烟草和元麦叶肉原生质体吸收尼古丁机制的研究

用EA_3-867纤维素酶分离的烟草(Nicotianatabacum)和元麦(Hordeumdistichon)叶肉原生质体,分别保温在尼古丁溶液中,用紫外分光光度计测定它们对尼古丁的吸收。结果表明,这两种原生质体能迅速地吸收尼古丁;原生质体吸收尼古丁的量,随介质尼古丁浓度的升高而增多;在生理学pH范围内,介质的pH对原生质体吸收尼古丁没有多大的影响。初步认为,这两种原生质体对尼古丁的吸收是一种简单的扩散机制。     

Cloning of two plastid division ftsZ genes from Nicotiana tabacum and their expression in E.coli

Two cDNAs of plastid division gene NtFtsZ1-1 and NtFtsZ1-2 are isolated from Nicotiana tabacum by RT-PCR and rapid amplification cDNA ends (RACE) method. Analysis of the deduced amino acid sequences encoded by NtFtsZ1-1 and NtFtsZ1-2 indicate that these two proteins possess the typical conservative motifs and GTP binding sites existing in all FtsZ proteins. The existence of putative plastid transit peptide in their N-terminal suggests that there are at least two transit-peptide containing FtsZ proteins in higher plants. Phylogenetic analysis based on amino acid sequences of FtsZ proteins also supports this interference. These two NtFtsZ genes demonstrate a similar expression pattern during the plant development, detected by Northern blot. Expression of NtFtsZ1-1 and NtFtsZ1-2 in E.coli interrupts the normal division process of host cells. These results suggest the diverse functions of FtsZ proteins in higher plants.     

烟草叶片蔗糖酶的化学修饰

采用PMSF、NBS、IAc、BrAc、SUAN、TNBS、PCMB、DTT等8种化学修饰剂在一定的条件下选择性修饰蔗糖酶,研究烟草蔗糖酶分子中氨基酸侧链基团与酶活性中心的关系.结果表明:PMSF、NBS、PCMB等3种化学修饰剂能显著地抑制酶的活力,而BrAc、IAc、TNBS、SUAN、DTT等对酶的活力影响不大.说明对丝氨酸残基、色氨酸残基和巯基的化学修饰后对酶的活力影响很大,而酶分子中的组氨酸残基及赖氨酸残基不在酶的活性中心,修饰后对酶的活力没有直接影响.     

Transformation of plant young proembryos by electroporation

It is first reported that plant young proembryos expressed exogenous reporter genes by electroporation. Young proembryos with 8–32 cells and globular proembryos with 250–400 cells could be isolated by enzymatic maceration combined with microdissection. After electroporation withGUS orGFP genes, the proembryos were cultured for 1–2 d in KM8p medium. At the field strength of electroporation 500–1500 V/cm, blue reaction of GUS or green fluorescence of GFP could be observed in the proembryos. The highest transient expression frequency of young proembryos (2.2%) was obtained at the field strength of 750 V/cm, whereas the highest frequency of globular proembryos (5.9%) was obtained at the field strength of 1 250 V/cm. Taking the proportion of transformed cells in the whole cells of proembryos as efficient transformation frequency, the efficient transformation frequency of the young proembryos was 7 times that of the globular proembryos.     

烟草与TMV不同互作中PAL活性变化

漂盘培育云烟87和三生烟,苗期接种TMV后0h,12h,24h,36h,48h,60h和72h分别取样测定其叶片和根系中PAL的活性,并以未接种TMV烟苗叶片和根系中PAL的活性为对照.结果表明：烟草与TMV非亲和性互作时,叶片和根系中PAL活性上升速度比烟草与TMV亲和性互作时PAL活性上升速度快;烟草与TMV无论是亲和性互作还是非亲和性互作,烟草叶片中PAL活性升高速度快于根系中PAL活性上升速度.     

农杆菌介导的大豆反义fad2-1基因转化烟草研究

以烟草无菌苗叶片为外植体,通过根癌农杆菌介导法,将大豆反义油酸脱饱和酶基因导入烟草中,经梯度卡那霉素筛选,获得可在含75mg/L卡那霉素选择生根培养基上再生的抗性植株75株,其中67株抗卡那霉素植株的PCR分析扩增出目的片段;RT-PCR分析表明该基因在烟草中表达.结果表明大豆反义油酸脱饱和酶基因在烟草基因组中整合和表达.这为大豆反义油酸脱饱和酶基因转入大豆和表达奠定基础,也为烟草基因工程育种提供了依据.     

烟草内源细胞分裂素变化与植株形态发育的初步研究

农杆菌介导将异戊烯基转移酶基因(isopentenyl transferase,ipt)导入烟草SD株系的叶肉细胞,经过抗性筛选和鉴定,获得转基因植株.酶联免疫吸附(ELISA)测定叶片内源玉米素核苷(ZR)、异戊烯基腺苷(iPA)和吲哚乙酸(IAA)的质量分数.结果表明:转基因株系的ZR和iPA的质量分数w不同程度地升高,IAA水平随之升高.高水平细胞分裂素导致叶片的气孔密度增加、气孔长度和花型大小减小,以及花粉萌发率降低.当w(iPA+ZR)与w(IAA)的比值大于2时,成年植株上部侧枝茎端生长发育异常,出现丛生状小叶.本文讨论了细胞分裂素水平和细胞分裂素与生长素的比值对转基因烟草生长发育的影响.     

利用TRV过表达和沉默系统验证植物油脂合成相关基因的功能

为探究烟草脆裂病毒过表达和沉默系统进行油脂相关基因研究的可能性,该研究克隆了拟南芥的WRI1和FAD2基因,分别构建了TRV过表达和沉默载体. 利用瞬时侵染技术侵染本氏烟草,取材料进行RT-PCR和脂肪酸含量检测. 结果显示在过表达WRI1基因的本氏烟草中,侵染叶和非侵染叶的WRI1基因及其相关参与脂肪酸合成基因ACP1、KAS1和BCCP2等都有明显上调,且脂肪酸含量检测结果显示分别增加16%和28%. 在沉默FDA2基因的本氏烟草植株中,发现多不饱和脂肪酸含量分别减少约25%和24%. 因此,利用TRV系统对油脂相关基因进行研究是可能的.