Study On The Biological Function Of High Photosynthetic Gene NRPC2 In Rice

Photosynthesis is one of the most important biological processes on Earth.It provides the energy consumed by almost all living things and regulates the composition of the planet's atmosphere.The mechanism of plant regulation of photosynthesis is extremely complex,and a large number of genes are involved in it.GLK?GOLDEN2-LIKE?is an essential plant-specific nuclear transcription factor that positively regulates chloroplast development and photosynthesis gene expression.Therefore,exploring the molecular mechanism of photosynthesis-related genes around GLK will further improve the whole photosynthesis regulation network of plants and play an active role in high photosynthetic crop breeding.In this study based on the research of NRPC1gene in rice,we identified an NRPC2 gene which interacted with GLK protein,and it can obtain high photosynthetic efficiency in rice when getting mutation.The main results of this study are as follows:1.CRISPR/Cas9 and pCMABIA1300-35S::NRPC2 vectors were constructed and transformed into Nipponbare rice to obtain NRPC2 knockout mutants and NRPC2-OE over-expression plants.The chlorophyll content of nrpc2 mutants and NRPC2-OE over-expressed plants in rice showed that nrpc2 had darker green leaves than WT,but the over-expressed plants had the opposite;the ultrastructure analysis showed that the chloroplast of nrpc2 mesophyll cells in rice had larger surface area than WT;the net photosynthetic rate and photosystem activity of nrpc2 leaves measured by Li-6800photosynthetic meter were also significantly higher than WT.2.On this basis,we studied the molecular mechanism of NRPC2 gene in rice.Firstly,RT-PCR assay showed that NRPC2 gene was mainly expressed in green tissues such as leaves,stems and sheaths,and it was induced by light.Subcellular localization confirmed that NRPC2 was mainly located in nucleus and cytoplasm.Secondly,yeast two-hybrid assay showed that NRPC2 mainly interacted with GCT-box domain of GLK.Finally,combined with transcriptome sequencing analysis and gene/protein expression,we found that those genes associated with photosynthesis or chloroplast formation were up-regulated in mutants and down-regulated in over-expression.3.In previous study,nrpc1 also has the characteristic of high photosynthetic efficiency?especially in spikelet shell?in rice.Thus,in order to understand the genetic relationship between NRPC1 and NRPC2,we obtained F₁ lines by two crosses between nrpc1&NRPC2-OE and nrpc2&NRPC1-OE.The results showed that F₁ plants were restored to over-expression phenotype and the net photosynthetic rate of leaves decreased,which proved that NRPC1 and NRPC2 genes had genetic complementarity.Therefore,we further obtained the double mutant plant nrpc1nrpc2 by two crosses between nrpc1&nrpc2.Phenotypic and functional identification showed that nrpc1nrpc2 had darker green leaves and panicles than either of the single mutant.The photosynthetic rate and expression of related genes was significantly enhanced.Statistical analysis of agronomic traits of the mutant plants showed that the grain size of nrpc1nrpc2 plants were significantly larger than that of the single mutant,and the1000-grain weight,yield per plant and yield of plots were also increased in rice.In conclusion,combining phenotype and functional mechanism,we infered that NRPC2 may act as a negative regulator of GLK in rice.It negatively regulates the transcriptional activation of GLK downstream photosynthesis and chlorophyll synthesis-related genes in a complementary way with NRPC1.Therefore,it affects plant chloroplast development and photosynthesis,and yield formation ultimately.