ФИЗИОЛОГИЯ РАСТЕНИЙ, 2011, том 58, № 1, с. 111-117
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
DIFFERENTIAL GENE EXPRESSION IN LEAVES AND ROOTS OF WINTER RAPE IN RESPONSE TO PHOSPHORUS STARVATION © 2011 Ling Qin*, **, Chun-Lei Zhang*, Bin Zhang***
*Crop Physiology and Sustainable Agriculture Division, Institute of Oil Crops Research,
Chinese Academy Agricultural Science, Wuhan, China **Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan, China ***High-Tech Research Center, Shandong Academy of Agriculture Sciences, Jinan, China
Received January 6, 2010
Genes induced by phosphorus deficiency in leaves and roots of winter rape (Brassica napus L.) seedlings were isolated and analyzed. The mRNA differential display technique was used to visualize cDNA fragments derived from the phosphorus-tolerant cv. Zy 821 and the phosphorus-sensitive cv. No. 1182 grown under conditions of phosphorous starvation. Approximately 2000 cDNA bands were visualized by differential display using 78 primer pair combinations. A total of 61 phosphorus-starvation-induced cDNA fragments differentially expressed were isolated. Among these, 40 were derived from roots and only 21 from leaves. Sequence analysis of 16 fragments revealed that they represented distinct cDNAs. A subsample of five cDNAs was analyzed by semi-quantitative RT-PCR, which showed that they were truly different products. Transcripts coding for enzymes involved in photosynthesis (thylakoid membrane phosphoprotein) and root hair initiation (xyloglucan endotransglycosylase), and two regulators (RNA-binding/nucleic acid-binding mRNA and a structural constituent of ribosomal mRNA) were found to be differentially expressed. These results show that the mechanism of cv. Zy 821 adaptation to P starvation is complex. Although its P-tolerance trait is controlled by a major gene, many other genes influencing P acquisition and remobilization, carbon and secondary metabolism, developmental processes, and regulatory pathways are also involved.
Key words: Brassica napus — DDRT-PCR — phosphorus starvation — semi-quantitative RT-PCR — gene expression
INTRODUCTION
Phosphorus (P) is one of the most important nutrients for plant growth and development. The concentration of P in the soil solution is often as low as 2—10 mM [1]. Due to the low availability of soluble P in many soils, plants developed morphological, physiological, and molecular mechanism to tolerate low phosphate availability [2]. It is thought that these responses to P starvation are coordinated by both general stress-related and P-specific signaling cascades. Many genes induced by P deficiency have been isolated and characterized to clarify the system ofgenetic regulation underlying adaptation to P starvation. Transcription elements related to the response to P deficiency have been isolated [3]; however, the current knowledge is not sufficient to explain the details of the network regulating gene expression in response to low P.
Abbreviations: CA — carbonic anhydrase; DDRT-PCR — differential display reverse transcription-PCR; EST — expressed sequence tag; TF — transcription factor; TMP14 — 14-kD thylakoid membrane phosphoprotein; XET — xyloglucan endotransglycosy-lase.
Corresponding author: Chun-Lei Zhang. Crop Physiology and Sustainable Agriculture Division, Institute of Oil Crops Research, Chinese Academy Agricultural Science, Wuhan, 430062 China. E-mail: clzhang@vip.sina.com
Rapeseed (Brassica napus) requires a sufficient supply of P during early growth, and thus it is very sensitive to P deficiency. Its adaptation to P deficiency includes highly coordinated modification of root development and metabolism resulting in increased root-to-shoot ratios [4]. Rapeseed roots exude large amounts of organic acids (malic and citric acids) [5] and phenols [6] into the soil. Concurrently, the expression levels of citrate and phosphoenolpyruvate carbox-ylase are strikingly increased in P-starved roots [7]. The transcription and activity of RNases [8] and acid phosphatases [9] are enhanced by P deficiency. At present, the molecular mechanisms of highly efficient P utilization and genes induced by P starvation are not very clear and need further study.
Analysis of expressed sequence tags (ESTs) is an efficient approach for identifying large numbers of plant genes expressed during different developmental stages and in response to a variety of environmental conditions [10]. The objectives of this study were to assess genes expressed in winter rape roots and leaves and to analyze global gene expression in roots and leaves under P-starvation stress. To achieve this goal, we (a) identified ESTs from roots and leaves in two different rape varieties grown under P-starvation (-P) conditions; (b) used the mRNA differential display tech-
nique to compare gene expression in roots and leaves grown under —P and P-sufficient (+P) conditions; and (c) confirmed the expression patterns of differentially expressed ESTs by semi-quantitative RT-PCR at various stages of winter rape development.
