The Identification And Regulation Of Structural Genes Involved In Catechins Biosynthesis Of Tea Plant([camellia Sinensis(L.)O.Kuntze])and Functional Verification Of Chalcone Isomerases

It has been concerned how the tea plant accumulates high levels of polyphenols,especially catechins.In recent years,lots of valuable imformation emerge with the application of genomics,transcriptomics,proteomics and metabonomics.However,due to the limitation of existing technical approaches,the unique nature of tea cultivars and especially the deficiency of structuaral gene functional validation,the understanding of catechins biosynthesis in tea plant is still obscured.This paper comprehensively analysed the phenylpropanoid and flavonoid metabolic pathways involved in biosynthesis of catechins in tea based on genome data,transcriptome data in NCBI,transcriptome sequencing and qRT-PCR technologies.Using gene cloning,genome-walking,prokaryotic expression and eukaryotic expression technologies,cloned and functioanally verified three genes in CHI family which were key genes to tea catechins biosynthesis.The above work provides valuble insights to learn about the biosynthesis,accumulation and regulation of catechins in tea plant.The main finding are as follows:1.In this paper,36 transcripts containing complete ORF of structural genes involved in polyphenol biosynthetic pathways were screened by blast against 10 tea plant transcriptome databases accessed in NCBI and validated by PCR cloning.The sequences of these 36 transcripts were validated by gene cloning,and 28 transcripts were functionally verified using reverse genetics methods.Twenty-nine genes contained complete ORFs were mapped into tea genome and we re-annoted the structural information of the structural genes involved in tea catechins biosynthesis.The number of transcripts do not show enhanced expression compared with Camellia reticulata,which accumulates lower amounts of polyphenols.Interestingly,the intron-retion events are found in CHI and DFR family,which may one of the reasons caused the abundant transcripts in these two families.2.The tissue-specific and inducible transcriptional profiles were generated by transcriptome sequencing or qRT-PCR analysis.The transcriptional expression results showed that the transcripts of members in same family that phylogenetically cluster into a same clade present an obvious bud-or root-specific expression pattern an obvious tissue-specific and development-dependent pattern in phenylpropanoid biosynthetic pathway.However,in the flavonoid biosynthetic pathway,most transcripts are abundant in buds and 2^ndleaves,and fewer in mature leaves and young roots.The inducible expression results indicate that sucrose and ABA had positive effects on gene expression in the whole pathway,while NaCl,mannitol,and IAA have negative impacts.SA only activated expression in up-stream genes of polyphenol biosynthesis pathways.Heat stress mainly up-regulated the related genes expression in short time.At normal light intensity,the red light down-regulats the most transcripts expression and this effect is strengthed with time increasing.The regulation of blue light on transcripts expression involved in polyphenol biosynthesis presents an increase-decrease pattern.At high light intensity,the red light influences the pathways by first down-regulating and then up-regulating some transcript expression,while the impact of blue light presents a decrease-increase-decrease pattern.3.In this work,three genes of CHI family were cloned and were named as CsCHIa,CsCHIb and CsCHIc.The phylogenetic analysis showed that they were belonged to Type I,Type IV and Type III of CHI family,respectively.We cloned the complete DNA sequences and promoter region of the three genes in tea genome,and analysed the gene structures as well as cis-acting elements.It was found that the CHI family had alternative splicing event caused by intron retention.The transcriptional profile of the CHI family was obtained by qRT-PCR technology,and the results presented a tissue-specific and inducible pattern.4.The functional verification of CHI family in vitro or vivo was carried out based on construction of heterologous expression vector,prokaryotic expression,protein purification,Gateway technology,transient expression in Nicotiana benthamiana and genetic transformation to Arabidopsis thaliana.The results in vitro showed that subcellular localization of three CsCHI proteins were mainly in cytoplasm,nucleus and endoplasmic reticulum.CsCHI could catalyze naringenin chalcone while the other two could not.The genetic transformation experiment showed that CsCHIa gene could improve the content of PAs and flavonol glycosides in seeds of transgenetic Arabidopsis thaliana.