenic tobacco BBC.BCH2 is tobacco endogenous beta-carotene 3-hydroxylase 2.Neoxanthin catalyzed by NXS,and neoxanthin is the final product of another branch in carotenoids metabolic pathway.Abscisic acid catalyzed by NCED1.In addition,compared with the wild tobacco CK,most genes expression are up-regulated of fatty acid or oil metabolic pathway in transgenic tobacco BBC’s leaves.This suggests that there was a significant association between astaxanthin enrichment and oil synthesis.This research revealed the regulating effect that autophagy participate in astaxanthin enrichment of H.pluvialis under stress-induced condition.That will enrich acknowledge of the regulative mechanism on astaxanthin biosynthesis.Two key enzyme genes from micro-algal which can control astaxanthin biosynthesis and transcription factor CsMADS6 from citrus that can modulate multi-enzyme genes of carotenoid pathway worked as the target.The enrichment pathway for astaxanthin has been successfully assembled in high biomass tobacco and tobacco new germplasm which can enrich high concentrations of astaxanthin has been successfully bred.The research findings of this paper provide theory and technique supports for the optimization system of astaxanthin production using H.pluvialis,using tobacco bioreactor sustainable and efficient to produce astaxanthin and helping multiple functions fortification products based on astaxanthin to commercialize.Our study also provides a scientific reference for analyzing complex metabolic engineering of synthetic biologists and improving agronomic traits in crop plants.