2.1. Plant materials and cultivation
We grew Chinese cabbage (B. rapa subsp. pekinensis) plants in soil (Professional soil: Dio Chemicals, Tokyo, Japan). We used KmP02, a clubroot-resistant (CR) double-haploid line derived from a CR F1 cultivar of Chinese cabbage (Hatakeyama et al., unpublished result), for our expression analyses and for constructing the full-length cDNA library. We also used Kyoto No. 3, Kigokoro 85, Okiniiri, Muso (Takii Seed Co. Ltd, Kyoto, Japan), and Chifu hakusai (Tohoku Seed Co. Ltd, Utsunomiya, Japan) for expression analyses. Seeds were sown in sterile soil in pots, moistened, and held at 4°C for 7 days in the dark to synchronize germination. The pots were then transferred to 22°C with a long-day photoperiod (16 h light/8 h dark). Except as noted in subsequent sections, all plants were grown under these conditions for the various stress treatments that we applied. Plants at the four-leaf stage were transferred individually into new pots and grown to the rosette stage.
2.2. Foliar treatment with chemicals
To determine the effects of chemical stress on gene expression, we sprayed the leaves with solutions of 5 mM salicylic acid, 0.5 mM benzothiadiazole, 1 mM ethephon, 0.1 mM methyl jasmonate, 0.025 µM paraquat, 0.1 mM abscisic acid, or 10 mM CuSO4. The plants were then placed in a growth chamber at 22°C under a 16 h light/8 h dark cycle. Leaves were harvested at 5, 10, and 24 h after treatment.
2.3. Drought, salt, and cold stresses
To create drought stress, the seedlings were transferred from the soil pots onto dry filter paper and allowed to air-dry. For salt stress, the seedlings were transferred into 250 mM NaCl solution. For cold stress, seedlings were transferred into a refrigerated chamber, in which the temperature was controlled at 4°C. Leaves were harvested at 5, 10, and 24 h after treatment.
To simulate wounding stress, the leaves were wounded with scissors. The plants were then placed in a growth chamber at 22°C under a 16 h light/8 h dark cycle. Leaves were harvested at 5, 10, and 24 h after treatment.
2.5. Ultraviolet radiation
Ultraviolet (UV-C) irradiation (0.1 kJ/m2) was supplied by a UV-C light. The plants were then placed in a growth chamber at 22°C under a 16 h light/8 h dark cycle. Leaves were harvested at 5, 10, and 24 h after treatment.
2.6. Root treatment with chemicals
The seedlings were transferred from soil pots into 0.5 mM benzothiadiazole solution or 1 mM ethephon solution. The roots were harvested 24 h after treatment.
2.7. RNA preparation
Total RNA was isolated using a modification of the method of Chirgwin et al
The tissues were ground to a fine in the presence of liquid nitrogen. The powder was then mixed with 10 volumes of GTC solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium N
-lauroylsarcosine, and 0.1 M 2-mercaptoethanol. The cellular debris was pelleted out (14000g
for 10 min at 4°C). About 2.4 ml of the supernatant was layered on top of 1.1 ml of 5.7 M CsCl cushion solution (5.7 M CsCl, 0.1 M EDTA) to create a step gradient and centrifuged at 240000g
for 2 h at 20°C. The RNA pellet was dissolved in a mixture of 10 mM Tris–HCl (pH 7.5) and 5 mM EDTA (pH 8.0). The supernatant was extracted with successive, equal volumes of phenol–chloroform and chloroform. The upper phase was collected and mixed with one-third of its volume of 8 M LiCl. The RNA was precipitated at 4°C overnight and centrifuged at 14 000g
for 30 min. The pellet was washed with 70% ethanol and dissolved in DEPC-treated water. Poly(A) + RNA was isolated by using Oligo(dT)-Latex (OligotexTM-dT30-Super; Roche, Tokyo, Japan) by following the manufacturer's instructions.
2.8. Construction of the cDNA library
Aliquots of total RNA extracted from the plant materials after each treatment were mixed equally to obtain one composite sample per treatment. The RNA mixture was used for the construction of an enriched full-length cDNA library based on the method of Kato et al
by using a vector-capping method. The resultant double-stranded cDNAs were ligated into a pGCAP10 vector. The primary library size was estimated at 2.4 × 106 colony-forming units (cfu) in the Chinese cabbage library.
