花生茎全长cDNA文库的构建与分析

为挖掘花生茎中重要的功能基因,了解花生茎生长发育的分子机理,以闽花6号为材料,利用SMART技术成功构建了花生茎不同生育时期混合的全长cDNA文库,并对文库的部分序列进行了测序及分析。结果表明,该文库原始库容为1.25×106cfu·mL-1,重组率达100%,插入片段大小为750~2 000 bp,平均插入片段大小为1 000bp,达到文库质量要求。随机挑选50个单克隆进行5'端测序,共获得50条有效序列。通过与NCBI非冗余蛋白质数据库进行比对和注释,获得已知功能基因或具推测功能的基因29个,未知功能基因9个,新基因12个,其中47个(94%)基因为花生未报道的基因。因此,该文库的构建为克隆和研究花生茎部重要表达基因,从分子水平上揭示花生茎的生长发育规律提供了基础。     

非生物胁迫诱导的棉花酵母双杂交文库构建及GhJAZ1互作蛋白筛选

非生物胁迫导致棉花生长发育受到抑制,严重影响棉花的产量和品质。为了深入探究棉花非生物逆境响应的分子作用机制并鉴定GhJAZ1的互作蛋白,本研究以陆地棉标准系TM-1为材料,采用Gateway重组技术构建了非生物胁迫诱导的陆地棉酵母双杂交cDNA文库,获得的初级文库总克隆数为1.2×10⁷ CFU,次级文库总克隆数为1.6×10⁷ CFU,重组阳性率100%,酵母文库滴度为5.0×10⁷ cells·mL^-1,酵母克隆平均插入片段>1 000 bp,符合建库标准,可用于后续酵母双杂交筛选。以GhJAZ1为诱饵,构建pGBKT7-GhJAZ1诱饵蛋白表达载体,经毒性检测及自激活活性检测,明确了诱饵质粒在酵母系统中无毒性和自激活活性,可直接用于酵母文库筛选。利用酵母双杂交筛选构建的非生物胁迫诱导的酵母cDNA文库,得到72个初筛阳性克隆,经测序、序列比对和酵母回转验证,获得11个与GhJAZ1相互作用的候选蛋白。选取其中4个候选蛋白,利用酵母双杂交进一步确认了GhJAZ1与候选蛋白的相互作用关系。本...     

花生AhMKK4基因的克隆、表达分析和亚细胞定位研究

为挖掘花生抗逆相关基因,本研究以花生品种花育20号为试验材料,根据cDNA文库中已知的促丝裂原活化蛋白激酶激酶(MKK)基因EST序列设计引物,通过RACE-PCR克隆得到AhMKK4基因。结果表明,AhMKK4基因序列全长1 434 bp,含有3'非编码区151 bp,5'非编码区317 bp,开放阅读框全长966 bp,编码一条含有322个氨基酸的蛋白序列。预测其分子量为36.74 kDa,属于MAPKK基因家族D组成员。亚细胞定位显示AhMKK4基因定位于细胞质和细胞核中。RT-qPCR分析发现,AhMKK4基因在根中表达量高于其他组织,说明该基因具有组织表达特异性;AhMKK4基因受JA和IAA诱导时表达量上调,受SA和ABA诱导时表达量下调,说明该基因可能参与到JA和IAA介导的信号转导途径;AhMKK4在盐胁迫下表达量上调,说明该基因可能参与花生对盐胁迫的适应性调控。本研究结果为花生抗逆育种研究提供了新的基因资源。     

盐生植物海马齿盐胁迫全长cDNA文库的构建与分析

为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下：采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN＋TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。     

