Analysis of promoters in transgenic barley and wheat

Advances in the genetic transformation of cereals have improved the prospects of using biotechnology for plant improvement, and a toolbox of promoters with defined specificities would be a valuable resource in controlling the expression of transgenes in desired tissues for both plant improvement and molecular farming. A number of promoters have been isolated from the important cereals (wheat, barley, rice and maize), and these promoters have been tested mostly in homologous cereal systems and, to a lesser extent, in heterologous cereal systems. The use of these promoters across the important cereals would add value to the utility of each promoter. In addition, promoters with less sequence homology, but with similar specificities, will be crucial in avoiding homology-based gene silencing when expressing more than one transgene in the same tissue. We have tested wheat and barley promoters in transgenic barley and wheat to determine whether their specificity is shared across these two species. The barley bifunctional α-amylase/subtilisin inhibitor ( Isa ) promoter, specific to the pericarp in barley, failed to show any activity in wheat, whereas the wheat early-maturing ( Em ) promoter showed similar activity in wheat and barley. The wheat high-molecular-weight glutenin ( HMW-Glu ) and barley D-hordein ( D-Hor ) and B-hordein ( B-Hor ) storage protein promoters maintained endosperm-specific expression of green fluorescent protein (GFP) in wheat and barley, respectively. Using gfp , we have demonstrated that the Isa and Em promoters can be used as strong promoters to direct transgenes in specific tissues of barley and wheat grain. Differential promoter activity across cereals expands and adds value to a promoter toolbox for utility in plant biotechnology.     

Effect of Compost and Manure Soil Amendments on Nematodes and on Yields of Potato and Barley: A 7-Year Study

A 7-year study located in Prince Edward Island, Canada, examined the influence of compost and manure on crop yield and nematode populations. The compost used in this study consisted of cull waste potatoes, sawdust, and beef manure in a 3:3:1 ratio, respectively. No plant-parasitic nematodes were detected in samples collected from windrow compost piles at 5- and 30-cm depths prior to application on field plots. Low population densities of bacterial-feeding nematodes were recovered from compost windrows at the 5-cm depth. Field plots of potato (Solanum tuberosum cv. Kennebec) received compost applied at 16 metric tonnes per hectare, or beef manure applied at 12 metric tonnes per hectare. An adjacent trial with barley (Hordeum vulgare cv. Mic Mac) received only the compost treatment. In both trials the experimental design was a complete randomized block with four replicates. Data averaged over seven growing seasons indicated that population levels of root-lesion nematodes (primarily Pratylenchus penetrans) were higher in root-zone soil in potato plots treated with either compost or manure compared to the untreated control plots. The soil amendments did not affect root-knot nematode (Meloidogyne hapla) population densities in the potato plots, but clover-cyst nematodes (Heterodera trifolii) were more numerous in the root-zone soils of barley treated with compost compared to the untreated plots. Numbers of bacterial-feeding nematodes (primarily Diplogaster lheritieri) were greater in soil in potato plots treated with manure and in soil around barley roots than in untreated plots. Total yields of potato tubers averaged over seven growing seasons increased by 27% in the plots treated with either compost or manure. Grain yields of barley also were increased by 12% when compost was applied. These results indicated that organic amendments increased crop yields, but the impacts on different nematode species varied and usually increased soil population levels.     

Salinity induced differences in growth,ion distribution and partitioning in barley between the cultivar Maythorpe and its derived mutant Golden Promise

Dry matter changes and ion partitioning in two near isogenic barley cultivars Maythorpe (relatively salt sensitive) and Golden Promise (relatively salt tolerant) were studied in response to increasing salinity. Although the growth of both cultivars was significantly reduced by exposure to NaCl, the effect was greater in Maythorpe, whilst Golden Promise maintained an increased ratio of young to old leaf blade. Golden Promise maintained significantly lower Na⁺ concentrations in young expanding tissues compared with Maythorpe. Partitioning of Cl^– was evident in that both varieties maintained lower Cl^– concentrations in mesophyll than in epidermal cells. Golden Promise maintained higher K⁺/Na⁺ and Ca²⁺/Na⁺ ratios in young leaf blade and young sheath tissues than Maythorpe when exposed to salt. Differences in ion partitioning and the maintenance of higher K⁺ and Ca²⁺ to Na⁺ ratios, especially in young growing and recently expanded tissues, would appear to be important mechanisms contributing to the improved salt tolerance of Golden Promise.     

