一株海洋来源铜绿假单胞菌Gxun-7角蛋白酶基因的克隆、表达及重组酶酶学性质

【背景】角蛋白酶是一类特异性降解角蛋白的水解酶,在动物饲料、生物肥料、医学、洗涤、制革及环境治理等方面具有重要的应用潜力。【目的】对前期从海洋环境筛选出的一株铜绿假单胞菌Gxun-7的角蛋白酶基因进行克隆、表达,并探究重组酶酶学性质,为角蛋白酶在工业生产中的应用奠定基础。【方法】以铜绿假单胞菌Gxun-7基因组推定的角蛋白酶基因为基础,设计引物克隆获得角蛋白酶基因kp2,构建重组表达质粒pET22b-kp2,并转化到E. coliRosettagamiB (DE3)中进行诱导表达,同时对重组表达菌株的表达条件进行优化。利用镍柱分离纯化重组角蛋白酶并研究其酶学性质。【结果】重组角蛋白酶的分子量约为33 kDa,最适温度和pH值分别为40 ℃和8.0,在温度30-60 ℃和pH 6.5-8.0具有较好的稳定性。金属离子Co²⁺、Cu²⁺和化学试剂十二烷基磺酸钠(sodium dodecyl sulfonate,SDS)、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)对酶活力有抑制作用,而Mg²⁺、K⁺、巯基乙醇和二硫苏糖醇(dithiothreitol,DTT)对酶活力有促进作用。重组角蛋白酶具有良好的耐盐性,在12.5%的NaCl作用下相对酶活为87.55%。以酪蛋白为底物时,酶的K_m值为60.92 mg/mL、V_max值为9.70 U/mL。【结论】海洋来源铜绿假单胞菌Gxun-7的重组角蛋白酶具有良好的温度、碱、盐稳定性,可应用于工业生产中。     

三峡水库消落带植被高程梯度分异及其对生境胁迫的响应

三峡水库蓄水运行形成的极端生境胁迫深刻改变了消落带植被结构和功能。在消落带横向断面高程梯度上,植被生境具有典型的空间异质性。选取三峡水库典型自然恢复消落带,通过野外调查和室内分析,揭示了消落带自然演替植被的群落构成、物种多样性和生物量随高程梯度的分异特征,系统分析了极端淹水、侵蚀-沉积、土壤环境等生境胁迫类型对消落带植被高程梯度分异的影响。结果表明：①研究消落带适生植被以草本为主,共有15科25种23属,其中禾本科种类较多,但单属单种、单优群落现象明显;一年生和多年生草本分别占52%和48%,且一年生草本多在消落带上部定居,多年生草本主要在消落带下部聚集。②物种多样性指数与高程呈正相关趋势,在145-150m范围内较低,在160-170m范围内较高。③植被生物量为199.68-1211.2g/m²,总体呈现随高程增加而显著增加的趋势;受多种生境因子的复合胁迫影响,生物量随高程存在局部波动。④水库水位变动形成淹水时长、出露时令、淹水强度等是影响消落带植被生物量高程梯度分异的主导因子;侵蚀/沉积过程改变土层厚度、根层土壤持水能力和肥力条件,对植被生长产生重要影响;土壤水分和氮是植被生长的限制性因子。因此,优势生境适宜性物种选育、土壤基质保育和植被格局功能优化是三峡水库消落带植被恢复和生态功能重建的重要任务。     

黄土丘陵区坡面林-草边界土壤水分特征

研究了黄土高原丘陵区林-草边界上旱季和雨季土壤水分的空间分布变化特征.结果表明,旱季林地土壤水分的变异系数都小于草地,林地内各个层次土壤含水量差异不大,草地内各个层次土壤含水量差别较大;雨季林地土壤水分的变异系数都大于草地,林地内各个层次土壤含水量差别较大,而草地内各个层次土壤含水量差异不显著;林-草边界各个层次土壤水分的变异程度为弱变异或中等变异.林-草边界在旱季和雨季具有不同的影响域,旱季边界影响域为从林外0.4倍树高距离到林内0.4倍树高距离;雨季边界影响域为从林外0.4倍树高距离到林内0.8倍树高距离.因此,可将林-草景观划分为3个区：草地区,即由距林缘0.4倍树高距离处向草地方向延伸;林缘区,即由林外0.4倍树高距离到林内0.4倍树高距离(旱季)或0.8倍树高距离(雨季);林地区,即由林内0.4倍树高距离(旱季)或0.8倍树高距离(雨季)处向林内方向延伸.林-草边界水平方向上3个分区的土壤水分垂直分布呈现出不同的变化规律,而且旱季的规律特征与雨季相反.     

