基于φC31整合酶的高等真核生物基因组操作技术研究进展

位点特异性重组系统已经成为基因组操作的强有力工具。来自链霉菌噬菌体φC31的整合酶是丝氨酸重组酶家族的一员,可催化2个不同的识别位点attB和attP间发生有效的单向性重组。总结了φC31整合酶可能的重组机制以及在人类细胞、小鼠胚胎干细胞、果蝇和植物等真核生物基因组操作上的应用,并对该系统的优点和存在问题进行了分析。φC31整合酶系统有望成为高等生物基因功能分析、转基因标记删除、生物合成等研究领域最强有力的手段之一,为此也简要介绍了家蚕个体水平上φC31整合酶介导的盒式交换的研究进展,期望能为家蚕基因组的操作技术研究提供一些借鉴。     

A 15N-Labeling Study of the Capture of Deep Soil Nitrate from Different Plant Systems

The objective of this study was to determine the efficiency of different plant systems in capturing deep soil nitrate （NO3-） to reduce NO3- leaching in a field plot experiment using 15N labelling. The study was conducted on a calcareous alluvial soil on the North China Plains and the plant systems evaluated included alfalfa （Medicago sativa）, American black poplar （Populus nigra） and cocksfoot （Dactylis）. ^15N-labelled N fertilizer was injected to 90 cm depth to determine the recovery of ^15N by the plants. With conventional water and nutrient management, the total recovery of ^15N-labeled NO3--N was 23.4% by alfalfa after two consecutive growth years. The recovery was significantly higher than those by American black poplar （12.3%） and cocksfoot （11.4%）. The highest proportion of soil residual ~SN from the labeled fertilizer N （%Ndff） was detected around 90 cm soil depth at the time of the 1st year harvest and at 110-130 cm soil depth at time of the 2nd year harvest. Soil %Ndff in 0-80 cm depth was significantly higher in the alfalfa treatment than those in all the other treatments. The soil %Ndff below 100 cm depth was much lower in the alfalfa than those in all the other treatments. These results indicated that ^15N leaching losses in the alfalfa treatment were significantly lower than by those in the black poplar and cocksfoot treatments, due to the higher root density located in nitrate labeling zone of soil profile. In conclusion, alfalfa may be used as a plant to capture deep soil NO3- left from previous crops to reduce NO3- leaching in high intensity crop cultivation systems of North China Plain.     

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune,skin, and neuronal target cells

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.     

有机物料对平邑甜茶根系32P吸收动力学参数的影响

 研究了3种有机物料对平邑甜茶实生苗吸收根、生长根、褐色木质根磷吸收的影响。结果表明，3类根对磷的吸收均符合离子吸收动力学模型。有机物料的加入均不同程度地提高了Imax，而Km有所降低。不同处理间根系磷吸收动学力学参数有差异：在夏季各类根的Imax表现为鸡粪处理>羊粪处理>花生秧处理，秋季为羊粪处理>鸡粪处理>花生秧处理（Km值的大小顺序与之相反）。3类根的磷吸收动力学参数差异较大，吸收根其Imax是褐色木质根的7.4～8.7倍，生长根是褐色木质根的4.7～5.8倍。而褐色木质根的Km值是吸收根的1.1～1.5倍，是生长根的1.0～1.2倍。说明吸收根和生长根对磷的吸收潜力、与磷的亲和力均大于褐色木质根。夏季根系对磷的最大吸收速率与植株生物量呈极显著正相关，秋季未达到显著水平（褐色木质根除外）。     

含水量对14C标记秸秆和土壤原有有机碳矿化的影响

 【目的】研究土壤含水量与土壤有机碳矿化的关系。【方法】标准培养条件（25℃，100%空气湿度）下，应用14C示踪技术研究了6个水分梯度下（30%、45%、60%、75%、90%和105%WHC，WHC为最大田间持水量）添加物料和旱地土壤有机碳的矿化特征。【结果】在100 d的培养期内，添加物料和土壤原有有机碳的累积矿化量随含水量的提高而增加，且与含水量呈极显著正线性相关关系。在105%WHC的处理中，添加物料和土壤有机碳的累积矿化量均最大，为146 ?g·g-1和0.76 mg·g-1，是其它处理中添加物料和土壤有机碳矿化量的1.24～1.68和1.33～3.01倍。100 d内，添加物料的矿化率约20%～33%，土壤有机碳的矿化率约1.0%～3.1%。【结论】在30%～105%WHC的范围内，含水量对土壤原有有机碳矿化量的影响更明显，且渍水促进添加物料和旱地土壤有机碳的矿化。     

