Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.     

A STUDY OF THE CINNAMON-BREASTED ROCK BUNTING Fringillaria tahapisi tehepisi

Brown, C. J. &; Piper, S. E. 1988. Status of Cape Vultures in the Natal Drakensberg and their cliff site selection. Ostrich 59:126-136.

Ground, aerial and questionnaire surveys on the status of the Cape Vulture Gyps coprotheres in the Little and High Drakensberg mountain ranges of Natal were carried out from 1981–1983. The area supported at least 1325 Cape Vultures, 60% on the High Drakensberg in 17 nesting colonies (mean of 31 birds per colony) and 38 roosts (mean of 7 birds). In the Little Drakensberg 17 sites were found, six of which were confirmed nesting colonies (mean of 53 birds) and seven were roosts (mean of 16 birds). A minimum of 215 nests was recorded while the actual number of breeding pairs was probably about 325. In the Natal Drakensberg 84% of nests and 77% of roosting birds occupied ledges that faced between east and south, on the lee side of the generally westerly and northwesterly “Bergwind”. Nesting ledges in the High Drakensberg were on average about 100 m lower than roosting ledges. The northern Drakensberg supported the largest numbers of Cape Vultures (19 birds per 10 km of cliffs), while the smallest numbers were recorded in the south (8 birds per 10 km of cliffs). In the south smallstock farming predominates and the availability of carrion is higher than in the north where mainly largestock are farmed. The use of poisons (mainly strychnine) for killing mammalian predators on farms is considered to be the main reason for fewer birds occurring in the south, and is the main threat to Cape Vultures in the Natal Drakensberg.     

Distribution and speciation of Mn in hydrated roots of cowpea at levels inhibiting root growth

The phytotoxicity of Mn is important globally due to its increased solubility in acid or waterlogged soils. Short‐term (≤24 h) solution culture studies with 150 µM Mn were conducted to investigate the in situ distribution and speciation of Mn in apical tissues of hydrated roots of cowpea [Vigna unguiculata (L.) Walp. cv. Red Caloona] using synchrotron‐based techniques. Accumulation of Mn was rapid; exposure to 150 µM Mn for only 5 min resulting in substantial Mn accumulation in the root cap and associated mucigel. The highest tissue concentrations of Mn were in the root cap, with linear combination fitting of the data suggesting that ≥80% of this Mn(II) was associated with citrate. Interestingly, although the primary site of Mn toxicity is typically the shoots, concentrations of Mn in the stele of the root were not noticeably higher than in the surrounding cortical tissues in the short‐term (≤24 h). The data provided here from the in situ analyses of hydrated roots exposed to excess Mn are, to our knowledge, the first of this type to be reported for Mn and provide important information regarding plant responses to high Mn in the rooting environment.     

Predicting zinc bioavailability to wheat improves by integrating pH dependent nonlinear root surface adsorption

Aim

Our aim was to improve the prediction of Zn bioavailability to wheat grown on low-Zn soils. The classical approach that directly relates Zn in a certain soil extract to Zn uptake has been shown to be inadequate in many cases. We tested a stepwise approach where the steps of the uptake process are characterized with, respectively, Zn solid-solution distribution, adsorption of Zn to root surface, Zn uptake into root and Zn translocation to shoot.

Methods

Two pot experiments were done with wheat grown on nine low-Zn soils varying widely in pH, clay and organic matter content. Soluble Zn concentrations in two soil extracts (DTPA and CaCl₂) were measured. Free Zn ion concentrations in CaCl₂ soil extracts were determined with the Donnan Membrane Technique. These Zn concentrations were then related to plant Zn uptake following both the direct and the stepwise approach.

Results

In the direct approach, Zn in the DTPA extract was a better predictor for shoot Zn uptake than Zn in the CaCl₂ extract. In the stepwise approach, the relationship between Zn in CaCl₂ extracts and the root surface adsorbed Zn was pH-dependent and nonlinear. Root surface adsorbed Zn was linearly related to root Zn uptake, and the latter was linearly related to the shoot Zn uptake. The stepwise approach improved the Zn uptake prediction compared to the direct approach and was also validated for different wheat cultivars.

Conclusions

The adsorption of Zn on the root surface is pH dependent and nonlinear with respect to the soil Zn concentration, and a useful proxy for bioavailable Zn over a wide range of soils.     

Using an Activity to Simulate the Dangers of Multitasking with Technology while Walking

People are increasingly trying to multitask while walking. Text messaging while walking is a significant area for concern. The number of text messages sent is expected to be more than 8 trillion in 2012. Texting is becoming so commonplace that people use this technology while engaged in other activities. The dangers of multitasking have hit the streets as people are becoming involved in more accidents while walking as they text on mobile phones. This simulation activity measures a student's ability to multitask by texting and walking. The activity simulates walking in the real world and is best suited for middle school students. Additional activities, after completing the simulation, will challenge middle school students to learn more about multitasking globally through scientific inquiry researched on the Internet. The students will use language arts skills to generate a report or presentation to share with classmates. Their findings will give the entire class a cultural perspective on accident statistical comparisons between different countries.     

Addressing STEM Concepts through a Food Safety Activity

The activity described in this article engages students in learning about proper meat storage procedures by using the Internet. They apply their knowledge practically through creation of a pamphlet to be displayed in their kitchen at home and a follow-up report on their parents’ kitchen procedures; they may also participate in a classic food storage experiment. This activity is well suited for elementary school children in the second through fourth grades and promotes all basic STEM (science, technology, engineering, and math) concepts. Kitchen activities require all these concepts for successful and safe outcomes. The examination of food storage devices references engineering, and technology is used for the research and pamphlet creation. The section on bacteria introduces life sciences, and physical sciences come into play through the discussion of heat inducing change in the meats. Math skills are integral to the reference table containing temperatures and times.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.