Global transcriptome profiles ofCamellia sinensis

during cold acclimation

文章第一部分：背景、实验结果、结论、关键词

背景：茶多么多么重要。。。在冬天，茶需要经历低温去提高自身的抗冻能力。低温中分子水平上发生的变化还没有被广泛研究。为了阐明低温胁迫的分子机理，我们使用了转录组（RNA-seq）和数字表达谱digital gene expression(DGE)技术来低温过程中基因表达谱。

实验结果：五千多万条RNA-Seq reads——assembled into 216,831transcripts，with an average length of 356 bp and an N50 of 529 bp
——1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated
——这其中包括：寒冷传感相关、信号转导基因、冷敏感转录因子基因、细胞膜稳定相关基因、osmosensing 响应、解毒酶基因。

DGE和荧光定量PCR分析证实了来自转录组的结果是正确的。通路分析：carbohydrate metabolism pathway、calcium signaling pathway
might play a vital role in tea plants’ responses to cold stress

结论：a global survey of transcriptome profiles of tea plants in response to low,non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process

Keywords: Camellia Sinensis, Cold Acclimation, RNA-Seq, DGE, Genome-wide Expression Profiles, Tea Plants
下面是正文内容：
cold acclimation (CA) 

When plants sense the cold temperature, a series of protective mechanisms are triggered.

当植物感受到低温时，一系列自我保护的生物机制被触发。

A group of cold-related genes has been reported to regulate these aforementioned changes.

一群低温相关的基因已经被报道与上述这些变化的调节有关。
Moreover, changes in geneexpression have been demonstrated to occur during CA in a wide range of plant species, and hundreds of cold inducible genes have been identified
而且，

A previous study showed that when the average air temperature decreases to around 7°C, tea plants undergo the CA process, and after
the average air temperature increases to over 9°C, tea plants start the de-acclimation process

使用组学的研究方法去研究CA过程中的分子机制，这是。。。的关键
RNA-Seq 。。。improved the efficiency and speed of gene discovery。
转录组测序技术...提高了基因发现的效率和速度。
Digital gene expression(DGE) is a tag-based sequencing approach according to which short tags are generated by endonuclease. 
The expression level of genes in the sample is measured by counting the number of tags generated from each transcript 

This study demonstrates the first attempt to use a combination of RNA-Seq and DGE to study the transcriptome profiles in tea plants and thereby gain a deeper insight into the molecular mechanism of CA.

Results and discussion
above 10°Ctea-plant leavesCK
relatively low temperatures
lowest point CA1
average temperature reached above 10°CA3

RNA-Seq andde novo assembly

We performed RNA-Seq analyses for CA1, CA3 and CK using the Illumina HiSeq2000 genome analyzer.

De novo assembly was performed with the trimmed reads using Trinity（Trinity was specially developed for de novoassembly from short-read RNA-Seq data, which has been shown to be the best singlek-mer assembler）

In total, 226,026 transcripts were reconstructed.

After removing the redundant transcripts caused by small variations as described in the previous study,a final set of 216,831 transcripts were obtained. The average transcript size is 356 bp, and the N50 is 529 bp。

A combination of dataset 1 and dataset 2 was also generated, which we called dataset 3, representing all available RNA-Seq data for C.sinensis.

Although more transcriptome sequences could be produced from de novoassembly using dataset 3 than dataset 1, the mapping ratio could not be improved (Table 2), indicating that the additional transcripts from dataset 3 are most likely transcripts that are expressed in tissues other than the leaves of tea plants.

Functional annotation of C. sinensis transcriptome

To predict and analyze the function of the assembled transcripts, non-redundant sequences were submitted to a BLASTx (E-value≤10-5) search against the following databases: the NCBI’s NR database, UniRef90,the Arabidopsis Information Resource (TAIR,version 10),KEGG and Clusters of Orthologous Groups from 7 eukaryotic complete genomes (KOG).

We found that about one third of all non-redundant transcripts had significant homology with genes in either the NR or UniRef90 databases

拟南芥·A BLAST search against genes from Arabidopsis produced more definitive annotations and helped us to evaluate the quality and coverage of our assembled transcripts.

A BLAST analysis of the assembled transcripts against the KEGG database showed that 21,194 transcripts were annotated with corresponding Enzyme Commission (EC) numbers and assigned to the reference canonicalKEGG pathways

A search against the KOG database reported that 41,341 transcripts had the best hits when theE-value was less than or equal to 10-5.

Since some transcripts could be assigned multiple KOG functions, altogether 46,291 functional annotations were produced and all hit transcripts were grouped in 25 categories

In total, 72,967 transcripts got the best hits with known proteins in at least one of the five databases and 16,430 transcripts had similarity to proteins in all of the five databases。

To functionally categorize the assembled transcripts,gene ontology(GO) terms were assigned to each transcript based on the best BLASTx hit from the NR database using Blast2GO。

Identification of genes involved in cold acclimation
RSEM

DESeq package（The DESeq package [28] and the winflat program were then applied to identify differentially
expressed genes. ）

nes were activated than repressed
during the CA process. Dozens of cold-regulated or coldrelated genes were found in this differential expression list,
including cold sensor or signal transduction genes, coldresponsive transcription factor genes, plasma membrane
stabilization related genes, osmosensing-responsive genes
and detoxification enzymes genes.

Cold sensor or signal transduction genes

The signal transduction pathway plays a pivotal role in the response to the stress of low temperatures 。It is well known that Ca2+acts as a key messenger in regulating growth and developmental processes and plays a crucial role in stress signaling, i.e. cold stress.

Cold stress could activate Ca2+channels to increase the cytosolic Ca2+level, and then trigger phospholipase C and D, producing inositol...

Table 5 KOG functional classification of C. sinensis
transcripts

Subsequently, many signaling pathways are triggered.

接着，许多信号通路被触发了。

In this study, 13 genes, which were annotated as CDPKs, CBL, calmodulin, CAMTA, MAPK and phospholipase, were identified as being involved in signal transduction upon low temperature stress.
Among these genes, 9 (4 calmodulin genes, 2 CDPK genes, 1 CAMTA gene and 2 phospholipase genes) were up-regulated in CA1,
whereas 4 (1 phospholipase gene, 1 calmodulin gene, 1 CBL gene and 1 MAPK gene) were down-regulated.

Plant protein kinases belong to a large superfamily,some of which have been known to play a central role in cellular signaling, for example CDPKs and MAPKs.
In addition, a growing body of evidence has shown that receptor-like kinases (RLKs) are involved in the perception of environmental signals.

Histidine kinases(HKs), being localized to the cellular membranes and endoplasmic reticulum, are the major signaling molecules
and are involved in the two-component signaling pathways that mediate plant-sensed environmental signals and regulate the downstream environmental stress response.
组氨酸激酶，位于细胞膜和内质网，是主要的信号分子并且

In this study, 27 RLKs genes and 2 HKs genes were differentially expressed and all of these were up-regulated in
CA1 samples, which indicates that protein kinases play an important role in the CA process in tea plants.

在此研究中，27 RLKs基因和2个组氨酸激酶基因被差异表达了。同时，所有的这些基因在CA1样本中都是正调控，这也就显示了：蛋白激酶在茶树的CA过程中起到了重要的作用。

Cold-responsive transcription factor genes
真核生物转录起始十分复杂，往往需要多种蛋白因子的协助，转录因子与RNA聚合酶Ⅱ形成转录起始复合体，共同参与转录起始的过程。

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