ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 1, с. 103-110
^^^^^^^^^^^^^^^ ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^^^^
СТАТЬИ
УДК 581.1
IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN Alternanthera philoxeroides UNDER DROUGHT STRESS USING SUPPRESSION SUBTRACTIVE HYBRIDIZATION1
© 2015 D. Jia*, 2, B. Zhang**, 2, P. P. Zhang***, J. Y. Zhang***, Y. H. Liu***,
J. S. Wang***, R. Y. Ma*
* College of Agriculture, Shanxi Agricultural University, Taigu, China ** College of Horticulture, Shanxi Agricultural University, Taigu, China *** College of Life Science, Shanxi Agricultural University, Taigu, China Received April 22, 2014
The current study was conducted by using suppression subtractive hybridization (SSH) to identify the differentially expressed genes in Alternanthera philoxeroides (Mart.) Griseb. under drought stress and thus to explore the molecular mechanisms of drought tolerance. The mRNA was extracted from the roots of drought-treated and well-watered A. philoxeroides to construct SSH cDNA library. Positive clones were selected for sequencing and further analyzed by BLAST for screening non-redundant and homologous expressed sequence tags (ESTs). Then these ESTs were put into the Gene Ontology database for functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) for metabolic pathways analysis. Four cDNA fragments, ZFP (zinc finger protein), HSP70 (heat shock protein 70), CAT (catalase), and TPS (trehalose-6-phosphate synthase), were randomly chosen for RT-PCR analysis. In the SSH cDNA library, 286 positive clones picked up randomly were sequenced and finally 269 sequences were available. After cluster analysis of the ESTs, 82 unigenes were obtained, in which 63 genes displayed a high homology to known sequences. KEGG analysis found that these genes were involved in 14 metabolic pathways, such as the pathways of plant hormone signal transduction and biosynthesis of secondary metabolites. The expressions of the above four cDNA fragments were all up-regulated in A. philoxeroides under drought stress. This study presented a basis for studying the drought tolerance mechanism of A. philoxeroides, which provided a theoretical basis for managing the spread of this plant.
Keywords: Alternanthera philoxeroides - drought stress - suppression subtractive hybridization - unigene - differential expression genes - Gene Ontology - Kyoto Encylopedia of Genes and Genomes
DOI: 10.7868/S0015330315010091
INTRODUCTION
Alternanthera philoxeroides (Mart.) Griseb. (Ama-ranthaceae) is an invasive plant native to South America [1], which currently has spread to other areas of South America and also to the continents of North America, Asia, and Australia as well as some of the adjacent island countries [2]. In the 1930s, it was introduced into China initially as a forage crop. Since A. philoxeroides demonstrates very strong reproductive
1 This text was submitted by the authors in English.
2 First authors Dong Jia and Bing Zhang contributed equally to this work.
Abbreviations'. CAT - catalase; EST - expressed sequence tag; HSP70 - heat shock protein 70; KEGG - Kyoto Encyclopedia of Genes and Genomes; SSH - suppression subtractive hybridization; TPS - trehalose-6-phosphate synthase; ZFP - zinc finger protein.
Corresponding author. Rui-yan Ma. College of Agriculture, Shanxi Agricultural University, Taigu, 030801 China; fax. 0354628-9555; e-mail. maruiyan2004@163.com
abilities, it brings a great threat to the biological diversity, ecosystem, and social economy in the invasion regions and has become one of the worst weeds in many parts of the world [2].
Notably, A. philoxeroides, as one of the rather few amphibious plants, displays strong adaptive ability to different water environment, ranging from water to semi-aquatic even to drier terrestrial habitats [3]. Adventitious roots of A. philoxeroides seedlings could grow long, thick, and eshy during drought stress [3]. This is one reason for the rapid invasion of A. philoxeroides. Many physiological, cellular, and molecular processes are responsible for the survive of plants under water-decit conditions [4]. The previous study by our laboratory indicated that the morphological and anatomical structure of A. philoxeroides varied under different drought-stress conditions. These phenotype changes in response to environmental heterogeneity, often called phenotypic plasticity, are usually involved in genetic diversity. Meanwhile, it has been evidenced
that the adaptations are also involved in the expression of cascade ofgenes and interactions among their products [4]. Therefore, differential expression of genes may contribute to the modifications of morphology and structure that allow A. philoxeroides survive under different environment in China. But the information on genes associated with drought stress responses of A. philoxeroides is limited at present.
