orfX基因整合表达在阿维链霉菌工业育种中的应用

阿维链霉菌（Streptomyces avermitilis）基因组中含有orfX基因，通过增加orfX基因剂量可以提高阿维菌素的产量。本研究以pIJ8600为出发质粒，构建了含红霉素启动子（ermEp*）和orfX结构基因DNA片段的整合型表达载体pWJ827。通过接合转移将pWJ827导入阿维菌素的工业菌株BIB9903中，得到稳定高产的重组菌株BIB0827。高效液相（HPLC）分析结果表明，与出发菌株BIB9903相比，BIB0827发酵产物阿维菌素产量提高了1.5倍，达到3350μg/mL。传代稳定性实验证明，BIB0827传代10代后依然稳定，在工业生产上有较大的应用价值。     

透明颤菌血红蛋白基因(vgb)表达对委内瑞拉链霉菌秦岭变种生长代谢及瑞拉菌素效价的影响

委内瑞拉链霉菌秦岭变种(Streptomyces venezuelae var.qinlingensis)是瑞拉菌素(Zuelacmycin)的产生菌,为进一步探讨透明颤菌血红蛋白基因(vgb)表达对秦岭变种以及瑞拉菌素产率的影响,利用红霉素抗性基因启动子在委内瑞拉链霉菌秦岭变种中表达vgb基因,并在5 L发酵罐中研究了工程菌株与原始菌株的生长代谢特性及瑞拉菌素的效价.结果表明:在高溶氧条件下vgb基因表达并未对菌丝体生长和氨基氮的消耗产生明显的影响,但提高了对还原糖的消耗,工程菌株发酵瑞拉菌素的效价比原始菌株降低8%;在低溶氧条件下,vgb基因的表达可促进菌丝体的生长和次级代谢,并提高了还原糖的消耗,工程菌株发酵瑞拉菌素的效价比原始菌株提高45%.本研究首次利用VHb蛋白基因表达提高了委内瑞拉链霉菌秦岭变种对氧的利用率和瑞拉菌素的效价,为进一步推动瑞拉菌素工业化生产提供基础资料.     

耐热链霉菌的分子育种改造

BIB0830是一株可将泰乐菌素转化为乙酰异戊酰泰乐菌素（AIV）的耐热链霉菌工业菌株。以pIJ8600为载体分别构建了acyB1-B2基因整合型表达载体pBIB425和带红霉素突变启动子ermEp*的红霉素抗性ermE基因整合型表达载体pBIB304。通过大肠杆菌-链霉菌属间接合转移分别将pBIB425、pBIB304和pBIB308（耐热链霉菌的nsdA基因中断载体,本实验室保存）导入工业菌株BIB0830,抗性筛选分别得到接合子BIB425、BIB304和BIB308。PCR验证均为阳性克隆。摇瓶发酵结果表明：与出发菌株BIB0830相比,BIB425和BIB308泰乐菌素转化率分别提高了14%和22%,BIB304无明显影响。     

提高耐热链霉菌转化泰乐菌素能力的分子改造与育种

BIB0830是将泰乐菌素转化为乙酰异戊酰泰乐菌素(AIV)的耐热链霉菌(Streptomyces thermotolerans)工业菌株。以pIJ8600为载体分别构建了acyB1-B2基因整合型表达载体pBIB425和带红霉素突变启动子ermEp*的红霉素抗性ermE基因整合型表达载体pBIB304。通过大肠杆菌(Escherichia coli)-链霉菌属间接合转移分别将pBIB425、pBIB304和pBIB308(耐热链霉菌的nsdA基因中断载体)导入工业菌株BIB0830,抗性筛选分别得到接合子BIB425、BIB304和BIB308。经PCR验证均为阳性克隆。摇瓶发酵结果表明,与出发菌株BIB0830相比,BIB425和BIB308泰乐菌素转化率分别提高了14%和22%,而BIB304则无明显影响。     

利用PCR介导的基因置换技术构建阿维链霉菌bkdF-突变株

PCR介导的基因置换技术是一种利用λ噬菌体Red重组系统在微生物中进行基因中断的新方法。实验利用该方法快速构建基因中断载体，并成功地置换了阿维链霉菌（Streptomyces avermitilis）bkdF基因。先合成1对长度分别为58和59nt的引物，其5’端的39nt序列分别与bkdF基因两侧同源，3’端则分别与安普霉素抗性标记盒（aac（3）Ⅳ＋onT）两侧序列一致。以该引物扩增的PCR产物电转化能表达λRed重组酶且含有目标质粒的大肠杆菌（Eschcrichia．coli）菌株BW25113／pU790／pXW1224，获得了阳性重组质粒pXW1230。将该质粒中的大小为4．0kbBglⅡ目的片段插入基因置换载体pHZl35l，再接合转移至阿维链霉菌BJBM9903，筛选得到表型为Apma^RThio^S的接合子。PCR验证并对发酵产物进行HPLC和MS分析，证实该接合子为敞dkdF^-突变株。     

