基因工程技术在烟草研究中的应用现状与展望

介绍转基因、RAPD分子标记、基因芯片、mRNA差异显示以及蛋白质组学等基因工程技术在烟草中的应用,指出这些技术不仅在烟草遗传图谱建立、系统分析、物种及品系的鉴定、转基因烟草鉴定和检测等方面发挥着重要作用,而且在改善烟草品质和建立烟草基因组学方面也有广阔的应用前景,并从抗病性、抗虫性、抗旱性、抗除草剂等方面介绍了基因工程技术在烟草抗逆性中的研究现状,对转基因烟草的安全性问题进行了探讨。     

利用病毒诱导的基因沉默(VIGS)技术快速鉴定两个马铃薯晚疫病抗性相关EST片段EL732276和EL732318的功能

马铃薯晚疫病是由致病疫霉[Phytophthora infestans(Mont.)deBary]引起的第一大作物病害,目前已严重限制了全球马铃薯的生产和发展。马铃薯晚疫病抗性分子机理的研究一直是抗病育种中的热点问题。本研究利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术,在本氏烟草(Nicotina benthamiana)中沉默两个马铃薯晚疫病水平抗性相关的基因片段EL732276和EL732318。目的基因沉默后的烟草接种晚疫病菌P.infestans,根据病斑生长速率LGR(length grow rate)来了解这两个基因是否参与烟草对晚疫病的抗性反应。结果表明,目的基因片段EL732276和EL732318被成功地插入烟草脆裂病毒(Tobacco rattle virus,TRV)载体中。利用携带八氢番茄红素脱氢酶基因(phytoene desaturae,PDS)的TRV重组载体病毒接种烟草,感染病毒的烟草植株表现光漂白现象,说明VIGS体系有效。用携带目的片段的重组载体病毒TRV:EL732276和TRV:EL732318感染烟草植株,一个月后,烟草叶片接种晚疫病菌P.infestans,从接种P.infestans的烟草叶片上可以看出,感染TRV空载体的对照烟草叶片上病斑扩展速率非常缓慢,而目的片段EL732276和EL732318沉默后的烟草叶片可见明显的水浸状病斑和少量白色的晚疫病菌菌丝。进一步的分析表明TRV:EL732276重组载体病毒侵染的烟草接种P.infestans后LGR值极显著上升,TRV:EL732318病毒侵染的烟草接种P.infestans后LGR值显著上升,表明EL732276和EL732318同源基因在烟草中沉默后,烟草对P.infestans的抗性显著降低。研究结果认为,病毒诱导的基因沉默技术可在本氏烟草中初步快速鉴定马铃薯抗病相关基因的功能。     

马铃薯Y病毒CP基因RNAi载体构建与干涉效果测定

RNA沉默（RNAi）介导的植物抗病毒研究是近年来引起广泛关注的一项植物抗病毒基因工程策略。马铃薯Y病毒（PVY）在世界范围内引起马铃薯、烟草等重要经济作物的病害,并且造成严重的经济损失。本文以PVY的外壳蛋白（CP）基因为模板扩增300bp和354bp两个片段,正向和反向分别插入植物表达载体pROKⅡ的35S启动子下游,从而构建了以PVY的CP基因为靶标的RNAi植物表达载体pROKY300,转入农杆菌EHA105中,以农杆菌渗入法在本氏烟中瞬时表达与wyCP同源的hairpin RNA。结果表明,瞬时表达的hairpin RNA有效干涉了PVY侵染。     

烟草病毒病及其检测鉴定技术

简介了病毒及烟草病毒的分类,烟草病毒病的检测鉴定技术。重点叙述酶联免疫吸附反应（ELISA）在烟草病毒检测中的应用。     

利用RNA沉默抑制子HC-Pro提高基因表达的策略

利用植物生物反应器生产药用蛋白具有独特的发展优势,但外源基因在受体内会发生基因沉默现象而影响基因的表达。由于基因沉默抑制子能有效抑制外源基因的沉默反应,本研究利用沉默抑制子蚜传辅助因子(helper component-proteinase,HC-Pro)转基因烟草有效提高了外源基因的表达。采用叶片注射法将携带表达载体p35S-30B-GFP的农杆菌(Agrobacterium tumefaciens)重悬液分别侵染野生型烟草(Nicotiana bentamiana)和HC-Pro转基因烟草(N.bentamiana),通过蛋白电泳检测和Western blot检测技术对绿色荧光蛋白(GFP)表达进行比较。研究结果表明,与野生型烟草Nb相比,HC-Pro转基因烟草中GFP的表达水平显著增加。本研究为基因沉默抑制子在植物生物反应器中的应用提供了理论依据。     

