Study On Mg-protoporphyrin â…¨ Monomethyl Ester Cyclase Gene And Development&Application Of SSR Marker From Phyllostachys Edulis

Photosynthesis is the basic way to obtain the energy, food and oxygen in the biologicalworld. Chlorophyll is the main carrier for absorption of light energy in plant photosynthesis.Chlorophyll biosynthesis takes15-step reaction, involving15kinds of enzymes, among whichMg-protoporphyrin Ⅸ monomethyl ester cyclase (MPEC) plays an important role in theformation of isocyclic ring. Plants will appear the phenomenon of death if the gene encodingMPEC was knocked out. The published draft genome of moso bamboo laid a good foundationfor the study of gene function, development and application of molecular marker.Phyllostachys edulis, an important economical bamboo, was selected for the isolation ofMPEC homologous gene. The spatio-temporal expression patterns of MPEC in Ph. edulis wereanalyzed, and the gene function was verified through transgenic technology. SSR molecularmarkers in the genes of related with photosynthesis pathway were developed according to thegenome of Ph. edulis and used to analyze the genetic diversity of bamboos in Phyllostachysgenus. The results were as follows:First, Isolation of PeMPEC gene A homologous gene of MPEC was abtained from thefull length cDNA library of Ph. edulis by alignment method and designed as PeMPEC(Genbank: FP092998). The cDNA of PeMPEC was1474bp including5′untranslated region(UTR)23bp,3′UTR206bp and an open reading frame (ORF) of1248bp which encoded aprotein with415aa. The corresponding genomic sequence of ORF was1854bp including5exons and4introns. The analysis of protein structure showed that PeMPEC had one conservedleucine zipper domain and two copies of the EXnDEXRH motif. The result of sequencealignment demonstrated that PeMPEC shared73.6%homology with that of Arabidopsisthaliana, and more than85.0%homology with those of gramineous plants such as Oryza sative,which indicated that MPEC had a high conservative in evolution.Second, Spatio-temporal expression pattern of PeMPEC Semi-quantitative PCRanalysis showed that PeMPEC was transcribed at high level in leaf which is main photosynthesis organ and could not be detected at all in root. The result of real-timequantitative PCR suggested that the expression of PeMPEC was induced by darkness andsupressed with increase of light from100μmol m-2s-1to1500μmol m-2s-1. Hightemperature (42℃) also could suppress the gene expression. However, it was induced by lowtemperature (4℃) in a short time(0.5h) and then was suppressed by the prolonged treatment.Thhough expression of PeMPEC was very weak in etiolated seedlings, it was increasedgradually with the treatment of light (200μmol m-2s-1) and reached to80%of the controlafter8h treatment.Third, Ectopic expression of PeMPEC in A. thaliana The sense and antisenseexpression vectors of PeMPEC were constructed and transferred into A. thaliana. The analysisof phenotype showed that there was no obvious difference between sense transgenics and wildtype. However chlorophyll deficient leaf with light yellow was found in the antisensetransgenic lines which grew slowly. The semi-quantitative PCR analysis confirmed thatPeMPEC had been expressed in transgenic plants, among which the most abundant was insense line S-5and the least was in antisense line A-4. The analysis of pigments showed thatchlorophyll content in sense plants was20%higher than that of of wild type, while that ofantisense plants all was reduced, especially for that of line A-2was only58%as that of wildtype. The data of chlorophyll fluorescence parameters showed that Fv/Fm, Y(Ⅱ) and ΔI/Ivalue of the antisense line A-1and A-2decreased significantly (p<0.05) compared with thoseof wild type, however there were no significantly different between parameter values of senselines and those of wild type. These indicated that the chlorophyll content of transgenic plantshas been influenced by PeMPEC which involved in the regulation of chlorophyll biosynthesis.Forth, Functional complementation analysis of PeMPEC PeMPEC was transferredinto Crd1(SALK-024716C) which is a T-DNA mutant of A. thaliana, the phenotype oftransgenic plants was similar to that of wild type. The semi-quantitative PCR of transgenicplants confirmed that PeMPEC had been integrated into genome and its expression was mostabundant in line M-4and least in line