基因工程菌表达率对细菌总蛋白和包涵体中目的蛋白含量的影响

测定了猪生长激素基因工程菌不同表达了效率时细菌总蛋白的含量以及包涵体中重组猪生长激素占包涵体总蛋白的比值。结果表明：随着表达效率的提高，细菌总蛋白含量缓慢增加，最后稳定在一定水平上，当表达效率增加，包涵体中重组猪生长激素占包涵体中总蛋白的百分数也增加，两者上升超势基本一致。     

大肠杆菌BL21(DE3)中表达重组蛋白的研究

为了研究在大肠杆菌BL21 (DE3)中重组蛋白的表达特点,探讨重组蛋白在大肠杆菌中表达时可溶性蛋白与包涵体出现的特点,从表达载体中表达了植物ACC合成酶.经SDS-PAGE电泳与双波长扫描分析,确定目的蛋白随表达时间、菌液密度的增加而增加.但是其最佳表达时间为2.5 h,菌液密度OD600为0.6,IPTG的最佳浓度为0.6 mmol/L,此时目的蛋白含量占全菌蛋白含量之比最高.植物ACC合成酶在大肠杆菌中表达时以包涵体的形式存在,未能检测到可溶性蛋白的表达.     

大肠杆菌BL21(DE3)中表达重组蛋白的研究

为了研究在大肠杆菌BL2 1(DE3)中重组蛋白的表达特点 ,探讨重组蛋白在大肠杆菌中表达时可溶性蛋白与包涵体出现的特点 ,从表达载体中表达了植物ACC合成酶 .经SDS PAGE电泳与双波长扫描分析 ,确定目的蛋白随表达时间、菌液密度的增加而增加 .但是其最佳表达时间为 2 5h ,菌液密度OD6 0 0 为 0 6 ,IPTG的最佳浓度为 0 6mmol L ,此时目的蛋白含量占全菌蛋白含量之比最高 .植物ACC合成酶在大肠杆菌中表达时以包涵体的形式存在 ,未能检测到可溶性蛋白的表达     

重组苏芸金杆菌杀虫晶胞蛋白的高效表达

重组质粒pSC66d(Cry IAb601)转化大肠杆菌DH5a后,接种TCB培养基,经SDS-PAGE电泳和电镜分析,晶胞蛋白基因在大肠杆菌细胞中得到高效表达,表达产物分子量约130KD,在细菌细胞中形成卵圆形包涵体.比较了重组菌株在不同培养基中的生长及晶胞蛋白的表达水平,结果说明重组菌株在LB及2×YT培养基中的生长及表达均较低,在TCB培养基中细菌生长良好,晶胞蛋白高效表达,表达量可达细菌总蛋白的63.4%,较出发菌株GH9601增加17.8倍.测定了纯化的晶胞蛋白对4种鳞翅目昆虫的杀虫活性.     

重组鲤鱼生长激素的试验制备与纯化

采用高效表达鲤鱼生长激素的大肠杆菌基因工程菌株E .coli(pBVcGH8)进行发酵培养 ,经温度诱导可产生一分子量约为 3.6 3× 10 -2 0 g的特异表达产物 ,该表达产物主要以包涵体的形式存在 ,表达量占菌体总蛋白的 2 9.2 %。表达产物经包涵体收集、洗涤等初步纯化后 ,纯度可达 85 .0 %。再经变性与复性后 ,用阴离子交换层析进一步分离纯化 ,最终可获得纯度为 96 .0 %的重组鲤鱼生长激素。用该纯化过的重组鲤鱼生长激素注射罗非鱼 ,有明显的促生长作用     

