Salt tolerance conferred by over-expression of OsNHX1 gene in Poplar 84K

OsNHX1 gene (Na^＋/H^＋ antiporter gene of Oryza sativa L.) was introduced into Poplar 84K with Agrobacterium tumefaciens-mediated transformation. PCR, Southern and Northern blot analysis showed that OsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 2OO mmol/L NaCI, while the Na^＋ content in the leaves of the transgenic plants grown at 2OO mmol/L NaCl was significantly higher than that in plants grown at 0mmol/L NaCI. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.     

OsNHX1基因转化84K杨的研究

采用农杆菌介导的方法开展了水稻Na /H 逆向转运蛋白基因OsNHX1转化84K杨的研究.建立了84K杨的叶外植体高频再生系统,经诱导不定芽及诱导生根阶段卡那霉素连续筛选,获得了大量卡那霉素抗性转化植株.PCR检测和Southern杂交检测表明,OsNHX1基因已成功整合到84K杨基因组.耐盐实验表明,转基因植株能在200 mmol NaCl条件下正常生长.     

Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus： The reduced Na^＋ level is the basis of the increased salt tolerance

Soil salinity is one of the major factors reducing plant growth and productivity. The detrimental effects of salt on plants are a consequence of both a water deficit resulting in osmotic stress and the excess so- dium ions on critical biochemical processe…     

Effect of Cl- on photosynthetic bicarbonate uptake in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803

Photosynthetic inorganic carbon utilization was investigated in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803 grown in standing culture. Photosynthetic rates for the two algae reached about 10 times the theoretical CO₂ supply rate at low dissolved inorganic carbon (DIC) of 100 μmol/L, and the rates were unaffected by the addition of 20 mmol/L Na⁺, indicating that the two algae possessed Na+-independent HCO^-₃ utilization for photosynthesis under low DIC. Their photo- synthetic rates at low DIC were inhibited by higher Cl^- and the degrees of inhibition were increased with the rise of CI^- concentration, and in the presence of Diphenylamine-2-carboxylate (DPC), a reported Cl^- channel inhibitor, the rates decreased by 74%－82%, implying that putative DPC-sensitive Cl^- channels participate in Na+-independent HCO₃^- uptake for photosynthesis. The experiment of intracellular 14C-DIC accumulation for photosynthesis showed that internal DIC pools decreased by about 80% with 200 μmol/L DPC and by 64%－70% with 100 mmol/L Cl^-. The experiment of chlorophyll a fluorescence quenching showed that initial rates and extents of fluorescence quenching obviously decreased with 200 μmol/L DPC or 100 mmol/L Cl^-. The two experiments gave further evidence that putative DPC-sen- sitive Cl^- channels participate in Na+-independent HCO^-₃ uptake for photosynthesis in the two algae grown in standing culture.     

Effect of fatty acids on polyamine contents and Na+/H+ antiport activity in PM vesicles isolated from roots of barley under salt stress

With 200 mmol/L NaCl treatment on barley cultivar “Jian 4” (Hordeum vulgare L. cv. J4) seedlings for 6 d, the contents of covalently and noncovalently conjugated polyamines (PAs) and activities of H⁺-ATPase in plasma membrane (PM) vesicles isolated from the roots decreased remarkably. Moreover, the activity of Na⁺/H⁺ antiport was detected first in PM vesicles. The results showed that the decrease in the contents of membrane phospholipid, noncovalently conjugated PAs and activity of H⁺-ATPase caused by NaCl could be restored partially by application of 1 mmol/L stearie acid (C_16:0) and linoleic acid (C_18:2), and C_18:2 was more effective than C_16:0 In addition, a reduction in the contents of covalently conjugated PAs was only reversed partially in the presence of C_18:2 Furthermore, Na⁺/H⁺ antiport activity was strengthened by exogenous C_16:0 and C_18:2 and C_18:2 was more effective than C_16:0. The correlative analysis suggested that, after application of C_16:0 and C_18:2 under salt stress, there was a significant positive correlation existing among phospholipid content, noncovalently conjugated PA levels, H⁺-ATPase activities and Na⁺/H⁺ antiport activities, indicating that one of the mitigative mechanisms of exogenous fatty acids on salt injury was to improve membrane phospholipid and PA contents, leading to an enhance in membrane integrity and a change in charge status of PM vesicles, so the activity of membrane-associated enzyme H⁺-ATPase was increased and synthesis of Na⁺/H⁺ antiport protein was activated.     

