| Carotenoids are important photosynthetic pigments with many physiological functions,nutritional benefits and high commercial value.Astaxanthin is one of the carotenoids with powerful antioxidant activity,known for its numerous health benefits to humans and animals.Over the years,natural astaxanthin as well as other carotenoids have been in increasing demand year by year and can no longer meet the needs of the market,at the same time,how to improve the synthesis ability of carotenoids in organisms is also one of the current research hotspots.Chlamydomonas reinhardtii is a kind of single-celled eukaryotic green alga with clear genetic background,rapid growth,mature genetic manipulation and has a relatively complete biosynthesis pathway of carotenoids.Therefore,it is expected to be transformed into an ideal bioreactor for producing astaxanthin,?-carotene and other pigments.However,due to the low gene expression of some key enzymes such as ?-carotene ketolase gene bkt and ?-carotene hydroxylase gene crt Z,C.reinhardtii cannot produce a large quantity of astaxanthin and other carotenoids.The objective of this study was to improve the content of astaxanthin and other carotenoids in C.reinhardtii by introducing and efficiently expressing genes of rate-limiting enzymes of astaxanthin synthesis in the carotenoid biosynthesis pathway in C.reinhardtii,and by single-factor as well as multi-factor stress treatments.The main results are as follows:(1)?-carotene ketolase gene bkt from H.pluvialis,HSP70A-RBCS2 promoter,RBCS2 terminator,and vector containing expression cassette of ?-carotene hydroxylase gene crt Z from H.pluvialis preserved in the laboratory were used to successfully construct C.reinhardtii expression vector p64 DBKT which contained both expression cassettes of foreign gene bkt and gene crt Z.(2)The vector was transformed into C.reinhardtii by glass bead grinding.After exploring the transformation factors,it was determined that the optimum transformation conditions were vortex time of 35 s,plasmid amount of 5 ?g and antibiotic screening concentration of 75 ?g/m L.(3)After the resistance screening and successfully obtaining the C.reinhardtii transformant BZ3,PCR and RT-PCR were used to confirm that the gene bkt and the gene crt Z had been integrated into the genome of C.reinhardtii and expressed stably.(4)The culture conditions were optimized for wild-type C.reinhardtii WT and the transformant BZ3.It was determined that WT and BZ3 both grew well with high biomass under 4500 lx which was the optimum light intensity of normal culture.It was also found that the introduction of foreign genes did not affect the growth of C.reinhardtii.(5)The carotenoid content in wild-type C.reinhardtii WT and the transformant BZ3 was detected and analyzed by HPLC and UV spectrophotometer.The results showed that the highest content of astaxanthin in BZ3 reached 1.46±0.12 mg/g FCW(Fresh cell weight),which was 2.03 times of that in WT under the same condition;the maximum value of ?-carotene content reached 126.82±7.58 ?g/g FCW,which was 3.76 times of that in WT;the highest content of total carotenoids reached 0.58±0.04 mg/g FCW,which was 1.35 times of that in WT.These results indicated that foreign gene bkt and gene crt Z could be expressed normally in C.reinhardtii and had a positive effect on the synthesis and accumulation of carotenoids.(6)C.reinhardtii WT and BZ3 were subjected to single and multi-factor stress treatments of high light,high salt,and nitrogen deficiency,respectively,in order to explore the optimum stress condition to increase the carotenoid content.The results showed that the carotenoid content in BZ3 was always higher than that in WT cultured under the same conditions of single-factor,two-factor and three-factor stress.Astaxanthin and total carotenoids in BZ3 were significantly accumulated under the three-factor stress condition(salt concentration of 0.3 M),and astaxanthin content reached 2.61±0.14 mg/g FCW after 1 day,which was 1.78 times of that in WT under the same condition;the total carotenoid content reached the highest accumulation of0.93±0.04 mg/g FCW,which was 1.82 times of that in WT.At the same time,the maximum production of astaxanthin and total carotenoids of BZ3 reached 18.27±1.01mg/L and 7.44±0.32 mg/L,respectively,under this three-factor stress condition.Besides,the accumulation of ?-carotene in BZ3 reached the maximum value of 182.09±11.88?g/g FCW on the second day under the two-factor stress condition of high salt(salt concentration of 0.2 M)and high light,which was 3.11 times of that in WT under the same condition,and the maximum production of ?-carotene of BZ3 reached 1.38±0.09mg/L under this two-factor stress condition.Therefore,in this study,the three-factor stress condition(high light,nitrogen deficiency,salt concentration of 0.3 M)was considered to be the best stress condition for transgenic C.reinhardtii BZ3 to accumulate astaxanthin and other carotenoids;the two-factor stress condition(high light,salt concentration of 0.2 M)was the best stress condition for C.reinhardtii transformant BZ3 to accumulate ?-carotene.In this study,the foreign ?-carotene ketolase gene bkt and hydroxylase gene crt Z were successfully integrated into the genome of C.reinhardtii by transgenic technology and were stably expressed.The introduction of foreign genes of key enzymes increased the production of astaxanthin and other carotenoids in C.reinhardtii cells.Besides,single-factor and multi-factor stress treatments further promoted the synthesis and accumulation of carotenoids in C.reinhardtii,and the content of astaxanthin,?-carotene and total carotenoids was significantly increased in cells of C.reinhardtii.This study provides theoretical clues for exploring the metabolic pathways of astaxanthin and other carotenoids in C.reinhardtii cells,and lays a foundation for their commercial development.It is expected that C.reinhardtii will become a cost-effective and highyield bioreactor for astaxanthin and other carotenoids. |
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