猪轮状病毒VP7蛋白在E.coli中的表达纯化

为制备猪轮状病毒VP7蛋白的多抗,构建大肠杆菌表达载体pQE-30-VP7,表达猪轮状病毒VP7蛋白,纯化重组蛋白VP7并对其进行鉴定。利用RT-PCR方法扩增出猪轮状病毒VP7全长基因1062 bp,克隆到pMD18-T载体中,利用SalⅠ和HindⅢ消化重组DNA进行酶切鉴定,定向克隆到表达载体pQE-30中,转化E.coli BL21(DE3)株,经PCR和酶切鉴定阳性质粒,经IPTG(终浓度为1 mmo L·L-1)37℃进行诱导,Western blot和间接免疫荧光检测目的蛋白的反应原性。酶切和序列表明成功构建原核表达系统,重组大肠杆菌经IPTG诱导后,我们发现该重组蛋白主要以包涵体形式存在于细菌中,大小约为43 KDa,Western blot和间接免疫荧光分析表明该VP7重组蛋白可与Po RV阳性血清发生了特异性反应。     

猪白细胞介素18成熟蛋白基因克隆及在不同原核表达系统中的表达

RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18成熟蛋白基因的cDNA,克隆到pGEM-T载体,构建重组质粒pGEM-T pIL18,转化E.coli JM109感受态细胞,取PCR和酶切鉴定为阳性的重组质粒进行序列测定.测序结果表明,pIL-18成熟蛋白基因核苷酸长度为474 bp,编码157个氨基酸.将其分别克隆到表达载体pQE30、pET-28a、pGEX6P-1中,构建重组表达质粒pQE-mpIL18、pET-mpIL18、pGEX-mpIL18.用IPTG诱导表达,重组菌菌体裂解物SDS-PAGE可检测到相对分子质量分别为19 000、21 000、45 000的重组蛋白.经薄层扫描分析,pQE-mpIL18、pET-mpIL18、pGEX-mpIL18菌体裂解物分别占菌体总蛋白的17%、28%、27%,均以包涵体形式存在.     

猪血管紧张素转化酶2重组蛋白诱导表达和纯化条件的优化

[目的]本文旨在探索猪血管紧张素转化酶2(ACE2)在大肠杆菌中最佳表达条件和纯化蛋白的最佳方法。[方法]将已构建好的猪重组表达载体p ET-32a-ACE2转染至大肠杆菌BL21(DE3),通过改变IPTG诱导浓度以及诱导时间来对诱导表达条件进行筛选,实现目的蛋白高效表达后选用Ni~(2+)-NTA亲和层析和KCl染色切胶2种方法,对获得的重组猪p ET-32aACE2融合蛋白进行纯化,SDS-PAGE和免疫印迹等分析鉴定表达产物。[结果]当IPTG浓度为1 mmol·m L-1、诱导时间为10 h时,ACE2蛋白在BL21(DE3)中表达量最高,以包涵体形式存在。表达产物的相对分子质量约为100×103,与预期目的蛋白大小相符。选用的Ni~(2+)-NTA亲和层析和KCl染色切胶2种方法纯化重组p ET-32a-ACE2融合蛋白包涵体,均能得到可溶性的重组蛋白,但KCl染色切胶法纯化获得的可溶性重组蛋白浓度及纯度都更佳,纯化蛋白经Western blot鉴定具有良好的抗原性。[结论]本试验建立猪p ET-32a-ACE2重组蛋白诱导表达的最佳条件:IPTG浓度1 mmol·m L-1,诱导时间为10 h。并且以KCl染色切胶法纯化该重组p ET-32a-ACE2融合蛋白包涵体效果更好。     