Overexpression of phospholipase <Emphasis Type="Italic">D</Emphasis>α gene enhances drought and salt tolerance of <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The cDNA of AtPLDa （Arabidopsis thaliana Phospholipase Da） gene was introduced into P. tomentosa （Populus tomentosa） under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDa gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type （WT） plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants （control） were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCI treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDa expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants （control） were treated with PEG6000 and NaCI separately. The contents of chlorophylls and the activities of some anti- oxidant enzymes （superoxide dismutase, guaiacol peroxidase and catalase） as well as malondialdehyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDa gene can enhance the drought and salt tolerance in transgenic P. tomentosa plants.     

The short C-terminal hydrophilic domain of NhaH Na^＋/H^＋ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na^＋/H^＋ antiporter plays key roles in maintaining low cytoplasmic NaNa^＋ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na^＋/H^＋ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues （^395PLIKKLGMI403） existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaH△C. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coil KNabc, in which three main Na^＋/H^＋ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coil KNabc/nhaHAC decreased both Na^＋/H^＋ and Li^＋/H^＋ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na^＋, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.     

转AlNHX1基因大豆后代遗传稳定性及耐盐性分析

为了获得耐盐性有所提高的转AlNHX1基因大豆后代材料,以已获得的转AlNHX1基因的6个株系中的3个株系后代为研究对象,通过分别对这3个株系转基因大豆各后代进行PCR分子检测并结合耐盐性鉴定,以分析外源基因在转基因大豆中遗传稳定性和耐盐性.结果表明:AlNHX1基因在转基因植株的后代中遗传;选取3个株系中部分阳性植株做耐盐性检测,结果表明:转基因大豆耐盐性状均好于野生型大豆.在150mmol/L NaCl胁迫下,转基因大豆叶片中维持了相对较高的K+/Na+比值,相对含水量较野生型提高了9%,而渗透势降低了39%,表明转基因大豆具有较好的吸水和保水能力;在盐胁迫下,超氧化物歧化酶(SOD)与过氧化物酶(POD)活性较野生型大豆分别提高了45%与69%.综上,通过耐盐筛选获得的转AlNHX1基因大豆具有较强的耐盐性.     

Reduced Na+ and K+ permeability of K+ channel in plasma membrane isolated from roots of salt-tolerant mutant of wheat

The Na⁺ and K⁺ permeability of K⁺ channel in plasma membrane, isolated from roots of the salt-tolerant mutant of wheat, was lower than that of wild type in 100 mmol/L KCl and NaCl solution. The opening frequency of K⁺ channel of the mutant reduced more significantly than that of wild type in two kinds of solution mentioned above. It is assumed that the reduction of opening frequency mainly contributes to the Na⁺ and K⁺ permeability of K⁺ channel of the mutant. The electric conductance of single-channel of the mutant was similar to that of wild type and the main difference between them was exhibited as the opening frequency. Their K⁺/Ka⁺ selectivity of K⁺ channel had no significant difference. The K⁺/Na⁺ selectivity of the mutant and wild type was 3.35 and 3.18 respectively.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

不同浓度锰胁迫对向日葵幼苗叶片生理生化特性的影响

为研究锰胁迫对向日葵幼苗的影响,以向日葵幼苗为实验材料,通过沙培盆栽法研究不同Mn~(2+)浓度胁迫下向日葵生长和生理生化特性的变化。结果显示:随着Mn~(2+)浓度的增加,向日葵幼苗叶片中丙二醛(MDA)、可溶性糖(SS)以及游离脯氨酸(Pro)的含量都呈现上升趋势;而幼苗叶片叶绿素、蛋白质(Pr)含量则呈现先升高再下降的趋势。Mn~(2+)浓度为100 mmol/L时,向日葵幼苗叶片的总叶绿素、叶绿素a和叶绿素b含量达到峰值,而后随着Mn~(2+)浓度的继续增加而显著下降;Mn~(2+)浓度为200 mmol/L时,蛋白质含量最高,之后急剧下降。综上可见,低浓度Mn~(2+)对植株有显著的促进生长作用,细胞代谢加快;高浓度Mn~(2+)对植株有显著的抑制作用,可导致细胞代谢紊乱,甚至植株死亡。向日葵可以种植在被低浓度重金属污染的土壤中,一方面可以加快土壤净化,另一方面可以促进植株的生长发育。     