中科院研究人员通过hrdB反向生物工程提高工业菌株的抗生素产量

中科院微生物研究所张立新小组通过对阿维链霉菌的hrdB基因进行反向生物工程,成功地进一步提高了阿维菌素工业生产菌株中的阿维菌素产量。这一研究结果发表在《Proceedings of the National Academy of Sciences of the United States ofAmerica 》上。     

肉桂地链霉菌接合转移体系的构建及nsdA基因中断对其次级代谢的影响

以pSET152和pHL212为出发质粒，通过供体大肠杆菌ET12567（pUZ8002）和S17-1属间接合转移肉桂地链霉菌（Streptomyces cinnamonensis），首次构建并优化了肉桂地链霉菌的接合转移系统。利用PCR介导的基因置换技术快速构建了肉桂地链霉菌nsdA基因 （negative regulator of Streptomyces differentiation）中断载体，通过接合转移导入肉桂地链霉菌工业菌株BIB2005，筛选得到一株遗传稳定表型为AmR、KmS的接合子BIB309。PCR分析结果显示该接合子即为nsdA中断突变株。与出发工业菌株相比，在摇瓶水平上nsdA中断突变株BIB309莫能菌素产能提高了2.7倍。     

链霉菌Snea253菌株的变异分析及对根结线虫活性影响

为明确委内瑞拉链霉菌(Streptomyces venezuelae)自发突变对南方根结线虫活性的影响。本研究利用继代培养法选育Snea253突变株,对形态分化及相关基因进行分析,并测其代谢产物的生物活性。获得6株菌落形态差异大的自发突变菌株,其中不产色素的3株突变菌株Snea253-G、Snea253-B和Snea253-F杀线虫活性分别为61.0%、45.5%和35.0%;产色素的3株突变株Snea253-A、Snea253-H和Snea253-J杀线虫活性分别为48.7%、37.2%和37.9%。ssgA、bldC、bldD、whiB、whiE和whiI基因克隆测序结果显示各突变株基因序列没有差异,可能此6个基因没有直接参与控制Snea253菌株的自发突变。链霉菌自发突变不但使其形态发生变异,而且也影响了其代谢产物的生物活性,研究为从形态上筛选优良菌株提供了相关理论依据。     

阴沟肠杆菌B29菌株脂多糖合成基因（waaL,waaG）缺失突变株的构建及分析

脂多糖（LPS）在革兰氏阴性细菌中的功能作用在不同菌株中已有研究,但尚未有对肠杆菌属LPS结构与功能的报道。本研究试图构建能引起肥胖的阴沟肠杆菌B29菌株waaL和waaG基因的缺失突变株,以了解突变体菌株产生的LPS结构与活性与野生菌株产生的LPS的差异。根据同源重组技术原理,利用广宿主自杀性质粒构建成敲除载体pKNG101△waaL和pKNG101△waaG,转化E.coli SM10菌株,并与B29菌株进行双亲杂交,再以抗生素和蔗糖筛选条件筛选waaL和waaG基因缺失突变株;利用多重PCR、ERICPCR、PCR-DGGE、DNA测序结果均证实突变株B29△waaL和B29△waaG构建成功;最后,对野生菌株B29与两个突变株生物学特性的差异进行了比较。银染实验结果表明野生型B29菌株的脂多糖为光滑型结构,突变株的LPS结构明显缺失O抗原,B29△waaG突变株同时还缺失了外部核心部分;用鲎试剂法检测LPS的内毒素活性显示,突变株的内毒素活性与野生型相比有较大幅度下降;B29△waaL突变株的生长速率与野生型相当,而B29△waaG突变株的生长速率则相对降低。本研究成功构建两种内毒素活性下降的突变株,为下一步利用突变株研究B29菌株在肥胖发生中的机制奠定基础。     