猪肌生成抑制素基因成熟蛋白编码序列的表达与纯化

 猪肌生成抑制素基因myostatin (MSTN)的cDNA去除信号肽后对成熟蛋白编码序列PCR扩增出1.2 kb片段,将该片段与pMD18-T载体连接,转化JM109受体菌细胞,筛选阳性克隆测序分析,结果表明与设计序列完全一致。将该克隆载体的质粒DNA用带有BamH I和Sal I内切酶识别序列的另一对引物进行PCR扩增,将回收的1.2 kb PCR目的片段定向克隆到pET28a（+）表达载体上,成功地构建了猪肌生成抑制素成熟蛋白编码的原核表达载体。对成功构建表达载体阳性克隆在LB液体培养基中用IPTG诱导表达, SDS-PAGE凝胶电泳显示,重组菌表达的MSTN蛋白是以包涵体的形式表达的;SDS-PAGE凝胶经薄层扫描仪扫描分析,表达的MSTN包涵体蛋白占菌体不溶性蛋白含量的27.9%,表达的MSTN分子量为41 451.3D. 因为所构建的表达载体中含六聚组氨酸标签,则用His-trap亲和柱进行纯化后,纯度可达92.5%. 该试验为获得较好的猪肌生成抑制素基因抗原、制备抗体打下了良好的基础。     

猪肌生成抑制素基因成熟蛋白编码序列的表达与纯化

猪肌生成抑制素基因myostatin(MSTN)的cDNA在去除信号肽后,对成熟蛋白编码序列PCR扩增出1.2kb片段,将该片段与pMD18-T载体连接,转化JM109受体菌细胞,筛选阳性克隆,并测序分析,结果表明其与设计序列完全一致。将该克隆载体的质粒DNA用带有BamH 和Sal 内切酶识别序列的另1对引物进行PCR扩增,将回收的1.2kbPCR目的片段定向克隆到pET28a(+)表达载体上,成功地构建了猪肌生成抑制素成熟蛋白编码的原核表达载体。对成功构建的表达载体阳性克隆在LB液体培养基中用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,SDS-PAGE凝胶电泳显示,重组菌表达的MSTN蛋白是以包涵体的形式表达的;SDS-PAGE凝胶经薄层扫描仪扫描分析,表达的MSTN包涵体蛋白占菌体不溶性蛋白含量的27.9%,表达的MSTN分子质量为41.4513ku。因为所构建的表达载体中含六聚组氨酸标签,用His-trap亲和柱进行纯化后,纯度可达92.5%。     

Strawberry vein banding virus P6 protein intracellular transport and an important domain identification

Strawberry vein banding virus(SVBV)-infected strawberry cells contain cytoplasmic inclusions with isometric particles.To identify the components of the inclusions,green fluorescent protein(GFP)was fused to the carboxy-terminus(C-terminus)of SVBV open reading frames,these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves.Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies(IBs).To investigate P6 subcellular localization,P6-GFP was ectopically expressed in N.benthamiana leaves by agroinfiltration and then stained with 4′,6-diamidino-2-phenylindole(DAPI).We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes.To further determine the location of P6 IBs in the cytoplasm,and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum(ER),the microfilament marker protein(GFP-ABD2-GFP),microtubules marker protein(m Cherry-MAP65-1)and ER marker protein(m Cherry-HDEL)were separately coexpressed with P6-GFP and into N.benthamiana leaves by agroinfiltration,exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum.Meanwhile,coinfiltration of P1 and P6 indicated the P6colocalized with the P1 protein at periphery of cells.The P6 protein contains one C-terminal nuclear localization signal(NLS)region,a P6 protein mutant with a deleted NLS did not localize in the nucleus,did not form IBs,and was unable to facilitate exogenous GFP expression.These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions.In summary,the mobile P6 IBs are associated with ER,microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.     

