利用CRISPR-Cas9技术敲除MAS1基因的质粒构建与酶切方案优化的研究

CRISPR-CAS9技术是新一代基因编辑技术,可简便快捷地对哺乳动物细胞进行指定基因的敲除,实现沉默外源基因表达的目的。但传统的CRISPR-CAS9技术提供的酶切方案没有特异性,致使酶切效率不稳定,影响胶回收率以及后续实验。本实验通过改变酶切体系大小,以及酶切反应时间,继而通过胶回收率及测序检测的方法验证最佳酶切条件,最后通过荧光定量PCR和Western blotting实验检测目的基因的表达量,确认是否成功敲除目的基因。研究结果说明:BbsⅠ酶与px459质粒比值为1μL:500 ng,酶切体系为50μL,酶切时间为45 min时,酶切效果最佳、胶回收率最高,且此时目的基因在细胞内的表达量为0,确定该实验条件下目的基因被成功敲除。     

甘肃小苏干湖盐沼湿地盐地风毛菊叶形态-光合生理特征对淹水的响应

盐沼湿地植物叶片功能性状对淹水的响应分析, 有助于探究植物叶片可塑性机制与光合生理特征间的内在关联性, 对深入理解盐沼湿地植物的生境抗逆性策略具有重要意义。根据小苏干湖湖水泛滥区静水持留时间长短分别设置: 轻度淹水区(静水持留30-90天)、中度淹水区(静水持留90-150天)、重度淹水区(静水持留150-210天) 3个试验样地, 以盐地风毛菊(Saussurea salsa)为研究对象, 研究了小苏干湖盐沼湿地盐地风毛菊叶片功能性状对淹水的响应。结果表明: 随着静水持留时间的增加, 轻度淹水区盐地风毛菊形态上采用小比叶面积(SLA)的肉质化小叶模式, 光合生理上具有高实际光合效率(Y(II))和低调节性能量耗散的量子产额(Y(NPQ))的协同变异; 重度淹水区盐地风毛菊形态和光合生理上则采用与轻度淹水区完全相反的协同变异策略; 在3个样地中, SLA与Y(II)、光化学淬灭(QP)和Y(NPQ)间均呈极显著相关关系; 叶绿素a含量和叶绿素b含量与调节性能量耗散的量子产额(Y(NO))均呈显著正相关关系。小苏干湖湖水泛滥区静水时空演变格局影响下, 盐地风毛菊种群通过改变叶面积、叶厚度和SLA等叶片形态特征, 适时调整叶片Y(II)和Y(NPQ)等光合生理特征, 实现植物叶片光合碳同化产物的收支平衡, 表现出对水盐异质性环境较强的耐受性, 反映了盐沼湿地植物在极端生存环境下的叶片可塑性和抗逆性机制。     

碳排放影响下的中国省域土地利用效率差异动态变化与影响因素

土地利用效率是衡量区域经济社会系统运行质量的重要参数。在构建了考虑非期望产出的土地利用效率评价指标体系基础之上,综合运用Super SBM-undesirable DEA和多元统计等研究方法,分析了2003—2016年碳排放影响下的中国省域土地利用效率的时空特征及其影响因素。研究结果表明:中国区域土地利用效率处于中低水平,碳排放非期望产出降低了15%的土地利用效率水平;与全国发展水平空间格局一致,土地利用效率省际差异在空间上呈现出自东向西逐渐递减的分异特征;2003—2016年,土地利用效率演化呈现出"U"型演进特征,区域差异呈现收敛态势;不考虑非期望产出的土地利用效率水平呈现下降态势;非期望产出和能源消费投入过大成为土地利用效率提升的主要限制因素。经济发展水平、对外开放程度以及固定资产投资对土地利用效率的提升起到了显著的正向作用;现阶段城镇化水平的增加对土地利用效率的提升具有微弱促进作用,且仅在东部地区具有显著性。基于松弛变量冗余度和影响因素分析,针对不同区域土地利用效率低效的差异性因素,提出了相应的对策措施。     

农业化学投入强度替代效率及其影响因素

采用SE-DEA和空间基尼系数,研究了2000—2016年我国农业化学投入强度替代效率,并运用Tobit模型分析了我国及各地区替代效率的驱动因素.结果表明: 我国农业化学投入强度替代的综合效率和规模效率呈现出“N”字型趋势,纯技术效率则波动上升,替代效率的地区差异明显,东部地区更倚重替代要素的投入规模;各年份全国省域差异最高的是综合效率,其次为纯技术效率,规模效率的区域差异性最小,地区内部省域差异逐渐减小,东部地区省域差异最大;农业化学投入替代效率受农业技术水平、农户收入水平、农业生产结构、农业发展程度和工业化水平的显著影响,各地区受各因素影响差异明显.推动农业化学投入替代效率,要从提高农业化学投入强度替代要素的投入效率、调整农用工业发展结构、关注农民收入水平和修订农业化学投入强度替代的补贴政策等方面入手.     

