| Photosynthetic efficiency is an important factor affecting plant yield and biomass.Before,our research group cloned and named a high-efficiency gene NRPC(negative regulator of photosynthesis and chloroplast development)related to rice leaf color and compared them with Arabidopsis thaliana AtNRPC1.The leaf and silique of Arabidopsis atnrpc1 mutant showed dark green color and it was found that the Arabidopsis AtNRPC1 can interact with the transcription factor AtGLKs.In view of the previous results of our research group,Arabidopsis AtNRPC2 and AtNRPC3 were been cloned,the function and expression pattern of AtNRPC2 and AtNRPC3 were analyzed.The phenotype identification,photosynthetic pigment content,photosynthetic rate,yield and biomass of Arabidopsis atnrpc2,atnrpc3 mutants and AtNRPC2-OE,AtNRPC3-OE overexpression strains were carried out.Thus,the biological functions of AtNRPC2 and AtNRPC3 in Arabidopsis were thoroughly investigated and the effects of AtNRPCs on the growth and development of Arabidopsis were systematically analyzed.Furthermore,MtNRPCs of Medicago truncatula were cloned by homologous comparison of OsNRPC1,as well as the function and expression pattern of it had been analyzed.Based on the Medicago truncatula MtNRPCs genome sequence amplification to obtain Medicago sativa L.MsNRPCs,the Medicago sativa L.MsNRPCs CRISPR / Cas9 knockout vector was constructed,a complete and mature Medicago sativa L.transgenic system was established,and the successful transformation of MsNRPC1 and MsNRPC2 double knockout vectors Seedlings of Medicago sativa L..Above all,Arabidopsis AtNRPC2 and AtNRPC3 based on the homologous sequence of the rice OsNRPC1 were been cloned.Through yeast two-hybrid experiments,AtNRPC2 interacting with AtGLK1 and AtNRPC3 interacting with AtGLK1 and AtGLK2 were been demonstrated respectively.Real-time fluorescence quantitative PCR indicates that AtNRPC2 and AtNRPC3 are specifically expressed in the wild-type plant tissues at mature stage,these two genes have different expression patterns.Later,CRISPR/Cas9 knockout and overexpression vectors for AtNRPC2 and AtNRPC3 in Arabidopsis were constructed,atnrpc2,atnrpc3 mutants and AtNRPC2-OE,AtNRPC3-OE overexpression lines were obtained by transgenic technology.Comparing these knockout mutants and over-expressing plants with wild type,it was found that the leaves and siliques of atnrpc2 mutant and atnrpc3 mutant were greener than wild type and the atnrpc2 mutant had darker green leaves than the atnrpc3 mutant.Comparing AtNRPC2-OE and AtNRPC3-OE with knockout mutants and WT,the overexpression lines are significantly shorter and the leaves and siliques are yellow-green.The determination of photosynthetic pigment content found that in different growth periods of Arabidopsis,compared with the wild type,the photosynthetic pigment accumulation of rosette leaves and carob(at mature stage)of atnrpc2 and atnrpc3 mutants increased significantly.The photosynthesis ability of the rosette leaves of the mutant was also significantly enhanced,while AtNRPC2-OE and AtNRPC3-OE were the opposite.It was found that the fresh weigh of atnrpc2 and atnrpc3 per plant were 2.27 g and 2.01 g,which were significantly higher than the 1.61 g of WT,the fresh weigh of AtNRPC2-OE and AtNRPC3-OE per plant were 1.37 g and 1.22 g,which were significantly lower than WT and mutants.The dry weight of each plant of atnrpc2 and atnrpc3 were 0.61 g and 0.54 g,which were significantly higher than the 0.33 g of WT,the dry weight of each plant of AtNRPC2-OE and AtNRPC3-OE were 0.19 g and 0.25 g,which is significantly lower than WT and mutants.Consistent with those results were the number of silique and thousand-grain weight of these five strains.These results indicate that AtNRPC2 and AtNRPC3 negatively regulate Arabidopsis biomass and yield.Due to the high productivity,high feed value,potential role in soil improvement and soil protection,Medicago sativa L.has become one of the most valuable legumes around the world.The development of Chinese Alfalfa industry is relatively backward,requiring large imports to meet domestic demand.The high light efficiency breeding of Alfalfa is very important for improving the yield and quality.Medicago truncatula MtNRPC1,MtNRPC2 and MtNRPC3 were cloned by homology alignment of OsNRPC1 in rice.The qPCR tissue-specific expression indicated that MtNRPC1,MtNRPC2 and MtNRPC3 had different expression patterns during the mature stage of wild type plants.It was proved that MtNRPC1,MtNRPC2 and MtNRPC3 proteins interact with MtGLK1 protein by yeast two-hybrid.Therefore,MsNRPC1 and MsNRPC2 double-knockout CRISPR/Cas9 vectors and MsNRPC3 single-knockout vectors for Medicago truncatula were constructed respectively.MsNRPC1 and MsNRPC2 double knockout vectors and MsNRPC3 single gene knockout vectors into Alfalfa were transformed by Agrobacterium-mediated genetic transformation technology.The identification of hygromycin-labeled primers proves that the double knockout vectors of MsNRPC1 and MsNRPC2 has been successfully transformed into wild-type Alfalfa.In summary,AtNRPC2 and AtNRPC3 interact with AtGLKs.AtNRPC2 and AtNRPC3 negatively regulate the expression of genes required for chlorophyll synthesis and light collection by AtGLKs,thereby affecting Arabidopsis chloroplast development and photosynthesis.There is no functional redundancy in AtNRPCs.Similar to the previously identified homologous gene AtNRPC1 in the research group,AtNRPC2 and AtNRPC3 not only affect chloroplast development but also cause significant increase in biomass and yield of Arabidopsis.It will have a positive effect on plant high-efficiency breeding to increase plant yield.In addition,experiments have shown that the MtNRPCs of Medicago truncatula were specifically expressed in the tissues of wild-type plants.MtNRPC1,MtNRPC2,MtNRPC3 proteins and MtGLK1 proteins can interact with each other.Alfalfa seedlings which successfully transformed with MsNRPC1 and MsNRPC2 double knockout vectors were obtained through the Agrobacterium-mediated method basing on the establishment of a systematic and mature Medicago sativa L.transgenic system.It created important genetic materials for subsequent Medicago sativa L.high-efficiency breeding. |
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