Methodology Case Study (I)

2.5 Case Study (I)

Candidate genes involved in tanshinone and salvianolic acid biosynthesis acquired by “ingredient difference phenotypic cloning” method.

This example is desired to concretely introduce how to use the new method or strategy for cloning of functional genes involved in secondary metabolism. The advantages of high speed, high flux, and without need for clear genetic information of the method in gene cloning will be shown in details in this chapter.

2.5.1 Materials and Methods

2.5.1.1 Plant Material and Preparation of mRNA

The S. miltiorrhiza used for cDNA library construction was collected from Shangluo, Shanxi province. Hairy roots were grown in MS medium, and on day 18, postinoculation was dealt with YE and Ag + according to the reference [ 43 ] and harvested after 24 h. For RNA extraction, 5–10 g of hairy root material was frozen and stored in liquid nitrogen immediately after harvest.

2.5.1.2 Microarray Production

Microarray production was performed as described previously [ 22 ] . Briefl y, the source of the clones arrayed was an S. miltiorrhiza root tissue cDNA library. The library was constructed in ZAP Express vector (Stratagene, La Jolla, CA). Eight thousand seven hundred and thirty-six individual phage clones were picked randomly and amplified by polymerase chain reaction (PCR) using the M13 and BK universal primers with the GeneAmp PCR system 9700 (Perkin Elmer, Foster City, CA). PCR products were purified using MultiScreen filter plates (Millipore) and eluted in 100 m L of 0.1 × TE (pH 8.0). After analysis by agarose gel electrophoresis, 4,354 samples were dried to completion, resuspended in 15 mL 50% DMSO (approximately 1g/L) , and then transferred to a 384-format plate to be subsequently used for spotting. Amplified cDNAs were spotted in duplicate onto silylated microscope slides (CEL Associates, Houston, TX) using a 16-pin print head and a custom-built arraying robot. After arraying, the slides were air-dried and stored in the dark. Each of the microarray experiments was performed in duplicate with the dyes reversed.

2.5.1.3 Preparation of Fluorescent Dye-Labeled DNA, Hybridization, Scanning, and Data Acquisition

cDNA labeled with a cyanine fl uorescent dye (Cy5 or Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction [ 44 ]. Specifically, double-stranded cDNAs (containing the T7 RNA polymerase promoter sequence) were synthesized from 101g total RNA using the cDNA synthesis system according to the manufacturer’s protocol (Takara). A T7-oligo (dT) primer (50-AAACGACGGCCAGTGATTGTAATACACTCACTATAGGCGCTTTTTTTTTTTTTTTTT3) was used instead of the polyT primer provided in the kit.

After completion of double-stranded cDNA synthesis, cDNA products were puri fied using a PCR purification kit (Qiagen) and eluted with 60 m L elution buffer. One-half of the eluted double-stranded cDNA products was vacuum evaporated to 8 mL and used as a template in 20 m L in vitro transcription reactions at 37 °C for 3 h using the T7 RiboMAX Express large-scale RNA production system (Promega). The amplified RNA was purified using an RNeasy minikit (Qiagen). Klenow enzyme labeling strategy was adopted after reverse transcription. Briefly, 2mg amplified RNA was mixed with 2 lg random hexamers, denatured at 70 °C for 5 min, and cooled on ice.

Then, 4 mL of first-strand buffer, 2 mL of 0.1 M DTT, 1 m L 10 mM dNTP, and 1.5 mL SuperScript II (Invitrogen) were added. The mixtures were incubated at 25 °C for 10 min, then at 42 °C for 60 min. The cDNA products were purified using a PCR purification kit (Qiagen) and vacuum evaporated to 10 m L. The cDNA was mixed with 2 lg random hexamers, heated to 95°C for 3 min, and snap cooled on ice. Then, 10 mL buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (Amersham Pharmacia Biotech) were added to final concentrations of 120 mM dATP, 120 mM dGTP, 120 mM dTTP, 60 mM dCTP, and 40 mM Cy-dye. Klenow enzyme (1m L; Takara) was then added, and the reaction was performed at 37 °C for 60 min. Labeled cDNA was purified using a PCR purification kit (Qiagen) and resuspended in elution buffer. Labeled controls and test samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed with 30 m L hybridization solution (50% formamide, 19 hybridization buffer; Amersham Biosciences). DNA in the hybridization solution was denatured at 95 °C for 3 min prior to loading onto a microarray. Arrays were hybridized at 42°C overnight and then washed twice (0.2% SDS, 29 SSC at 42°C for 5 min, then 0.29 SSC for 5 min at room temperature). Microarray data were analyzed using GenePix Pro 5.0 (Axon Instruments, Union City, CA). The scanned data were normalized by the global normalization method [ 45 ], which normalizes the image data between Cy3 and Cy5 channels by adjusting the total signal intensities of two images and removing unreliable spots. The unreliable spots were discarded based on the following screening. Spots containing clones that had poorly amplified or multiple bands, as well as those that were flagged because of a false intensity caused by dust or background on the array, were removed. Spots with <65 % of the spot intensity at >1.5-fold that of the background in both channels were ignored. Clones in one sample that had an average induction greater than two fold in another were determined as up- or downregulated. Data management and analyses were carried out using Microsoft Excel and Microsoft Access database. After normalization, we calculated the means and coefficients of variation for the observed signal intensities in each channel and the ratio of signals from two replicates.