Contaminant Removal of Domestic Wastewater by Constructed Wetlands： Effects of Plant Species

A comparative study of the efficiency of contaminant removal between five emergent plant species and between vegetated and unvegetated wetlands was conducted in small-scale （2.0 m×1.0 m×0.7 m, lengthxwidthxdepth） constructed wetlands for domestic wastewater treatment in order to evaluate the decontaminated effects of different wetland plants. There was generally a significant difference in the removal of total nitrogen （TN） and total phosphorus （TP）, but no significant difference in the removal of organic matter between vegetated and unvegetated wetlands. Wetlands planted with Canna indica Linn., Pennisetum purpureum Schum., and Phragmites communis Trin. had generally higher removal rates for TN and TP than wetlands planted with other species. Plant growth and fine root （root diameter ≤ 3 mm） biomass were related to removal efficiency. Fine root biomass rather than the mass of the entire root system played an important role in wastewater treatment. Removal efficiency varied with season and plant growth. Wetlands vegetated by P. purpureum significantly outperformed wetlands with other plants in May and June, whereas wetlands vegetated by P. communis and C. indica demonstrated higher removal efficiency from August to December. These findings suggest that abundance of fine roots is an important factor to consider in selecting for highly effective wetland plants. It also suggested that a plant community consisting of multiple plant species with different seasonal growth patterns and root characteristics may be able to enhance wetland performance.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori Strains

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkwormeggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs (P=0.1258),the percentages of G_0 transformed moths in fertile mothsand injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively).Thetransformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other twostrains,Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain),which had similar rate. Theseresults indicate that the transformation efficiency of the piggyBac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggyBac-like elements might bean important factor influencing the transformation efficiency of introduced piggyBac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.     

植物硼营养研究的重要进展与展望

硼是高等植物必需的矿质营养元素, 但是人们对硼行使生理功能及其分子机理的认识远落后于其它必需营养元素。近几年国际上植物硼营养的研究取得了一些重要的突破。首先是进一步明确了B-RG-II复合物的形成及其影响细胞壁结构和功能的分子机制, 并且发现B-RG-II的形成及其含量与陆生植物的进化密切相关。其次是在拟南芥中克隆了第一个植物硼转运子基因 BOR1, 并揭示了它的作用机理; 通过转基因实验证明了植物硼的高效吸收与水通道基因NIP5;1密切相关。进而通过大量的种质筛选, 从油菜、小麦、大麦及棉花等农作物中获得一批硼高效吸收利用的优异种质材料, 并开展了硼高效QTL定位和克隆。本文详细综述了以上几个方面的研究进展, 并对进一步的研究做了展望。     

动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展*

基因组编辑技术可以对DNA或RNA进行精准改造,极大地促进了生命科学的发展。CRISPR/Cas9系统在靶位点诱导DNA发生双链或单链损伤,细胞对损伤部位采用无供体模板的非同源末端连接(non-homologous end joining,NHEJ)或有供体模板的同源重组(homologous recombination,HR)修复。基于HR的基因组编辑策略通常被用于获得DNA的精准改造,而NHEJ在动物DNA损伤修复中起主导作用。为了提升HR效率,研究人员设计了多种方案,包括CRISPR/Cas9系统优化和DNA修复通路调控等。从DNA损伤修复途径、Cas9变体选择、sgRNA设计、供体模板设计、DNA修复途径相关蛋白功能调控、供体模板募集效率提升、细胞周期调控及编辑细胞生存效率提升等方面详细综述了相关研究成果,发现尚未开发出放之四海而皆准的HR提升策略,基于HR的基因组编辑需要针对具体案例制定个体化策略。旨在为动物基因组编辑中提升CRISPR/Cas9介导的HR效率研究提供理论参考,为动物基因功能分析、基因治疗和经济动物基因编辑育种提供帮助。     

探究新课改背景下提升高中生物教学效率的有效途径

随着高中新课改的不断深化,如何构建高效的课堂教学成为众多教育工作者研究的重点。本文以高中生物教学为例,分析指出当前高中生物课堂教学中存在的问题,并对如何提高高中生物课堂教学效率的途径进行了研究。