A large insert Thellungiella halophila BIBAC library for genomics and identification of stress tolerance genes

Salt cress (Thellungiella halophila), a salt-tolerant relative of Arabidopsis, has turned to be an important model plant for studying abiotic stress tolerance. One binary bacterial artificial chromosome (BIBAC) library was constructed which represents the first plant-transformation-competent large-insert DNA library generated for Thellungiella halophila. The BIBAC library was constructed in BamHI site of binary vector pBIBAC2 by ligation of partial digested nuclear DNA of Thellungiella halophila. This library consists of 23,040 clones with an average insert size of 75 kb, and covers 4× Thellungiella halophila haploid genomes. BIBAC clones which contain inserts over 50 kb were selected and transformed into Arabidopsis for salt tolerant plant screening. One transgenic line was found to be more salt tolerant than wild type plants from the screen of 200 lines. It was demonstrated that the library contains candidates of stress tolerance genes and the approach is suitable for the transformation of stress susceptible plants for genetic improvement.     

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.     

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.     

Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Culture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.     

Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies

Key message

Differentially expressed antioxidant enzymes, amino acids and proteins in contrasting rice genotypes, and co-location of their genes in the QTLs mapped using bi-parental population, indicated their role in salt tolerance.

Abstract

Soil salinity is a major environmental constraint limiting rice productivity. Salt-tolerant ‘CSR27’, salt-sensitive ‘MI48’and their extreme tolerant and sensitive recombinant inbred line (RIL) progenies were used for the elucidation of salt stress tolerance metabolic pathways. Salt stress-mediated biochemical and molecular changes were analyzed in the two parents along with bulked-tolerant (BT) and bulked-sensitive (BS) extreme RILs. The tolerant parent and BT RILs suffered much lower reduction in the chlorophyll as compared to their sensitive counterparts. Activities of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) and non-enzymatic antioxidant ascorbic acid were much higher in salt-stressed CSR27 and BT RILs than MI48 and BS RILs. Further, the tolerant lines showed significant enhancement in the levels of amino acids methionine and proline in response to salt stress in comparison to the sensitive lines. Similarly, the tolerant genotypes showed minimal reduction in cysteine content whereas sensitive genotypes showed a sharp reduction. Real time PCR analysis confirmed the induction of methionine biosynthetic pathway (MBP) enzymes cystathionine-β synthase (CbS), S-adenosyl methionine synthase (SAMS), S-adenosyl methionine decarboxylase (SAMDC) and serine hydroxymethyl transferase (SHMT) genes in tolerant lines, suggesting potential role of the MBP in conferring salt tolerance in rice variety CSR27. Proteome profiling also confirmed higher expression of SOD, POD and plastidic CbS and other proteins in the tolerant lines, whose genes were co-located in the QTL intervals for salt tolerance mapped in the RIL population. The study signifies integrated biochemical-molecular approach for identifying salt tolerance genes for genetic improvement for stress tolerant rice varieties.

     

Overexpression of SaRBP1 enhances tolerance of Arabidopsis to salt stress

Abiotic stresses are the major concern in recent years as their effect on world food production is constantly increasing. We have obtained salt tolerant Arabidopsis lines overexpressing SaRBP1 (Suaeda asparagoides RNA binding protein 1) of a Korean halophyte, S. asparagoides. Homozygous T3 Arabidopsis transgenic lines were developed and used for salt stress tolerance studies. The transgenic seedlings displayed tolerance to salt and mannitol compared to the wild type (WT) seedlings. Transgenic lines produced longer primary roots, more fresh weight, and higher number of lateral roots than WT. In planta stress tolerance assay results showed that the survival rates of transgenic plants were significantly higher than WT plants. Transgenic lines showed delayed germination under 200 mM NaCl stress. In addition, the transgenics showed higher water retention ability than WT. Subcellular localization results revealed that SaRBP1 was targeted to the cytoplasm. Northwestern blot analysis results confirmed the RNA binding property of SaRBP1. Quantitative Real-Time Polymerase Chain Reaction results revealed that many stress marker genes were upregulated by SaRBP1 overexpression. Thus, our data demonstrate that SaRBP1 overexpression lines are tolerant to salt stress. Hence, this is the first report for the functional characterization of SaRBP1, a novel RBP gene isolated from S. asparagoides cDNA library.     

