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He Y.,Chinese Academy of Agricultural Sciences | He Y.,National Citrus Engineering Research Center | He Y.,National Center for Citrus Variety Improvement | Chen S.,Chinese Academy of Agricultural Sciences | And 20 more authors.
Scientia Horticulturae | Year: 2011

Transgenic plants of Jincheng orange (Citrus sinensis Osbeck) and Newhall navel orange (C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange (Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease. © 2011 Elsevier B.V.


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.,Southwest University | Jiao B.,National Citrus Engineering Research Center | Tang H.,Wuhan University
RSC Advances | Year: 2014

As a new, water-soluble material, graphene oxide (GO) has gained growing interest for sensing applications. Particularly interesting is the interaction of nucleic acids with GO. Recently, it was found that short single-stranded DNA (ssDNA) had weaker affinity to GO than long ssDNA. This property makes it possible to prepare a novel bioassay platform for metal ions, antibiotics, and nuclease detection via the DNA(RNA) cleavage reaction. While practical analytical applications have been successfully demonstrated, few studies are focused on the mechanism of this phenomenon. In this work, we use fluorescence spectroscopy to deeply investigate the binding mechanism of ssDNA with GO to reveal the reason for this affinity difference caused by DNA length. Through computing with literature models, the main binding force, the binding constant, and number of binding sites between ssDNA and GO are obtained. Besides, our results show that the binding constant of short ssDNA with GO is much lower than that of long ssDNA with GO, which is the strongest evidence to prove the affinity difference between short ssDNA and long ssDNA with GO. Finally, based on these basic understandings of the interaction between ssDNA and GO, we develop a GO based biosensor for S1 nuclease and an inhibitor of S1 nuclease with satisfying results. © 2014 The Partner Organisations.


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.,Southwest University | Jiao B.,National Citrus Engineering Research Center
RSC Advances | Year: 2014

An ultrasensitive and straightforward fluorescent sensing platform for S1 nuclease activity has been developed based on S1 nuclease-induced DNA strand scission and the difference in affinity of cytochrome c (Cyt c) for single-stranded DNA (ssDNA) containing different numbers of bases. This sensing platform consists of a negatively charged fluorophore carboxyfluorescein (FAM) labeled 20-mer ssDNA (20F) and a positively charged protein Cyt c. In the absence of S1 nuclease, the electrostatic and hydrophobic interaction between 20F and Cyt c makes the FAM be in close proximity to Cyt c, resulting in efficient quenching of the fluorescence of FAM. Conversely, and very importantly, in the presence of S1 nuclease, it cleaves 20F into small fragments. The introduction of Cyt c into the sensing solution results in weak quenching of the fluorescence of FAM due to the relatively weak electrostatic interaction between the fragmented ssDNA chains and Cyt c, making the FAM move away from Cyt c, and thus the fluorescence intensity gradually increases with increasing concentration of S1 nuclease. S1 nuclease can be detected in a range of 4.0 × 10-3 to 4.0 × 10-2 units per mL with a detection limit of 8.3 × 10-4 units per mL and good selectivity. Moreover, the sensing system is used for the detection of an inhibitor of S1 nuclease with satisfying results. © 2014 the Partner Organisations.


Zhang W.-W.,Chinese Academy of Agricultural Sciences | Zhang W.-W.,Southwest University | Fu X.-Z.,Chinese Academy of Agricultural Sciences | Fu X.-Z.,National Citrus Engineering Research Center | And 12 more authors.
Scientia Horticulturae | Year: 2013

