Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

Generation of Large Numbers of Independently Transformed Fertile Barley Plants

A rapid, efficient, and reproducible system to generate large numbers of independently transformed, self-fertile, transgenic barley (Hordeum vulgare L.) plants is described. Immature zygotic embryos, young callus, and microspore-derived embryos were bombarded with a plasmid containing bar and uidA either alone or in combination with another plasmid containing a barley yellow dwarf virus coat protein (BYDVcp) gene. A total of 91 independent bialaphos-resistant callus lines expressed functional phosphinothricin acetyltransferase, the product of bar. Integration of bar was confirmed by DNA hybridization in the 67 lines analyzed. Co-transformation frequencies of 84 and 85% were determined for the two linked genes (bar and uidA) and for two unlinked genes (bar and the BYDVcp gene), respectively. More than 500 green, fertile, transgenic plants were regenerated from 36 transformed callus lines on bialaphos-containing medium; albino plants only were regenerated from 41 lines. T0 plants in 25 lines (three plants per line) were analyzed by DNA hybridization, and all contained bar. Most contained the same integration patterns for the introduced genes (bar, uidA, and the BYDVcp gene) as their parental callus lines. Transmission of the genes to T1 progeny was confirmed in the five families analyzed by DNA hybridization. A germination test of immature T1 embryos on bialaphos-containing medium was useful for selecting individuals that were actively expressing bar, although this was not a good indicator of the presence or absence of bar. Expression of bar in some progeny plants was indicated by resistance to the herbicide Basta. The T1 plants were in soil approximately 7 months after bombardment of the immature embryo.     

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

根癌农杆菌介导小麦幼胚遗传转化的影响因素

利用携带pC3301质粒(含bar和gus基因)的超毒根癌农杆菌菌株EHA105对普通小麦(Triticum aestivum L.)扬麦158进行了遗传转化,对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、幼胚预培养时间、乙酰丁香酮(AS)浓度、洗涤用液及筛选方式等影响转化的几个重要因素进行了讨论.对从294个小麦幼胚外植体中转化得到的5株成活植株进行了PCR和Southern blot分析,结果表明其中2株小麦基因组中整合了外源DNA,转化频率为0.68%.     

Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells.

The rha1 gene from Arabidopsis encodes a small GTP binding protein belonging to the Ypt/Rab family. Transgenic Arabidopsis plants containing the promoter region of the rha1 gene fused to the beta-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the root tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl beta-D-glucuronide and a newly developed GUS substrate: Sudan II-beta-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by whole-mount mRNA in situ hybridization. A hypothesis for the function of the Rha1 protein is discussed.     

Temporal and spatial expression analysis of gamma kafirin promoter from Sorghum (Sorghum bicolor L. moench) var. M 35-1

Different cis acting elements of gamma kafirin gene from Sorghum bicolor var. M 35-1 were amplified and cloned using different combination of the primers. The amplified promoter was replaced with CaMV35S promoter of vector pCMBIA-1304 and resultant vector contained beta-glucuronidase (gus) gene under the control of amplified gamma-kafirin promoter. The resulting fusants were then transformed in to different explants of sorghum via particle bombardment. The regulation of uid gene expression was analyzed to find out the minimum required 5' regulatory sequence and cis acting elements for the efficient expression. However no gus expression was detected in leaves of micropropagated plants, scutellum and calli at any stage of growth. The expression of gus, with pKaf gus-P4 gene construct, was detected in immature embryos and endosperm 20 days after pollination (DAP). The result suggest that at least three motifs (two GCN4 and one prolamin box) besides TATA and CATC boxes are required for the efficient expression of the kafirin gene of sorghum. The study shows that PCR based isolation of different motifs and regions can be used as an alternate to deletion analysis for observing the role of various motifs and their importance in the gene expression and regulation.     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。     

香蕉凝集素基因启动子的分离、序列分析及鉴定

香蕉是世界上最重要的水果之一,由于香蕉果实是利用转基因方法生产重组药用蛋白或有价值的化合物的理想器官,构建能在香蕉果实中高水平表达异源蛋白质的表达载体是非常有意义的。而一个高效表达的载体,启动子则是其最重要元件之一,因此,果实特异性表达启动子的获得是香蕉作为生物反应器的前提。香蕉凝集素是一种在香蕉果实中大量存在的蛋白质,其基因被证明在果肉组织中大量表达。利用染色体步移法克隆到香蕉凝集素基因5′端上游的一段长702bp的序列,经序列测定及软件分析表明,该序列具有典型的启动子结构。此序列置换植物表达载体pBI121的CaMV35S启动子,构建植物表达载体,命名为pBIL2,该启动子下游为gus基因。利用基因枪法转化香蕉的根、叶和果实薄片,对gus基因的瞬时表达进行测定,结果表明所获得的凝集素基因启动子,只在香蕉果肉中瞬时表达,该启动子的表达具有果实特异性,并且表达量较高。     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as ‘Koshihikari’, which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the ‘Koshihikari’ genome using our system. Approximately 5–10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic ‘Koshihikari’ rice lines with reduced content of two major seed allergen proteins, the 33 and 14–16?kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system. Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.