青稞中β-1,3葡聚糖酶的纯化及部分性质研究

β-1,3-葡聚糖酶[EC．3．2．1．39]存在于大多数的高等植物中,它们由一个小的基因家族编码,在植物不同的生理活动中起着重要的作用。采用NaCl抽提、硫酸铵分部沉淀和2步离子交换法和分子筛从青稞的胚芽中提纯得到了一种β-1,3-葡聚糖酶。在非变性电泳中纯化的β-1,3-葡聚糖酶用银染只有一条蛋白带,运用底物进行的活性染色时在相同位置也只显示一条酶活性带。此活性染色可以直接在电泳胶板上快速鉴定和检验β-1,3-葡聚糖酶。纯化的β-1,3-葡聚糖酶在还原及非还原条件下的SDS-PAGE变性电泳中,呈现一条分子量为32kD的主要蛋白带及2条低分子量的弱带,表明此酶无链内二硫键存在。等电聚焦分析显示其等电点为8．1。上述结果表明纯化得到的是一种碱性β-1,3-葡聚糖酶。     

转基因大麦中gfp基因的染色体位置及其表达

通过对大麦小孢子进行基因枪轰击获得4株转绿色荧光蛋白基因(gfp)的植株(A、C、D、E),以gfp基因为探针进行荧光原位杂交(FISH)研究转化植株中转基因插入位置和基因表达。4个株系在染色体7L(5HL)的不同位置都有一个插入点,而E株系在染色体5S(7HS)还有第2个插入点。所有的转基因T0代植株都是半合子并在T1、T2代发生分离。D株系GFP未表达,但FISH和PCR分析表明gfp基因已成功插入其染色体。各株系在根尖和花粉中的GFP表达水平不同：C株系在花粉表达强而在根尖表达中等;A株系在花粉中等表达而在根尖表达较淡;E株系则在根尖高表达,花粉中等表达。A和C株系在根尖和花粉的GFP分离都表现单位点特性,而E株系的根尖分离表现重叠作用(15：1)特征,但在花粉中表达GFP的频率低。PCR结果和3个分离株系的根尖表达结果一致。D和E株系的GFP表达不正常可能和加基因插入位置或基因的结构有关。     

小麦-大麦2H异代换系的鉴定

通过基因组原位杂交、重双端体测交及RFLP分析,解析了来自小麦品种"中国春"(Triticum aestivumL.cv."Chinese Spring"(CS))×大麦品种"Betzes"(Hordeum vulgare L.cv."Betzes")杂种后代15份材料的遗传组成,鉴定出6个二体异代换系;对与"中国春"重双端体DDT2A、DDT2B及DDT2D测交的F1代花粉母细胞减数分裂中期染色体构型进行观察,同时以小麦第二部分同源群短臂探针psr131进行RFLP分析,鉴定出一套遗传稳定的小麦-大麦2H二体异代换系2H(A)、2H(B)和2H(D).小麦第二部分同源群短臂探针psr131可作为追踪大麦2H染色体的RFLP标记.从代换系的生长势及其他农艺性状看,大麦2H染色体对小麦染色体2B和2D的补偿作用较好.通过考种观察到携带大麦α淀粉酶抑制蛋白基因的2H染色体导入小麦后,淀粉品质发生了改变,外观品质由原来"中国春"的半粉质转变为代换系的半角质.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Effects of barley straw (Hordeum vulgare) on freshwater and brackish phytoplankton and cyanobacteria

A short-term laboratory study was conductedto investigate the effect of barley strawin controlling several common phytoplanktonand cyanobacterial species. Following aone-month incubation of barley straw incoarsely filtered fresh Potomac River andbrackish Patuxent River waters, the growthof six autotrophic taxa was followed inculture. Barley straw slurry reduced theyield of three taxa (Ankistrodesmusfalcatus, Chlorella capsulata, Isochrysis sp.) in comparison withcultures not receiving the slurry. Although no significant changes in growthwere detected with three other taxa (Cyclotella sp., Prorocentrumminimum, freshwater Pseudanabaenasp.), some patterns indicated potentialimpacts of the barley straw. First, ahigher addition of straw to Cyclotella sp. resulted in a lower biomassaccumulation than in cultures receivinglower levels. Second, the bloom-formingdinoflagellate Prorcentrum minimumwas apparently stimulated at low barleystraw levels, perhaps suggesting conditionsassociated with the straw(metals-chelation, bacterial-producednutrients) might stimulate dinoflagellategrowth. Third, species shifts wereobserved in two of the cultures, withbarley straw favoring shifts from Isochrysis to a Cyclotella sp. –Thalassiosira sp. mixture and shiftsfrom Pseudanabaena to a Pseudanabaena – Scenedesmus mixture. These results provide new records for thesusceptibility of freshwater and brackishphytoplankton taxa to barley strawexposure, including species-specificresponses and shifts in species dominancein mixed assemblages.