新疆无苞芥锌指蛋白基因的克隆及表达分析

利用同源克隆法从新疆无苞芥中克隆获得1个锌指蛋白基因(OpZFP)。序列分析表明,OpZFP基因的开放阅读框为684bp,推测编码含227个氨基酸的蛋白质。生物信息学分析显示,OpZFP蛋白含有1个典型的C2H2型锌指结构,在C端含有一个可能具有转录抑制功能的EAR结构域。系统进化树分析表明OpZFP编码产物与拟南芥AtZFP1、琴叶拟南芥AlZFP1的进化关系较近。分离了OpZFP基因2 095bp的启动子序列,发现该启动子与拟南芥AtZFP1基因的启动子序列只有84.4%的相似性,启动子分析表明二者存在多处不同的顺式作用元件。半定量RT-PCR分析表明,OpZFP在根、茎、叶、花和果荚中均有表达,在根中的表达量最高。OpZFP基因受高盐、干旱和低温等胁迫的诱导表达,表明该蛋白涉及多种胁迫相关的信号传导途径。     

过表达秋茄C2H2型锌指蛋白基因KcZFP提高烟草耐盐性

基因转录调节是植物对非生物胁迫适应机制的一个重要方面,转录调节因子在胁迫信号转导途径中调节下游基因的表达,在建立植物对胁迫适应性过程中起到重要作用.锌指蛋白是功能多样的转录调节因子蛋白家族,家族成员在植物响应非生物胁迫方面扮演着重要角色.本研究以秋茄C2H2型锌指蛋白编码基因KcZFP为目的基因,在烟草中过表达KcZFP,分析C2H2型锌指蛋白在植物耐盐性中的作用.研究结果显示:转基因株系中,KcZFP表达量显著提高.过表达KcZFP的烟草植株的耐盐性明显提高,在200 mmol/L NaCl处理的条件下,KcZFP过表达烟草中脯氨酸水平远高于野生型植株.对光合作用参数比较分析显示,在KcZFP过表达植株中净光合速率受盐胁迫的影响小于野生型植株,光合系统在一定程度上得到了保护.研究结果说明KcZFP作为转录调节因子参与了植物的渗透调节,对植物的耐盐性具有贡献.     

新疆无苞芥类钙调素蛋白基因OpCML13的克隆与分析

通过对无苞芥幼苗cDNA文库的随机克隆测序分析,获得1条与拟南芥编码类钙调素13(calmodulinlike13,CML13)高度相似的EST(GenBank登录号为JZ152285)。利用RT-PCR技术从无苞芥cDNA中克隆了该基因,命名为OpCML13,该基因开放阅读框(ORF)为447bp,编码148个氨基酸。生物信息学分析表明,OpCML13蛋白的二级结构含有4个Ca2+结合基序(EF-hands)结构,是一个亲水蛋白,无信号肽,无跨膜区域,为非跨膜蛋白;同源建模发现该蛋白包含8个α-螺旋结构和10个β-转角。系统进化树分析表明,OpCML13编码产物与拟南芥AtCML13和AtCML14进化关系较近,属于同一进化分支。半定量RT-PCR结果显示,OpCML13基因在无苞芥的不同组织中都有表达,在根中表达量最高;高盐胁迫处理6~24h,OpCML13基因的表达明显增强,随后逐渐恢复正常表达水平;低温、干旱和ABA处理6h后基因表达明显上调,并且一直保持较高的表达水平。研究表明,OpCML13基因在无苞芥逆境胁迫中可能起着重要作用。     

烟草NTHK1基因提高杨树根系的耐盐性

以受体杨树‘107号’和转入NTHK1（Nicotiana tabacum histidine kinase-1）基因的‘18-1’及‘18-4’的水培苗为材料,不同浓度的NaCl胁迫12d后,发现‘18-1’及‘18-4’的根长和根重显著大于‘107号’。以离体根段为材料,在400mmol·L-1NaCl溶液中胁迫60min后,‘107号’的K＋外渗量比‘18-1’和‘18-4’分别高49.34%和19.68%;在400mmol·L-1KCl或NaCl胁迫30min,‘107号’的电解质外渗率（REL）显著大于‘18-1’和‘18-4’;等渗的30%PEG对离体根段的REL影响很小。在400mmol·L-1NaCl溶液中添加200～400mmol·L-1的KNO3或KCl能显著增加离体根段的REL,并使不同基因型的REL差异更大。这表明,测定离体根段的K＋外渗量和REL可以快速鉴定不同基因型杨树的耐盐潜力。     

