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Cloning of Protein Phosphatase Type-2C Gene ZmPP2Cα of Zea mays.L and Its Expression under Drought Stress

Author: LiFuHua
Tutor: LiWanChen
School: Sichuan Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: maize drought stress serine / threonine protein phosphatase 2C ZmPP2Ca real-time fluorescence quantitative PCR expression property
CLC: S513
Type: PhD thesis
Year: 2007
Downloads: 320
Quote: 1
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Abstract

Drought is one of the most widespread abiotic constraints to maize production in developing countries. Average annual yield losses due to drought in tropical rainfed environments have been estimated at around 17%, as high as 60% in southern Africa, and 37% in Asia. In China, about 60% of maize zones are harmed by drought, and annual yield losses are estimated at more than 15%-20%, only inferior to the losses caused by plant diseases and insect pests. Significant difference of drought tolerance has been found among maize inbred lines.However, Drought tolerance is believed to be the result of cooperative interactions among multiple morphological, physiological and biochemical characters. Moreover, different genotypes may have different responses to drought stress. Along with interference of environment, it is usually difficult to evaluate drought tolerance under ordinary conditions. The identification of candidate genes is helpful to improve the efficiency of breeding for increased drought tolerance by marker-assisted selection and to improve drought tolerance of maize varieties.In previous study, we identified 3 tolerant (81565、N87- and R09) and 2 sensitive (200B and ES40) inbred line from 57 parent lines of commercial maize hybrids under strict drought stress. Based on the results, the method of mRNA differential display was used to isolate differential expression genes from a drought tolerant maize inbred line ’81565’ under drought stress and well-watered control. Two down-regulated expression gene fragments(MD1 and MD2) and one up-regulated expression gene fragment were obtained under drought stress. Sequence analysis and homology alignment showed that MD2 had 88% similarity with gene PP2C, encoding serine / threonine phosphorylase 2C in extremely drought tolerant Sporobolus stapfianus. In this study, the full length cDNA sequence of gene fragment MD2 was cloned with the method of in silicon cloning and reverse transcription PCR(RT-PCR), and the putative amino acid sequence of the gene was identified by sequence alignment and the conserved domains analysis. Its expression property on the level of mRNA was detected among the inbred lines with different drought tolerance under drought stress by real-time fluorescence quantitative PCR (FQ-PCR). Its expression property in different maize tissues were detected by FQ-PCR.The main results were as follows:1. The cDNA sequence of a differential displayed fragment MD2 was used as the querying probe and highly homologous EST sequences were blasted in dbEST database of maize at Genbank, The downloaded EST sequences were aligned and assembled into a 1731 bp contig by Seqman program of DNAstar software. To validate the correction of the in silicon sequence, the specific primers were designed and used to amplify the complementary sequences of the assembled contig by RT-PCR, According to the sequencing contig, open reading frames (ORF) were analysed by ORF Finder program at NCBI website. The deduced amino acid sequence was aligned in SWISSPORT database at NCBI website and analysed for function domains by Simple Modular Architecture Research Tool (SMART). The results showed that two different ORF of the contig in length, QH1 and QH2, were amplified by RT-PCR, Sequence analysis and homology alignment showed that The longer QH1 sequnece was the same as the in silicon cloning cDNA sequence, The QH2 was a shorter nucleotide fragment removed 213 bp from the QH1 sequence. It was speculated that the two fragments were alternative splicing products of the assembled contig. According to QH1 and QH2 sequences, one open reading frame (ORF) of 1164 bp and one of 954 bp were found using ORF Finder program at NCBI website, respectively. These ORF encode 388 and 317 amino acid residues, respectively. SWISSPORT alignment showed that the putative amino acid sequences have 34% similarity with PP2C genes in mouse, human and yeast, and 35 % similarity with 2 PP2C genes (ABA-Insensitive 1, ABI1 and ABA-Insensitive 2, ABI2) in arabidopsis. By running the SMART program a PP2C-like catalytic domain of serine / threonine protein phosphatase 2C was found at the C-end, and several residues such as MED, DGH, DG, D and R combining with divalent metal ions and phosphate ion were conserved. All these results indicated that the two ORF we cloned encode the protein of serine / threonine protein phosphatase 2C. Because no significant sequence similarity were found between the two ORF and the 2 reported PP2C genes in maize [ZmPP (AF213455) and ZmPP2C (AY621066)], the ORF was identified as a new member of PP2C gene family in maize, nominated as ZmPP2Ca and registered at GenBank with accession number EF 195257.2. The expression of the QH1 and QH2 transcripts of ZmPP2Ca were detected by RT-PCR. The results showed that the two transcripts in the leaf tissue were expressed under well-watered control, but the expression amount of QH1 was higher than that of QH2. The above result suggests that the QH1 transcript may have been extracted before maize material was treated with PEG6000, possibly not as the result of the exposure of the plant material to drought stress. Under drought stress, mainly QH1 was involved in maize response to drought stress.3. The real-time fluorescence quantitative PCR (FQ-PCR) was used to detect expression difference of ZmPP2Ca among 3 drought-tolerant (81565, N87-1 and R09) and 2 drought-sensitive (200B and ES40) inbred lines under drought stress and well-watered control. Maize seedlings were grown in the growth chamber in sandy loam soil. When soil absolute water content was about 12%, uniform seedlings with nine leaves were treated with 16% polyethylene glycol 6000 (PEG-6000) to simulate drought stress of -0.5 MPa osmotic potential, while another seedlings were watered with the same volume of fresh water and used as control. At 3, 6, 12, 24 and 48 h after drought treatment, leaf samples were taken from the same leaf position and used for total RNA isolation with Trizol. The real-time fluorescence quantitative PCR results showed that down-regulated expression was confirmed in the 3 drought-tolerant lines (81565, N87-1 and R09) and decreased to the lowest at 6 h of drought treatment. On the other hand, the 2 drought-sensitive lines (200B and ES40) displayed up-regulated expression under drought stress. Referring to the important role of protein phosphokinase, gene ZmPP2Ca, encoding protein phosphatase catalyzing the reverse reaction that phosphokinase catalyzes, was believed to be related with the respnose of maize to drought stress.4. Total RNA were isolated from root, shoot, leaf and sheath of maize inbred line 81565 under well-watered control. The technique of FQ-PCR was used to detect the expression of gene ZmPP2Ca in different maize tissues. The results showed that the ZmPP2Ca was expressed in all tissues examined. The relative expression amount of the gene was more abundant in leaves and shoots, and showed 6.86-fold and 2-fold higher compared with roots. The expression of ZmPP2Ca was little high in sheaths than that in roots. According to the above result, It was speculated that the expression pattern of ZmPP2Ca belongs to organ-constituted expression.

Cloning of Protein Phosphatase Type-2C Gene ZmPP2Cα of Zea mays.L and Its Expression under Drought Stress

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