转Na+/H+antiporter(Nhap)基因烟草植株的获得及耐盐性鉴定

Nhap基因编码一种质膜Na /H 逆向转运蛋白,在耐盐方面具有重要的研究价值。实验利用根癌农杆菌(Agrobac-teriumtumefacience)介导的叶盘法将Nhap基因转入烟草(Nicotianatobacum),经PCR和RT-PCR检测已获得转Nhap基因烟草的再生植株。同时从发芽率、存活率、叶绿素含量、Na 含量及耐盐性表现进行了F1代转基因烟草的耐盐性检测。结果表明,转基因烟草的发芽率、存活率、叶绿素含量均较对照高,Na 含量较对照有所下降,并且转基因烟草在200mmol/LNaCl处理下,仍能正常存活、生长,而对照烟草的生长却受到抑制。即转基因F1代烟草的耐盐性得到了显著的提高。     

转基因技术在玉米育种中的应用

介绍了应用转基因技术在改善玉米抗性方面培育出了抗虫、抗除草剂及抗病毒的转基因玉米,并提出转基因技术还用于改良玉米的品质。在基因转化途径方面主要分析了农杆菌介导法、基因枪法和花粉管通道等技术。     

昆虫杆状病毒研究和应用新进展

杆状病毒(Baculovirus)是昆虫专一性的病原病毒。自80年代开发了杆状病毒作为外源基因的表达载体系统以来,该技术不断完善并得到了广泛应用。近年来杆状病毒除应用于表达系统和生物杀虫剂外,在表面展示系统、作为哺乳动物基因转移的载体、固定外源蛋白质以及作为生物纳米材料等方面的研究出现了一些新动向。     

PTLF-PTAG-IR对转基因烟草开花的抑制效应

PTLF（LEAFY同源基因）和PTAG（AGAMOUS同源基因）是杨树中控制花发育的重要基因,为了解RNA干涉同时抑制PTLF和PTAG对花发育的影响,本研究采用根癌农杆菌（Agrobacterium tumefaciens）介导法将35S：：PTLF-PTAG-IR导入烟草（Nicotiana tabacum）。经PCR检测获得了15株阳性转化株系,进一步的Southern分析证实PTLF-PTAG-IR基因已整合到烟草基因组中。荧光定量分析结果显示,转基因烟草内源NFL（LEAFY同源基因）和NAG1（AGAMOUS同源基因）的表达量均低于野生型。对开花时间调查的结果表明：与野生型烟草相比,53.3%的转基因株系开花受到完全的抑制,26.7%的转基因株系开花时间平均推迟34.3d,仅20%的转基因株系与野生型在同一时期开花。另外,对转基因烟草花器官的观察发现花瓣形态,雄蕊着生方式、数目等方面发生变异,这些研究结果表明过量表达35S：：PTLF-PTAG-IR对烟草花发育起到抑制作用,本研究将为利用转基因手段获得不育材料提供参考。     

优化的VHb基因和融合杀虫基因在烟草中的表达

将人工合成的、密码子优化后的透明颤菌血红蛋白(VHb)基因(vgbM)与含有融合杀虫基因(GFMcryIA和CPTI)表达载体pGBIF4ABC构建成双价基因植物高效表达载体pGBIF4ABCVHB,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum),经PCR及Southernblot检测,证实了双价基因在32株转基因烟草基因组中整合。Westernblot检测证实了vgbM基因的表达,杀虫实验表明,融合杀虫基因表达的毒蛋白具有杀虫活性,转基因烟草平均每株干重高于非转基因烟草植株8%。表明VHb基因和融合杀虫基因烟草在烟草中的表达可使烟草可增产,又具抗虫性。     

Free and conjugated phytosterols in cured tobacco leaves: influence of genotype, growing region, and stalk position