无苞芥NAC转录因子基因OpNAC026的克隆与分析

在新疆耐逆植物无苞芥幼苗cDNA文库的随机克隆测序结果中,发现1条与拟南芥NAC转录因子基因AtNAC026高度相似的5′端EST序列(GenBank登录号为JZ151854),对该克隆进行3′端测序,拼接得到一条全长1 327bp的cDNA序列,该序列包含一个906bp的最大开放阅读框(ORF),推测编码301个氨基酸。根据该ORF序列设计引物,利用RT-PCR技术对其进行克隆,将该基因命名为OpNAC026(GenBank登录号为KM457621)。理化性质分析表明,OpNAC026蛋白是一个无跨膜区域的亲水蛋白,在N端具有一段保守结构域;蛋白质二级结构预测显示,OpNAC026蛋白包含54个α-螺旋和12个β-转角。系统进化树分析表明,OpNAC026基因与拟南芥AtNAC026和AtNAM进化关系最近。实时荧光定量PCR(qRT-PCR)分析显示,OpNAC026基因在无苞芥的各组织中都有表达,在叶中表达量最高;高盐胁迫处理24h、干旱12h、ABA 6h、4℃低温8h处理均可明显诱导OpNAC026基因的表达。研究表明,OpNAC026基因可能参与无苞芥抗逆机制的调控。     

过量表达AtNHXS1新基因提高烟草耐盐性的研究

拟南芥液泡膜上的Na⁺/H⁺逆向转运蛋白是由 AtNHX1 基因编码的一种重要的植物耐盐性因子。 AtNHXS1 是利用DNA改组(DNA shuffling)技术对 AtNHX1 基因进行定向分子进化获得的新基因。利用农杆菌介导的叶盘法将该基因转入烟草中,经过潮霉素和PCR鉴定,得到了10个独立的转基因株系。对其中两个PCR阳性株系进行Southern blot 鉴定,确定 AtNHXS1 以单拷贝的形式成功地插入到烟草的基因组中。荧光定量PCR分析表明, AtNHXS1 基因可以利用烟草的转录体系正确转录。在盐处理下,随着盐浓度的提高,植株不同组织部位 AtNHXS1 基因的表达均有不同程度的提高,其中叶片上调趋势最明显。耐盐性试验结果表明,盐处理下,转基因烟草的长势明显优于野生型。400 mmol/L NaCl 处理下,野生型烟草完全死亡,转基因烟草生长受到抑制,但是仍然能够正常生长。研究结果表明, AtNHXS1 新基因能够显著提高烟草的耐盐性。     

锌指蛋白基因ZNF191

锌指是Ｍｉｌｌｅｒ等人 (1 985年 )最初在爪蟾转录因子ＴＦ ＩＩＩＡ中发现的一种特殊结构 ,即在蛋白质一级结构上 ,存在着Ｃ Ｘ2 4 Ｃ Ｘ12 Ｈ Ｘ3 Ｈ的序列单元 (Ｃ :半胱氨酸 ;Ｈ :组氨酸 ;Ｘ :任意氨基酸 ) ,其中的 2个半胱氨酸和 2个组氨酸通过配位键与一个锌离子结合 ,成手指状结构 ,这类序列单元被称为锌指模体 ,含有锌指模体的蛋白质称为锌指蛋白。研究表明 ,锌指蛋白作为一类转录因子而广泛地参与基因表达的调控。这种调控作用主要是通过它与特定的ＤＮＡ序列 (如启动子 )和蛋白质的结合而实现的。锌指蛋白与胚胎发育和细胞分化…     

转LEA基因烟草的耐盐性分析

目的:验证柽柳LEA基因的功能,为通过基因工程手段培育耐盐植物提供基础资料。方法:对转LEA基因烟草当代(T0)和子一代(T1)分别进行不同浓度的NaCl胁迫处理,研究转基因烟草的耐盐性。结果:转基因烟草T0代组培苗耐受NaCl的临界浓度为230mmol/L,而对照耐受NaCl的临界浓度为130mmol/L以下;T1代幼苗耐受NaCl的临界浓度为150mmol/L,对照耐受NaCl的临界浓度为100mmol/L以下;在临界浓度转基因烟草的T0代、T1代根系发育良好,生长量明显高于非转基因对照烟草。结论:柽柳LEA基因的转化提高了烟草的耐盐性。     

