Dissertation
Dissertation > Agricultural Sciences > Gardening > Vegetable gardening > Root vegetables ( taproot class ) > Radish

Cloning and Expression of Responsive Genes Related to Cadmium Stress in Radish (Raphanus sativus L.)

Author HeXiaoYan
Tutor GongYiQin;LiuLiWang
School Nanjing Agricultural College
Course Olericulture
Keywords Raphanus sativus L. Cadmium Gene Cloning Real-time PCR DDRT-PCR Expression analysis
CLC S631.1
Type Master's thesis
Year 2011
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The increasingly severe heavy metal pollution constitutes a great threat to global environment and human health. Cadmium (Cd) is not an essential nutrient for plants, which is recognised to be the heavy metal posing the most threat to agricultural food quality. The excessive Cd entered water-soil-biological cycle will be taken up by plants and concentrated in roots, stems, leaves and fruits which will not only affect the growth and development of the plant directly, but also accumulate in the human body from ingestion of food containing Cd which will ruin the health. Radish (Raphanus sativus L.)belongs to Brassicaceae family and is a very important worldwide root vegetable originated from China. The uptake, accumulation, transport mechanisms of cadmium-responsive genes of heavy metal-binding protein, some key synthases and the protein which can transport heavy metal are not fully understood, in this paper, we use radish as materials, cloning and expression analysis cadmium-responsive genes under Cd stress, and by mRNA differential display technology to isolate and identify the differentially expressed genes in radish under Cd stress, it’s valuable to understand heavy metal stresses and characterize their functions by transferring them into plants. The results were summarized as follows:In this study, the heavy metal-binding protein genes RsPCS and RsMT2 form radish were isolated using T-A clone. The full-length RsPCS DNA sequence was 2710 bp, including nine exons and eight introns, contained a complete open reading frame(ORF) of 1458 bp, which encoded a peptide of 485 amino acids with a molecular weight of 54.3 kDa and a theoretical pI of 6.36. RsMT2 contained a complete open reading frame of 252 bp, encoding a peptide of 83 amino acids. The RsMT2 protein had 16 Cys residues distributed at protein in N terminal and C terminal in this peptide (19.3% of the total residue number), and a conserved sequence form of CC, CXC, CXXC at the end, and seemed to belong to the typeⅡMT family. Semi-quantitative RT-PCR and real-time quantitative PCR results showed that the RsPCS and RsMT2 expressed in different organs, including petals, flower bud, leaves and roots, the expression quality of the RsPCS and the contents of the PCs in leaves were increased after 6 hours under Cd2+stress; however, the expression quality of the RsPCS in roots were decreased. It means that RsPCS was regulated by Cd at the transcriptional level.With the increase of cadmium concentration, the expression of RsMT2 increased, the results showed that RsMT2 expression was also induced by cadmium stress.Several heavy metal transporters were also cloned, sush as:IRT1, MTP1 and members of the CAX famLiy genes, the full DNA and cDNA sequences of them were cloned as respectively. With the bioinformatics method, the results inferred that the RsIRT1 protein is a membrane protein which has one signal peptide, eight transmembrane motifs, and it might be localized in the plasma membrane. Expression analysis by semi-quantitative RT-PCR showed that expression of the gene expression can be induced under Fe deficiency and Cd stress, while no expression under normal Fe level. It’s suggested that the IRT1 involved in the Fe and Cd uptake and transport.A gene were obtained which was highly homologous with AtMTP1 form radish, designed RsMTP1. It has six transmembrane domains, and there is a variable region between transmembrane domains 3 and 4 that is histidine-rich. Real-time PCR analysis showed that AtMTPl transcripts were highly relative expression in flower buds, were low in roots and petals, and were least in leaves; the expression of RsMTPl in leaves was indued under Cd stress, with the time of Cd stress, the relative expression of the RsMTP1 was increased, after 96 hours with an increase of 2.15-fold compared with controls;however, in roots, there was no obvious change under Cd stress in different times.The results indicated that the RsMTP1was tissue-specific expression.Based on the primers derived from the conserved sequences of the CAX genes in different crops, the genomic DNA and cDNA sequance of two members of the CAX family were amplified by PCR and RT-PCR of’Nau-RG’, named RsCAX1 and RsCAX2. RsCAX1 protein and RsCAX2 protein are membrane proteins which have elven transmembrane motifs. The CAX genes expression levels were analyzed by means of real-time PCR, transcription analysis showed that RsCAX1 transcript levels were the highest in leaves, but RsCAX2 transcript levels were higher in flower buds than that in other tissues. RsCAX1 gene expression in roots and RsCAX2 in leaves can be induced under Cd stress.Differential-display reverse transcription-PCR (DDRT-PCR) was applied to isolate of genes whose transcription was altered in radish under Cd stress in different concentrations after 96 h. A total of 52TDFs were identified; from these bands, fragments corresponding to 29TDFs were cloned.25TDFs showed high sequence similarity to the genes of known or putative function were transcriptional factors, expression regulators, and stress responding and transport facilitation genes, as well as genes involved in cellular metabolism and organization and the photosynthetic process. The results showed that plants rapidly and simultaneously change the expression of a set of genes to cope with Cd stress, suggesting that a multitude of processes are implicated in Cd stress response.

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