Many studies have been performed to evaluate the effects of sink demand on photosynthesis using artificial defruiting experiments. However, information is scarce regarding photosynthesis changes in field citrus trees with naturally varying sink demands. In this study, we compared photosynthesis in three types of shoots of 'Newhall' navel oranges (Citrus sinensis Osbeck), including no fruit bearing shoots, one-fruit bearing shoots and two-fruit bearing shoots, in field conditions. The results showed that the highest sink demand of shoots corresponded to the lowest net photosynthetic rate (Pn). The determination of stomatal conductance (Gs), substomatal CO2 concentration (Ci) and transpiration rate (E) suggested that the Gs and E were not significantly different between these three types of shoots, while the Ci increased as the sink demand increased. Further analysis of leaf morphology features and nutrient status showed that the higher fruit load shoots had smaller leaf area, length, width, thickness and dry weight, and also lower concentrations of nitrogen, phosphorous and potassium. The quality comparison of fruits on one-fruit bearing shoots and two-fruit bearing shoots suggested that there was no difference in internal quality such as total soluble solids (TSS), titratable acidity (TA) and the TSS/TA ratio except for some external features. Overall, our data indicated that the reduction of Pn with sink demand increasing was mainly caused by limited development and a lower nutritional supply in source leaves. This limitation may be derived from competition between fruit and adjacent leaves under a high fruit load. This finding provides new insights into sink demand effects on photosynthesis in field conditions and useful guidance for citrus orchard management practices. © 2012 Elsevier B.V.


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.-N.,Southwest University | Jiao B.-N.,National Citrus Engineering Research Center
RSC Advances | Year: 2015

In this work, a novel fluorescent method for protein detection has been developed based on terminal protection of small molecule-linked DNA by target protein and the difference in affinity of graphene oxide (GO) for single-stranded DNA (ssDNA) containing different numbers of bases in length. A probe ssDNA, which is labeled carboxyfluorescein (FAM) at the 5′ end and a small molecule at the 3′ end, is designed for the detection of target protein. In the absence of target protein, the probe ssDNA can be hydrolyzed into mononucleotides by Exo I. The introduction of GO into the sensing solution results in weak quenching of the fluorescence of FAM due to the weak affinity of the short FAM-labeled oligonucleotide fragment to GO. Conversely, and very importantly, in the presence of target protein, the specifically binding of target protein to the small molecule of probe ssDNA can protect probe ssDNA from the Exo I-catalyzed digestion. Then the adsorption of the probe ssDNA on GO makes FAM close proximity to GO surface resulting in high efficiency quenching of fluorescence of FAM, and the fluorescence intensity gradually decreases with increasing concentration of target protein. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 1 to 80 ng mL-1 with a detection limit of 0.81 ng mL-1. Besides satisfactory sensitivity, the developed strategy also shows high selectivity, excellent reproducibility, and low cost, implying that this technique may have great potential applications in the future. © The Royal Society of Chemistry.


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.,Southwest University | Jiao B.,National Citrus Engineering Research Center
RSC Advances | Year: 2015

As the extracellular nuclease of Staphylococcus aureus (S. aureus), micrococcal nuclease (MNase), which preferentially digests single-stranded nucleic acids, can be used as the standard to identify S. aureus and can be used to evaluate the pathogenicity of S. aureus. So the assay of MNase is of high importance. However, traditional methods for the assay of MNase activity have intrinsic limitations such as sophisticated synthesis processes, the need for functionalizing thiol (or dye)-modified oligonucleotide probes or critical operation conditions. Herein, a simple and convenient fluorescence sensing platform for MNase activity has been developed based on N-methyl mesoporphyrin IX (NMM)/G-quadruplexes. In the absence of MNase, the G-rich single stranded DNA (ssDNA) folds into a quadruplex in the presence of monovalent ions, thus greatly enhancing the fluorescence of NMM (a specific G-quadruplex binder). In the presence of MNase, the G-rich ssDNA was digested into small fragments. As a result, the fluorescence of the solution decreases with increase of MNase activity. Under the optimized conditions, the fluorescence intensity exhibits a linear correlation to MNase concentration in a wide range of 1.2 × 10-4-2.4 × 10-3 units per mL with a detection limit of 7.1 × 10-5 units per mL and good selectivity. The detection limit is much better or at least comparable to previous reports. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays. © The Royal Society of Chemistry 2015.


