Cloning And Expression Analysis Of Genes Related To Shikimate Pathway In Camellia Sinensis L.

Tea plant(Camellia sinensis(L.)O.Kuntze)was a perennial woody plant.The leaves can be made for processing into tea.As one of the world's three largest beverage,tea's special ingredient,catechins and theanine were not only formed the Xianshuang and mellow taste of tea,but also played a role in health function.Therefore the research about theanine and catechins had always been a hot topic in the field of tea.'Baiye 1' is a tea varietie of AnJiBaiChais and a temperature-sensitive nature mutant.All the time,the reason of its unique whitening and re-greening phenomenon need to be further elucidated.In this experiment,Based on the analysis of the changes in chloroplast structure,amino acids and catechins content during the albino,the shikimate pathway-related genes were cloned:phosphoenolpyruvate transporter(CsPPT),DAHP synthase(CsDAHP),shikimate dehydrogenase(CsSDH)and chorismate mutase(CsCS).Then combined with changes in genes' transcription of'Baiye l',we made the preliminary analysis of the reasons for albino.Specific results are as follows:(1)Observing the chloroplast with the transmission electron microscope and testing the content of theanine,catechin,amino acid was teated during Pre-albinsim stage,Albinism stage and Re-greening stage in 'Baiye 1'.It was found that the content of chlorophyll decreased during Albinism stage.The internal structure of chloroplast was collapsed and the grana lamellae structure was damaged.While the chloroplast envelope was still complete.And the content of theanine increased sharply while catechins decreased.There is a negative correlation between the content of theanine and catechin.The content of various amino acid increased generally,but the growth rate of different amino acid were diverse.(2)The open reading frame(ORF)of CsPPT(GenBank accession number:KJ652972)from Camellia sinensis 'Baiye l'was obtained using RACE technology.The deduced ORF of CsPPT has 1 227 nucleotides,encoding 408 amino acids.Bioinformatic analysis showed that the molecular weight of the predicted protein was 44.7 kDa.The phylogenetic tree of CsPPT protein was built and suggested that it had high homology with phosphoenol pyruvate transporter protein related to potato,tomato and tobacco.Subcellular localization of CsPPT protein displayed that it was located in the chloroplast.TMHMM analysis showed that CsPPT protein belonged to transmembrane proteins.QRT-PCR analysis results showed that the expression of CsPPT has obvious tissue specificity,which was higher in flower,followed by buds,leaves,and tender stems,but lower in roots.The expression decreased obviously during the albino but increased during the greening.(3)The open reading frame(ORF)of CsDAHP from Camellia sinensis 'Baiye l'was obtained using RACE technology.The deduced ORF of CsDAHP has 1 620 nucleotides,encoding 539 amino acids.Bioinformatic analysis showed that the molecular weight of the predicted protein was 59.6 kDa and the theoretic isoelectric point was 10.16.The phylogenetic tree of CsDAHP protein was built and suggested that it had high homology with DAHPS related to lettuce,tomato,Petunia,tobacco,potato and tomato.Subcellular localization of CsDAHP protein displayed that it was located in the chloroplast.QRT-PCR analysis results showed that the expression of CsPPT has obvious tissue specificity,and it was higher in flower,followed by buds,leaves,and tender stems,but lower in roots.And during the albino its expression rate has droped.(4)The open reading frame(ORF)of CsSDH and CsCS from Camellia sinensis'Baiye l'was obtained using RACE technology.The deduced ORF of CsDAHP has 1 569 nucleotides,encoding 522 amino acids.Bioinformatic analysis showed that the molecular weight of the predicted protein was 56.7 kDa and the theoretic isoelectric point was 10.16.Poylogenetic tree of CsSDH had been built And showed that it had affiliation with soybean,grape,populustrichocarp and castor.The deduced ORF of CsCS has 1 314 nucleotides,encoding 437 amino acids.Bioinformatic analysis showed that the molecular weight of the predicted protein was 47.2 kDa and the theoretic isoelectric point was 10.16.Poylogenetic tree of CsCS had been built and had close affiliation with grape and tomato.QRT-PCR analysis results showed that the expression of CsSDH and CsCS has obvious tissue specificity,and it was higher in bud,followed by leaves,flower and tender stems,but lower in roots.And those two gene's expression had little relationship with albino.