大豆Lea5基因过表达提高烟草抗旱和耐盐性的研究

大豆Lea5基因是LEA基因家族成员之一。为分析大豆Lea5基因的功能,构建了大豆Lea5基因植物过表达载体p BI121-Lea5。通过农杆菌介导法转化烟草,获得TL-06、TL-09、TL-17和TL-32等4个转大豆Lea5基因烟草株系。RT-PCR分析表明大豆Lea5基因在4个转基因烟草株系的T2代植株均有不同程度的表达。以转基因TL-09和TL-32株系T2代植株为材料,进行了干旱和盐渍处理,结果表明,2个转大豆Lea5基因烟草株系T2代植株对干旱和盐渍的抗性显著强于野生型烟草,说明大豆Lea5基因可以提高植物抗干旱和盐碱的能力。     

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。     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

Subcellular aspects of differentiation of microspore mother cells ofNicotiana tabacum

Summary Differentiation of microspore mother cells inNicotiana starts with a period of active vesiculation in archesporial cells which is attended by a reduction of ribosome population. Vesiculation has been interpreted to cause cell reorganization and with their presumed hydrolytic functions the vesicles are reckoned to eliminate macromolecules associated with the sporophytic phase and result in the formation of cells capable of gametophytic functions. However, a part of cell cytoplasm is preserved in the form of membrane inclusions, which are bi- or multimembranous structures. Therefore, the rationale behind elimination and preservation of part of ribosomes remains to be understood. Another important feature of cell reorganization, the significance of which is quite elusive, is the dedifferentiation of organelles mitochondria and plastids. The former become electron dense and lose their cristae and the latter become cup-shaped and show regression of lamellae.     

Varietal differences in potassium uptake by excised roots ofNicotiana tabacum

Summary The rate of potassium uptake by two varieties of burley tobacco (Nicotiana tabacum) differed by as much as two-fold when excised roots were absorbing K from solutions of 2.5 × 10^–4 M KCl. With increasing substrate levels of KCl the difference between varieties decreased and no difference was observed at a KCl concentration of 1.6 × 10^–2 M. The K-absorption by excised roots of several varieties from solutions of 5 × 10^–4 M KCl was compared.Contribution of the Department of Agronomy, Kentucky Agr. Exp. Sta., Lexington, and published with the approval of the Experiment Station Director.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

Amino acid analysis ofin vivo and androgenic anthers ofNicotiana tabacum

Summary Glutamine was the only amino acid present in significantly greater amounts in aqueous extracts of androgenic as compared with non-androgenic and mature anthers of Burley tobacco. Serine was not detectable in androgenic extracts and a role for this amino acid in pollen embryogenesis is questioned.     

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.     

Cell division and chromosome numbers in the tissue culture ofNicotiana tabacum

The callus tissue derived from tobacco pith and subcultured for 5^1/2 years on a solid synthetic modium revealed considerable differences in cell division activity depending on the age of the subculture and diurnal rhythm. The callus cells exhibited different level of ploidy among which an aneuploid condition nearer to diploid (2n=30–50) predominated. Chromosomal bridges and other structural rearrangements (lagging chromosomes, fragments) were observed in ana-and telophase.     

Regeneration of chlorophyll chimeras from leaf explants ofNicotiana tabacum L.

Chimeral plants with variegated leaf blades were obtained by induction of organogenesis in primocultures of leaf explants ofNicotiana tabacum L. cv. Burley 49, chlorophyll mutation White Seedling. Only green plants regenerated from primocultures of explants taken from dark green leaf areas of the chimeras. The possibility of a multicellular initiation of chimera regeneration from tissue cultures is discussed.     

Androgenesis in vitro in anther cultures of chlorophyll mutants ofNicotiana tabacum

The course of androgenesisin vitro was investigated with anther cultures of two chlorophyll mutants of Nicotiana tabacum. A different sensitivity to the hormonal composition of the medium was revealed between the cultures White Seedling and Sulfur; the stimulatory effect of kinetin on the frequency of androgenesis was observed only in White Seedling cultures. In addition to green plants, “aurea” (mutation Sulfur) or “albino” (mutation White Seedling) phenotypes also differentiated in both cultures. The possible causes of variability in the participation of green and mutant forms are discussed.     

Pathogenesis-related proteins in plants and tissues ofNicotiana tabacum transformed byAgrobacterium tumefaciens

Large amounts of pathogenesis-related (PR) proteins were found inNicotiana tabacum crown gall tissue, following transformation of normal tobacco cells withAgrobacterium tumefaciens. In contrast, PR proteins were not detected in leaves of grafted plants that had been recovered from crown gall tissue even though these plants were still transformed as shown by their inability to form roots and ability to produce octopine. No difference was observed in susceptibility to virus infection between untransformed and transformed plants grafted onto identical rootstocks. The results are discussed in relation to physiological factors controlling PR protein induction and virus resistance.