Expression Variation Of The Astaxanthin Biosynthesis Genes In Phaffia Rhodozyma

Astaxanthin,a carotenoid synthesized by Phaffia rhodozyma,is quite valuable in the commercial industry.It has a number of biological functions including antioxidation,antitumous effect enhancing the immune system and coloring,thus it is widely used in aquaculture,medicine,health care products,cosmetics and so on.However,the productivity of the astaxanthin produced by P.rhodozyma strains is insufficient to meet the requirements of mass production.Therefore,it's significant to explore the development of synthetic route and adjusting mechanism of astaxanthin on the natural astaxanthin resources in P.rhodozyma strains.The focus of this paper is to learn more about the metabolic rule of astaxanthin and find the relationship between the carotenoids synthesis and expression of related genes.The following procedures were done:1.The production specificity of 12 P.rhodozyma strains in the early and late periods of incubation was analyzed in this section.In addition,according to the results above,the relationship between cellular anabolism and carotenoid production in JMU-VDL668,JMU-MVP14 and JMU-N3 has been lucubrated.It is found that: JMU-VDL668 grows slowly,and has a low yield of astaxanthin(0.31 mg/g DCW).?-carotene and ?-cryptoxanthin were not detected by HPLC.JMU-MVP14 grows comparatively slowly,but has a high yield of astaxanthin(5.2 mg/g DCW).It can accumulate masses of ?-carotene,?-cryptoxanthin and other unknown carotenes.JMU-N3 grows relatively fast and the biomass reached 13.45 g/L.It doesn't yield astaxanthin,but it can accumulate other kinds of carotenoids.The total carotenoids reach 0.53 mg/g(DCW).2.SYBRGreen Real-time PCR assay system has been established in this part established.First,the RT-PCR primers for ?-actin,gpd,18 S,idi,crtE,mm crtI,am crtI,mm crtYB,am crtYB,asy and crtR were designed.And then the standard curves of each gene were constructed.After optimization,all 11 genes can well amplify in the system.Finally,the software geNorm was used to test the expression stability of three commonly used ruler genes(?-actin,gpd,and 18S).The order of stability of the 3 genes in P.rhodozyma cells is: ?-actin> gpd> 18 S.3.The synthesis of carotenoids and the expression of related genes in different growing periods of JMU-VDL668,JMU-MVP14 and JMU-N3 were analyzed in this part.It is found that:(1).The expressions of idi,crtE,crtYB,crtI and asy genes in JMU-MVP14 are clearly positively related to the synthesis of carotenoids,which indicates that this kind of gene regulation profile can promote the product formation.The expression quantity of asy is 3 to 18 times greater than JMU-VDLL668 and JMU-N3.This may be the reason why JMU-MVP14 is a high-yield astaxanthin strain.(2).idi,crtYB,crtI and asy genes in JMU-VDL668 also showed a simultaneous increase in the carotenoid synthesis;and crtE has a higher expression in the whole growth process.The gene expression level of crtI was low in the early stage,increasing after 84 h.it is not sufficient to analyze the strain of crtI mRNA to translate phytoene dehydrogenase.(3).The expression of crtE,crtYB,crtI,asy were higher than JMU-MVP14 and JMU-VDL668,but the corresponding carotenoids yield was lower.Gene expression and carotenoid biosynthesis changes dynamically at different times,thus it is difficult to analyze its expression pattern.Using Real-time quantitative PCR method is not available to detect the crt R and am crtI gene fragment was amplified,which inferred the strain may also exist with other forms of regulation.