Studies On Transcriptome And Gene Expression With Regard To Chlorophylls Metabolism Pathway In Photosensitive Albino Tea Plant

Albino tea plants are rare mutant tea resources that grow albino young leaves under certain environmental conditions,owing to lack ofchlorophylls.The albino tea shoots are usually white,yellow,or white and green alternating in color.According to the difference in albino inducing factors,the albino tea plants can be classified into light-sensitive albino tea cultivars and temperature-sensitive ones.However,the albino mechanism has not been well understood,especially for the light-sensitive albino tea plant,because it has not been long since the light-sensitive albino tea was discovered.In view of this,the gene transcriptome in leaves of light-sensitive albino tea cultivar 'Huangjinya' were investigated using transcriptome sequencing technique and digital gene expression general technique,under open field and sunshading conditions,with comparison to normal green leaf tea cultivar 'Jinxuan' as control.The expression patern of genes involved in biosynthesis and degradation of chlorophylls under open and sunshading conditions in cultivar 'Huangjinya' were profiled by real-time polymerase chain reaction(RT-PCR)method.Magnesium protoporphyrin IX methyltransferase(CsCHLM)and mg-protoporphyrin IX monomethyl ester cyclase(CsCRD),two genes involved chlorophyll biosynthesis pathway,were cloned from light-sensitive albino tea cultivar 'Huangjinya'.The major results of the study were summarized as follows:1.Construction of transcriptome.A transcriptome database was established by RNA-seq and digital gene expression(DGE)method using tested tea cultivar'Huangjinya' and control cultivar 'Jinxuan'as test material with the genomic data of tea cultivar 'Yunkang 10' as reference genome for sequence comparison.A total of 508789634 high quality sequences were obtained after filtration,in which the average Q30(%)(the percentage of bases whose accuracy is above 99.9%)was 92.2%,and the average GC content is 44.78%.A total 95,531 gene functional annotations were obtained by comparing with GO database,KEGG data,eggNOG database and swiss-prot database.DGE test showed that the differentially expressioned genes(DEG)were ranged from 939 to 9307 between treatments.Based on the results of the DGEs,genes with high expressed abundance and significantly differentiated expression,including magnesium chelatase H subunit(CHLH),Mg-protoporphyrin ?methyltransferase(CHLM),Mg-protoporphyrin IX monomethyl ester cyclase(CRD)and protochlorophyllide reductase(POR)were screened for subsequent study.2.Tea leaf pigments.Tea leaf pigments in shoots with one leaf and a bud from cultivar 'Huangjinya' were detected.It shows that contents of chlorophylls a and b in'Huangjinya' were significantly lower than those of the control cultivar "Jinxuan",and contents of carotenoids including neoxanthin,violaxanthin,lutein and ?-carotene in'Huangjinya' were' also lower than "Jinxuan".However,chlorophylls content in'Huangjinya' was significantly increased after shading treatment.The total content of chlorophylls gradually accumulated with the extension of shading time.It was found that ratio of chlorophylls a/b in the shaded 'Huangjinya' was greatly decreased,which is beneficial for its absorbing light energy and promoting photosynthesis.After shading treatment,neoxanthin and ?-carotene in leaves of 'Huangjinya' were also increasingly accumulated.3.With the increase of natural light intensity,the expression of genes involving in chlorophylls biosynthesis pathway including CHLM,DVR and CHLG in'Huangjinya' changed from active state to inhibited state.At the same time,the shading treatment with different durations had different effects on the gene expression.When the shading treatment on 'Huangjinya' lasted for 14 days,the expression of genes involving in the chlorophyll biosynthesis pathway was significantly reduced.However,there was no significant change in the expression of these genes in 'Jinxuan'.However,after 40 days of shading treatment,the expression level of the genes involving in chlorophylls biosynthesis in 'Huangjinya' was significantly increased to a level as high as normal cultivar such as 'Jinxuan'.The genes involving in chlorophyll degradation pathway such as CLH,RCCR and SGR were significantly highly expressed in 'Huangjinya' compared to 'Jinxuan'.It is concluded that low expression of genes involving in the chlorophyll biosynthesis pathway accompanying with high expression of genes involving in the chlorophyll degradation pathway is considered to be the reason for the less accumulation of chlorophylls in leaves of'Huangjinya'.4.Cloning of gene CsCHLM.The gene CsCHLM was cloned from cultivar'Huangjinya',which has a full length cDNA sequence 976bp,and can encodes 322 amino acid(Genebank:MG893509).Bioinformatics analysis shows that molecular weight of the predicted protein was 34855.96 Da,with a theoretical isoelectric point pH6.26.It was predicted by ChloroP that the protein contained a chloroplast transporter peptide,which was generated in the nucleus and then transferred to chloroplasts.BLAST analysis in NCBI shows that the amino acid sequences encoded by CsCHLM had the conservative structural domain PLN02585 of the eukaryotic CHLM family,and were highly similar to CHLM sequences of Actinidia chinensis(86%similarity),Nicotiana tabacum(80%)and Capsicum annuum(80%).5.Cloning of CsCRD gene.CsCRD gene was cloned from cultivar 'Huangjinya',with a full length 1217bp and an opening reading frame(ORF)1215bp which encodes 404 amino acids.The cloned gene was defined as CsCRD(Genebank:MH091705).Bioinformatics analysis showed that molecular weight of the predicted protein was 46868.99Da,with a theoretical isoelectric point pH8.56.It was predicted that using FoldIndex software that the CsCRD was a disordered protein.It was predicted by ChloroP that the protein contained a chloroplast transporter peptide,which was generated in the nucleus and then transferred to chloroplasts.BLAST analysis in NCBI shows that the amino acid sequences encoded by CsCRD had the conservative structural domain PLN02508 of the Mg-protoporphyrin IX monomethyl ester cyclase family,and were highly similar to CRD sequences of Ricinus,Actinidia chinensis and Durio zibethinus,with a homology 85%or above.