Baseline Sensitivity of Populations of Phytophthora capsici from China to Three Carboxylic Acid Amide (CAA) Fungicides and Sequence Analysis of Cholinephosphotranferases from a CAA‐sensitive Isolate and CAA‐resistant Laboratory Mutants

During 2006–2008, 572 isolates of Phytophthora capsici were collected from seven provinces in China, and their sensitivities to three carboxylic acid amides (CAA), dimethomorph, flumorph and pyrimorph were determined. Of these isolates, 90 isolates without a history of exposure to CAA fungicides (CAAs) were used to set up the baseline sensitivity. Baseline EC₅₀ values ranged from 0.122 to 0.203 (mean ± SD, 0.154 ± 0.022) μg ml^?1 for dimethomorph, from 0.301 to 0.487 (mean ± SD, 0.373 ± 0.043) μg ml^?1 for flumorph and from 0.557 to 0.944 (mean ± SD, 0.712 ± 0.082) μg ml^?1 for pyrimorph, respectively. The other 482 isolates were tested with a single discriminatory dose and were completely inhibited at 0.5 μg ml^?1 of dimethomorph. Four CAA‐resistant mutants were generated by repeated exposure to dimethomorph in vitro. As compared to the parental wild‐type isolate, the four CAA‐resistant mutants showed similar fitness in hyphal growth, sporulation in vitro and pathogenicity in vivo. Mutants resistant to CAAs in the in vitro assay caused visible lesions on pepper stems or roots treated with the recommended dose of dimethomorph. Previous studies upon the mode of action of CAAs suggested that these fungicides maybe inhibit phospholipid biosynthesis and that the primary target could be the cholinephosphotranferase (CPT), which is referred to aminoalcoholphosphotransferases (AAPTs). We sequenced and analyzed two CPT (AAPT1 and AAPT2) genes in P. capsici. Based on the cDNA sequence, we found that the AAPT1 and AAPT2 gene span 1538 and 1459 bp and were interrupted by five and three introns, respectively. There was no difference between the parental wild‐type isolate and the four CAA‐resistant mutants in the amino acid sequences of AAPT1 and AAPT2 gene. So, it was assumed that the resistance to dimethomorph was not due to mutations in the amino acid sequence of these two possible target genes.     

Microbiological purification of L-arabitol from xylitol mother liquor

As a rare sugar alcohol, L-arabitol can be used in food and can prevent extra fat deposits in the intestinal tract. Commercially, L-arabitol is prepared from pure L-arabinose by hydrogenation, which needs a high temperature and high pressure, leading to a high production cost for Larabitol. Therefore, this study describes a novel L-arabitol production method based on biological purification from the xylitol mother liquor, a cheap and readily available raw material that contains a high concentration of Larabitol. First, a novel Bacillus megaterium strain was screened that can utilize xylitol, sorbitol, and mannitol, yet not L-arabitol. The isolated strain was inoculated into a medium containing the xylitol mother liquor under formulated culture conditions, where a high L-arabitol yield (95%) and high purity (80%) were obtained when the medium was supplemented with 50 g/l of xylitol mother liquor. Upon further purification of the fermentation broth by ion exchange and decolorization, L-arabitol was crystallized with a purity of 98.5%.     

樟子松人工固沙林天然更新特征

采用比较分析与野外调查的方法。对沙地樟子松人工林天然更新特征进行了研究,能够进行天然更新的引种地区天然更新在时间上是不连续的,与特定年份的某些降水因子密切相关;在空间上主要有3种更新方式;林隙更新,林缘更新,阔叶林下更新;天然更新幼树生长情况与人工林的生长差异不大。     

Effect of vegetation on runoff-sediment yield relationship at different spatial scales in hilly areas of the Loess Plateau, North China

Whether vegetation reduces soil loss by reducing runoff volume or rather by changing runoff-sediment yield relationship has received little attention. Base on the observed data from monitoring stations and the published data from other research, this issue is addressed at different scales in hilly areas of the Loess Plateau, North China. At the plot scale, vegetation helps reduce soil loss not only by reducing runoff volume, but also by changing the runoff-sediment yield relationship, resulting that the sediment-reduction rate is higher than the runoff-reduction rate. At the watershed scale, gully erosion and mass wasting process are dominant. Vegetation measures are insufficient to control local mass movement, implying that sediment availability remains high even after vegetation is established. It is also hard for slope vegetation to change the capacity of the sediment transport system at the watershed scale. Therefore, vegetation cannot change the runoff-sediment yield relationship at the watershed scale. This implies that vegetation reduces sediment yield only by reducing runoff volume and the sediment-reduction rate approximates the runoff-reduction rate at the watershed scale. Other slope measures for soil conservation such as terraces are considered to have the same effect on the runoff-sediment yield relationship as the vegetation. Several case studies involving different spatial scales are presented and confirm this conclusion.     

A new point mutation in the iron–sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum

Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid‐resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid‐sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild‐type strains, BR and GSM (SdhB gene in the wild‐type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron–sulfur subunit of SDH. These results suggest that resistance based on non‐conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.     

水稻恶苗病菌对苯并咪唑类杀菌剂抗药性分子机理研究初探

用真菌β-微管蛋白基因的丰余寡聚核着酸引物B1和B3,扩增了一段871bp的水稻恶苗病菌Fusariummoniliforme的β微管蛋白基因片段,进行了克隆和DNA序列测定,并根据该序列设计了Fmoniliformeβ-微管蛋白基因的特异性测序引物。经过对恶苗病菌对多菌灵具有不同抗性水平菌株的β-微管蛋白基因核着酸序的比较研究,表明Fmoniliforme的β微管蛋白的165,198,200和257位置氨基酸末发生突变,在克隆的片段内也未发现能引起氨基酸改变的核着酸突变。说明该菌对多菌灵产生抗性的分子机理与目前已知的其他真菌有所不同,有待进～步研究。