Construction Of Binary Vector Research Articles

Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

Genomic analysis and development of infectious clone of a novel carlavirus infecting blueberry.

A new blueberry virus was discovered using high throughput sequencing (HTS). Using sequence identity values, phylogenetics, serological and biological properties, we propose the virus, putatively named blueberry virus S (BluVS) to be a distinct species within the genus Carlavirus (family Betaflexiviridae). The genome was analyzed in depth and an infectious clone was developed to initiate studies on virus pathogenicity. Agroinfiltration of the binary vector construct produced severe systemic symptoms in Nicotiana occidentalis. Back inoculation using sap from agroinfiltrated N. occidentalis produced identical symptoms to the recipient plants (N. occidentalis) and virus purification yielded flexuous carlavirus-like particles. However, unlike BlScV, BluVS caused symptomless infection in Chenopodium quinoa and reacted weakly to BlScV antibodies in enzyme-linked immunosorbent assay (ELISA). Collectively those results provided evidence for the distinct speciation of BluVS. The availability of an infectious clone provides tools for future studies on the biology of the virus.

Targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes in tomato confers resistance to parasitic weed Phelipanche aegyptiaca.

Phelipanche aegyptiaca and Orobanche spp. are obligate plant root-parasitic weeds that cause extensive damage in agricultural crop plants. Their germination requires exposure to strigolactones (SLs) exuded by the host plant roots. Here we studied genes in the host plant tomato involved in SL exudation and their impact on parasitic weeds. We provide evidence that CRISPR/Cas9-mediated targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes, ABCG44 (Solyc08g067610) and ABCG45 (Solyc08g067620), in tomato significantly reduces SLs in the root exudate and abolishes germination of the root-parasitic weed P. aegyptiaca. Based on genome sequence similarity between ABCG44 and ABCG45, a 20-bp target sequence in their exon region was selected to design single guide RNA targeting both genes using CRISPR/Cas9. The plant binary vector constructs harboring the specific Cas9 and single guide RNA were transformed into tomato. Selected T0 mutated tomato plants showed different types of deletions at both gene loci. Genotype analysis of T1 plants suggested stable inheritance of the introduced mutations without any potential off-target effects. The phenotype of Cas9-mutated plants included increased shoot branching and growth of axillary buds, and reduced length of primary stems. Interestingly, reduced germination of P. aegyptiaca resulted from a decrease in the SL orobanchol in the root exudate of Cas9-mutated plants; however, orobanchol content in the root extract was unchanged compared to control plants. Moreover, in single and double ABCG mutants, expression of the SL-biosynthesis genes CCD8 and MAX1 decreased. The current study offers insights into CRISPR-mediated mutagenesis of ABCG genes, which could serve as an efficient control method to prevent root-parasitic weed germination.

Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method

<p>Cold shock protein (Csp) is an essential bacterial protein for increasing abiotic stress tolerance, especially cold stress. Several studies discovered that overexpression of the gene successfully improves the tolerances of several types of plant not only under cold stress, but also other abiotic stresses, e.g. hot and drought conditions. The objectives of this study were to construct a binary vector containing the LcCsp gene modified from Lactobacillus casei and transform the gene into Nipponbare rice genome. The native LcCsp gene sequence, however, has low GC content (46.7%) while rice as transformation target plant has 52% GC content. The native LcCsp gene sequence, therefore, was optimized to the level close to 52.7% similar to GC content of the rice genome. This LcCsp gene was synthesized by using DNA printing technology (gBlocks® Gene Fragments Entry, IDT). The synthetic LcCsp gene was successfully inserted into the pCAMBIA1300-int binary vector driven by Ubiquitin1 promoter and NOS terminator. The T-DNA cassette was successfully transformed into Nipponbare rice genome by Agrobacterium tumefaciens strain LBA4404 using immature embryo transformation protocol. A total of 51 T0 Nipponbare lines survived from hygromycin selections and 21 lines were successfully acclimatized. Molecular analysis of the candidate lines showed that all Nipponbare transgenic putative lines contain the LcCsp gene demonstrating high transformation efficiency of 11.8%. The rice lines resulted from this study should be further analyzed and might be useful for developing rice transgenic lines tolerance to heat, drought, or saline stress condition.</p>