Although phytosterols in tobacco leaves have specific effects on tobacco quality, there is little research on the distribution of free and conjugated phytosterols in various tobacco leaves. In this paper, we explored the content and composition of phytosterols in representative cured tobacco leaves by gas chromatography of TMS-ether derivatives. We found that phytosterol contents in tobacco leaves ranged from 1.0 to 2.5 mg/g of dried leaf tissue, depending on different types of tobacco leaves. The majority of phytosterols (75-85%) were conjugated as ester and glycosides, with only about 15-25% existing in the free form. Furthermore, the genetic variability gives rise to the significant differences among different tobacco types with phytosterol levels: the contents of phytosterols in tobacco leaves decreased in the order of flue-cured tobacco, Oriental tobacco, Burley tobacco, cigar tobacco, and Maryland tobacco. At the same time, the tobacco curing process leads to a difference in phytosterol existing-form distribution in some variation laws.     

转碱蓬SsNHX1基因烟草的耐盐抗旱性研究

烟草是重要的模式植物和经济作物,盐害和干旱两种环境因子对其生长发育、产量和品质都危害很大。为了提高烟草的耐盐抗旱性,本研究利用农杆菌介导的遗传转化法在烟草中过量表达了碱蓬液泡膜Na~+/H~+逆向转运基因SsNHX1,对转基因烟草的耐盐及抗旱性进行表型鉴定和各项生化指标的检测,以期得到耐盐抗旱表性良好的SsNHX1转基因烟草。表型分析发现,SsNHX1基因过表达株系L1和L5的抗盐能力比野生型显著提高,表现为盐胁迫条件下仍能保持旺盛的生长且根系的伸长未受抑制。SsNHX1过表达株系在叶片和根系中积累了更多的Na~+和K~+,同时Na~+含量增长速率较快,而K~+含量降低速率较缓,并可维持较高的叶片相对含水量和叶绿素含量,及较低的丙二醛含量和相对电导率。干旱胁迫发现,过表达株系受干旱胁迫程度更小,并在复水后迅速恢复正常生长。同时,过表达株系的丙二醛含量和相对电导率显著低于野生型,且维持了较高的叶片相对含水量及叶绿素含量。这些结果说明SsNHX1基因在烟草中过量表达后,降低了盐胁迫和干旱胁迫对烟草根系及细胞膜的损伤,并通过调节离子含量、降低细胞的渗透势,维持了叶片较高的相对含水量和叶绿素含量,最终提高了烟草的抗盐和抗旱性。     

GENETIC ANALYSIS OF CHLORIDE CONCENTRATION IN ORIENTAL TOBACCO GENOTYPES

A high chloride concentration in oriental tobacco leaf has a detrimental effect on tobacco quality and its burning properties. In order to determine the inheritance of chloride concentration in oriental tobacco leaf, eight tobacco genotypes showing genetic variability for chloride concentration on the basis of our previous investigation were crossed in a diallel program. Parental genotypes and their F1 hybrids were evaluated in greenhouse experiment using randomized complete block design with three replications. Each replication consisted of two pots (one plant per pot). Cultivation took place in plastic plant pots which contained 10 kg soil from the fields of the northwest Iran where the oriental tobacco type is planted. Standard agrotechnical practices for this tobacco type were applied during growth. Tobacco was harvested when it reached technical maturity then it was sun-cured. Percentage of chloride in cured leaves was determined as defined by CORESTA (Cooperation Center for Scientific Research Relative to Tobacco, Paris, France). Analysis of variance was performed to determine the effect of genotypes on chloride concentration and showed significant variability among genotypes for this trait. Diallel analysis showed that general combining ability (GCA) and specific combining ability (SCA) effects for chloride concentration were highly significant indicating that both additive and dominant gene effects were important in controlling the chloride concentration. However, the mean square for specific combining ability was greater than those of general combining ability that shows the predominance of the non-additive genetic effects in controlling the chloride concentration. Therefore, the interpopulational breeding strategy using the heterosis effect could give superior gains to this character.     