Effect of Fertilization on Tomato Bacterial Wilt Biocontrolling and Soil Health Restoration Using FAME Analysis

The effect of applying biological organic fertilizer (BOF) on bacterial wilt incidence of tomatoand soil microbial community under continuous cropping was studied. The results showed that all the tomatoeswere infected by bacterial wilt in the control. The infection rates of tomatoes in the treatments with un-com-posted BOF and decomposed BOF were 55 and 50% respectively. Fatty acid methyl esters (FAME) analysisindicated soil microbial community changed sensitively after applying BOF. Soil FAME total content, relativefungi content and ratio of fungi to bacteria were significantly increased in both BOF treatments. The soil odd-number fatty acid proportion changed after applying BOF, aC15: 0, iC17 . 0 decreased, while cyC17 . 0 in-creased in soil odd-number fatty acid proportion. BOF application would strengthen soil health and diseasesuppression. The content of C16: 111c in soil microbial community was obviously increased after decomposedBOF application. It indicated that the growth of AM fungi could be enhanced with decomposed BOF applica-tion. FAME microbial biomarkers could be used for an indicator of soil health and disease suppression. Odd-number fatty acid proportion was a sensitive indicator of the effect of applying un-composted and decomposedBOF respectively on soil health regulating.     

自然生草改变苹果园土壤根际细菌群落的组成与结构

【目的】为了研究自然生草对果园土壤微生物群落的组成、结构及时空变化特征的影响,进而对增强果园土壤肥力、改善理化性状、促进果树生长发育方面产生的积极作用。【方法】利用Miseq高通量测序技术对自然生草(TR)与清耕(CK)条件下,根际土壤细菌群落进行测定与分析。【结果】结果表明,与清耕相比,自然生草明显提高果实膨大期土壤0~20cm、20~40cm的土层的有机质、全氮、碱解氮、速效磷及速效钾含量;降低土壤pH值。自然生草比清耕明显提高土壤细菌群落的丰度、种类丰富度和多样性指数(Shannon Index)。在门级水平上,宏基因组检测样本中共有37个门,变形菌门是比较明显的优势菌群,其次为酸杆菌门、浮霉菌门、放线菌门;在属分类级别上,宏基因组检测样本中共有470类菌属,芽单胞菌属(Gemmatimonas)、鞘氨醇单胞菌属(Sphingomonas)、类似牙球菌属Blastocatella是优势菌属。自然生草明显提高Ohtaekwangia、Chryseolinea的比例,降低Gaiella、化螺菌属(Nitrospira)比例;韦恩图显示细菌群落自然生草与清耕共有470类菌属,自然生草有63类独特的菌属,清耕有72类独特的菌属。【结论】自然生草提高苹果园土壤细菌群落的多样性,改良群落的结构和组成,明显提升土壤的养分状况。     

MsxC基因在斑马鱼肌间刺和大侧肌发育中的作用

肌间刺存在于有些真骨鱼类的肌间隔中。已有研究表明，作为Hox基因家族成员，MsxC（muscle segment homeobox C）基因的表达模式与唇鱼骨肌间刺形成有一定的相关性。为了进一步探究MsxC基因在肌间刺形成中的作用，本研究利用CRISPR/Cas9基因编辑技术，建立了斑马鱼MsxC基因纯合突变体（MsxC-/-）。结果表明，与野生型斑马鱼相比，MsxC-/-斑马鱼尾部的髓弓小骨和脉弓小骨平均缩短15%以上，差异极显著，而躯干部的肌间刺长度则无明显差异；MsxC-/-斑马鱼大侧肌肌隔周围白肌细胞分布疏松，且排列混乱，越靠近尾部差异越明显；MsxC-/-突变体红肌面积增大，越靠近尾部，红肌面积增大越明显，尾部红肌面积增大2.22%（p<0.01），而躯干靠前部位面积增大0.97%（p<0.05）。以上结果表明，MsxC基因的缺失既影响了肌间刺的形成，同时也影响了大侧肌的发育。     

