Activity of promoters of carnation etched ring virus and dahlia mosaic virus in tobacco protoplasts and transgenic plants

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants.     

Comparative expression of β-glucuronidase with five different promoters in transgenic carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) root and leaf tissues

Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the β-glucuronidase (GUS) gene driven by one of five promoters: Cauliflower mosaic virus 35S (35S) and double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) from Agrobacterium tumefaciens or the rooting loci promoter (rolD) from A. rhizogenes. Five independently transformed carrot lines of each promoter construct were assessed for GUS activity. In leaves, activity was highest in plants with the D35S, 35S and UBQ3 promoters, while staining was weak in plants with the mas2 promoter, and only slight visual staining was present in the leaf veins of plants containing rolD promoter . Strong staining was seen in the lateral roots, including root tips, hairs and the vascular tissues of plants expressing the 35S, D35S and UBQ3. Lateral roots of plants containing the rolD construct also showed staining in these tissues while the mas2 promoter exhibited heightened staining in the root tips. Relatively strong GUS staining was seen throughout the tap root with all the promoters tested.. When GUS expression was quantified, the UBQ3 promoter provided the highest activity in roots of mature plants, while plants with the D35S and 35S promoter constructs had higher activity in the leaves. Although plants containing the mas2 promoter had higher levels of activity compared to the rolD plants, these two promoters were significantly weaker than D35S, 35S and UBQ3. The potential for utilization of specific promoters to target expression of desired transgenes in carrot tissues is demonstrated.     

Promoters derived from banana bunchy top virus-associated components S1 and S2 drive transgene expression in both tobacco and banana

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.     

Construction of hybrid promoters of caulimoviruses and analysis of their activity in transgenic plants

By the techniques of DNA shuffling, PCR, and restriction-ligation, chimeric forms of cauliflower (Brassica oleracea) mosaic virus (CaMV), dahlia (Dahlia pinnata) mosaic virus (DMV), and carnation (Dianthus caryophillus) etching ring virus (CERV) promoters were obtained at various combinations. Twelve chimeric promoters were cloned into pCambia binary vectors comprising the reporter GUS gene, and their activities in transgenic tobacco (Nicotiana tabacum) plants were determined fluorimetrically. 35S promoter and those of DMV (442 bp) and CERV (371 and 501 bp) were used as controls. Seven of analyzed promoters displayed higher and seven promoters lower activity in transgenic tobacco plants than 35S promoter. The highest activity was characteristic of natural DMV promoter, and the least one — natural CERV promoter 501 bp in size. The CERV promoter 371 bp in size was approximately similar in strength to 35S promoter.     

Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants

To develop strong promoters for protein over-expression in both dicots and monocots, we constructed a new family of chimeric promoters using sequences of the Commelina Yellow Mosaic Virus (CoYMV), of the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter. The chimeric promoters were cloned upstream from the gusA reporter gene. The constructs were used in transient expression experiments, via DNA-coated gold particle delivery to tobacco leaves and maize endosperms. The results showed that candidates among the chimeric promoters could drive expression of the reporter gene to very high levels in the dicot plant tobacco, and all chimeric promoters showed higher expression in maize endosperm than the maize γ-zein promoter used as reference for the monocot expression. Expression cassettes were then used in stable tobacco transformation. Determination of GUS activity throughout growth of the primary transformants showed that two promoters (MPr1163 and MPr1165) could drive expression three to five-fold higher than the highly efficient enhanced 35S promoter. The use of MPr1163 was additionally validated for successful heterologous protein production of human lactoferrin in tobacco via agroinfiltration.     

不同启动子调控转GhCDPK1基因烟草的抗逆性研究

为探究不同启动子对陆地棉GhCDPK1基因抗逆功能的影响,该研究克隆了长度为824bp和1 524bp的2个拟南芥RD29A的启动子序列,分别构建了35S启动子和2个RD29A启动子驱动的GhCDPK1融合表达载体,并利用农杆菌介导法转化烟草,分析了其驱动的转GhCDPK1基因烟草,在逆境胁迫处理后的表型变化,叶绿素、丙二醛(MDA)和脯氨酸含量,过氧化物酶(POD)和超氧化物歧化酶(SOD)活性以及细胞膜透性的生理变化。结果显示:RD29A启动子驱动的转GhCDPK1基因烟草,比35S启动子驱动表现出更强的耐逆性,其叶绿素含量、脯氨酸含量以及POD、SOD活性都高于35S启动子,而MDA含量与细胞膜的通透性低于35S启动子,且1 524bp的RD29A2启动子片段驱动转GhCDPK1基因烟草的耐胁迫能力比824bp启动子片段更强。     

不同调控序列作用下GUS基因在烟草中瞬时表达活性

以pB1121为出发质粒,利用烟草泛素启动子Ubi．U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片,检测瞬时表达活性,研究不同调控序列对外源基因表达的调控作用。结果表明：CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍：双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当;烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1．5倍;Ubi．U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高,其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍,为CaMV35S独立作用时的10倍。     

不同调控序列作用下GUS基因在烟草中瞬时表达活性的研究

以pBI121为出发质粒, 利用烟草泛素启动子Ubi.U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片, 检测瞬时表达活性, 研究不同调控序列对外源基因表达的调控作用。结果表明: CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍; 双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当; 烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1.5倍; Ubi.U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高, 其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍, 为CaMV35S独立作用时的10倍。     

Promoters pro-SmAMP1 and pro-SmAMP2 from Wild Plant <Emphasis Type="Italic">Stellaria media</Emphasis> for the Biotechnology of Dicotyledons

Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.