"红灯"甜樱桃对秋季叶施15N-尿素的吸收、分配及利用特性

在田间条件下以7年生“红灯”甜樱桃/大青叶为试材,探讨了甜樱桃不同枝类叶片对晚秋叶施^15N-尿素的吸收和翌年的分配及利用特性,研究了晚秋叶面施氮对果实品质的影响。结果表明：晚秋对甜樱桃叶面施用浓度为1.00%的^15N-尿素,施用总量的16.30%1-7.27%随落叶脱离树体,其余82.73%8-3.70%进入树体。在翌年,叶片回流贮藏的氮素化合物可迅速转移到新生器官中,不同处理间的^15N分配运转特性存在差异。长枝叶片吸收、回流贮藏的氮素化合物在翌年铃铛花期主要供应至新梢,促进营养生长;随着物候期的推移,表现出明显的极性分配特性。短枝叶片吸收、回流的氮素化合物主要运至花、果等生殖器官中,用于甜樱桃开花、结果。验证试验表明：叶施尿素处理甜樱桃果实单果重较对照增加21.84%;总糖含量、糖酸比分别提高11.98%、21.84%,同时叶施尿素促进了己醛、2-己烯醛（、E）-2-己烯-1-醇、苯甲醛4种果实重要香气成分的合成。     

尿素与复合氮肥增效剂配施对水稻氮素利用的影响

采用^15N-尿素进行盆栽和田间试验，研究了复合氮肥增效剂用量、尿素与复合氮肥增效剂配施对水稻生长、籽粒产量和氮素吸收利用的影响。结果表明，适宜用量（施氮量的20％）的复合氮肥增效剂能显著促进水稻幼苗生长发育；尿素全量配施复合氮肥增效剂不影响水稻生长，能显著提高水稻植株Ndff％、氮吸收总量，氮素利用率和^15N的吸收量,尿素减量5％～15％（即减少施氮7．8～23．7kg／hm^2）配施复合氮肥增效剂基本上不影响水稻生长、籽粒产量和吸氮总量，能显著提高氮素的农学效率、生理效率和氮素利用率；植株吸氮总量、净吸收氮量和^15N总吸收量与不施增效剂的处理相当或有所提高。尿素减量达30％以上配施复合氮肥增效剂，对水稻植株生长和氮素吸收利用产生明显不利影响。尿素与施氮量20％的复合氮肥增效剂配施，不影响大田水稻植株生长和单季产量，能提高氮素利用率，节省氮肥投入达15％。     

青蒿组方对球虫感染雏鸡血液中CD4^+和CD8^+T淋巴细胞的影响试验

为研究青蒿组方中药对鸡免疫力的影响,试验选取14日龄三黄鸡120只,平均分为4组:感染给药组(1组),感染不给药组(2组),不感染给药组(3组),不感染不给药组(4组).感染组人工接种柔嫩艾美耳球虫孢子化卵囊.接种后第4、7、10 d运用流式细胞仪测定各组鸡血液中CD4^+、CD8^+T淋巴细胞值及二者的比值.结果:1组CD4^+、CD8^+T淋巴细胞值及其比例在接种后第4、7、10 d均高于2组,差异显著(P<0.05);2组CD4^+、CD8^+T淋巴细胞平均比值低于其他3个组.结论:青蒿组方中药可促进鸡血液中CD4^+、CD8^+T淋巴细胞生成,进而增强机体的免疫功能.     