MATERIALS AND METHODS
Plant materials and treatment. Winter rape (Brassi-ca napus L.) cultivars used in this study were No. 1182, a P-sensitive variety, and Zy 821, a P-tolerant variety. Seeds were directly sown in separate sand beds in mid-September. The P starvation was initiated at the four-leaf stage. Half of the seedlings were irrigated with a P-starvation solution (0 mg/l KH2PO4 replaced with KCl) and half with a normal P-sufficient solution (136 mg/l KH2PO4) as a control. Leaves and roots from +P and —P seedlings were harvested separately at 6, 12, 24, 48, and 72 h; 5, 7, and 9 days after treatment, rapidly frozen in liquid nitrogen, and stored RNA extraction at —70°C.
RNA extraction. Total RNA was extracted from plant tissue at various developmental stages using Tri-zol Reagent ("Invitrogen", United States). Each sample was incubated with DNase I ("Promega", United States) for 30 min at 37°C. After extraction with phenol : chloroform : isoamyl alcohol (25 : 24 : 1), RNA was precipitated with ethanol and redissolved in dieth-yl pyrocarbonate-treated water. RNA samples were merged into eight different pools. Agarose gel electrophoresis was used to test the integrity and purity of the extracted RNA. RNA was quantified by the UV spectrophotometry analysis.
mRNA Differential Display-PCR (DDRT-PCR).
Anchored primers (HT10M: 5'-AAGCTTTTTTTTTT-TA/G/C-3') and arbitrary primers (No. B0301-B0326) were synthesized by "Sangon", Shanghai (www.sangon.com). Total RNA was used for reverse transcription. For each RNA sample, three reverse transcription reactions were set up, each containing one of the three different HT10M primers (where M may be G, A, or C) anchored to the beginning of the polyA+ tail. First-strand cDNA was synthesized using the Reverse Transcription System ("Promega") according to the technical bulletin.
Each 20 |l PCR reaction was set up in a 0.2-ml microcentrifuge tube containing 10.8 |l sterile H2O, 2.0 |l 10x PCR reaction buffer, 0.2 |l 25 mM mixed dNTPs, 2.0 |l anchored primer, 2.0 |l arbitrary primer, 1.5 |l cDNA, and 0.3 |l ExTaq DNA polymerase (5 U/| l). PCR reaction conditions were as follows: 40 cycles of 95°C for 30 s, 38°C for 2 min, and 72°C for 60 s, followed by 1 cycle of 72°C for 7 min. PCR reaction products were stored at —20°C.
Urea-denaturing polyacrylamide gel electrophoresis. DDRT-PCR products were separated on a 6% urea-denaturing polyacrylamide gel run at 60 W until the xylene cyanole dye front reached the bottom.
DNA fragments were visualized by silver staining according to the Silver Sequencing System Technical Manual ("Promega").
Cloning and sequencing of differentially expressed cDNA fragments. The gel bands representing differentially expressed cDNA fragments were excised with a knife and incubated for 60 min in 20 |l TE (pH 8.0) at 37°C. The extract served as the template for re-amplification with the same pair of primers used in DDRT-PCR. PCR products were run on a 2% agarose gel to remove short oligonucleotides and residual dNTPs, and then the desired band was excised and cleaned using a UNIQ-10 column DNA recovery kit ("Sangon"). Finally, the purified and digested DNA was directly cloned into a pMD18-T vector ("Takara", Japan) and transformed into Escherichia coli DH5a cells treated with 100 mM CaCl2. Blue/white screening was used to select positive clones. The positive clone plasmid was extracted with a Plasmid DNA Extraction Kit and identified by PCR and Eco RI and PstI restriction digestion, followed by sequencing. The sequences thus obtained were deposited to the GenBank (www.ncbi.nlm.nih.gov). Using the BLASTX algorithm, DNA sequences were translated into the deduced amino acid sequences and searched against the nonredundant protein database in GenBank.
Semi-quantitative RT-PCR. A two-step semiquantitative RT-PCR method was used to measure gene expression in rape under P-starvation conditions. Total RNA (about 5 | g) was used to synthesize firststrand cDNA in a 20 |l reaction volume containing 100 units of Superscript II reverse transcriptase ("Invitrogen"), 0.5 |M oligodT primer, 20 units RNase inhibitor, 10 mM DTT, 1 mM of each dNTP, and 4 |l 5x reverse transcription buffer. The yield of cDNA was measured according to the PCR signal generated from the internal standard housekeeping gene p-actin (GenBank accession no. AF111812) amplified in 20— 29 cycles starting with the cDNA solution. The volume of each cDNA pool was adjusted to give the same exponential phase PCR signal strength for p-actin.
Relative RT-PCR was performed to measure the expression of bnl1, bnl4, bnl8, bnr2, and bnr8 mRNAs (the corresponding GenBank accession nos. GR505445, GR505446, GR505447, GR505444, and GR505443). Primer sequences, optimal PCR annealing temperatures (T), and expected fragment sizes are listed in table 1.
RESULTS mRNA differential display after P starvation
Good quality RNA from leaves and roots
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