2.9. Sequencing of both ends of the BBRAF cDNA clones
The plasmid DNA of each clone was prepared from bacterial cultures by using a NucleoSpin Multi-96 Plus Plasmid kit (Nihon Genetix Co.). End sequencing of 10 000 clones was carried out with an ABI 3700 capillary sequencer (Applied Biosystems, Foster City, CA, USA). The BBRAFpGCAP10F primer (5′-ACTGCTCCTCAGTGGATGTT-3′) and the BBRAF-polyT primer [mixture of equal amounts of 5′-T(30)AA-3′, 5′-T(30)AT-3′, 5′-T(30)AC-3′, 5′-T(30)AG-3′, 5′-T(30) CA-3′, 5′-T(30)CT-3′, 5′-T(30)CC-3′, 5′-T(30)CG-3′, 5′-T(30)GA-3′, 5′-T(30)GT-3′, 5′-T(30)GC-3′, and 5′-T(30)GG-3′] were used for forward and reverse sequencing, respectively.
2.10. Trimming and assembly of sequence data
Raw sequence data (chromatograms) were base-called using version X of the Phred software and vector sequences were then detected by using cross_ match. We trimmed off low-quality regions (for which the average quality score of a five-base window was <20) and the vector sequences of both ends of each read. Poly-A sequences of 15 or more bases were also trimmed off if found at the end of 5′ expressed sequence tags. Sequences that were shorter than 30 bases and left behind after the trimming process were also omitted from further analysis. The expressed sequence tags were assembled using version X of the PCAP.REP software without mate-pair constraints27
as the insert size of each clone could not be assigned a priori
. All sequences were submitted to the DNA Databank of Japan (DDBJ).
2.11. Annotation of the sequences
We aligned the above sequences with known Arabidopsis
sequences in the TAIR9 transcript database (http://www.Arabidopsis.org/
) using a BLASTN search (e
-value < 0.1) to estimate the degree of homology to Arabidopsis
genes. To detect genes that did not exist in the Arabidopsis
database, we used a BLASTN search with e
2.12. Quantitative real-time reverse-transcription PCR
We treated 2-week-old Chinese cabbage plants at the rosette stage with 100 μM jasmonic acid or 1 mM salicylic acid for 2, 5, 10, or 24 h (n = 5 per duration, with two replicates). The plants were then frozen in liquid nitrogen and total RNA (2 µg) was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and an RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA). Quantitative real-time reverse-transcription PCR was carried out with the Power SYBR Green PCR Master Mix (Applied Biosystems) by using the first-strand cDNA as a template and an ABI Prism 7900HT sequence detector (Applied Biosystems). Expression of BrACT2 was used for normalization (i.e. values for all other genes were divided by the activity of this gene). Nucleotide sequences for the B. rapa gene-specific primers were as follows: BrVSP2 (forward, 5′-GACTC CAAAACGGTGTGCAAA-3′; reverse, 5′-AGGGTCTCG TCAAGGTCAAAGA-3′); BrLOX2 (forward, 5′-TCCCCA CTTCCGCTACACC-3′; reverse, 5′-AATACTTTCCGGGC CAGAAAC-3′); BrPR1 (forward, 5′-TACGCTCAAAACTA CGCCGA-3′; reverse, 5′-GAAAGGTCCCCGCTACTTCC-3′); BrBGL2 (forward, 5′-GCAGAACATCGATAGAGC GGT-3′; reverse, 5′-TGAATGTCCCACTCGAAGGC-3′); and BrACT2 (forward, 5′-ACCCAAAGGCCAACAGA GAG-3′; reverse, 5′-CTGGCGTAAAGGGAGAGAACA-3′).
2.13. Accession numbers
The GenBank accession numbers for the genes mentioned in Section 2.12 are as follows: BrPR1 (BBRAF03K11), BrBGL2 (BBRAF10P08), BrVSP2 (EX103556), BrLOX2 (EX100417), and BrACT2 (BBRAF03F20).