| Plant molecular pharming/farming is the use of transgenic plants to produce human-made pharmaceutical proteins on an agricultural scale.Compared with microbial or animal cell culture,it has the advantages of low cost,high safety and easy mass production.In addition to being one of the most important major food crops in the world,rice is also currently being used as a bioreactor for molecular pharming through Agrobacterium-mediated DNA transformation.Rice seeds have been used as plant bioreactors for the production of many recombinant proteins,vaccines and antibodies.Fibroblast growth factor 21?FGF21?is important for the early treatment of metabolic diseases such as hyperglycemia,obesity and liver cancer,and is expected to become a new generation of drugs for the treatment of diabetes,obesity and fatty liver.Microbial expression of FGF21 protein has the disadvantages of easy formation of inclusion bodies in cells,lack of post-translational modifications and high purification costs.At present,the transcriptome studies of rice mostly focus on biotic and abiotic stresses,and little is known about the whole genome functional changes caused by this DNA transformation method.For the sake of biosafety,it is necessary to understand the effect of expression of foreign genes on rice.In this study,the rice seed bioreactor platform was firstly established by using japonica rice from cold region in northern China,and then rice seeds were used to express the human FGF21 gene.After FGF21positive rice plants and seeds were obtained,the effects of FGF21 on rice metabolism were analyzed from the aspects of agronomic traits,microstructure,physiological level,transcription level and protein level.The results could lay a foundation for more efficient and safe expression of foreign proteins in rice bioreactors.The main results obtained in this study are as follows:1.A pre-requisite for the high efficiency transformation technique is a robust tissue culture system.The higher percent of callus induction and regeneration in different rice varieties was mainly depended on the type and concentration of PGRs.In this paper,the optimal combination of PGRs for Dongnong 428,Longjing25,Longdao 17,Kongyu 131 and Kendao 19 were 1 mg·L-12,4-D+2 mg·L-1 6-BA+4 mg·L-1 KIN+0.2 mg·L-1 NAA,1 mg·L-1 2,4-D+4 mg·L-1 6-BA+4mg·L-1 KIN+0.5 mg·L-1 NAA,2 mg·L-11 2,4-D+4 mg·L-11 6-BA+4 mg·L-1 KIN+0.02 mg·L-1NAA,1mg·L-11 2,4-D+4mg·L-1 6-BA+2mg·L-1 KIN+0.2mg·L-1 NAA,1mg·L-11 2,4-D+4mg·L-16-BA+2 mg·L-1 KIN+0.02 mg·L-11 NAA,1 mg·L-1 2,4-D+6 mg·L-1 6-BA+4 mg·L-1 KIN+0.5mg·L-1NAA,2 mg·L-1 2,4-D+2 mg·L-1 6-BA+4 mg·L-1 KIN+0.2 mg·L-1NAA,respectively.2,4-D was used for induction medium,and 6-BA,KIN and NAA were used for differentiation medium.Among them,Dongnong 427 and Longdao 14 have the highest ability to culture,so they are used as the next step in genetic transformation system research.2.Agrobacterium strain EHA 105 containing the plasmid pCAMBIA1301 was used for transformation.The frequency of transient transformation was expressed as the ratio between the number of calli showing GUS expression and the total number of calli kept for staining.The highest transformation efficiency in Dongnong 427 was obtained when calli were immersed in0.272 OD600?optical density determined at 600 nm?for 10 min.While it was best for calli of Longdao 14 to be infected with 0.592 OD600 for 20 min.Infected calli of the two varieties were co-cultivated on two pieces of sterile filter paper moistened with 1 mL liquid co-cultivation medium for three days.The expression of gus gene was confirmed by PCR analysis of plants of both varieties.3.Plant expression vectors were constructed and Agrobacterium-mediated transformation of rice was performed to obtain T0 plants.PCR-and RT-PCR assays yielded 50 transgenic positive plants and collected T1 seeds.In the second and third years of planting,T2 and T3 generation seeds were obtained for western blot analysis.There were 6 western blot positive lines in T2 generation and 12 western blot positive lines in T3 generation.Analysis of agronomic traits of transgenic and wild-type plants and seeds showed that grain weight of mature transgenic seeds was significantly lower than that of wild-type seeds,and there was no significant difference in the rest.The T3transgenic positive seeds were obtained for physiological analysis.There were significant differences in starch and sucrose content and metabolism-related enzyme activities between immature and mature seeds of transgenic FGF21 rice and wild rice.Scanning electron microscopy analysis revealed that there were differences in the glume structure of mature seeds between transgenic FGF21 rice and wild rice,and the transgene was less regulated than the wild-type vascular bundle.Subcellular localization analysis showed that FGF21 protein was distributed in cytoplasm and nucleus.4.Transcriptomic analysis of immature seeds of wild-type?CK?and transgenicFGF21 rice?T1,T2,and T3 strains?:KEGG pathway analysis revealed that 1,762 differentially expressed genes?DEGs?were mainly involved starch and sucrose metabolism,plant-pathogen interactions,endocytosis,pentose and glucuronide interconversion and galactose metabolism.Twelve candidate genes were selected from six pathways.The differential expression of candidate DEGs deduced from RNA-Seq was confirmed by qRT-PCR,demonstrating the accuracy and reliability of RNA-Seq data.By analyzing the transcriptional levels of the above five pathways,it was shown that FGF21 translocation could affect rice starch and carbohydrate metabolism,and then affect seed material accumulation,but FGF21 translocation did not cause rice immune response and stress response.5.Lablefree proteomics studies were performed on mature seeds of the wild-type?CK?and transgenic line T1.Among them,there were 1298 differential proteins,426 up-regulated proteins,and 872 down-regulated proteins in the CK group;there were 1209 differential proteins in the T1group,500 up-regulated protein,709 down-regulated proteins;CK and T1 shared a total of 1135differential proteins?426 up-regulated and 709 down-regulated?.The number of differential proteins after screening?fold change>1.5 and p value<0.05?was 437.The differential proteins were analyzed by GO enrichment.As a result,a total of 1355 differential proteins were annotated to 35 entries,of which 259 were up-regulated and 1096 were down-regulated.Pathway analysis was performed on the identified differential proteins and the identified 996 proteins?227differential proteins?were annotated to 89 pathways.The first 10 were significantly different?p<0.05?,including starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,lysine degradation,aminoacyl-tRNA biosynthesis,tryptophan metabolism,peroxisome,pantothenate and CoA biosynthesis,unsaturated fatty acid biosynthesis?biosynthesis of unsaturated fatty acids?,fatty acid metabolism and valine,leucine and isoleucine biosynthesis.Nine genes were selected from starch and glucose metabolism,amino sugar and nucleotide sugar metabolism,and peroxisomal pathways for qRT-PCR analysis.The number of up-regulated genes was 4,the number of down-regulated genes was 5,which was consistent with proteome changes,proving that the proteome data is reliable.Western blot analysis of the two enzymes OsAGPS1 and OsCht5 in CK and T1,T2 and T3 were consistent with proteomic results. |
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