华南8号木薯及其四倍体块根淀粉及蛋白表达谱的差异分析

【目的】通过对华南8号木薯及其四倍体块根淀粉含量与结构及蛋白质组学分析,揭示染色体加倍对木薯块根淀粉含量与结构的影响及其蛋白质调控机制。【方法】植后10个月收获木薯块根,采用空、水重测定块根鲜薯的淀粉含量,分光光度法分别测定淀粉中支链淀粉与直链淀粉的比例,采用Excel 2013和DPS v7.05统计软件对数据进行分析,差异显著性标准采用新复极差法,通过扫描电镜对块根淀粉体的大小、形态及数量进行显微结构观察,利用蛋白质印迹（Western blot）技术对参与淀粉合成与降解的酶表达水平进行验证,采用双向电泳技术分离块根蛋白质,Delta 2D软件分析差异倍数在2.0以上的差异蛋白质点,并通过基质辅助激光解析电离飞行时间质谱技术（MALDI-TOF-TOF-MS/MS）鉴定差异蛋白质,结合KEGG数据库将其按照功能进行分类。【结果】木薯染色体加倍后,块根干物率、淀粉含量及单株鲜薯重均显著下降,分别比二倍体降低了12.18%、11.41%和35.34%;而淀粉中直链淀粉和支链淀粉比例无显著变化;淀粉体形态无明显差异,主要为球形,不规则球体、椭球体也有存在,大小较均一,淀粉体排列较为疏松,空间间隙较大,视野内淀粉体数量减少;蔗糖磷酸合成酶（SPS）表达水平降低,β-淀粉酶表达水平升高,而颗粒结合型淀粉合成酶I（GBSSI）表达水平无显著变化;经软件分析得到的20个表达差异显著的蛋白质点中上调表达的有2个,下调表达的有18个;经质谱技术成功鉴定到其中的19个,1个下调表达的蛋白质点16未得到匹配,17个下调表达的蛋白质其功能涉及到碳代谢及能量代谢（4个）、结构蛋白（3个）、DNA和RNA代谢（2个）、分子伴侣（2个）、HCN代谢（2个）、抗氧化与解毒（1个）、蛋白质合成（1个）及未知功能（2个）;2个上调表达蛋白质为茎特异性蛋白TSJT1。【结论】参与碳代谢及能量代谢、核酸代谢、分子伴侣等7个代谢途径相关蛋白质表达水平下调;淀粉体疏松排列,数量减少;参与淀粉代谢合成过程的SPS表达水平的下降,参与淀粉降解过程的β-淀粉酶表达水平的升高。表明四倍体块根淀粉合成能力降低,分解能力提高,从而降低了块根的淀粉含量,而直链淀粉与支链淀粉比例无显著变化。     

鸡粪菌剂对复垦土壤上油菜产量和品质的影响

以复垦土壤和油菜为试验材料,采用随机区组设计,以化肥为基肥,将鸡粪、菌剂配施。试验结果表明:鸡粪和菌剂单独施用都能提高油菜的产量、品质和油菜的养分。但总体来说,在油菜产量的增加、品质的改善和养分的提高方面,鸡粪和菌剂按一定比例配比施用,效果更显著。其中,处理11油菜产量、还原糖含量、全氮、全钾达到最高,分别比CK增加183%,76.3%,66.1%,77.1%;硝酸盐含量最低,比CK降低64%;其他各项指标也明显高于CK。     

施氮量对春玉米产量及其早衰因子的影响

辽西北地区是玉米早衰的高发地区。通过大田定位施肥试验,对春玉米生育后期穗位叶叶绿素含量、保 护酶活性、根系干重和叶片枯黄比例进行调查,结果表明,随着施氮量的增加,叶绿素含量、根系干重都呈增加的趋 势,黄叶比例逐渐下降;SOD、POD 活性在灌浆期、乳熟期和蜡熟期随玉米生育期的推进呈逐渐降低的变化趋势;施 氮量240 kg/hm2可明显提高SOD、POD 活性,叶绿素含量、根系干重都达到较高值,黄叶比例最低,有效延缓了玉米 叶片的衰老速度,籽粒产量最高达到9 284 kg/hm2,与无肥对照比较增产40.09%。     