2.5.1.4 Sequence Analysis

cDNA clones with different expression in the microarray experiments were sequenced using the Applied Biosystems dye terminator cycle sequencing Read Reaction kit and a 3130 DNA sequencer. Vectors or imprecise nucleotides, such as polyT and polyA, and double peaks were removed. EST assembly was performed to obtain unigenes using Staden Package (gap4) software ( http://staden.sourceforge*net ). The sequences were fi rst compared with the GenBank database using BLASTX(http://www.ncbi.nlm.nih*gov/BLAST/). Genes with score values higher than 80 and identity values higher than 35 % were designated as significant homologous genes. Unigenes with E-values [ 5 ] were designated as unknown. The undesignated genes were then identified by comparison with sequences in the nonredundant nucleotide and EST databases of GenBank using the BLASTn algorithm. Gene ontology (GO) (http://www.geneontology*org ) was used to describe gene and gene product attributes. All genes were classi fi ed in terms of biological process, cell component, and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome*jp/keg) [ 46 ] was used to identify biochemical pathways associated with hairy root development stages.

2.5.2 Results

2.5.2.1 Microarray Experiments

Microarrays were used to examine gene expressions quantitatively after hairy root dealt with YE + Ag + . Previous research has shown that after dealt with YE + Ag + , the secondary metabolite of tanshinone’s accumulation increased dramatically [ 47 ] . In total, 4,354 unsequenced ESTs were picked from a cDNA library constructed from S. miltiorrhiza root tissue, ampli fi ed by PCR, and arrayed in duplicate on chemically modified microscope slides by using a robotic printing device. Experiments of comparing gene expression difference in two groups of hairy root YE + Ag + and control were performed. In each experiment, one mRNA population (target) was labeled with Cy3 and the other with Cy5. The labeled targets were then mixed and hybridized simultaneously to a microarray. To exclude artifacts, the researchers performed a reciprocal labeling experiment with each pair of targets, using the same techniques used in the first experiment except that the labels were exchanged.

Statistically, only genes for which we had 8 data points (two duplicates per slide, two replications, and dye-swap experiments) were considered, and approximately 201 cDNA clones were selected for further analyses.

2.5.2.2 Detection of Differentially Expressed Genes

Analysis of the microarray data revealed significant changes in transcript levels of those genes for which the expression varied by more than two fold were considered to exhibit significant changes in expression. A total of 201 genes (significant at single test, P < 0.05) were differentially expressed in hairy roots after 24 h’ dealt with YE + Ag + compared with that of control. After sequencing and correction for redundancy (performed by sequence alignment), 196 unique differentially expressed cDNA clones were identified. Sequence alignment of the cDNA clones identi fi ed as differentially expressed by microarray analysis revealed that several shared a high degree of sequence similarity. A total of 181 cDNA clones were successfully sequenced and acquired 130 unique genes. Among the 130 unique differentially expressed genes, 107 were categorized as genes of known function, 3 were homologues with low similarity.

2.5.2.3 Expression Profile of Genes Involved in Tanshinone and Salvianolic Acid Biosynthesis

The genes differentially expressed after dealt with YE + Ag + were then further analyzed using GO and KEGG pathways. On the basis of GO and KEGG analysis, candidate differentially expressed gene out of 130 unique genes were analyzed and notated. The researchers identi fi ed five genes involved in tanshinone biosynthesis: acetoacetyl-CoA thiolase (SmAACT, GenBank accession no. EF635969), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (SmCMK, GenBank accession no. EF534309), isopentenyl diphosphate isomerase 2 (SmIPPI, GenBank accession no. EF635967), farnesyl diphosphate synthase (SmFPS, GenBank accession no. EF635968), and ent-kaurene synthase (SmKSL, GenBank accession no. EF635966); one gene involved in salvianolic acid biosynthesis. These genes were upregulated after dealt with YE + Ag + (Table 2.3 ).

2.5.3 Discussion

The “ingredient difference phenotypic cloning” being a useful and powerful method mainly involved transcript-profiling analysis of S. miltiorrhiza hairy root under the YE + Ag + treatment could display differential regulation of secondary metabolism-related genes in S. miltiorrhiza . This method helps to identify more genes involved in biosynthesis of secondary metabolites of tanshinone and salvianolic acid if conditions satisfied. At the same time, the power of microarrays as a useful tool for novel gene discovery in “ingredient difference phenotypic cloning” method has been demonstrated in this study. In addition, because the cDNA clones were obtained from a recombinant cDNA library originating from the root of S. miltiorrhiza , the arrays are not representative of the entire transcriptome.

Nevertheless, the data obtained provide an important and novel description of the expression of a large number of S. miltiorrhiza genes.