Transgenic Function Validation Of GLK Regulated By Rice NRPC1/NRPC2 Gene

Photosynthetic efficiency is an important factor affecting rice yield.In the preliminary study of this laboratory,two rice high-efficiency genes NRPC1(negative regulator of photosynthesis and chloroplast development 1)and NRPC2(negative regulator of photosynthesis and chloroplast development 2)were cloned,it was found by yeast double-hybrid experiments that NRPC1 interacts with GLK1 and GLK2,and NRPC2 also interacts with GLK1 and GLK2.On this basis,the glk1glk2 mutant,glk1glk2nrpc1 mutant and GLK1/GLK2 overexpression material were obtained by transgenic technology,and hybridized with NRPC material.The chlorophyll content and photosynthetic related gene expression analysis were performed on the obtained material to determine the relationship between NRPC and GLK.The upstream and downstream relationship clarified the regulation mechanism of chloroplast development and chlorophyll synthesis.Using RNA-Seq and bioinformatics analysis,the downstream genes regulated by NRPC1 gene were screened and the expression of downstream related genes was detected by quantitative analysis.Firstly,the CRISPR/Cas9 knockout and overexpression vectors of GLK1/GLK2 were constructed,and the glk1glk2 mutant,glk1glk2nrpc1 mutant and GLK1/GLK2 overexpressing lines were obtained by transgenic technology.The chlorophyll content and electron microscopy analysis were performed.The chloroplast development status and the expression analysis of photosynthetic related genes revealed that the nrpc1 mutation did not restore the yellowing phenotype caused by the glk1glk2 mutation,the chloroplast development was still abnormal,and the expression level of photosynthetic related genes was still significantly down-regulated,indicating that the NRPC gene is located upstream of the GLK gene;The NRPC1/NRPC2 overexpression materials were hybridized with GLK1/GLK2 overexpression materials.After phenotypic identification and chlorophyll content determination,it was found that overexpression of GLK gene restored the yellowing phenotype caused by NRPC1/NRPC2 overexpression,and its chlorophyll content was restored the wild type level,thereby further confirming that the NRPC gene is located upstream of the GLK gene;expression analysis of wild type,glk1glk2 mutant and GLK1/GLK2 overexpression materials in four-leaf stage and heading stage showed that NRPC1/NRPC2 gene was hardly expressed in glk1glk2 mutant and expressed in late development of GLK1/GLK2 overexpression lines.It was highly induced;the GLK1/GLK2 overexpressing line in the background of nrpc1/nrpc2 mutant was obtained by hybridization,and the chlorophyll content and photosynthetic rate was significantly increased,and the dark green phenotype were maintained in the later stage.The photosynthetic related genes expression have a significant increasing.Then,by RNA-Seq analysis technique and bioinformatics analysis,the nrpc1 mutant was screened for a gene whose expression was up-or down-regulated compared to wild-type expression compared to wild-type expression.Comparison of the two resulted in 102 candidate differentially expressed genes.Among them,the expression levels of 80 genes were highest in nrpc1,followed by wild type and overexpression.Twenty-two genes had the lowest expression in nrpc1,higher in wild type,and highest in overexpression.In order to further confirm the function of the candidate differentially expressed genes and the involved biological metabolic pathways,GO analysis was carried out,and it was found that the candidate differential genes were mainly concentrated in photosynthesis-related sites such as photosynthesis,photoreaction centers,chloroplasts,and thylakoids,but also involved in the middle.The immune system process related to plant resistance;and KEGG Pathway analysis,found that the photosynthesis-related genes have the highest enrichment rate and are enriched in plantpathogen interactions.RT-qPCR was used to verify the expression of the diseaseresistant genes,and the expression of disease-resistant genes was up-regulated in the context of nrpc1 mutants.The bacillus blight fungus was inoculated,the resistance of the nrpc1 mutant material was significantly enhanced,and the NRPC1 overexpressing material was more susceptible.In summary,the NRPC gene is located upstream of the GLK gene and has a negative feedback effect on the expression of the GLK gene.The two genes are regulated to maintain the normal expression of chlorophyll synthesis and chloroplast development related genes in rice.To maintain the normal phenotype of rice.The NRPC1 gene is not only closely related to chloroplast development and photosynthetic reactions,but also has some regulation on resistance-related genes.Mutation of NRPC1 gene can increase the resistance of rice to Xanthomonas oryzae.These results laid the foundation for studying the genetic relationship between NRPC1 gene and GLK gene,high-efficiency breeding of plants and rice resistance to Xanthomonas oryzae.