Transcript Analysis Of Phase-related Differential Expressed Genes Of Gracilaria Lemaneiformis In Different Phase And Developmental Atage

Gracilaria/Gracilariopsis lemaneiformis is the important agarophytic red algawhose life history includes one gametophytic phase and two sporophytic phases, i.e.tetrasporaphyte and carposporaphyte which is parasited on the branches of femalegametophyte. The thalli of tetrasporaphyte and male/female gametophyte havesamilar appearance and can’t be identified until they are matured. Until now, the genemunipulation during this complicated life cycle procedureare still obscur, with littleand preliminary studies. This thesis is trying to study the genetic control of sex andphase differentiation for G. lemaneiformis using rt-PCR technique to further analysethe positive clones screened from the previous SSH libraries. Hope these data willgive more information and hints for other sex determination studies for red algae.Abundant reports show that the quality of inner reference gene for RT-qPCR willplay important role in the consequence of gene expression analysis. For thisexperiments, the commonly used reference genes is especially doubtful duringdifferent phase and development stage of G. lemaneiformis,so they must be testifiedby experiment. We tested9candidate genesfor their expression level in4stagesamples, which is ACT, Rbc-L, GAPDH, ITS1,18S,28S, CoI, PEB, NR. Resultswere analysed using geNorm and NormFinder to evaluate the stability of geneexpression and Rbc-L and CoI are seletcted as the most suitable inner reference genesto analyse the differentially expressed genes in the different phase and developmentstage of G. lemaneiformis.In the previous work, to get differentially expressed genes in the different phaseand different development stage of G. lemaneiformis，we established5suppressionsubtractive hybridization libraries and the positive clones were re-screened usingdot-blot hybridization and then sequenced. This thesis clustered the1873sequenced clones and obtained365available sequences, including118contigs and247single sequences which are not clustering into any contigs. Then sequenceannotation was done using Blast2GO, which makes subtractive hybridization resultsbecome complete and clear. Because of the time limitation，this thesis select onlyeight sequences to do the quantitative PCR analysis. Including all four sequences ofthe female gametophyte library and three sequences of the male gametophyte libraryand contig-44from multi-library mixed. Relative expression of contig-3, contig-52,contig-60and7310are similar to the basic SSH library screening result, whichconsistent female gametophyte large number of expression. Sequence7310is relatedto the cells or embryonic development,which expression amount in the femalegametes is far greater than tetrasporophytes and male gametes body, while in matureand immature female gametes body there is no obvious expression differences. Thissequence is probably related to development of female gametophyte cells in earlystage for the expression of female characteristics.Gracilaria981is the screened strains for enduring higher temperature. Afterlong-term cultivation it is proved that theGracilaria981has high speed of vegetativegrowth and low mature rate and,therefore, less breakage.In order to explore therelationship of this characteristics and gene regulated of growth, we screened seventetrasporophytes specific expressed genes from established suppression subtractivehybridization library for further screening analysis. Although these genes were provedno relationship with sexual reproduction, the growth-related characteristics of thesegenes support that Gracilaria981has the growth advantages.