Validation and high-resolution mapping of a major quantitative trait locus for alkaline salt tolerance in soybean using residual heterozygous line

Alkaline soil restricts soybean plant growth and yield. In our previous study, a major alkaline salt tolerance quantitative trait locus (QTL) was identified in soybean on chromosome 17. In this study, the residual heterozygous line (RHL46), which was selected from a population of F6 recombinant inbred lines (RILs) derived from a cross between an alkaline salt-sensitive soybean cultivar Jackson and a tolerant wild soybean accession JWS156-1, was used for validation and high-resolution mapping of the QTL. In a large segregating population (n = 1,109), which was produced by self-pollinating heterozygotes of RHL46, segregation of alkaline salt tolerance showed a continuous distribution, and the tolerant plants were predominant. Linkage mapping analysis revealed a major QTL with a large dominant effect for alkaline salt tolerance, and the highest LOD score was detected between the single sequence repeat (SSR) markers GM17-12.2 and Satt447. Furthermore, 10 fixed recombinant lines carrying chromosome fragments of different lengths in the QTL region were selected from the RHL46 progeny. Phenotype evaluation and SSR marker analysis of the recombinant lines narrowed down the QTL to a 3.33-cM interval region between the markers GM17-11.6 and Satt447 with a physical map length of approximately 771 kb. High-resolution mapping of the alkaline salt tolerance QTL will be useful not only for marker-assisted selection in soybean breeding programs but also for map-based cloning of the alkaline salt tolerance gene in order to understand alkaline salt tolerance in soybean and other plant species.     

Organic substances responsible for salt tolerance inEruca sativa

Responses of a salt tolerant and a normal population of an oilseed crop,Eruca sativa Mill. were assessed after four weeks growth in sand culture salinized with 0 (control), 100, 200, or 300 mol m^?3 NaCl. The salt tolerant plants produced significantly greater dry biomass than the normal population. The populations did not differ significantly in leaf osmotic potential, relative water content and leaf soluble proteins. However, the tolerant population accumulated significantly greater amounts of soluble sugars, proline and free amino acids in the leaves compared with the non-tolerant population. It is established that leaf soluble sugars, proline, and free amino acids are important components of salt tolerance inEruca sativa.     

Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment

To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50–57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.     

苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆

以耐盐的苜蓿中华根瘤菌(\%Sinorhizobium meliloti) \%042B为材料,制备其总DNA,经过限制性内切酶\%Eco\%RⅠ的部分酶解,利用电洗脱方法回收15～25kb大小的DNA片段。以碱法制备载体质粒pLAFRⅠ,用\%Eco\%RⅠ将其切成线状,然后用T\-4DNA连接酶将回收片段与线状载体连接,利用包装蛋白进行包装后,感染大肠杆菌(Escherichia coli)S17\|1,构建了042B的基因文库。以固体亚硝基胍作为诱变剂处理出发菌株,在05mol/LNaCl的条件下,从2000个菌落中筛选得到12株042B的盐敏感突变株,以其中稳定的盐敏突变株GZ17为受体菌,利用两亲本杂交将含有042B的DNA片段的pLAFRⅠ重组质粒转移到GZ17中,在含有四环素和05mol/LNaCl的基本培养基上筛选出能够耐盐的阳性克隆,获得了与耐盐有关的7kb长的DNA片段。对该片段进行亚克隆,最终获得了4kb与耐盐有关的片段。     

桑蚕金属硫蛋白基因在大肠杆菌中的克隆和表达

用\%Bam\%HⅠ和\%Sac\%Ⅰ双酶切质粒pCM1\|1,获得酵母的MTI基因片段,用非放射性地高辛标记作为探针。提取桑蚕肥苏蚕卵的总DNA,分别用\%Eco\%RⅠ、\%Bam\%HⅠ和\%Hin\%dⅢ酶切,与MTI探针进行Southern杂交,出现较强的杂交信号。然后用\%Eco\%RⅠ完全酶切桑吞的总DNA,电洗脱法回收1～6kb的染色体片断,与\%Eco\%RⅠ酶切的M13-载体以3∶1比例连接,转化受体菌DH5α。筛选到4 000多个白色转化子,与探针MTI进行Southern杂交筛选阳性转化子,选择到有强杂交信号的三个转化子［编号为T1(pZHC\|1)\,T5(pZHC\|5)\,T7(pZHC\|7)\]。用12种限制性内切酶对pZHC\|5重组质粒进行酶切分析表明插入片段约12kb,在基因内有一个\%Hin\%dⅢ位点。抗性测定表明受体菌DH5α在含有50mmol/L CuSO\-4的培养基上生长,在含有52mmol/L CuSO\-4的培养基上不生长,而转化子确能在含有52mmol/L CuSO\-4以上的培养基上生长。上述研究结果表明12kb左右的插入片段含有桑吞的金属硫蛋白基因。     

Relationships between the osmoadaptation strategy, amino acid composition of bulk protein, and properties of certain enzymes of haloalkaliphilic bacteria

