Functional Analysis Of High Quality Storage Protein Gene AhAMA1 In Amaranthus Hypochondriacus And Its Genetic Transformation In Chlorella Pyrenoidosa

Chlorella pyrenoidosa is a new resource food with high level of protein content and complete amino acid composition.However,such algae accumulate lower levels of sulfur-containing amino acids such as methionine and cystine,two kinds of essential amino acids for human.Therefore,it is so necessary to increase the accumulation of sulfur-containing amino acids in C.pyrenoidosa,which will improve the algal protein quality of and also benefit the development of high-quality microalgae foods with balanced nutrition and essential amino acids.Amaranthus hypochondriacus L.is an annual crop used for food,feed and vegetables with its seed having high level of protein and abundant nutrients.The Ah AMA1 protein derived from mature seeds of A.hypochondriacus is an excellent storage protein with well-balanced nutrition and high levels ofeight essential amino acids.Ah AMA1 is also a non-allergic protein for human.In this paper,the Ah AMA1 gene encoding a high-quality storage protein was isolated from developing seeds of A.hypochondriacus followed by examination of its spatial and temporal expression profiles.Bioinformatics tools were used to analyze the physical-chemical properties and phylogenetic evolution of Ah AMA1 protein.The constitutive plant expression vector of Ah AMA1 gene was constructed,and then used for genetic transformation of C.pyrenoidosa by particle bombardment in order to develop the novel strains of C.pyrenoidosa with high-quality protein and essential amino acid nutrition.In addition,Agrobacterium-mediated infiltration was used for the transiently express of Ah AMA1 in the leaves of Nicotiana benthamiana.Analysis of the effects of Ah AMA1 heterologous expression on the physiological and biochemical traits such as protein,oil and starch content in tobacco leaves was performed to evaluate whether Ah AMA1 gene could be used for genetic improvement of protein content and nutritional quality in plant vegetative organs having large biomass.The main findings are as follows:1.The ORF sequence(915 bp)of Ah AMA1 gene was isolated from the developing seed of A.hypochondriacus L.cv.SX-04 using the homologous cloning technique.Ah AMA1 encodes a 304 amino acid protein with a molecular weight of 34.96 k D and a theoretical isoelectric point(p I)of 6.65.Secondary structure prediction and 3D structural simulations showed that the protein consisted of alpha helix(11.51%),extended chain(36.18%),beta turn(4.93%)and random coil(47.37%),with its function form as homodimers.-44-2.Quantitative expression profiling revealed that Ah AMA1 gene expressed in roots,stems,leaves andseeds,with higher expression in the late stage of seed development,which coincided with the rapid enrichment of seed protein.3.A constitutive plant expression vector Ah AMA1-GFP-p BI121 was successfully constructed,which contains a kanamycin resistance gene as the selection marker,and the reporter genes of GUS and GFP.4.The Ah AMA1-transient expression tobacco leaves were obtained by Agrobacterium-mediated infiltration.Biochemical analysis on such tobacco leaves showed that the content of eight essential amino acids increased in a balanced manner,amd moreover,protein content in the leaves of Ah AMA1 expression was significantly increased to 22.39%(w/w),which was twice as much as that of wild-type tobacco leaves.No significant difference in oil and starch contents were detected between the Ah AMA1 expressed and the wild-type tobacco leaves.This indicates that the Ah AMA1 gene can be used to be efficient expression in heterologous host tissues,promoting the accumulation of high-quality storage proteins,and consequently leading to the genetic improvement of the nutritional quality in high-biomass plant vegetative organs.5.The purified microalgal strain used here was identified to be C.pyrenoidosa FACHB-09 by 18 S r DNA sequence analysis.The biomass of C.pyrenoidosa was measured to determine the effects of different culture conditions and factors on the growth of C.pyrenoidosa FACHB-09.The optimal culture conditions and parameters for this microalgal strain were established,including LED warm white light(photoperiod of12 h/12 h),temperature at 30 ?,light intensity at 4000 Lx,initial p H=8 and so on.Na NO3 was the best nitrogen source for this strain,The daily growth of algae cells was as high as 0.78 g·L-1 when the algae grew in the full nitrogen culture.6.The concentration of kanamycin(1000 ?g·m L-1)was determined for screening the positive transformants of C.pyrenoidosa FACHB-09.The Ah AMA1-GFP-p BI121 vector was introduced into the algal strain FACHB-09 cells by particle bombardment.Molecular examinations on DNA and RNA as well as reporter genes showed that the Ah AMA1 gene was successfully integrated into the genome of C.pyrenoidosa FACHB-09 and expressed efficiently.Finally,the stable transgenic algal cell lines were obtained.7.Comparative analysis of cell morphology,biomass and protein content in the wild-type and transgenic C.pyrenoidosa FACHB-09 showed that there was no significant difference between the transgenic and wild-type microalgae.However,the protein content in the transgenic lines was as high as55.94%,which was 45.98% higher than that of the wild-type algae.The eight essential amino acids including isoleucine,leucine,lysine,methionine,phenylalanine,threonine,tryptophan and proline in the algae powder of the transgenic C.pyrenoidosa FACHB-09 were increased by 32%,47%,42%,62%,31%,14%,18% and 25%,respectively,compared to the wild-type algae.This demonstrates that the high-quality storage protein Ah AMA1 gene derived from higher plant A.hypochondriacus can be used for genetic improvement of the single-celled C.pyrenoidosa to significantly improve the nutritional quality of the microalgae.In summary,the high-quality storage protein gene Ah AMA1 was isolated from A.hypochondriacus,and the function of this gene was characterized by transient heterologous expression in tobacco leaves.More importantly,Ah AMA1 gene was successfully introduced into C.pyrenoidosa by genetic engineering,and the excellent cell lines of engineered C.pyrenoidosa were developed,which can produce high levels of protein and well-balanced eight essential amino acids.The present findings provide a theoretical basis and excellent germplasm for the development of functional microalgae foods.