蜡梅非特异性脂转移蛋白基因nsLTP家族3个成员的分子特征及非生物胁迫应答分析

为了探索脂转移蛋白在蜡梅(Chimonanthus praecox)应对非生物胁迫过程中的可能功能,在构建蜡梅花cDNA文库及EST分析的基础上,通过对文库cDNA克隆全长测序,得到了3个蜡梅非特异性脂转移蛋白的基因(nsLTPs),分别命名为CpLTPI.1、CpLTPI.2和CpLTPI.3,GenBank登录号为FJ889521、FJ904082和FJ904083.它们的cDNA全长分别为611,1016和656 bp,ORF分别为360,360和351 bp,5'和3'端的非翻译区的长度不等,存在的调控基序有所不同.3个基因的编码蛋白均具有植物nsLTP的典型结构,即4对二硫键,4个α-螺旋,有1个可结合和容纳脂肪酸分子的类似口袋状的疏水结构,分子量约为9kD,为nsLTPI类.定量RT-PCR分析和比较蜡梅叶片中3个基因对非生物胁迫的应答情况,结果表明,3个nsLTP基因在蜡梅幼苗中的表达受干旱、ABA、低温和NaCl处理的影响程度不同,表明这些基因可能在蜡梅幼苗的水分平衡、耐受低温、离子代谢等过程中发挥着不同的作用.     

镉诱导的蚯蚓全长cDNA文库的构建与初步鉴定

采用人工土壤法，研究重金属镉(Cd2+)对成熟赤子爱胜蚓（Eisenia fetida）的急性毒性,表明蚯蚓对镉具有较强的耐受性。Trizol法提取蚯蚓RNA后，采用Clontech公司的CreatorTM SMARTTM cDNA文库构建试剂盒，成功构建了低剂量Cd2+（200 mg•kg-1）诱导的蚯蚓cDNA文库，其库容量为3.02×105 pfu/mL,扩增后滴度为8.67×109 pfu/mL，重组率为97.3％, 绝大多数的cDNA插入片段≥0•5 kb,平均长度≈1•0 kb。所够建的cDNA质粒文库容量及插入片段大小符合合格文库要求, 为进一步筛选蚯蚓体内解毒、免疫相关基因，寻找生物标志物奠定了基础。     

苦瓜Ⅲ型过氧化物酶Prxs基因及其启动子的克隆与表达分析

过氧化还原蛋白(Prxs)是广泛存在于植物体内重要的抗氧化酶。为深入研究Prx基因在苦瓜非生物胁迫中的作用,从苦瓜叶片均一化文库中获得McPrx基因的cDNA全长序列,并命名为McPrx(KJ722768.1),该cDNA序列全长1 068 bp,开放阅读框972 bp,编码324个氨基酸,属于ClassⅢ基因家族成员。采用基因组步移法分离获得McPrx基因5'上游1 237 bp的启动子调控序列,运用Plant CARE对其进行顺式作用元件分析,结果显示该序列除含有CAAT-box、TATA-box等核心启动子元件,还具有激素、抗病与抗逆应答元件,表明McPrx基因的表达可能受多种外界环境条件的调控。qRT-PCR分析表明,McPrx在根、茎、雌花等器官中的表达量存在极显著差异。其中在茎中表达量最大,在雌花中表达量最低,说明该基因的表达具有器官表达特异性;低温胁迫1 h时,McPrx表达量显著上调,胁迫3 h时McPrx表达量达到最大,随后下降,说明McPrx响应了低温胁迫的应答。本研究结果为进一步研究McPrx的生物学功能及其应用奠定了基础。     

甘蔗锌指蛋白基因的克隆和表达分析

在对甘蔗(Saccharum officinarum)叶片全长cDNA文库测序的基础上,获得1个编码锌指蛋白基因的全长cDNA序列,命名为Sc-zf.生物信息学分析表明,该基因全长1 189 bp,编码171个氨基酸,具有zf-A20和zf-AN1两个锌指结构域;构建了包含Sc-zf开放读码框的原核表达载体,经IPTG诱导,目的基因原核表达产物大小约为18.3 kD;经定量PCR技术分析,该基因在黑穗病菌(Ustilago scitaminea)、水杨酸(SA)、H2O2、NaCl和PEG胁迫下表达特性不同,受到黑穗病菌和NaCl的诱导,PEG的抑制,但也受SA和H2O2的影响.     