Haloalkaliphilic microorganisms isolated from soda lakes were compared in terms of the amino acid composition of bulk protein and the reaction of a number of key enzymes to salts and pH of the medium. In the extremely haloalkaliphilic bacterium Natroniella acetigena (selt-in osmoadaptation strategy), acidic amino acids (glutamic and aspartic) made up 30.91 mol % of the total of bulk protein amino acids. In the moderate haloalkaliphiles Tindallia magadiensis, Halomonas campisalis, and Halomonas sp. AIR-2 (compatible-solutes osmoadaptation strategy), the proportion of acidic amino acids (24.36, 23.15, and 23.58 mol %, respectively) was lower than in N. acetigena but higher than in the freshwater Acetobacterium paludosum (20.77 mol %). The excess of acidic amino acids over basic amino acids (lysine and arginine) increased with the degree of halophily. The enzymes of haloalkaliphiles proved to be tolerant to salts and high pH values, although the degree of tolerance varied. The activity of N. acetigena CO dehydrogenase was maximum in the presence of 0.7 M NaCl, but it was virtually independent of the NaHCO₃ concentration. The hydrogenase and CO dehydrogenase of T. magadiensis exhibited maximum activity in the absence of NaCl; the Co dehydrogenase was most active at 0.25 M NaHCO₃, and hydrogenase activity was only weakly dependent on NaHCO₃ in the concentration range of 0–1.2 M. The nitrate reductases of H. campisalis and Halomonas sp. AIR-2 were active in broad ranges of NaCl and KCl concentrations; the activity maxima were recorded at moderate concentrations of these salts. The pH optima of most of the studied enzymes of haloalkaliphiles were in the alkaline zone. Thus, it was shown that the amino acid composition of bulk protein is determined by the osmoadaptation strategy employed by the bacterium. A correlation was found between the salt tolerance of enzymes and the proportion of acidic amino acids in the bulk protein. The ability of enzymes to function at high pH values is one of the mechanisms of adaptation of microorganisms to high pH values.     

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni–NTA (Ni²⁺-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.     

Identification of a major QTL allele from wild soybean (Glycine soja Sieb. & Zucc.) for increasing alkaline salt tolerance in soybean

Salt-affected soils are generally classified into two main categories, sodic (alkaline) and saline. Our previous studies showed that the wild soybean accession JWS156-1 (Glycine soja) from the Kinki area of Japan was tolerant to NaCl salt, and the quantitative trait locus (QTL) for NaCl salt tolerance was located on soybean linkage group N (chromosome 3). Further investigation revealed that the wild soybean accession JWS156-1 also had a higher tolerance to alkaline salt stress. In the present study, an F₆ recombinant inbred line mapping population (n = 112) and an F₂ population (n = 149) derived from crosses between a cultivated soybean cultivar Jackson and JWS156-1 were used to identify QTL for alkaline salt tolerance in soybean. Evaluation of soybean alkaline salt tolerance was carried out based on salt tolerance rating (STR) and leaf chlorophyll content (SPAD value) after treatment with 180 mM NaHCO₃ for about 3 weeks under greenhouse conditions. In both populations, a significant QTL for alkaline salt tolerance was detected on the molecular linkage group D2 (chromosome 17), which accounted for 50.2 and 13.0% of the total variation for STR in the F₆ and the F₂ populations, respectively. The wild soybean contributed to the tolerance allele in the progenies. Our results suggest that QTL for alkaline salt tolerance is different from the QTL for NaCl salt tolerance found previously in this wild soybean genotype. The DNA markers closely associated with the QTLs might be useful for marker-assisted selection to pyramid tolerance genes in soybean for both alkaline and saline stresses.     

Effect of Salt Stress on Growth,Na+ Accumulation and Proline Metabolism in Potato (Solanum tuberosum) Cultivars

Potato (Solanum tuberosum) is a major crop world-wide and the productivity of currently used cultivars is strongly reduced at high soil salt levels. We compared the response of six potato cultivars to increased root NaCl concentrations. Cuttings were grown hydroponically and treated with 0 mM, 60 mM and 180 mM NaCl for one week. Growth reduction on salt was strongest for the cultivars Mozart and Mona Lisa with a severe senescence response at 180 mM NaCl and Mozart barely survived the treatment. The cultivars Desiree and Russett Burbank were more tolerant showing no senescence after salt treatment. A clear difference in Na⁺ homeostasis was observed between sensitive and tolerant cultivars. The salt sensitive cultivar Mozart combined low Na⁺ levels in root and stem with the highest leaf Na⁺ concentration of all cultivars, resulting in a high Na⁺ shoot distribution index (SDI) for Mozart as compared to Desiree. Overall, a positive correlation between salt tolerance and stem Na⁺ accumulation was found and the SDI for Na⁺ points to a role of stem Na⁺ accumulation in tolerance. In stem tissue, Mozart accumulated more H₂O₂ and less proline compared to the tolerant cultivars. Analysis of the expression of proline biosynthesis genes in Mozart and Desiree showed a clear reduction in proline dehydrogenase (PDH) expression in both cultivars and an increase in pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Desiree, but not in Mozart. Taken together, current day commercial cultivars show promising differences in salt tolerance and the results suggest that mechanisms of tolerance reside in the capacity of Na⁺ accumulation in stem tissue, resulting in reduced Na⁺ transport to the leaves.