Identification Of Genes Involved In Response To Chromium Stress With DD-PCR And RNA-SEQ In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.) belongs to the Brassicaceae family and is an important worldwide biennial herb vegetable crop with high nutrition and economic values. As an important export vegetable, radish has long cultivation history and rich resource varieties. In recent years, its planting area had increased rapidly. For its high growth adaptability, yield and root nutrient, radish is widely cultivated in China, Japan, Korea and other Southeast Asian countries.Chromium (Cr) is one of the most toxic heavy metals in the environment and is not necessary for the plant biological structure and metabolic activity. Accumulation of chromium in plants can toxic the plants and finally endanger human life through food chain. Cr can stay in the soil for thousands of years, therefore, how to control and reduce the heavy metal pollution of the environment and human has become an increasingly attentioned issue both in China and other countries.In this study, we studied the genotype variance analysis of24radish varieties under different concentration of chromium at first. Then we identified the differentially expressed genes under Cr stress using mRNA differential display PCR (DDRT-PCR), for the analysis of plants mechanism of absorption and transportation. To investigate genes of radish in response to Cr treatments, genome-wide transcriptional profiling of the without (control) and with Cr (Ⅵ) solution (800mg·L-1) treatment were performed using the High-throughput RNA-sequencing (RNA-Seq) approach. Moreover, the expression patterns of20selected genes were confirmed by quantitative real-time PCR (qRT-PCR), and the results acquired showed general agreement with the Solexa data. QRT-PCR was also used in the expression of radish roots and leaves in different concentrations as Cr (0mg·L-1), Cr (200mg·L-1), Cr (400mg·L-1), Cr (800mg·L-1) through seven Cr related genes. Gene ontology (GO) and KEGG enrichment were used to analysis was performed to predicate functions of the identified genes. The main findings are as follows:1. Genotypic differences of different radish cultivars under Cr stressHydroponic and flame atomic absorption spectrometry methods were used to study the difference of24varieties under chromium treatments. The results showed that there are significant differences between different radish varieties under400mg·L-1and800mg·L-1Cr (VI) treatments. Under400mg·L-1Cr (VI) treatment, the range of chromium amont in different varieties is from658.4μg·g-1to6531tyzm. Under800mg·L-1Cr () treatment, the range of chromium in different varieties was from2095tyzmto8608tyzm. According to the results, we can identify that ’Nau-RG’ is high accumulation variety and ’Nau-YH’is low accumulation variety.2. Differentially expressed gene identification using DDRT-PCR under Cr stressDDRT-PCR was used to isolated genes of radish varieties ’NAU-XZH’ treated in different concentrations of Cr (0,50,200mg·L-1), and ’Nau-XBC in the same concentration (800mg·L-1) at different times (0h,24h,48h). Isolated a total of26different fragments,21of these bands have high homology with known functional genes. From these fragments, six were related to stress, eight associated with the metabolism of cells and tissues, five involved in transcription and translation regulation and two signal transduction fragments. These results showed that Cr stress was a complex process and the function was affected in many physiological processes.3. Differentially expressed gene identification using RNA-Seq under Cr stressThe research for the study was on research of radish varieties’Nau-RG’treated in different concentrations of Cr (0,800mg·L-1) treatment for48h. From the differentially expressed genes,20heavy metal related genes were identified by quantitative PCR. Gene Ontology (GO) and KEGG enrichment were used to analyse predicate functions of the identified genes. The main findings are as follows:(1) The most differentially expressed reads with a log2ratio>2or <-2(P<0.05) were analyzed further, revealed2985genes with significantly different expression levels between the libraries, among which1424genes were up-regulated and1561genes were down-regulated.(2) AmiGO software was used to classify the GO function. These GO enriched genes mainly involved in primary metabolic, response to stimuli, cell, the cell composition, cell membrance and the catalytic reaction.(3) KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in protein processing in endoplasmic reticulum, starch and sucrose metabolism, amino acid metabolism, glutathione metabolism, cytochrome P450and so on. Among them, some important genes encoding enzymes or proteins involved in sulfur transport, GSH metabolism, secondary metabolites biosynthesis, MAPK signaling pathway and metabolism of xenobiotics by cytochrome P450and metallothionein protein biosynthesis pathway were activated, and these pathways were linked, directly or indirectly through signaling pathways, to biosynthesis of metal transports and detoxification molecules.(4)β-actin was used as the reference genes for the quantitative real-time PCR (qRT-PCR). The expression patterns of20selected genes were confirmed by qRT-PCR, and the results acquired showed general agreement with the Solexa data. QRT-PCR was also used in the expression of radish roots and leaves in different concentrations as Cr (0mg·L-1), Cr (200mg·L-1), Cr (400mg·L-1), Cr (800mg·L-1) through seven Cr related genes. The results showed that these genes expression in root is higher than the leave and with the increasing of Cr amont, the expressions in root and leave declined. Taken together, this study revealed the complex changes at the transcriptional level during Cr stress of radish roots and provided a new insight into the molecular mechanism of chromium detoxification in radish.