Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.     

来源于Pyrococcus furiosus的耐高温α-淀粉酶基因在衣藻叶绿体中的表达

来源于Pyrococcusfuriosus的耐高温α-淀粉酶是一种重要的酒精工业用酶,在植物中表达耐高温α-淀粉酶可以大大降低用植物秸秆生产酒精的成本。选择衣藻叶绿体基因组同源片段clpP-trnL-petB-chlL-rpl23-rpl2和壮观霉素抗性基因,构建了来源于Pyrococcusfuriosus的耐高温α-淀粉酶基因的衣藻叶绿体表达载体p64A。通过基因枪将其导入衣藻叶绿体中,经壮观霉素抗性(100mg/L)筛选,获得了9个抗性衣藻转化子。转化子经过抗性继代筛选后,经PCR、Southernblot检测分析及暗培养,证实耐高温α-淀粉酶基因已整合到衣藻叶绿体基因组中并得到表达。酶活性检测表明,转基因衣藻表达产物具有耐高温α-淀粉酶活性,每克鲜重衣藻最高达77.5u。实验结果证明在植物叶绿体中表达工业酶制剂是可行的。     

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.     

OBPC Symposium: Maize 2004 &; beyond—Recent advances in chloroplast genetic engineering

Summary The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrols) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

高等植物叶绿体表达重组蛋白研究进展

近年来,基因工程技术发展迅速,许多重组蛋白得以表达。其中利用植物生物反应器表达特异药物蛋白为人类一些重要疾病的预防和治疗提供了新途径。植物叶绿体遗传转化和表达系统成为目前植物生物反应器的研究热点。因结构和遗传上的特殊性,高等植物叶绿体在重组蛋白表达方面具有独特优势,外源基因表达量高、定点整合,而且叶绿体母系遗传特性保证了生物安全性。很多重要药用蛋白质在植物叶绿体中表达成功。烟草作为高等植物叶绿体转化模式植物,在疫苗抗原、抗体等药物蛋白和其他重要重组蛋白表达方面取得显著进展。高等植物叶绿体遗传转化也为叶绿体基因的表达和调控机制的研究提供新的技术和方法。文中从叶绿体遗传转化原理、载体构建、重组蛋白和重要药物蛋白在叶绿体中的表达以及重组蛋白表达对植物代谢和性状影响等多个角度,对高等植物叶绿体遗传转化体系研究的新进展进行了综述,以期为叶绿体表达平台的开发和重要药用蛋白质的表达提供新思路。     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast

A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark. The CTBVP1 fusion protein was expressed in C. reinhardtii chloroplast and accounted for up to 3% of the total soluble protein. The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins. These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.     

Tissue-specific,light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.     

Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella

Chloroplast genetic engineering is an environmentally friendly approach, where the foreign integrated gene is often expressed at a higher level than nuclear transformation. The cry1Ab gene was successfully transferred into the cabbage chloroplast genome in this study. The aadA and cry1Ab genes were inserted into the pASCC201 vector and driven by the prrn promoter. The cabbage-specific plastid vectors were transferred into the chloroplasts of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by their resistance to spectinomycin and streptomycin. According to antibiotic selection, the regeneration percentage of the two cabbage cultivars was 4-5%. The results of PCR, Southern, Northern hybridization and western analyses indicated that the aadA and cry1Ab genes were not only successfully integrated into the chloroplast genome, but functionally expressed at the mRNA and protein level. Expression of Cry1Ab protein was detected in the range of 4.8-11.1% of total soluble protein in transgenic mature leaves of the two species. Insecticidal effects on Plutella xylostella were also demonstrated in cry1Ab transformed cabbage. The objectives of this study were to establish a gene transformation system for Brassica chloroplasts, and to study the possibility for insect-resistance in dicot vegetables using chloroplast gene transformation.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.     

Overexpressions of dihydroxyacetone synthase and dihydroxyacetone kinase in chloroplasts install a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco using modified Gateway entry vectors

Dihydroxyacetone synthase (DAS) and dihydroxyacetone kinase (DAK) are two key enzymes for formaldehyde assimilation in methylotrophic yeasts. In order to using a Gateway LR recombination reaction to construct a plant expression vector that contains the expression cassettes for the das and dak genes and allow the proteins encoded by the two target genes to be localized to the chloroplasts of transgenic plants, the entry vector pEN-L4*-PrbcS-*T-gfp-L3* contained the tomato rbcS 3C promoter (PrbcS) with its transit peptide sequence (*T) and a GFP reporter gene (gfp) was constructed in this study. To verify the applicability of pEN-L4*-PrbcS-*T-gfp-L3*, we generated an entry vector for the dak gene by replacing the gfp gene in this entry vector with the dak gene. We also generated an entry vector for the das gene by replacing the gus gene in another entry vector (pENTR*-PrbcS-*T-gus) with the das gene. Using these entry vectors and pK7m34GW2-8m21GW3, we successfully constructed the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus and the pKm-35S-PrbcS-*T-dak-PROLD-PrbcS-*T-das expression vectors. Our results showed that high expression of GUS was achieved in leaves, and the expressed GFP, DAS and DAK proteins could be targeted to the chloroplasts after the two expression vectors were used to transform tobacco. The overexpressions of DAS and DAK in the chloroplasts successfully created a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco. By utilizing these expression vectors, we not only successfully expressed two target genes with one transformation but also localized the expressed proteins to chloroplasts via the transit peptide sequence (*T). Therefore, the construction of pEN-L4*-PrbcS-*T-gfp-L3* establishes a technique platform that provides a convenient means for chloroplast genetic engineering.     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明, 模式植物拟南芥叶绿体中含有大约4 000多种蛋白质, 目前只分离得到1 000多种, 其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5'端长178 bp的DNA片段, 与绿色荧光蛋白(GFP)基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察, GFP只在叶绿体中特异表达。实验结果表明, AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明,模式植物拟南芥叶绿体中含有大约4000多种蛋白质,目前只分离得到1000多种,其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5端长178bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察,GFP只在叶绿体中特异表达。实验结果表明,AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。