Biological Function Of High Photosynthetic Gene AtNRPC1 In Arabidopsis

In nature,photosynthesis is the energy source for the growth of all things.It can use solar energy to convert inorganic matter into organic matter and use it for the metabolism of organisms.In the early stage of the laboratory,a mutant of rice dark green ear nrpc1(negative regulator of photosynthesis and chloroplast development 1)was obtained.The leaves and ears of the mutant showed a dark green phenotype,and the photosynthetic efficiency was enhanced.The mechanism of the gene has a certain research basis.Using the homologous alignment,the homologous gene AtNRPC1 of the NRPC1 gene was amplified from Arabidopsis thaliana,and the corresponding mutant and overexpressing transgenic lines were purchased.The obtained mutant and overexpressed plants were identified to obtain homozygous mutants and highly expressed overexpressing strains,which were then used as research materials.In this experiment,chlorophyll content determination,photosynthetic rate determination,protoplast observation,chloroplast submicroscopic structure observation,yeast double-hybrid interaction analysis,dual luciferase reporter system analysis,RNA-seq analysis,Western-Blot and other experimental techniques were used.Explore the function of the AtNRPC1 gene.The results are as follows:(1)The content of chlorophyll a,chlorophyll b and carotenoid in atnrpc1 of the three leaves of the mutant atnrpc1,the wild-type Col-0 and the overexpression AtNRPC1-OE were significantly increased,which were about 40% higher than Col-0;The content of chlorophyll a,chlorophyll b and carotenoid in AtNRPC1-OE overexpressing lines were significantly decreased;The same phenomenon was observed in the silique,and the contents of chlorophyll a,chlorophyll b and carotenoid in atnrpc1 weresignificantly increased.The content of chlorophyll a,chlorophyll b and carotenoid in AtNRPC1-OE overexpressing strain was significantly lower than that in Col-0.(2)Determination of maximum photochemical efficiency(Fv/Fm)and net photosynthetic rate(Pn)of photosystem II,Fv/Fm and Pn of atnrpc1 were increased by 2.7% and21%,respectively,compared with Col-0;AtNRPC1-OE The Fv/Fm and Pn of the overexpressing lines were lower than Col-0 by 3.4% and 30%,respectively.(3)Three-week-old atnrpc1,Col-0,AtNRPC1-OE plant which leaf protoplasts were extracted and observed.The chloroplast of atnrpc1 was larger than Col-0,while the chloroplast of AtNRPC1-OE was smaller than Col-0.At the same time,the rosette leaves of three plants were taken for electron microscopy to observe the submicroscopic structure of chloroplasts.The chloroplasts of the mutant atnrpc1 were densely distributed,the chloroplasts were large and the thylakoids were folded into basal granules,and the chloroplast structure was more developed.AtNRPC1-OE chloroplast individuals are the smallest and the inner capsules are thinly stacked,and the granules are stratified.(4)For the fresh weight of biomass,dry weight of biomass,number of silique and 1000-grain weight,the above indicators in atnrpc1 increased significantly compared with Col-0,reaching 36%,47%,16%,11%;AtNRPC1-OE the above indicators were significantly lower than Col-0.(5)The bolting time of atnrpc1,Col-0,and AtNRPC1-OE plants were counted.The bolting time of atnrpc1 increased,two days later than Col-0,and the bolting time of AtNRPC1-OE decreased,three days earlier than Col-0.(6)Analysis of the tissue-specific expression of AtNRPC1 gene,which is mainly expressed in rosette leaves,stem leaves and silique,and expressed in other tissues with low or no expression;at the same time,the expression of this gene is also affected by photoperiod the effect,and peaked at 6h of illumination.(7)Subcellular localization of the AtNRPC1 gene is mainly expressed in the nucleus and cytoplasm.(8)The NRPC1 interaction protein GLKs have been screened out in rice by rice.In order to analyze the interaction between AtNRPC1 and AtGLKs,a yeast double hybrid vector was constructed.There is an interaction between AtNRPC1 and AtGLKs,but the interaction between AtNRPC1 and AtGLK2 is weak.(9)Using thedual luciferase system to detect the regulation of AtGLK1,AtGLK2 and AtNRPC1 transcription factors on the photosynthetic-related gene promoter,it was found that AtNRPC1 can inhibit the transcriptional activity of AtGLK1 on the photosynthetic-related gene promoters Lhcb2 and Chl27.(10)The RNA-seq technology was used to analyze the downstream genes of AtNRPC1.The common differential genes of atnrpc1,Col-0 and AtNRPC1-OE were screened,115 genes were up-regulated or down-regulated?The flowers genes(eg,AG,FLC,SPL15,etc.)and genes involved in chlorophyll metabolism were correlated by GO analysis and KEGG analysis.Genes.(11)Verification of the expression of some photosynthesis and chlorophyll synthesis related genes,combined with Western-Blot results,the expression levels of Lhca1,Lhcb2,PsaD,POR ect.in atnrpc1 were significantly higher than those in wild type.The increase in sex and the consistent protein level results;the expression levels of the above genes in AtNRPC1-OE were significantly lower than those in the wild type,and the protein levels were consistent.(12)The above-mentioned RNA-seq screened flowering genes were verified,and six flowering-related genes of AG,FLC,AGL42,SPL15,SEP1 and TCP1 were obtained,which accorded with the phenomenon that the over-expressed plants were flowering in advance.(13)Construction of AtGLKs-OE / Col-0,AtGLKs-OE/AtNRPC1-OE transgenic plants,the flowering time of AtGLK1-OE / Col-0 and AtGLK1-OE/AtNRPC1-OE was prolonged compared with the control.In summary,the mutation of AtNRPC1 gene causes significant changes in the expression and protein accumulation of downstream photosynthetic related genes,and chloroplast development is also affected.At the same time,AtNRPC1 gene also has an effect on flower development.These results laid the foundation for studying the molecular mechanism of AtNRPC1 gene and the high photosensitivity of plants.