IN GEL DETECTION OF A HIS-TAGGED TRANSGENE FOLLOWING THE SEPARATION OF CRUDE PLANT PROTEIN EXTRACTS WITH SDS PAGE

In the present study, we visualised sundew His6-tagged chitinase protein in crude protein extracts of transgenic tobacco plants following protein separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis by detecting chitinase activity as well as the His-tag. A short sequence encoding six histidines was fused downstream of the DNA sequence encoding the last amino acid of the mature protein. Following binary vector construction and plant transformation, a set of 10 transgenic plants was analysed for transgene expression. Except for one, all transgenic plants exhibited the presence of sundew chitinase protein of ~52 kDa, which was different from the calculated molecular weight of ~32 kDa. Clear identification of DrChitHis protein was performed with a Ni2+:NTA complex conjugated to a fluorescent dye and visualized using light laser-based scanner. A subsequent endochitinolytic activity assay using a N-fluorescein-labelled chitin substrate confirmed that the two transgenic lines with the strongest expression of DrChitHis protein had endochitinolytic activity 6.4 and 6.7 times higher than non-transgenic control.

Transcription factor NnDREB1 from lotus improved drought tolerance in transgenic Arabidopsis thaliana

Dehydration responsive element binding factor (DREB) is believed to be a stress-tolerance enhancer in plants. In the present study, a cold-binding factor (CBF)/DREB homologous gene NnDREB1 (XP_010242642.1) was isolated from lotus roots using rapid amplification of cDNA ends (RACE) and reverse transcription (RT)-PCR methods. Analysis of the deduced amino acid sequence and phylogeny classified NnDREB1 into the A-1 group of the DREB1 subfamily. Expression profiling using a quantitative PCR method revealed that NnRDEB1 was significantly induced by NaCl, mannitol, and polyethylene glycol, but not by low temperature and abscisic acid. To evaluate function of NnRDEB1, Arabidopsis thaliana was transformed with the NnDREB1 gene in a binary vector construct. The transgenic plants exhibited higher resistance to drought compared with the wild-type plants in terms of survival rates, dry and fresh masses, and chlorophyll content. In addition, overexpression of NnDREB1 resulted in higher germination rates compared with the wild type plants on MS medium containing mannitol. The expressions of downstream target stressrelated genes, including cold-regulated15B (COR15B), rare cold inducible 2B (RCI2B) and repeat domain 26 (RD26), were activated in the transgenic plants. Taken together, the results suggest that NnDREB1 might be an important protein in lotus root drought tolerance.

Transgenic rice expressing the cry2AX1 gene confers resistance to multiple lepidopteran pests.

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34µgg(-1) leaf fresh weight and with 80-92% levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6ngg(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.

Overexpression of NnDREB2, isolated from lotus improves salt tolerance in transgenic Arabidopsis thaliana

DREBs are believed to participate in plant responses to adverse environmental factors by activating down-stream stress-inducible genes. In this study, we isolated an NnDREB2 from a salt-resistant lotus species using RT-PCR and RACE methods. Expression profiling by real-time PCR technique revealed that NnDREB2 enhanced transcriptional level when treated with NaCl and exogenous ABA, and while its expression was not significantly changed with mannitol or 4 °C treatments. NnDREB2 was transformed into Arabidopsis with a binary vector (SN1301) construct to identify its function. After selection of ‘positive’ transgenic plants, transgenic and non-transgenic plants (wild type plants) were treated with 250 mM NaCl. We found that the plants overexpressing NnDREB2 led to higher salt resistance than that of the wild type plants according to their survival rates. In addition, NnDREB2 expression exhibited higher germination rates and better root growth than in control plants on MS medium containing various concentration of NaCl. Down-stream target stress-related genes were also analyzed, and we observed that NnDREB2 overexpression activated stress-responsive genes such as PIP1-2, PIP2-5 and PIP2-7 in transgenic Arabidopsis plants. Totally, our findings suggested that the NnDREB2 participated in an ABA-dependent pathway, and might play an important role in plant for salt stress adaptation by directly binding with the DRE element tnemeto regulate down-stream gene expression in salt-resistant lotus.

Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision.

Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice.

