Research Of Improving Tobacco Photosynthetic Efficiency By Regulating The Expression The Genes Of ClpC1 And POR

Chlorophylls are indispensable substances in photosynthesis and participates in the light reaction of photosynthesis.They are mainly synthesized in the chloroplasts,which requires the participation of NADPH and is subject to multiple regulation.ATP and NADPH produced in the light reaction in photosynthesis are used for carbon assimilation in the dark reaction.The ratio of ATP to NADPH produced in the light reaction is lower than that of ATP to NADPH consumed in carbon assimilation,so there is a relative surplus of NADPH,which inhibits photosynthesis.It is believed that from the perspective of energy utilization,enhancing chlorophyll synthesis can increase the ability of light capture,meanwhile increase the consumption of NADPH,and therefore improve the photosynthetic efficiency,and finnally increase the biological accumulation.Glutonyl-trna reductase?GluTR?and NADP?H?-protochlorophyllide oxidoreductase?POR?are the key enzymes in the chlorophyll synthesis pathway,and GluTR is negatively regulated by the chloroplast protease ClpC1.In this study,the photosynthetic efficiency of tobacco was improved by editing ClpC1 gene and overexpressing PORA and PORB genes.The results of current study are detailed as followed:1.Two genes,PORA and PORB from tobacco were cloned,and the overexpression vectors are constructed.Meanwhile,the gene editing vector of ClpC1 was constructed.By using agrobacterium-mediated transformation method,transgenic tobacco plants with these three vectors were obtained by dialanine phosphine screening,Bar gene Aurum-labeled immunostrip tests,PCR detection and Southern blotting analysis.2.The sequencing analysis of ClpC1 gene editing vector T1-generation plants showed that among the 6 positive plants,there were 4 gene editing forms,in which 2single base substitutions and 2 kinds base deletions?1 base and 4 bases respectively?.Three of the positive plants were found to be the same CACG base deletions and resulted.Frameshift mutation and premature translation termination.Four base deletion mutants were selected for further experiments.3.In two kinds overexpressed plants,the expression of PORA and PORB were both higher than those in the wild type.In comparison of T1-generation plants from three vectors,the net photosynthetic rate ordered as follows:PORB overexpressed?OE-PORB?>ClpC1 gene editing?K-ClpC1?>overexpressing PORA?OE-PORA?;the total chlorophyll content was ordered as:OE-PORB>OE-PORA>K-ClpC1;and dry weight per unit leaf area was ordered as:OE-PORB>K-ClpC1>OE-PORA.In comparism to wild type plants,the net hotosynthetic rate of K-ClpC1,OE-PORA and OE-PORB plants increased 47.3%,44.1%and 29.2%,respectively;the dry weight per unit leaf area increased by 39.1%,10.7%and 44.2%,respectively;the total chlorophyll content increased by 117.2%,205.5%and 254.5%,respectively.4.The contents of NADPH and ATP inchloroplasts were tested.It was found that NADPH/ATP in T1-generation plants was significantly lower than that in wild type under normal conditions.While treated with NaHCO₃ as the sole inorganic carbon source for 10 min,the ratio of wild-type increased,whereas the ratios of T1 plants were significantly decreased and lower than that in wild-type plants,which were significantly from those of unreated and wild type plants.The results indicated that the NADPH utilization rate in the three groups of T1 plants was enhanced.In summary,the results show that the editing of ClpC1 gene or the over-expression of POR genes can consume excessive NADPH,enhance the synthesis of chlorophyll,and thus improve the photosynthetic efficiency of transformed plants.It provides a feasible pathway for improving of crop photosynthetic efficiency and breeding new crop cultivars with high photosynthetic efficiency.