小黑杨PsnHB22基因在烟草中的遗传转化研究

HD-Zip转录因子在光信号转导、非生物胁迫、叶片发育等方面发挥重要的作用,HB22转录因子是HD-ZipⅠ亚家族的成员之一。为研究PsnHB22基因的功能,从小黑杨（Populus simonii×P.nigra）cDNA中克隆PsnHB22基因并构建植物表达载体进行烟草（Nicotiana tabacum）的遗传转化,以获得该基因过量表达的转基因株系。对转基因株系进行PCR、qRT-PCR分子检测后观察表型,结果显示在营养生长时期,转基因烟草叶片窄小并且株高显著低于野生型对照。测定转基因烟草及野生型叶片的叶绿素含量,发现转基因烟草叶绿素含量显著高于野生型。由此推测PsnHB22基因在植株高生长、光合作用及叶片的形态建成等过程中起着重要的作用。     

PsnHB13与PsnHB15在小黑杨中的遗传转化与功能分析

PsnHB13与PsnHB15为小黑杨（Populus simonii×P. nigra）周期蛋白PsnCycD1;1过表达株系转录组差异基因。为探究PsnHB13与PsnHB15基因功能,使用InterPro工具对PsnHB13与PsnHB15保守结构域进行分析,通过STRING数据库对PsnHB13和PsnHB15蛋白质关联网络分析,酵母双杂交验证互作蛋白,统计转基因植株叶片长宽比、叶片和茎部干质量与鲜质量比值,并对PsnHB13过表达小黑杨进行转录组分析。结果显示PsnHB13与PsnHB15分属于2个不同亚家族,PsnHB13主要包含HDZip Ⅰ结构域,PsnHB15主要包含HDZip Ⅲ结构域。PsnHB13和PsnHB15蛋白质关联网络分析,均筛选到10条互作蛋白,且PsnHB15对家族蛋白具有较高的互作概率。酵母双杂交结果显示PsnHB15与PsnHB13互作,PsnHB13与PsnCycD1;1互作。PsnHB13与PsnHB15过表达小黑杨株系在幼苗初期叶片长宽比均明显增加。PsnHB13过表达株系茎部干质量与鲜质量比值显著增加（P<0.05）。PsnHB13...     

鸡α干扰素基因遗传转化烟草研究

利用植物生物反应器生产外源药用蛋白近年来备受关注,本研究通过农杆菌介导法,将人工合成的鸡α干扰素基因（ChIFN-α）转化烟草（Nicotiana tabctcum）无菌苗叶盘。对抗性植株进行的GUS活性鉴定,PCR和RT-PCR检测表明,ChIFN-α基因已整合到烟草基因组中并具有转录活性,ELISA检测和细胞病变（CPE）抑制试验表明转基因烟草表达的干扰素蛋白具有抗病毒活性。     

紫杉烷13α-羟基化酶基因的克隆及其转化烟草的研究

本研究利用东北红豆杉(Taxus cuspidata )cDNA文库作为模版,通过PCR技术扩增得到我国东北红豆杉紫杉烷13α-羟基化酶(Taxane 13α-hydroxylase,简称13OH)的全长cDNA,将PCR产物克隆到pGM-T载体后测序结果表明该序列长度为1458bp。同源性比较分析结果表明：其碱基序列与已经报道的东北红豆杉(Taxus cuspidata)的13OH基因的一致性为99.38%,其氨基酸序列同与已经报道的东北红豆杉的13OH氨基酸序列的一致性为99.18%。将获得的cDNA全长序列正向插入到含有GUS报告基因的pCambia1305.1后成功地构建出东北红豆杉13OH植物表达载体pC13OH,通过电击法把pC13OH转入根癌农杆菌GV3101中。利用该工程菌株对普通烟草进行了转化,在潮霉素选择压力下获得了完整的再生植株。利用13OH基因特异引物,通过PCR技术筛选到4株阳性再生植株。在这4株再生植株中,有3株植株GUS报告基因的组织化学染色呈现阳性反应,表明该植物载体表达载体中与13OH相融合的GUS基因成功地得到了表达。本研究为今后深入研究13OH基因在烟草中的表达和开展紫杉烷13α-羟基化酶基因对红豆杉细胞的转化以及研究作用于该基因的小RNA调节子打下了基础。     

六棱大麦HVA1基因在烟草中遗传转化的研究

本研究依据HVA1基因序列克隆六棱大麦HVA1基因cDNA片段,构建Ubiquitin启动子驱动下的植物表达载体pCAMBIA1300-HVA1。然后通过三亲杂交法将重组质粒PCAMBIA1300-HVA1转入农杆菌LBA4404,并采用农杆菌介导法转化烟草。经PCR,PCR-Southern blotting和RT-PCR检测表明HVA1基因已整合进烟草基因组,并在转录水平上获得表达。功能验证的结果显示,转基因植株叶片的保水率提高了近1倍,暗示转基因烟草具有一定的抗旱潜力。     