基于NIR-SVM对鸭梨褐变病果的识别

本文针对水果内部品质评定分类存在主观性强和一致性差的特点,提出了一种新的鸭梨褐变识别分类方法,该方法是利用近红外光谱仪获取正常鸭梨和褐变鸭梨近红外光谱并对其进行分析的基础上,应用支持向量机(SVM)算法的识别原理建立正常鸭梨和褐变鸭梨的分类识别模型。在多项式核函数下对试验样品的识别准确率为95%。研究结果表明NIR-SVM用于对于鸭梨褐变病果的无损检测识别是可行的。     

Biological response of rats fed with tofu treated with high hydrostatic pressure

Emerging technologies for food preservation have arisen in recent years, such as high-pressure (HP) hydrostatic treatment, and the biological response for this kind of food preservation is not well-known. Forty female rats (six weeks old) were used in the experiment to evaluate the biological effects of HP treatment of tofu. The animals were divided into groups that were fed with tofu (untreated), tofu treated with HP, and conventional food (as control) for 28 days. The glucose level, mineral content (calcium, potassium, zinc, and magnesium), shinbone maximum shear force, weight of the body, and weight of organs (heart, liver, spleen, and kidneys) were analyzed. The biological response for the rats was that significant differences were found in the calcium amount determined on the serum of the rats fed with untreated tofu and those fed with tofu treated with HP, and the calcium amount was lower on the rats fed with tofu treated with HP. Also, there were significant differences in the weight of the liver, and it was lower in the rats fed with tofu treated with HP. It was quite remarkable how the weight of the body and organs were smaller in the rats fed with tofu in comparison to the weight of the control rats. In the other components assayed no significant differences were found. HP produces a potential effect on tofu as it is observed in the rats response to the tofu treated with HP.     

Interaction of iron chelates with several soil materials and with a soil standard

Frequently the effectiveness of iron (Fe) chelates is low because they can be retained or destroyed by soil materials. The high cost of these Fe fertilizers makes it necessary to study soil material reaction with Fe chelates. Commercial Fe chelates with EDTA, EDDHA, and EDDHMA as ligands and their standards, prepared in the laboratory, were shaken for one hour with various soil materials [amorphous Fe(III) oxide, acid peat, calcium (Ca)‐montmorillonite and calcium carbonate (CaCO₃)] and with a soil standard made in the laboratory. After agitation, the chelate‐soil mixtures were filtered and the micronutrients and chelated Fe that remained in solution were determined. Among the soil materials used, amorphous Fe(III) oxide and acid peat had the greatest affect on the amount of chelated Fe remaining in solution. The type of chelating agent was the next major factor that affected the availability of soluble Fe following reaction with the soil materials. Another factor was the commercial formulation of the Fe chelates. The chelates comprised of EDDHA or EDDHMA maintained the highest percentages of chelated Fe in solution after interaction with the solid phases, except for the acid peat. The last soil material, acid peat, retained more chelated Fe for the Fe chelates with EDDHA or EDDHMA than with EDTA as the chelating agent. The commercial Fe‐EDDHA chelates had greater losses of chelated Fe than their standard after interaction with all the solid phases. The commercial Fe‐EDDHA chelate (Sequestrene) and the commercial Fe‐EDDHMA chelate (Hampirón) solubilized the highest amount of copper (Cu) from soil standard. This was attributed to the presence of by‐products in the commercial formulations since the Fe‐EDDHA standard did not have Cu in solution after the interaction. Therefore, the commercial Fe chelate by‐products are able to form Cu‐complexes which could affect chelated Fe and its availability to plants.     