GFP叶绿体转化烟草茎叶降解对土壤微生物数量及土壤酶活性的影响

以GFP叶绿体转基因烟草及对照烟草（非转基因烟草）为材料,在实验室条件下研究了叶绿体转基因烟草及对照烟草（非转基因烟草）在茎叶降解过程中及完全降解后对土壤微生物主要类群（细菌、真菌、放线菌）的影响,并对相关的土壤酶活性进行了分析。结果表明,在烟草茎叶降解过程中及完全降解后：（1）叶绿体转基因烟草及对照烟草（非转基因烟草）对微生物的数量影响不显著;（2）土壤微生物总量相比为细菌〉放线菌〉真菌;（3）叶绿体转基因烟草茎叶降解对土壤酶活性没有显著影响;（4）GFP基因没有水平转移到土壤微生物基因组中。以上结果显示GFP叶绿体转化烟草茎叶降解对土壤微生物数量及土壤酶活性没有显著的影响。     

镁肥施用对皖南烟区烤烟生长发育及产质量的影响

采用田间试验研究了Mg肥施用对皖南烟区烤烟生长发育及产质量的影响。结果表明:适量施用Mg肥能有效促进烤烟生长发育,提高烟叶产量,同时较好协调烟叶中主要化学成分,改善烟叶品质;施Mg量为300 kg/hm2Mg SO4·7H2O时,烤烟的农艺性状、干物质积累及产质量达最佳。     

3S技术在许昌数字烟草农业中的发展策略

阐述了＂数字烟草＂的基本内涵、许昌＂数字烟草＂的技术系统的组成和数字烟草农业在许昌应用前景,在分析基于3S技术许昌烟草生产特点和实施＂数字烟草农业＂的必要性和重要性,提出发展许昌数字烟草农业的基本策略。     

烟草与丛枝菌根真菌的共生效应研究进展

丛枝菌根(Arbuscular mycorrhiza,AM)真菌是陆地生态系统中广泛存在的一类专性共生土壤微生物,是根系土壤区域中重要的功能菌群之一。AM真菌可侵染植物根系形成丛枝菌根共生体,改变植物根系形态和改善营养状况,从而提高宿主植物的生长发育、产量、质量和抗逆性。目前从烟草根系土壤分离报道的AM真菌已达13属54种,显示出烟草(Nicotiana tobacum L.)栽培的潜在AM真菌资源较为丰富。围绕烟草与AM真菌的共生效应,总结了影响AM真菌侵染和定殖烟草根系的主要因素,阐述了AM真菌对烟草生长、抗性生理及品质的影响,并对PGPR与AM真菌的协同作用进行了简要回顾,最后讨论了该领域存在的不足及今后展望;旨在为菌根技术运用于烟草栽培提供参考。     

硼对烟草生理特性及品质影响的研究进展

硼是烟草生长过程中不可缺少的微量元素,参与烟草的生长发育和生理生化反应,并最终影响烟叶的品质和质量。本文综述了硼对烟草细胞组织结构、光合特性、酶活性、核酸和蛋白质及品质的影响,并从硼肥用量和硼肥利用率等方面进行了展望。     

Impact of alleles at the Yellow Burley (Yb) loci and nitrogen fertilization rate on nitrogen utilization efficiency and tobacco-specific nitrosamine (TSNA) formation in air-cured tobacco

Tobacco-specific nitrosamine (TSNA) formation in tobacco is influenced by alkaloid levels and the availability of nitrosating agents. Tobacco types differ in their potential for TSNA accumulation due to genetic, agronomic, and curing factors. Highest TSNA concentrations are typically measured in burley tobaccos. One of the main genetic differences between burley and all other tobacco types is that this tobacco type is homozygous for recessive mutant alleles at the Yellow Burley 1 (Yb(1)) and Yellow Burley 2 (Yb(2)) loci. In addition, burley tobacco is typically fertilized at higher nitrogen (N) rates than most other tobacco types. This study utilized nearly isogenic lines (NILs) differing for the presence of dominant or recessive alleles at the Yb(1) and Yb(2) loci to investigate the potential influence of genes at these loci on TSNA accumulation. Three pairs of NILs were evaluated at three different nitrogen fertilization rates for alkaloid levels, nitrogen physiology measures, and TSNA accumulation after air-curing. As previously observed by others, positive correlations were observed between N application rates and TSNA accumulation. Recessive alleles at Yb(1) and Yb(2) were associated with increased alkaloid levels, reduced nitrogen use efficiency, reduced nitrogen utilization efficiency, and increased leaf nitrate nitrogen (NO(3)-N). Acting together, these factors contributed to significantly greater TSNA levels in genotypes possessing the recessive alleles at these two loci relative to those carrying the dominant alleles. The chlorophyll-deficient phenotype conferred by the recessive yb(1) and yb(2) alleles probably contributes in a substantial way to increase available NO(3)-N during curing and, consequently, increased potential for TSNA formation.