高产、优质、多抗玉米新品种豫玉31的选育与示范推广

豫玉31(漯单97-1)是河南省漯河农科所用自选系漯12作母本,外引系丹340作父本育成的竖叶大穗中早熟玉米单交种.该品种夏播生育期96d.1997～1998年河南省夏玉米区域试验平均单产7695.0kg/hm²,比对照种豫玉12增产13.5%.两年20点次试验证明,该品种表现高产、稳产、优质、多抗、适应性广、增产潜力大,适宜在黄淮海夏玉米区套种或直播种植.种植密度45000～60000株/hm².制种时父母本同期播种或父本浸种后与母本同期播种,行比1:5～6。     

miR-31-5p对山羊毛囊干细胞增殖和凋亡的影响

【目的】长江三角洲白山羊是我国及世界上唯一能生产优质笔料毛的山羊品种,课题组前期转录组测序结果表明：在优质笔料毛与非优质笔料毛个体皮肤组织中,MAP3K1的表达水平存在显著差异。探究优质笔料毛性状形成过程中与MAP3K1相互作用的关键miRNAs及其对山羊毛囊干细胞增殖和凋亡的影响,为长江三角洲白山羊的分子选育提供理论依据。【方法】通过生物信息学网站（StrBase、miRDB、TargetScan、miRWalk、DAVID、KEGG、RNAhybrid）预测、筛选与MAP3K1具有靶向关系的miRNAs,利用在线网站Venny 2.1绘制韦恩图。通过构建miR-31-5p过表达载体,MAP3K1、RASA1野生型和突变型双荧光素酶报告基因载体,验证miR-31-5p与MAP3K1、RASA1之间的靶向关系,并结合qPCR和Western Blot技术检测过表达后miR-31-5p对MAP3K1、RASA1 mRNA和蛋白表达水平的影响。为探究过表达miR-31-5p后对细胞增殖、凋亡的影响,分析了转染miR-31-5p后毛囊干细胞内增殖相关基因（PCNA, CDK1, CCND2）、抗凋亡基因（Bcl-2）和促凋亡基因（Bax）的mRNA和蛋白表达水平;同时结合CCK-8,EdU,流式细胞术等方法验证过表达miR-31-5p对毛囊干细胞活力、细胞周期以及凋亡的影响。【结果】通过数据库共同预测到3个可能与MAP3K1相作用的miRNAs,结合现有miRNAs在皮肤和毛囊细胞上研究,最终选用评分相对较高的miR-31-5p作为研究对象。转染miR-31-5p后检测细胞内的miR-31-5p的相对表达量,发现miR-31-5p表达含量极显著高于对照组及空白载体组（P<0.01）;双荧光素酶报告基因结果显示过表达miR-31-5p可促使MAP3K1活性升高（P<0.01）,结合TargetScan、KEGG数据库预测发现miR-31-5p可靶向MAPK信号通路中位于MAP3K1的上游抑制因子RASA1。过表达miR-31-5p抑制了RASA1的活性（P<0.01）;同时,qPCR及Western Blot表明：过表达miR-31-5p后显著抑制RASA1的mRNA和蛋白表达,促进了MAP3K1的表达（P<0.01）。CCK-8结果显示过表达miR-31-5p后提高了细胞增殖能力（P<0.01）,通过EdU染色发现过表达miR-31-5p后,EdU阳性细胞率显著高于空白组（P<0.01）,促进细胞增殖;细胞周期数据说明过表达miR-31-5p后, G1/G0期细胞所占比例为52.23%,显著低于Control组（56.81%,P<0.01）,减缓了G1/G0期细胞阻滞,而S期和G2/M期差异不显著,但仍有上升趋势。通过细胞凋亡试验发现过表达miR-31-5p组活细胞率为93.8%,总凋亡率为4.9%,而空白组活细胞率仅为90.1%,总凋亡率为8.41%,说明在过表达miR-31-5p后细胞凋亡率明显下降（P<0.05）;最后检测miR-31-5p对增殖和凋亡相关基因的影响,发现过表达miR-31-5p后显著提高了增殖相关基因、抗凋亡基因（Bcl-2）的mRNA和蛋白表达水平（P<0.05）,降低了促凋亡基因（Bax）的mRNA和蛋白表达水平。最终根据所研究出的结果绘制miR-31-5p在毛囊干细胞中的分子作用机制图。【结论】 miR-31-5p通过靶向抑制MAPK信号通路中的RASA1,上调MAP3K1表达水平,进而促进毛囊干细胞增殖并抑制其凋亡,为进一步阐明调控长江三角洲白山羊优质笔料毛性状的分子形成机制提供理论依据。