Analysis of expressed sequence tags (ESTs) has been demonstrated to be an appropriate strategy for identifying genes involved in specific biological functions in model plants [5]. Subtractive suppression hybridization (SSH) is a valuable tool for molecular investigations of differentially expressed genes [6], which has been widely applied in the researches of plants [7-9]. Besides, the GO (gene ontology) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis provides an opportunity to predict the functions of newly isolated putative gene sequences. The objective of current study was to identify the differentially expressed genes in the roots of A. philoxeroides under drought stress by constructing and analyzing of SSH cDNA library. Bioinformatics analyses of the ESTs allowed us to detect the function or pathways implicated by the differentially expressed genes and further explore the biological processes that enable the plant to withstand drought stress.
MATERIALS AND METHODS
Plant materials and treatment. Alternanthera philoxeroides (Mart.) Griseb. from South China was planted in our laboratory under controlled greenhouse conditions. A total of 100 plants growing to the length of7-8 internodes were selected, and only 3-4 internodes at the basal section were remained for the following research since the basal internodes of A. philoxeroides show relatively stronger survival ability and grow sprouts and roots more quickly. All the leaves of A. philoxeroides were removed before experiment. Then these plants were divided into two groups randomly. Plants of one group were grown hydroponically (water) as a control group (Driver), and the other plants were grown in sand (water content of 5 to 15%) as the treatment group (Tester). Sand moisture content was evaluated by weighing every day. All plants were grown in the light incubator (PRX-450C, "Haishu Saifu Experiment Instrument Co.", China) at 26 ± 1°C with 14 h light/10 h dark, light intensity of 3000 ± 200 lx, relative humidity of 85 ± 5%. The drought treatment lasted for 30 days, when the roots grew to about 10 cm, then they were collected, frozen in liquid nitrogen, and stored at -80°C.
Total RNA extraction and mRNA isolation. Total RNA was extracted from collected roots of both drought-treated and well-watered plants (control) using Trizol reagent ("Invitrogen") according to the manufacturer's instructions. The mRNA was purified from the total RNA using an Oligotex mRNA
Mini kit ("TaKaRa") according to the manufacturer's instructions and then precipitated by 0.1 volume of 3 M ammonium acetate (pH 5.2) and 1.0 volume of isopropanol.
SSH cDNA library construction. The concentrated mRNAs were applied to synthesize cDNAs according to the user manual of PCR-SelectTM cDNA subtraction kit ("Clontech", United States). SSH library was constructed using the cDNAs of drought-treated plant and control plant as "tester" and "driver", respectively. Two rounds of hybridization and PCR amplification were performed to normalize the abundance of cDNAs and to enrich the differentially expressed cDNAs. Both cDNAs from drought-treated and control plants were first digested with Rsal at 37°C for 1.5 h, and then the tester was divided into two equal parts and ligated to different adapters. The two parts of tester were mixed with excess driver separately for the first round of subtractive hybridization, products from which were mixed and used for the second round of subtractive hybridization together with the fresh degenerated driver. Then the subtracted cDNAs were subjected to two rounds of PCR, products of which were cloned into pGEM®-T easy vector ("Promega", United States) and transformed into competent DH5a Escherichia coli cells ("TaKaRa") to construct a subtracted library. Positive clones were selected by culturing the bacteria on Luria-Bertani (LB) agar plates containing ampicillin (50 mg/mL), X-Gal (50mg/mL), and isopropyl P-D-1-thiogalactopyra-noside (IPTG; 100 mg/mL). Plates were incubated at 37°C until small colonies were visible, and then incubated at 4°C until blue/white staining could be clearly distinguished. The positive clones were incubated overnight on 96-well cell culture plates containing LB liquid medium at 37°C and then stored in 20% glycerol. The bacterial suspension was frozen in liquid nitrogen and stored at -70°C until use.
cDNA sequencing and analysis. The cDNA sequencing was completed in "Beijing Biological Technology Co., Ltd", China. The vector and adaptor sequences presenting at 5' and 3' ends were removed by using PHRED program to clean the raw ESTs. Trimmed sequences with the length larger than 100 bp were selected for BLASTn and BLASTx analyses against the non-redundant nucleotide and protein sequence databases of NCBI (www.blast.ncbi.nlm.nih.gov/Blast.cgi), respectively. The e-value = 1e-5 was selected as the threshold. Sequences that passed through the set quality parameters were put into Gene Ontology database for functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database for metabolic pathways analysis.
Se
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