Assaying poly(ADP-ribose) polymerase activity in plants by polarographic method

A new method has been developed to assay poly(ADP-ribose) polymerase (PARP) activity in plant tissues through determining the content of nicotinamide (NIC) produced by enzymatic reaction by linear sweeping polarographic method. The detection limit of NIC was 0.03μmol/L, the calibration graph was linear up to 5 Mmol/L ( r = 0.999). The recoveries were approximately in the range of 92% to 98% and the relative standard deviations were less than 6.6% . Moreover, NAD+ and other interference existing in the mixture after enzymatic reaction had been removed by simple pretreatment, thus PARP assays were not interfered. A rapid, simple, sensitive and reliable nonisotopic method is reported to assay PARP activity in plant tissues . The results show that the KmNAD+ value of PARP in maize ( Zea mays L.) seedlings is 59 and the optimum pH for PARP activity is 8.5. Moreover, physiological conditions affect PARP activity in plant tissues, which has not been reported previously. When tobacco ( Nico-tiana tobacum) suspension cells were stressed by NaCI at low concentrations (100, 200 mmol/ L), the PARP activity increased significantly; when the cells were stressed at high concentrations (400, 1 000 mmol/L), it decreased to or even below the control level. PARP activity in etiolated maize seedlings was higher than that in light-grown seedlings.     

Protective effects of nitric oxide on salt stress-induced oxidative damage to wheat (Triticum aestivum L.) leaves

The changes of chlorophyll and malondialdehyde (MDA) contents, plasma membrane permeability confirmed that 0.1 and 1 mmol/L sodium nitroprusside (SNP), a donor of nitric oxide (NO) in vivo, could markedly alleviate the oxidative damage to wheat (Triticum aestivum L.) leaves induced by 150 and 300 mmol/L NaCl treatments, respectively. Further results proved that NO significantly enhanced the activities of Superoxide dismutase (SOD) and catalase (CAT), both of which separately contributed to the delay of O ₂ ⁻ and H₂O₂ accumulation in wheat leaves under salt stress. Meanwhile, the accumulation of proline was apparently accelerated. Therefore, these results suggested that NO could strongly protect wheat leaves from oxidative damage caused by salt stress.     

Impact of gamma radiation and salinity on growth and K+/Na+ balance in two populations of Medicago sativa (L.) cultivar Gabès

The perennial Medicago sativa cv. Gabès is widely grown on saline soils in Tunisian oases. Morphological and physiological analyses of two populations (Mareth and Gannouch) were conducted in order to study the effect of salinity and gamma radiation (350 Gy) interaction on two populations of this species. It has been shown that the two irradiated populations of Medicago sativa are fairly tolerant to salt at growth phase compared to the non-irradiated. Exposure to gamma irradiation (350 Gy), alone or in combination with salt stress, increased significantly (p<0.001) shoot number, stem height and chlorophyll b pigment especially for the Gannouch population, while no change occurred for the Mareth population. The presence of salt to 9g/l affected significantly the root biomass and induced a reduction of shoot development of both control and irradiated alfalfa populations. For all treatments, plants of two populations remained able to produce and to allocate dry matter to the different organs. The survey of Na⁺ / K⁺ ratio showed that the growth of the aerial organs of two non-irradiated populations was at least determined by a selectivity in favour of the K+ ions (r² = 0.97 and r² = 0.59 for Mareth and Gannouch irradiated populations, respectively). However, the rather weak correlation detected for the irradiated populations, particularly for the irradiated Gannouch, seems to be the consequence of the effect of irradiation that improved potassium availability, essential element for growth and development. Results also showed that the two irradiated populations, especially the Mareth, accumulated Na⁺ ions in its photosynthetic organs. This accumulation was associated with an improvement of foliar water content at a level of salinity arrounding 5g/l. Such a mechanism reflects probably an inclusive behaviour of the plants and a good aptitude to use the dominant ions (Na⁺) for the osmotic adjustment. However, the non-irradiated populations are unable to adjust their internal osmotic potential. Consequently, irradiated plants have probably adapted to the osmotic stress by either closing their stomata or increasing the osmotic pressure of the leaf cells.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Expression analysis ofgdcsP promoter from C3-C4 intermediate plantFlaveria anomala in transgenic rice

ThegdcsP promoter isolated from C₃-C₄ intermediate plantFlaveria anomala was fused to the β-glucuronidase (GUS) gene. The chimeric gene was inserted into the binary vector pBin19 and introduced into the rice (Oryza sativa L.) cv. 8706 byAgrobacteriummediated gene transfer. GUS activity can be detected in leaf, leaf sheath, stem and root tissues via fluorometric GUS assay. However, no GUS activity was found in mature endosperm. Histochemical localization revealed that GUS expression was exclusively restricted to vascular tissues in transgenic plants. This promoter also showed spatial-temporal expression patterns that GUS expression declined significantly with the maturity of plants. These expression patterns make thegdcsP promoter extremely valuable in the applied biotechnology that needs target gene expression restricted to vascular tissues.     