Microflora of turfgrass treated with fungicides

Fungicides are used widely to manage high-quality turfgrass, but their use could adversely affect the composition of microbial populations. Fourteen fungicides, a nematicide, and five mixed fungicide programs were applied to turfgrass for 3 yr and were tested for their effects on numbers of bacteria, actinomycetes and fungi, and N concentrations. Combinations of fungicides suppressed fungi and stimulated bacteria and actinomycetes more than individual toxicants. Collective microbial groups were generally less affected by each fungicide than were individual species within each group. The rate of decrease of NH⁺₄ concentrations in fertilized turf (presumably via nitrification) was only slightly influenced by fungicides. The more harmful effects of some fungicides to turfgrass may involve induced acidity rather than direct suppression of specific microbial groups.     

改良B5培养基中加抗菌素对受芽孢杆菌污染葡萄试管苗的影响

该研究通过在改良B5培养基中加入不同浓度的青霉素、白霉素和先锋霉素观察和探讨了其对受芽孢杆菌(Bucillus.spp)污染葡萄试管苗生长发育的影响.结果表明培养基配制定容时,加入这三种抗菌素对芽孢杆菌的抑菌作用均达到了极显著水平.青霉素处理对促进葡萄试管苗根和茎生长效果最好,根数平均比对照增加了121.4%,株高平均增加了36%.青霉素和先锋霉素在高浓度下,对葡萄试管苗叶片产生不同程度的黄化.白霉素不同浓度处理都能降解葡萄试管苗叶片的叶绿素合成,造成试管苗叶片白化.青霉素,白霉素和先锋霉素处理的葡萄试管苗叶绿素含量分别为对照的80.88%、60.84%、79.32%.综合评价以青霉素中、低浓度处理最好.     

玉米间作花生冠层微环境变化及其与荚果产量的相关性研究

种植模式是影响花生冠层内透光率、光照度、温度、湿度等微环境的重要因素。本试验分别在2015年度和2016年度田间试验中设花生单作、玉米/花生宽幅间作2个处理,监测不同种植模式下花生结荚期后冠层透光率、光照度、冠层温、湿度的变化规律,并分析其与荚果产量的相关性。结果表明:1)与花生单作相比,玉米/花生宽幅间作显著降低了花生冠层的光照度、冠层顶部和中部的透光率及上午9:00—11:00的平均温度;增加了冠层平均湿度。2)花生冠层光照强度在晴天随时间推延而呈先升后降的单峰曲线,且单作显著高于间作;在上午光照强度上升期和下午光照强度下降期,单作和间作光照强度差值较大,而中午太阳直射期二者差值减小。间作降低了花生夜间和中午前后的冠层环境温度,二者温差最高可达4.9℃;增加了白天冠层相对湿度,二者湿度差最高达21.03%。3)本试验条件下,结荚期冠层环境温度、冠层光照度及饱果期冠层环境温度、冠层光照度均与花生荚果产量呈极显著正相关;冠层环境湿度则与荚果产量呈负相关关系,其中结荚期达到极显著水平。多元线性逐步回归分析得出,影响产量的重要环境因素为结荚期冠层光照度、结荚期冠层相对湿度、饱果期冠层相对湿度。通径分析得出,光照度除了直接影响产量外还有很大部分效应是通过影响冠层环境湿度进而影响花生荚果产量,说明间作条件下协调好光照度和冠层湿度的关系可提高光照度对产量的正面影响效应。本试验条件下,间作花生冠层光照度、透光率下降,冠层相对湿度升高,是限制花生荚果产量提高的主要气候生态因子。建议生产中间作为东西向种植,从而提高间作花生冠层上午9:00—11:00的有效光照度、适当降低冠层相对湿度,以期提高间作花生荚果产量。     

用遗传算法训练的人工神经网络识别番茄生理病害果

综合运用计算机视觉技术、遗传算法、人工神经网络技术，实现番茄生理病害果的自动识别。首先，通过计算机视觉系统获取番茄的图像，利用图像的圆度值判别空洞果，利用图像的果径变化特征判别变形果。其次，采用遗传算法训练的人工神经网络进行试验研究。试验表明，该方法能准确识别番茄的形状，满足分级的要求，对番茄生理病害果的识别准确率可以达到100%。