外源RNA干涉基因在烟草中的转化及表达

依据RNA干涉机制,以TMV复制酶基因为靶标基因,针对TMV 5个株系复制酶基因间高度同源序列设计引物,经RT-PCR反应获得靶序列,构建靶序列反向重复结构的RNA干涉双元载体.用根癌农杆菌介导将外源基因转化至烟草品种K326基因组中,培育RNA干涉转基因烟草.人工接种病毒验证转基因烟草中外源基因在植物抗病毒能力方面的表达效果,实时荧光定量PCR分析转基因烟草抗病毒能力.结果表明,实验培育的RNA干涉转基因烟草67%对TMV呈现高度抗性;荧光定量PCR分析显示,对TMV具高度抗性的转基因烟草中病毒复制酶基因转录产物mRNA存在很大程度的降解,证实了RNA干涉技术在培育抗病毒烟草品种中的效果.     

外源SOD和APX基因在转基因烟草中的表达与遗传

分析转超氧化物歧化酶基因（SOD）或抗坏血酸过氧化物酶基因（APX）烟草及其自交和杂交后代的叶片中超氧化物歧化酶（SOD）和过氧化物酶（POD）活性的结果表明：转基因烟草的SOD和POD活性在终花期最强,不同叶位叶中SOD活性差异不明显,POD活性以下部叶为最高;转基因烟草的SOD或POD活性显著高于近等基因的非转基因品系。杂交后代（F1、F2）的SOD活性能保持稳定,略高于亲本;自交后代（S1～S3）与自交亲本的SOD和POD活性相当。     

杜仲EuAFP1.2基因遗传转化烟草提高真菌病害抗性

本研究克隆了杜仲(Eucommia ulmoides)抗真菌蛋白EuAFP1.2基因,其CDS序列长为924 bp,编码307个氨基酸。构建植物过表达载体pCAMBIA1301-35S-EuAFP1.2,并遗传转化烟草(Nicotiana tabacum),筛选获得30株转EuAFP1.2基因植株,对转基因烟草植株与野生型及空载体转化烟草植株抗真菌病害能力进行比较。结果表明,在接种小孢根霉菌(Rhizopus microsporus)6 d后转EuAFP1.2烟草植株的相对病斑面积显著小于野生型与空载体转化植株的。接种前后转基因烟草的超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)、过氧化氢酶(catalase, CAT)活性均先升高后降低,在接种后24 h酶活性显著高于野生型和转化空载体植株的;而丙二醛(malondialdehyde, MDA)含量先下降后上升,接种后24 h迅速降低至14.89 nmol/g,显著低于野生型和转化空载体植株的;转基因烟草的脯氨酸含量在接种前后均显著高于野生型和空载体转化植株的。接菌前...     

烟草类锌指基因NtZFL基因的功能分析

从烟草cDNA中分离出一个类锌指基因NtZFL,开放读码框321 bp,编码106个氨基。QRT-PCR分析表明MV、H₂O₂、ABA、冷胁迫处理都提高了NtZFL在烟草的表达,Northern blot分析表明该基因在烟草的不同组织中具有组织特异性,在幼叶和花中表达量较高。亚细胞定位表明NtZFL蛋白是定位在细胞壁中的蛋白。NtZFL基因启动子驱动GUS基因转烟草植株显示在幼苗中整株有表达,但在根部和叶脉处表达量较高。     

Analysis of the Expression of a CycD3 Gene Isolated from Nicotiana tabacum

D-type cyclins (CycD) are thought to control the onset of cell division and responses to extracellular signals during G1 phase. In this study a cyclin D3 (CycD3) gene was isolated from Nicotiana tabacum. Southern blot analysis indicated that it was present in a single-copy in the N. tabacum genome, and Northern analysis showed that it was only expressed in organs and tissues containing dividing cells. Exposure to cold and salt stresses resulted in decreased CycD3 expression supporting the view that its expression is closely related to cell growth.     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

13C-NMR Analysis of Hydroxy-proline Arabinosides from Nicotiana tabacum

The vanillin dehydrogenase gene (ligV), which conferred the ability to transform vanillin into vanillate on Escherichia coli, was isolated from Sphingomonas paucimobilis SYK-6. The ligV gene consists of a 1,440-bp open reading frame encoding a polypeptide with a molecular mass of 50,301 Da. The deduced amino acid sequence of ligV showed about 50% identity with the known vanillin dehydrogenases of Pseudomonas vanillin degraders. The gene product of ligV (LigV) produced in E. coli preferred NAD⁺ to NADP⁺ and exhibited a broad substrate preference, including vanillin, benzaldehyde, protocatechualdehyde, m-anisaldehyde, and p-hydroxybenzaldehyde, but the activity toward syringaldehyde was less than 5% of that toward vanillin. Insertional inactivation of ligV in SYK-6 indicated that ligV is essential for normal growth on vanillin. On the other hand, growth on syringaldehyde was only slightly affected by ligV disruption, indicating the presence of a syringaldehyde dehydrogenase gene or genes in SYK-6.     

