Regularity Of Carotenoids Accumulation And Preliminary Analysis Of Function Of The Gene Encoding Carotene Hydroxylase In Tea Plant

Tea plant（Camellia sinensis（L.）O.Kuntze）is an evergreen perennial plants,and their fresh leaves are widely used in the processing of non-alcoholic beverage‘tea’.Tea is rich in beneficial to human body health of secondary metabolites,including tea polyphenols,theanine and carotenoids.Carotenoids are essential pigments of high plants.They are involved in the biological processes of plant photosynthesis,hormone synthesis,resistance to adversity,and participate in the quality formation of tea.At present,studies on carotenoids in tea plant mainly focus on aroma quality,and there are few studies on the molecular mechanism.In this paper,the C.sinensis cv.‘Longjing 43’and‘White leaf No.1’were used as material.The samples are mainly divided into two kinds,one is the leaves of different developmental stages（a bud with a leaf,mature leaf,old leaf）and tender stems,and the other is the leaves under low temperature treatment（4℃）.Carotenoids（lutein,β-carotene,andα-carotene）accumulation and the expression level of related genes of the samples were analyzed.Two carotenoid biosynthesis related genes CsCHXB and CsCYP97C1 were cloned from‘Longjing 43’.Their biological analysis was performed,and their expression levels were detected in different tissues and different stress conditions of tea plant.The main results are as follows:1.The content of carotenoids（lutein,β-carotene andα-carotene）in different developmental stages leaves and tender stems of‘Longjing 43’and‘White leaf No.1’were determined by UPLC.The results showed that lutein andβ-carotene were detected in mature leaves and a bud with a leaf.Lutein,β-carotene andα-carotene were contained in old leaves,and only lutein was detected in tender stems.The contents of lutein andβ-carotene in mature and old leaves were higher than a bud with a leaf.The content of carotenoids in tender stems was lower than leaves.There was a significant positive correlation between the carotenoids and the chlorophyll contents.The expression profiles of16 carotenoid biosynthesis-related genes were analyzed.The relative expression levels of CsPSY1,CsPDS,CsZ-ISO,CsZDS1,CsCRTISO,CsLCYe,CsLCYb1,CsZEP,and CsVDE in mature and old leaves were significantly higher than a bud with a leaf,and the expression level in tender stems was lower than the leaves.The expression profiling of carotenoid biosynthesis-related genes and analysis of carotenoid accumulation may help elaborate the regulatory mechanisms of carotenoid biosynthesis in tea plant.2.In this study,the main carotenoids,lutein andβ-carotene,in leaves of common green tea cultivar‘Longjing 43’and albino tea cultivar‘White leaf No.1’were detected after the low temperature treatment at 4℃.The results showed that the contents of lutein andβ-carotene in‘Longjing 43’were higher than that in‘White leaf No.1’,respectively.The chlorophyll content in two tea cultivars decreased and was significant positive correlation with two kinds of carotenoid content at low temperature.Under low temperature,the genes related to the biosynthesis pathway of methylerythritol 4-phosphate（MEP）and carotenoids all showed varying degrees of response with the changes of lutein andβ-carotene content.The relative expression levels of CsDXS1,CsPSY1 and CsCHXB were up-regulated and higher than other related genes.Promoter prediction results showed that the CsDXR,CsHDS1 and CsCHXB contain low-temperature response element LTR.This work will potentially helpful to understand the regulation mechanism of carotenoid pathway under low temperature treatment.3.In this study,a CsCHXB gene was cloned from the c DNA of‘Longjing 43’based on the genome data of tea plant.Its protein sequence characteristics and subcellular localization was analyzed.In addition,the expression profiles of the CsCHXB gene in different tissues and under high/low temperatures,drought and salt treatments in tea plant was analyzed by real-time quantitative PCR（RT-qPCR）.（1）Sequence analysis showed that the length of the CsCHXB open reading frame（ORF）is 1098 bp,encoding 365 amino acids.The amino acid sequence of CsCHXB protein contained histidine domain“HXXXXH”,“HXXHH”.Secondary structure prediction showed that the CsCHXB protein consists of37.26%α-helix,4.38%β-turn,13.70%extended strand and 44.66%random coil.The predicted three-dimensional structures indicated that CsCHXB protein containsα-helix andβ-turn.Subcellular localization analysis indicated that CsCHXB is targeted to the chloroplast.Promoter analysis indicated that the gene of CsCHXB contain light response,low temperature response,hormone response and drought-inducibility elements.（2）The expression profiles of the CsCHXB were detected in young leaves,mature leaves,old leaves,stems,flowers and roots by the RT-qPCR.The relative expression was the highest in old leaves and flowers.The expression level of CsCHXB was up-regulated under high temperature（38°C）,low temperature（4°C）,drought(200 g·L^-1PEG-6000)treatments,respectively.These results provide a foundation for the function study of CsCHXB gene in tea plant.4.The CsCYP97C1 gene was cloned from c DNA of‘Longjing43’based on the genome database of tea plant.Its protein characteristics and gene expression patterns were analyzed.In addition,the expression profiles of the CsCYP97C1 under abiotic stress treatments and different tissues in tea plant were detected by RT-qPCR.（1）Sequence analysis showed that the length of CsCYP97C1 open reading frame（ORF）is 1674 bp,encoding 557 amino acids.CsCYP97C1 belongs to the cytochrome P450 enzyme.Secondary structure prediction showed that the CsCYP97C1 protein consists of 45.70%α-helix,9.16%extended strand,3.59%β-turn and 41.47%random coil.Subcellular localization analysis indicated that CsCYP97C1 is targeted to the chloroplast.The promoter prediction analysis showed that the CsCYP97C1 promoter mainly contained light response elements and cis-acting regulatory element essential for anaerobic induction.（2）The relative expression of CsCYP97C1 was the highest in young and old leaves.The relative expression of CsCYP97C1 was up-regulated under high temperature（38℃）,low temperature（4℃）,drought(200 g·L^-1PEG-6000)treatments,respectively.