Identification And Functional Study Of NtiPGAM Involved In Glycolysis Pathway In Tobacco

Glycolysis,as a central metabolic process in all living organisms,can catalyze sugars into pyruvate and produce a small amount of ATP for body.In addition,glycolysis has many other functions.For example,it is also the basic inverse process of gluconeogenesis.The intermediate metabolites of gluconeogeniesis can participate in multiple metabolic pathways to produce a variety of key substances.Therefore,glycolysis is very important for organisms.The growth and development of plants cannot be separated from the normal operation of glycolysis.2,3-bisphosphoglycerate-independent phosphoglycerate mutase(i PGAM)is an enzyme that catalyzes the reversible interconversion of 3-phosphoglycerate and2-phosphoglycerate in glycolysis.Previous studies have shown that i PGAM is closely related to stomatal movement and vegetative growth in plants,however,the role of the i PGAM gene in the growth and development of tobacco(Nicotiana tabacum)has been unclear to date.In addition to being an important cash crop,tobacco has also been established as a model plant for basic biological research.Therefore,it is of great significance to study the role of i PGAM gene on growth and development of tobacco.In this study,we identified and functionalized Nti PGAM gene in tobacco.The main results were as follows:1.Identification,cloning and expression analysis of Nti PGAM gene in tobaccoIn order to identify the i PGAM gene in tobacco,we firstly cloned this gene.The amino acid sequences of Ati PGAM were compared with tobacco database,two coding sequences(m RNA＿61071＿cds and m RNA＿129134＿cds)were identified,which named Nti PGAMa and Nti PGAMb respectively.Analysis of the gene structure revealed that both of Nti PGAMa and Nti PGAMb genes had 9 exons and 8 introns.Using smart software to predict the protein domains of the two genes,it was found that both genes contained phosphoglycerate mutase domains.The results of multi-sequence comparison showed that Nti PGAMa and Nti PGAMb proteins had high sequence similarity with Arabidopsis thaliana,petunia and tomato,and had conserved catalytic sites of serine residues like other species.An evolutionary tree cluster analysis showed that Nti PGAM was the closest relative of petunia.Quantitative PCR results showed that Nti PGAMa and Nti PGAMb were expressed in 11 tissues of tobacco(root,stem,upper/middle/lower leaf,calyx,corolla,style,anther,thrum and ovary),and highly expressed in anthers.The results suggested that i PGAM gene may play an important role in anthers.Subcellular localization analysis showed that Nti PGAMa and Nti PGAMb genes were located in the nucleus and cell membrane.In addition,we constructed the prokaryotic expression vector of Nti PGAM gene,and finally selected 16°C as the induction condition to induce the expression of Nti PGAM protein.After nickel column affinity chromatography and gel filtration chromatography,we obtained the purified recombinant protein.After the accuracy of purified protein was determined by mass spectrometry,antibodies were prepared for further study.2.Creation and phenotypic observation of ipgam mutant plants of tobaccoWe used CRISPR/Cas9 system to produce ipgam mutunt plants.We designed sg RNAs that can simultaneously target the two genes.The location of sg RNAs for i PGAM gene were on the second and fourth exons.We constructed the CRISPR/Cas9 knockout carrier by Bsa I double enzyme digestion.After agrobacterium-mediated genetic transformation and sequencing analysis,single gene and double gene knockout mutants of i PGAM gene were finally obtained.Western blotting results showed that i PGAM protein was slightly down-regulated in the single-gene mutant plants.However,i PGAM protein expression could hardly be detected in the two-genes mutant plants.Leaf color observation showed that there was no significant difference between single-gene mutant plants and the wild type.However,the leaves of the two-genes mutant plants showed a light-dark Mosaic phenotype and abnormal pollen development.3.Preliminary study on the function of Nti PGAM geneIn order to explore the function of Nti PGAM gene,the leaf and reproductive organ phenotypes of the two-gene mutant plants were characterized by means of scanning and transmission electron microscope observation,GC/MS detection,chlorophyll fluorescence analyzer analysis,cytological staining and gene quantification.The causes of Mosaic phenotype and abnormal pollen development in tobacco were further explored.The results showed that the content of photosynthetic pigments(chlorophyll a,chlorophyll b and carotenoids)in the leaves of the mutant were decreased.The expression of genes related to chlorophyll synthesis and photosynthesis was down-regulated.The thylakoid lamelae in the leaves were thinner.The detection of chlorophyll fluorescence characteristics showed that the utilization rate of light energy of single and double-genes mutant plants decreased,and the value of NPQ reflecting the photoprotection ability of plants also decreased.In addition,the content of pyruvate and the expression of glutamate dehydrogenase,which catalyzed the production of α-ketoglutaric acid into glutamic acid,also decreased.By means of GC/MS,we further detected the glutamate content in the leaves of the double-mutant plants.The results showed the glutamate content decreased significantly,and the sucrose content and maltose content of the plants also showed a downward trend,indicating that the knockout of the target gene caused a disorder in the sugar metabolism of the plants.The observation of the morphology of reproductive organs showed that the mutant of Nti PGAM gene had no obvious effect on the morphology of flowers.However,compared to wide-type,ipagm mutant had shorter stamen and pistil,abnormal pollen,and the activity of pollen grains were significantly reduced.Using the overexpression method,we obtained overexpressed plants with Nti PGAMa and Nti PGAMb genes,respectively.The detection of the protein expression level in the plants showed that the target protein had a significant up-regulated expression trend.Under 4°C stress,it was found that compared with the wild type,the overexpressed plants showed a low sensitivity to low temperature.When the temperature returned to room temperature,the value of Fv/Fm(The maximum quantum yield of PS II reflects the potential maximum photosynthetic capacity of plants)of the overexpressed plants could return to the pre-stress state,while the wild type showed a damaged state that could not be recovered.In summary,the present study demonstrated Nti PGAM gene was involved in chlorophyll biosynthesis,pollen development and pollen tube growth.We believe that the results of this study will provide a useful reference for the role of i PGAM gene in vegetative growth and reproductive development of plants.