猪传染性胸膜肺炎放线杆菌缺失毒素1激活基因C(ApxIC)同时嵌合副猪嗜血杆菌外膜蛋白P2基因(ompP2)复合突变株的构建

猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)以及副猪嗜血杆菌(Haemophilusparasuis,Hps)是引起猪发病的2种病菌。本研究以APP血清5型分离株(SW1)为基础材料,通过构建重组转移载体pBOSKΔIC-1和pBOSKΔIC/ompP2,构成卡那霉素抗性基因(Kanr)和枯草芽胞杆菌(Bacillussubtilis)的果聚蔗糖酶基因(sacB)的正负双向筛选表达盒,并筛选获得SW1株缺失毒素I的激活基因C(ApxIC),同时嵌合Hps外膜蛋白P2基因(ompP2)的复合突变株SW1ΔApxIC/ompP2。该突变株经鉴定,与SW1亲本株相比,其缺失了大小为475bp的apxIC基因,同时嵌合有大小为1107bp的Hps ompP2基因,其基本生长特性与亲本株(SW1)没有显著区别;同时在体外,连续传代10代之后缺失的apxIC基因不会发生回复突变,嵌入的ompP2基因仍能够稳定遗传。本研究成功构建了猪传染性胸膜肺炎放线杆菌缺失apxIC基因并嵌合有Hps ompP2基因复合突变株,为今后研究APP和Hps新型二联疫苗打下基础。     

Silicon Effect on Nutrient Acquisition of Peanut (Arachis hypogaea L.) Under Aluminum Stress

Aluminum (Al) toxicity represents one of the main yield-limiting factors for crops in acid soils. Silicon (Si) is known to increase tolerance in higher plants. This study was conducted to determine whether treatment with Si could improve nutrient uptake by peanut under Al stress. Peanut (Arachis hypogaea L. cv Zhonghua 4) was raised with or without Si (1.5 mM) in the growth chamber under 0 and toxic Al (0.3 mM) levels. Aluminum stress significantly decreased the root- and total-dry weight by 52.4% and 32.0%, respectively. The content of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) was significantly decreased, but that of Al increased markedly in shoots and roots after Al exposure at seedling, flower-needle, and pod-setting stage. Silicon alleviates Al toxicity in peanut plants in relation to Al distribution and allocation of tissue P, K, Ca, and Mg by favoring the partitioning of dry mass to roots.     

强度耕作对^137Cs、^210Pbex和有机质剖面分布的影响

很少有关于直接测定强度耕作侵蚀引起土壤退化过程的报道。我们的目的是确定应用137Cs和210Pbex直接定量评价耕作侵蚀对土壤有机质(SOM)坡面运移影响的可能性。我们在黄土高原陡坡耕地上进行了50次犁耕活动,并将与其相临的另一块陡坡耕地作为对照。在对照坡地不同坡位,137Cs浓度均匀分布于上部0～30cm土层,而210Pbex浓度在坡上部和坡中部随土层深度增加呈现线性递减,在坡下部呈指数函数递减。0～30cm土层中土壤有机质含量显著大于30cm深度以下土层,并在坡中部和坡下部呈现与210Pbex类似的土壤剖面分布特征。与对照坡地比较,50次犁耕活动导致坡上部、坡中部0～45cm土层的SOM含量分别降低了38%和47%,坡下部0～100cm土层中的SOM含量却提高了18%。坡上部土壤剖面中137Cs浓度的加权平均值从1.48Bq/kg降低到0.29Bq/kg,坡中部从2.53Bq/kg减少到0.33Bq/kg,坡下部从1.48Bq/kg提高到2.81Bq/kg。210Pbex浓度的剖面加权平均值在坡上部从27.71Bq/kg下降到6.15Bq/kg,在坡中部和坡下部分别从35.46Bq/kg和25.53Bq/kg降低到1.57Bq/kg和19.40Bq/kg。137Cs和210Pbex的剖面分布与SOM在p<0.001水平呈显著相关,相关系数R2值在对照坡地为0.81～0.86,在犁耕实验坡地为0.86～0.91。实验结果表明,环境放射性核素137Cs和210Pbex在黄     

Irrigation Module and Sowing Date Affect Seed Cotton Yield,Quality, Productivity Indices,and Economics of Cotton in North-western India