Binary Vector Construction and Agrobacterium tumefaciens-mediated Transformation of Lysozyme Gene in Seaweed Kappaphycus alvarezii

Binary Vector Construction and Agrobacterium tumefaciens-mediated Transformation of Lysozyme Gene in Seaweed Kappaphycus alvarezii

Molecular cloning, characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice

The full-length cDNA encoding a glutamate dehydrogenase (GDH) which catalyzes the reaction of reductive amination of α-oxoglutarate (α-OG) to glutamate (the anabolic activity) and the reverse reaction of oxidative deamination of glutamate (the catabolic activity) was isolated from Sclerotinia sclerotiorum, we designated it as SsGDH. Bioinformatics analysis revealed that SsGDH had a typical GDH spatial structure and extensive homology with other fungal or bacteria GDHs. To evaluate its function in rice, rice (Oryza sativa L. cv. 'kitaake') was transformed with SsGDH in a binary vector construct by Agrobacterium-mediated transformation. Transgenic rice plants showed that transcripts and proteins of SsGDH accumulated at higher levels and GDH enzymatic activity was obviously higher in transgenic rice plants compared with the non-transformant rice plants (CK), though phenotype including plant height, fresh weight and dry weight became slightly weaker compared with CK under 50, 500 and 5,000 μM nitrogen gradient nutrient solution treatment (NH4NO3 as a nitrogen source) after introducing SsGDH into rice. For enzymatic activity assay in vitro, recombinant His6-SsGDH protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA agarose. Results suggested that recombinant His6-SsGDH protein had GDH activity using ammonium, α-OG, and L-glutamate separately as a substrate at two different concentrations, especially the affinity for ammonium was very high, and its Km value was only 0.28 ± 0.03 mM, indicating that SsGDH can assimilate more ammonium into rice. According to previous reports, transgenic plants expressing fungal or bacteria GDHs might show improved herbicide resistance. Basta resistance test showed that SsGDH expression in rice can significantly enhanced their tolerance to Basta than CK. In conclusion, our results may provide some clues for further investigation on nitrogen utilization via introducing exogenous GDHs from lower organisms into rice.

Binary Vector Construction and Agrobacterium tumefaciens-mediated Transformation of Lysozyme Gene in Seaweed Kappaphycus alvarezii

Ice-ice disease is the biggest problem in the cultivation of seaweed Kappaphycus alvarezii. The disease is caused by bacterial infection and induced by drastic changes of water quality. Lysozyme has the ability to break down bacterial cell wall. The purpose of this research was to construct of a binary vector pMSH1-Lys carrying chicken Lysozyme (Lis) gene and introduce pMSH1-Lis on K. alvarezii. The binary vector expression was transformed into Agrobacterium tumefaciens LBA4404 by triparental mating. Thallus was inoculated with A. tumefaciens carrying pMSH1-Lis and then the transformed thallus was selected by adding 20 mg/L hygromycin to the culture medium. PCR analysis showed that the construction of the binary plasmid pMSH1-Lys was established. Percentage of transformation of pMSH1-Lis on K. alvarezii was 23.56%, while the efficiency of regeneration was 11.32%. PCR analysis showed that three of the regenerated thallus contained Lysozyme gene. Thus, transgenic K. alvarezii was produced successfully and this can be useful for studying the mechanisms of seaweed defense against bacterial infection.

BioVector, a flexible system for gene specific-expression in plants

BackgroundFunctional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. The cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. Therefore, it is difficult both to assemble a gene and a promoter together and to modify the vectors in hand. Another disadvantage is that a long spacer from recombination sites, which may be detrimental to the protein function, exists between a gene and a tag. Multiple GATEWAY system only resolves former problem at the expense of very low efficiency and expensive for multiple LR reaction.ResultsTo improve efficiency and flexibility for constructing expression vectors, we developed a platform, BioVector, by combining classical restriction enzyme/ligase strategy with modern Gateway DNA recombination system. This system included a series of vectors for gene cloning, promoter cloning, and binary vector construction to meet various needs for plant functional genomic study.ConclusionThis BioVector platform makes it easy to construct any vectors to express a target gene from a specific promoter with desired intensity, and it is also waiting to be freely modified by researchers themselves for ongoing demands. This idea can also be transferred to the different fields including animal or yeast study.

Isolation and functional characterization of a salt responsive transcriptional factor, LrbZIP from lotus root (Nelumbo nucifera Gaertn)