Comparative proteomics analysis of <Emphasis Type="Italic">OsNAS1</Emphasis> transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> under salt stress

Salt stress is one of the major abiotic stresses in agricultural plants worldwide. We used proteomics to analyze the differential expression of proteins in transgenic OsNAS1 and non-transformant Brassica napus treated with 20 mmol/L Na₂CO₃. Total protein from the leaves was extracted and separated through a high-resolution and highly repetitive two-dimensional electrophoresis (2-DE) technology system. Twelve protein spots were reproducibly observed to be upregulated by more than 2-fold between transgenic and non-transformant B. napus. These 12 spots were digested in-gel with trypsin and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain the peptide mass fingerprints. Protein database searching revealed that 5 of these proteins are involved in salt tolerance: dehydrogenase, glutathione S-transferase, peroxidase, 20S proteasome beta subunit, and ribulose-1,5-bisphosphate carboxylase/oxygenase. The potential functions of these identified proteins in substance and energy metabolism, stress tolerance, protein degradation, and cell defense are discussed. The salt tolerance of the transgenic rapeseed was significantly improved by the introduction of the OsNAS1 gene from Brazilian upland rice of Oryza sativa (cv. IAPAR 9).     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Comparative expression analysis of three genes from the Arabidopsis vacuolar Na^＋/H^＋ antiporter （AtNHX） family in relation to abiotic stresses

Na~ /H~ antiporters (NHX) are ubiquitous transmembrane proteins that play a key role in salt tolerance of plants. In this study, the sequence of 3 Arabidopsis NHX gene (AtNHX2―4) were compared with other AtNHX members. Putative cis-elements analysis identified elements that have been associated with stress responses. The activities of the promoters AtNHX2―4 were studied in transgenic plants carrying corresponding promoter-β-glucuronidase (GUS) fusions. The AtNHX2 promoter-GUS analysis indicated that AtNHX2 was expressed in constitutive pattern with high GUS activity in roots and leaves. AtNHX2 promoter activity was not up-regulated by NaCl or abscisic acid (ABA), in contrast to the AtNHX1 promoter which was previously studied. The AtNHX3 and AtNHX4 promoters showed tissue-specific activities. Strong GUS activity was detected in roots and vascular bundles of the stele in plants carry-ing an AtNHX4 promoter-GUS fusion, and GUS activity increased under salt stress suggesting a func-tion related to salt tolerance. Transgenic plants carrying the AtNHX3 promoter-GUS fusion showed strong GUS activity in petals, stamens and tops of siliques, suggesting a possible role of AtNHX3 in flower and seed development. Results of histochemical analysis suggested that AtNHX2―4 are involved in divergent functions and are differentially regulated under abiotic stress. The structure of AtNHX4 was predicted to include 12 transmembrane regions and a NHX domain. Overexpression of AtNHX4 in Arabidopsis transgenic lines confers greater salt tolerance than in wild type plants. These results suggest that AtNHX4 may encode a putative vacuolar NHX that plays an important role in salt tolerance.     

Multiple transgenes Populus xeuramericana ＇Guariento＇ plants obtained by biolistic bombardment

A JERF36 regulation gene, a selection marker gene （NPT-Ⅱ）, and the foreign genes levansucrase （SacB）, Vitreoscilla hemoglobin （vgb）, and Binary coleopterus insect resistance （BtCry3A＋OC-I） were co-transferred into Populus xeuramericana ＇Guariento＇ using biolistic bombardment; 25 kanamycin resistant plants were obtained, The results of PCR and Southern hybridization showed that the foreign genes had been integrated into the genome of P, xeuramericana ＇Guariento＇ and 5 genes were all transferred into 7 poplar plants, The results of a BtCry3A ELISA experiment indicated that the BtCry3A gene was expressed in the 7 transgenic poplar plants, and these plants grew well on coastal saline land,