Abstract
Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea’s recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.
Highlights
Full-length cDNA (FL-cDNA) is the DNA complement to an mRNA sequence that covers the region near the 5 cap structure to the poly(A) tail [1]
The FL-cDNA sequences can improve the quality of genome annotation [3], and provide accurate transcription start sites (TSSs), which can increase our understanding of the transcriptional regulation and associated network because transcription factor-binding sites are located around TSSs [4]
To maximize the cDNAs included in this library, total RNA was isolated from nine different tissue types, including buds, tender stems, tender leaves, mature leaves, flower buds, flowers, fruit pericarps, roots, and bark
Summary
Full-length cDNA (FL-cDNA) is the DNA complement to an mRNA sequence that covers the region near the 5 cap structure to the poly(A) tail [1]. FL-cDNA makes it possible to manipulate gene expression in heterologous systems, aiding the functional analysis of genes by reverse genetics. Due to these advantages, FL-cDNA technology has been widely applied to the genomic researches of different plant species, including Arabidopsis [2], rice [5], soybean [6], corn [7], tomato [8], citrus [3], cotton [9,10], and wheat [11]. Sinensis (CSS.; Chinese type) have been reported, with 36,951 and 33,932 annotated protein-coding genes, respectively [20,21], and provide valuable resources for the functional characterization of tea genes. The protocols described in this study have been proven to be useful for functional characterization of tea genes
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