ABSTRACT

Delayed sowing and imprecise application of irrigation water to cotton has been the major hurdle in sustaining cotton yield in north-western India. Therefore, studies were initiated to ascertain the impact of heavy or normal level of presowing irrigation (PSI), scheduling time of first postsowing irrigation (POSI) under two sowing dates (SDs) on cotton arranged in a split block design replicated thrice. PSI_h (100 mm) recorded 23.2% higher seed cotton yield over PSI_n (70 mm) owing to better yield attributes and higher stand. First POSI at 4 weeks after sowing (WAS) resulted the highest yield (3072 kg ha^?1), while one at 6 WAS (POSI₆) recorded least due to poor population and reduced yield attributes. Water productivity under POSI₆ was lesser by 29.1%, 25.2%, and 16.2% as compared to POSI₃, POSI₄, and POSI₅, respectively. April sown crop out yielded the May sown cotton crop by 939 kg ha^?1 due to better yield attributes. Nitrogen factor productivity (NFP) among SD remained higher by 23.3% for April as compared to May sowing. PSI_h exhibited better NFP over PSI_n. POSI₆ recorded least NFP by 24.4%, 28.5%, and 16.3% as compared to POSI₃, POSI₄, and POSI₅, respectively and was indicative of relatively poor utilization of N under delayed schedules. Therefore, planting in April after heavy PSI and scheduling first POSI at 4 WAS is best strategy for sustaining cotton yield and maximizing farmer profitability.     

Trace Metal Contamination During Grinding of Plant Samples

Inductively coupled plasma-mass spectroscopic (ICP-MS) analysis of leaves from 22 cabbage crops in the Sa P? and B?c Hà districts of Láo Cai Province, North-Western Vi?t Nam, revealed unexpectedly high concentrations of chromium (Cr) and nickel (Ni). The concentrations were strongly linearly related (r² = 0.94), indicating sample contamination during grinding through a stainless-steel hammer mill. We tested this hypothesis in two ways. First, brown rice ground through the same mill was contaminated not only by Cr and Ni, but also cobalt (Co), iron (Fe) and molybdenum (Mo). Second, scanning electron microscopy/energy dispersive analysis of x-rays (SEM/EDS) of the ground samples revealed small fragments with co-located Fe, Cr and Ni, consistent with stainless steel wear fragments. Other grinders may perform differently and we suggest that quality assurance protocols for trace metal analysis of plants should include testing for grinder wear metals. Lastly, brown rice appears to be convenient for investigating contamination of plant tissues during grinding.     

Predicting the Classes and Distribution of Salt-Affected Soils in Northwest Libya

Sodicity and salinity can adversely affect soil structure and are common constraints to plant growth in arid regions. Current remote sensing techniques cannot distinguish between the various classes of salt-affected soils. Field and laboratory measurements of salt-affected soils are time-consuming and expensive. Mapping of the salt-affected soils can be used in soil conservation planning to identify regions with different degrees of limitations. There is a need to use existing field and laboratory measurements to create maps of classes of salt-affected soils. The objectives of this study are to classify salt-affected soils, use existing field data to interpolate and validate geospatial predictions of the classes of salt-affected soils using Geographic Information Systems (GIS), and create maps showing the different classes and distribution of salt-affected soils. The classification framework for salt-affected soils is based on electrical conductivity (ECe), soil pH and the sodium adsorption ratio (SAR), and provides four degrees of limitations to salt-affected soils: slight (normal soils), moderate (saline soils), severe (sodic soils), and extreme (saline-sodic soils). Spatial interpolation of the field data from northwestern Libya was verified by cross-validation, and maps of the salt-affected soils in the region were created. The majority of soils in this region of Libya are normal (slight degree of limitation). Twenty percent of the topsoil is saline-sodic (extreme degree of limitation). Land use recommendations and rehabilitation strategies can be developed from such maps of salt-affected soil classes. The methodology followed in this study can be applied to other arid regions around the world, particularly in developing countries where budgetary constraints limit detailed field and laboratory measurements of sodicity and salinity.     

模拟降雨条件下污染土壤中重金属元素径流迁移特征

探明污染土壤中重金属元素随地表径流迁移特征,对于防控重金属污染物的迁移扩散具有重要意义。通过室内模拟降雨试验,研究不同降雨强度下(55,100,120mm/h)污染土壤中Cd、Pb和Cr随地表径流迁移的特征。结果表明:径流中3种重金属元素总量在产流的前20min内逐渐降低,随后趋于稳定;产流初期(产流10min内)颗粒态重金属占径流重金属总量的比例均在80%以上;随产流时间延长,颗粒态Cd和Cr对径流重金属的贡献率快速降低,其颗粒态/溶解态的比值范围分别为47.0～0.4和12.9～0.4,而Pb则主要以颗粒态的形式存在,溶解态Pb对其随径流迁移的贡献率可忽略不计;随降雨强度增大,径流中总Cd和Pb含量显著降低,而径流中总Cr含量以及溶解态重金属含量无明显变化规律。     