M-1. The analysis of pigments showed that chlorophyll content in sense plants was22-50%higher than that of mutant and7-20%higher than that ofwild type. The measurement of chlorophyll fluorescence parameters showed that there were noobvious difference between transgenic plants and wild type for the value of Fv/Fm, Y(Ⅱ) andΔI/I, however significant difference (p<0.05) were presented between transgenic plants andmutant. These results indicated that the overexpression of PeMPEC could increased the contentof chlorophyll, which played an important role in keeping stability of PS Ⅰ and PSⅡ.Fifth, Western Blotting The prokaryotic expression vector of PeMPEC was constructedand transferred into Escherich coil (Rosetta-gamin B (DE3)). The high expression of solute proteinwas obtained with0.4mmol·L-1IPTG induced4h at28℃. The purification of protein showedthat a60kDa recombinant protein was isolated from E. coli and used to prepare polyclonalantibody. The total protein of root, stem, sheath and leaf were extracted respectively andseparated with SDS-PAGE. The polyclonal antibody of PeMPEC was used as probe forwestern blotting. The result suggested that PeMPEC was mainly detected in leaf tissue.Sixth, Characteristic of SSR in Ph. edulis genome Through genome-wide search, a totalof302615SSR loci were identified from the genome sequence of Ph. edulis, in which1Mbsequence appeared an average147.5SSRs. Mononucleotide and dinucleotide repeats SSR weredominant, accounting for87.33%. The most abundant motif in mononucleotide repeats is A/Taccounted for72.00%, and the most one in dinucleotide is AG/CT accounted for41.16%,followed by AT/AT, CG/CG motif repeats. The number of SSR was decreased gradually withthe increased length of SSR. The number of SSR with length of10-20bp was the most one,accounting for79.76%. When the bases were4-100bp between two SSR loci the number ofSSR was the most abundant, accounting for39.54%of that of compound SSR loci. Thenumber of compound SSR with4-100bp between two SSR loci was the most abundant,accounting for39.54%.Seventh, Polymorphism analysis of SSR in Phyllostachys A total of73primer pairswere designed and used to precede genetic diversity analysis of78samples from Phyllostachys,in which22pairs of primers were polymorphic, accounting for29.79%. A total64alleles were detected in78samples, there is an average of2.78alleles per primers. Genetic diversityparameters of22loci were calculated, the excepted heterozygosity was ranged from0.09-0.93with average value of0.44, the observed heterozygosity ranged from0.02~0.92with averagevalue of0.68, and the Shannon index ranged from0.25to1.3with average value of0.74. Allthe genetic parameters indicated that the genetic diversity level of Phyllostachys was lowerthan that of other species, it may be related with its asexual reproduction.Eighth, Auxiliary application of SSR in Phyllostachys classification The result ofcluster analysis showed78samples were clustered into3classes according to the geneticdistance. The samples clustered into the first and second classes belonged to Sect.Phyllostachys group and those of the third classs belonged to Sect. Heterocladae group.However, the result of STRUCTURE demonstrated that78samples were divided into4classes,which was consistent some but inconsistent portion with cluster analysis. Though the result ofSTRUCTURE method was not distinguished Sect Phyllostachys and Sect Heterocladae, itcould be used to classify the species and its variants. It was easy to distinguish SectPhyllostachys and Sect Heterocladae using cluster analysis, but difficult for the variants for thereason of genetic distance. Therefore, it was similar to the traditional taxonomy with highdegree of agreement by combining the results of two methods.PeMPEC was an essential gene in chlorophyll biosynthesis. This study provided a newgenetic resource for the regulate of chlorophyll biosynthesis through genetic engineering, and atheoretical basis for the breeding of new varieties with high photosynthetic efficiency. SSR lociin the genes related to photosynthesis pathway were not only provided the evidence fortaxonomy of Phyllostachys but also genetic resource for development of the loci related withphotosynthesis at molecular marker level, which had important value for revealing geneticpolymorphism of bamboo photosynthesis and screening the unique genetic factors.