不同有机肥对烤烟生长发育、产质量及青枯病的影响

 通过田间试验,比较研究了不同有机肥（精制有机肥和生物有机肥）与化肥配施对烤烟农艺性状、叶绿素含量、产质量及烤烟青枯病的影响。结果表明：与当地常规施肥（100%化肥）相比,精制有机肥、生物有机肥配施80%化肥,可促进烤烟生长,显著提高烤烟产质量,减轻青枯病的危害。与当地常规施肥（100%化肥）相比,在化肥减量20%的基础上,配施生物有机肥烤烟产量提高了4.5 %,产值提高了9.8%,上中等烟比例提高了8.4%,烤烟青枯病病情指数降低了47.3%;配施精制有机肥烤烟产量提高了5.3%,烤烟青枯病病情指数降低了15.8%。与配施精制有机肥处理相比,配施生物有机肥烤烟产值提高了9.5%,上中等烟比例提高了6.2%,烤烟青枯病病情指数降低了37.5%。     

Influence of different nitrogen application on flour properties,gluten properties by HPLC and end-use quality of Korean wheat

This study was performed to identify how the different levels of nitrogen application affected the variances of gluten properties and end-use qualities and the differences of variances among Korean wheat cultivars. Protein and dry gluten content, SDS sedimentation volume and water absorption of Mixolab increased as nitrogen application increased. This ratio of the increase was higher in Korean wheat cultivars for bread than in Korean wheat cultivars for noodles and cookies. The proportion of(α+β)-gliadin measured by reversed-phase high-performance liquid chromatography(RP-HPLC) increased, but the proportion of ω-and γ-gliadin decreased as the protein content increased. The Korean wheat cultivars for bread showed a high proportion of(α+β)-gliadin increase, the Korean cultivars for noodles had a high proportion of γ-gliadin decrease and the Korean wheat cultivars for cookies had a high proportion of ω-gliadin decrease. However, there was no variation of the component in the proportion of glutenin component measured by RP-HPLC, even though the protein content was increased, but all of the protein fractions measured by size exclusion(SE)-HPLC were increased. The soluble monomeric protein showed a high proportion of Korean wheat cultivars for bread by the increase of protein content. Bread loaf volume increased by the increase of protein content but there were no variances in the ratio of increase among Korean wheat cultivars. The cookie diameter decreased with the increase of protein content, and this ratio of decrease was the highest in Korean wheat cultivars for cookies. The hardness of cooked noodles also increased by the increase of protein content but there were no variations in springiness and cohesiveness. The decrease proportion of ω-gliadin affected the increase of bread loaf volume, the hardness of cooked noodles, and the decrease of cookie diameter.     

三门峡植烟土壤养分现状及动态变化分析

By analyzing soil nutrient data of tobacco fields in Sanmenxia City in 2007 and 2017,The results showed that the tobacco-growing soil in Sanmenxia tobacco-growing area was neutral and alkaline;Organic matter content is suitable to low level;The content of alkali-hydrolyzed nitrogen is higher;The content of available phosphorus is medium;The content of available potassium is higher;The average water soluble chlorine content is low;In line with high quality tobacco production standards.Compared to 2007,The proportion of soil PH value less than 7.0 slightly increased, alkaline characteristics are still prominent;The content of organic matter decreased slightly, and the proportion of soil suitable for tobacco planting decreased slightly;The proportion of the tobacco fields with high or very high fertility of alkali-hydrolyzed nitrogen increased obviously;The proportion of tobacco planting soil with low available P content was still larger;The average content of available potassium decreased slightly, but still belonged to the "rich" level;The average water soluble chlorine content was lower, and the proportion of tobacco fields with less than 30mg/kg increased by about 12%.We should pay more attention to the basic principle of "stabilizing nitrogen, improving potassium and enhancing fragrance", pay attention to the coordination of nitrogen, phosphorus and potassium ratio, do a good job of organic and inorganic combination, base fertilizer topdressing combination, centralized application combination, and vigorously promote base fertilizer strip application technology and other fertilization technologies.