外源胡敏酸对土壤有机质组分变化的影响及其与茶树铅有效性的关系

盆栽试验分析了胡敏酸输入土壤6个月后茶树根际和非根际土壤有机质SOM及其组分,包括水溶性物质WSS、富啡酸FA、胡敏酸HA以及土壤微生物生物量SMB的变化,并对不同土壤有机质组分碳含量和茶树不同组织铅含量作了相关性分析。外源胡敏酸设置3个浓度水平（0、300、600 mg kg-1）,铅设置2个浓度水平（0、300 mg kg-1）,试验共有6个处理。茶苗置于温室培养,6个月后分新叶、老叶、新茎、老茎、根收获。结果表明：根际并非总是富含有机质,未加铅处理根际SOM含量低于非根际;而加铅处理根际SOM含量高于非根际。HA加入土壤能够显著增加根际和非根际SOM和SMB的含量,但是加铅处理和不加铅处理增加幅度不同。高浓度HA显著增加了根际FA的含量,可能是HA部分裂解所致。非根际有机质各组分与茶树铅含量无显著相关关系;根际SOM、HA和茶树不同组织铅含量没有显著相关关系。根际WSS、FA与茶树嫩叶、老叶、嫩茎、老茎以及根部铅含量均存在显著或极显著正相关关系;而根际SMB与茶树各个组织铅含量存在显著负相关关系。总之,胡敏酸施入土壤改变了土壤有机质及其各个组分的含量,而根际某些土壤有机质组分的改变又显著影响了茶树不同组织铅的吸收和分布。     

Effects of humic acid on remediation of the nutritional deficiency of gerbera in hydroponic culture

Information is scant on the effect of humic acid (HA) on physiological, antioxidant and photosynthesis attributes of gerbera plants undergoing nutrient deficiency in culture solution. Gerbera plants cv. Malibu were grown in a factorial experiment based on a completely randomized design with 3 replications, using 3 different nutrient solutions [complete nutrient solution (NSc), 25% NSc (NS1), and 50% NSc (NS2)] treated with 2 levels of humic acid [0 (HA0) and 500 mg/l (HA1)].The interaction effect of HA and NS showed that HA improved the flower number in NSc, the transpiration in NS1+HA1, photosynthesis rate in NSc+HA1, stomatal conductance (gs) in NS2, mesophyll conductance of leaves in all NS levels and photosynthetic water use efficiency in NSc+HA1. The interaction effect of nutrient solution and HA on antioxidant activity was inconclusive, malondialdehyde content was the highest in NS2 and the lowest in NS1+HA1. The peroxidase activity increased in complete nutrient solution with and without HA and there were no differences among other treatments. Superoxide dismutase activity increased in NS1 and complete nutrient solution with HA and reached the highest in NSc. Humic acid was more effective in nutrient uptake, i.e., nitrogen, phosphorus, potassium, calcium, zinc, and iron (N, P, K, Ca, Zn, and Fe) in complete nutrient solution compared to NS1 and NS2. Conclusively, humic acid can compensate the nutrient deficiency stress of the culture solution in regards to protein synthesis, photosynthesis attributes regardless of the nutrient uptake of gerbera.     

华北平原灌溉农田的土壤水量平衡和水分利用效率

华北平原农业面临的主要问题是水资源短缺,地下水位持续下降。通过一维土壤水量平衡模型模拟了华北平原不同灌溉方式下农田耗水量和土壤水分深层渗漏的变化,并分析了作物的产量和水分利用效率。结果显示在正常的灌溉条件下,冬小麦季地下水的采补差额超过了200mm,某生育期一定程度的水分亏缺(返青期、拔节期或灌浆期)能明显减少冬小麦的耗水量,但没有明显减少作物的产量。因此,在一定程度上减少灌溉是可行的,但仍不能达到地下水资源的采补平衡。从长远来看,华北平原维持可持续的地下水灌溉开采,应减少冬小麦的种植面积、增加低耗水经济作物的比例。     

(A)ngstr(o)m-Prescott系数选取对参考作物蒸散量的影响-以黄河中游站点为例

利用黄河中游三个站点近50a的气象资料,研究了(A)ngstr(o)m-Prescott系数不同取值对参考作物蒸散量的影响.结果显示,当ngstrm-Prescott系数as和bs分别相差56%和93%时,引起的ET0差异月值在0.6%～26.2%,年值在173.1～197.4mm(18.2%～21.9%).ngstrm-Prescott系数不同取值对ET0的影响程度随地点而变化,即随海拔和日照时数的增加,对ET0的影响程度相应增大.因此在高海拔和高辐射地区,应对ngstrm-Prescott系数进行校正.尽管FAO56 PM推荐的(A)ngstr(o)m-Prescott系数在世界各地广泛应用,但依据本文结果尚不能确定其在中国能否直接使用.随着辐射数据的不断积累,有必要对这些系数进一步研究、验证和校正.