Basic leucine zipper transcription factor (bZIP) is involved in signaling transduction for various stress responses. Here we reported a bZIP transcription factor (accession: JX887153) isolated from a salt-resistant lotus root using cDNA-AFLP approach with RT-PCR and RACE-PCR method. Full-length cDNA which consisted of a single open reading frame encoded a putative polypeptide of 488 amino acids. On the basis of 78, 76, and 75 % sequence similarity with the bZIPs from Medicago truncatula (XP_003596814.1), Carica papaya (ABS01351.1) and Arabidopsis thaliana (NP_563810.2), we designed it as LrbZIP. Semi quantitative RT-PCR results, performed on the total RNA extracted from tips of lotus root, showed that LrbZIP expression was increased with 250 mM NaCl treatment for 18 h. Effects of low temperature on the expression of LrbZIP was also studied, and its expression was significantly enhanced with a 4 °C treatment for 12 h. In addition, LrbZIP expression was strongly induced by treatment with exogenous 100 μM ABA. To evaluate its function across the species, tobacco (Nicotiana tabacum L.) was transformed with LrbZIP in a binary vector construct. Transgenic plants exhibited higher resistance as compared with the control according to the results of the root growth, chlorophyll content and electrolyte leakage when exposed to NaCl treatment. In addition, LrCDPK2, LrLEA, and TPP also showed enhanced expression in the transgenic plants. Overall, expression of LrbZIP was probably very important for salt-resistant lotus root to survive through salt stress.

Agroinoculation of the cloned infectious cDNAs of Lettuce chlorosis virus results in systemic plant infection and production of whitefly transmissible virions

Lettuce chlorosis virus (LCV) is a single stranded, positive strand RNA virus that is solely transmitted by specific whitefly vectors (Bemisia tabaci biotypes A and B) but not by mechanical leaf-rub inoculation. The roles of viral encoded proteins involved in the infection cycle of LCV have not yet been characterized due to the lack of reverse genetic tools. We present here a report of the successful development of an Agrobacterium-mediated inoculation system for the cloned cDNA constructs of LCV. The cDNAs of both LCV RNAs 1 and 2 were engineered into binary vectors in which the expression of LCV RNAs was regulated under a Cauliflower mosaic virus (CaMV) 35S promoter. In addition, by engineering the sequence elements of the Hepatitis delta virus ribozyme and the nopaline synthase 3′ untranslated region immediately downstream of the last nucleotide of LCV RNAs 1 and 2 in the binary vector constructs, the in planta produced LCV transcripts were expected to bear authentic 3′ termini. Both constructs were transformed into Agrobacterium tumefaciens cells and infiltrated in Nicotiana benthamiana plants. Three to four weeks post-agroinoculation, the N. benthamiana plants developed typical interveinal chlorosis and LCV infection was detected in the systemic leaves by reverse transcription-PCR. Virions purified from the LCV-infected N. benthamiana plants were flexuous rod-shaped and were transmissible by both B. tabaci biotypes A and B following membrane feeding. These results support the conclusion that Agrobacterium-mediated inoculation of LCV binary vectors in N. benthamiana plants results in LCV infection and the production of biologically active, whitefly transmissible virions. This system represents an important tool for use with reverse genetics designed for the study of LCV gene functions.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08% among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Functional validation of Capsicum frutescens aminotransferase gene involved in vanillylamine biosynthesis using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures

Capsaicinoid biosynthesis involves the participation of two substrates viz. vanillylamine and C9–C11 fatty acid moieties. Vanillylamine which is a derivative of vanillin is synthesized through a transaminase reaction in the phenylpropanoid pathway of capsaicinoid synthesis. Here we report the functional validation of earlier reported putative aminotransferase gene for vanillylamine biosynthesis in heterologous system using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures. Molecular analysis tools comprising PCR and Southern blot analysis have shown the integration of the foreign gene in N. tabacum and C. frutescens calli cultures. The study shows the production of vanillylamine in transformed N. tabacum callus cultures and also the reduction of vanillylamine production when whole gene based antisense binary vector construct was used in transformation of C. frutescens callus cultures. Vanillylamine production, aminotransferase assay with Western blot analysis for crude proteins of transformants established the production of putative aminotransferase (pAMT) protein in alternate plant. The result is a clear evidence of involvement of the reported putative aminotransferase responsible for vanillylamine biosynthesis in capsaicinoid biosynthesis pathway, confirming the gene function through functional validation.

Dicistronic binary vector system—A versatile tool for gene expression studies in cell cultures and plants

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase ( gus) gene followed by an internal ribosome entry site and a firefly luciferase ( luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (At nhx1). Comparative studies with At nhx1 transgenic luc expressing tobacco cell cultures and pea plants ( Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.

Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo

While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of β-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 μmol H2S mg−1 protein min−1 and 0.92 μmol Cys mg−1 protein min−1, respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification.

Construction of Double Right-Border Binary Vector Carrying Non-Host Gene Rxo1 Resistant to Bacterial Leaf Streak of Rice

Rxo1 cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6- Rxo1, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.