烟草T-phylloplanin基因编码蛋白结构与功能的生物信息分析

利用美国国家生物技术信息中心(NCBI)网站所提供的相关信息,分析T-phylloplanin基因编码蛋白。该基因全长861hp,有一个完整的330hp的开放读框,编码110个氨基酸。该基因编码蛋白分子量为11.31kD,理论等电点为7.74。其氨基酸残基的不同区域分布有多N-糖基化位点、酪蛋白激酶Ⅱ磷酸化位点和N-肉豆蔻酰化位点,还有一个跨膜信号肽。T-phylloplanin基因编码蛋白与烟草叶片短柄腺毛分泌的抗性蛋白具有高度的同源性(93%),显示它在烟草抗性系统研究中有潜在价值。     

烟草ovate同源基因的全长cDNA克隆与序列分析

根据番茄中控制果实形状的主效数量性状基因ovate的序列,用生物信息学方法从茄科植物烟草中获得直系同源ovate基因(NTovate)的特异片段,经鉴定,此基因在烟草中至少有2个拷贝。在此基础上用cDNA末端快速扩增(RACE)方法,获得其中1个拷贝的1059bpNTovate全长cDNA序列。序列分析表明,NTovate cDNA序列编码352个氨基酸,其蛋白序列与番茄ovate蛋白序列和拟南芥ovate蛋白家族AtOFP7蛋白分别为70%和36%的序列一致率,而与此家族中其他蛋白以及水稻ovate蛋白仅在保守的ovate结构域有较低的同源性。此基因已在GenBank中登录(EU043369)。     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).     

烤烟主要农艺性状的遗传与相关分析

利用包括基因型与环境互作的加性-显性遗传模型,对14个烤烟品种(系)及其配制的41个杂交组合在4个环境下的7个农艺性状表现进行遗传分析。结果表明,株高、节距、腰叶宽主要受加性效应控制,叶数、腰叶长受显性×环境互作效应影响最大,茎围以加性×环境互作效应、显性×环境互作效应为主,产量以加性效应、显性×环境互作效应为主。适应当地生态条件的品种（系）具有较高的正向加性效应。许多组合的显性主效应及在各试验点的显性×环境互作效应在方向上不尽一致,杂交组合的选配宜针对特定的生态环境进行。性状相关分析表明,大多数成对性状的各项相关系数为正值,且多以加性遗传相关为主,可利用株高对产量进行间接选择。
     

Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L. and Nicotiana rustica L.

Soluble root exudates were collected from three plants (Nicotiana tabacum L., Nicotiana rustica L. and Zea mays L.), grown under axenic and hydroponic conditions, in order to study their metal-solubilizing ability for Cd and other cations (Cu, Fe, Mn, Ni, Zn). Nicotiana spp. and Zea mays L. root exudates differed markedly in C/N ratio, sugars vs. amino acids ratio and organic acids content. Metals from two soils were extracted with either root exudate solutions, containing equal amounts of organic carbon, or distilled water as control. In the presence or absence of root exudates, the solubility of Fe and Mn was much higher than of the four other metals tested. Root exudates increased the solubilities of Mn and Cu, whereas those of Ni and Zn were not affected. Root exudates of Nicotiana spp. enhanced the solubility of Cd. The extent of Cd extraction by root exudates (N. tabacum L. N. rustica L. Zea mays L.) was similar to the order of Cd bioavailability to these three plants when grown on soil. An increase in Cd solubility in the rhizosphere of apical root zones due to root exudates is likely to be an important cause of the relatively high Cd accumulation in Nicotiana spp.     

小黑杨基因组的初步组装及SSR信息分析

小黑杨是人们以小叶杨和欧洲黑杨为亲本培育出的杂交种,兼具双亲生长速度快、抗性强的优点。本研究通过二代测序技术,对小黑杨进行全基因组测序,初步组装出小黑杨基因组,并以此为基础,识别分析其SSR序列,为小黑杨品种划分、表型性状关联等提供参考。结果表明,共组装得到了366 876条总计368.96 Mbp的contig序列;对其中不小于2 000 bp的21 788条的非冗余contig进行SSR分析,共识别出18 111条SSR序列,其中一、二、三核苷酸重复基序较多。对得到的SSR序列设计引物,共得到12 838对引物,供今后实验使用。