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.,Southwest University | Jiao B.,National Citrus Engineering Research Center
Microchimica Acta | Year: 2016

It is known that the binding of certain proteins to small molecules in ssDNA/small-molecule chimeras protects the conjugated ssDNA from degradation by exonuclease I (Exo I). This has resulted in numerous methods to specifically detect the interaction between small molecules and proteins. We are presenting here an approach that utilizes the terminal protection strategy in combination with the formation of ssDNA-templated silver nanoclusters (AgNCs), thereby providing a fluorometric tool for the detection of such interactions. A C-rich ssDNA (type 5′-CCCCACCCCT-3′) was labelled with biotin at the 3′ end. In the absence of streptavidin (SA), the biotinylated ssDNA is hydrolyzed in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the AgNCs is prevented due to the lack of the DNA scaffold, and this results in weak fluorescence. Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. As a result, fluorescent AgNCs are being formed. Fluorescence is measured at excitation/emission wavelengths of 625/705 nm. The calibration plot for SA is linear in the 6 to 600 nM concentration range, with a 2.6 nM detection limit. The assay is simple, sensitive and affordable. Conceivably, the method may also be used to detect the binding of other small molecules to proteins. [Figure not available: see fulltext.] © 2016 Springer-Verlag Wien


He Y.,Southwest University | He Y.,National Citrus Engineering Research Center | Jiao B.,Southwest University | Jiao B.,National Citrus Engineering Research Center
Microchimica Acta | Year: 2016

Small molecule–protein interaction plays a critical role in a majority of chemical genetics, clinical diagnostics, and drug development. Herein, by using biotin–streptavidin interaction assay as a model, we describe a strategy for simple and accurately determining streptavidin (SA) activity by means of a coupled terminal protection strategy and G-quadruplex-based platform. A G-rich ssDNA [G3(T4G3)3] with a tail (T12) at the 3′ end (5′-GGGTTTTGGGTTTTGGGTTTTGGGTTTTTTTTTTTT-3′) has been adopted. Biotin was labeled at the 3′ end of the ssDNA. In the absence of streptavidin (SA), the biotinylated ssDNA is digested in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the G-quadruplex is prohibited due to the lack of the G-rich ssDNA, and this results in weak fluorescence of N-methyl mesoporphyrin IX (NMM). Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. Afterward, the G-rich region of the ssDNA folds into a stable G-quadruplex in the presence of potassium ions, thus strongly enhancing the fluorescence of NMM. This assay has a wide linear range and low detection limit. The performance of SA detection in 10 % serum sample has also been investigated. The method not only provides a universal platform for monitoring small molecule–protein interaction but also shows great potential in chemistry, biology, and medicine. [Figure not available: see fulltext.] © 2016 Springer-Verlag Wien


Li X.,Southwest University | Yi S.,Southwest University | Yi S.,National Citrus Engineering Research Center | Zheng Y.,Southwest University | And 3 more authors.
Intelligent Automation and Soft Computing | Year: 2015

To develop a quick, accurate and reliable technique for studying phylogenetic relationship of Citrus, FTIR (Fourier transform infrared spectroscopy) technique was used. The petals spectra of eighteen varieties of citrus germplasms were investigated by FTIR. Pretreatment methods of raw spectra (2000–500 cm − 1) were composed of baseline correction, normalize and first derivative (Savitzky-Golay). We used One-way ANOVA (analysis of variance) and Tukey’s HSD (honestly significant difference) to extract effective wave bands, where the spectral absorbance values of different citrus germplasms were significantly different. The results showed that 2000∼1831 cm − 1, 1763∼1595 cm − 1, 1517∼1090 cm − 1, 1035∼1024 cm − 1, 950∼935 cm − 1, 861∼784 cm − 1, 744∼721 cm − 1 and 653∼608 cm − 1 were the effective wave bands. HCA (hierarchical cluster analysis) was adopted to classify citrus germplasms based on the above eight effective wave bands. It was found that eighteen citrus varieties were classified into six subgroups. The results of classification and citrus phylogenetic relationships between six subgroups were consistent of results from Morphology, Biochemistry, Cytology and Molecular Biology. The overall results demonstrated that fourier transform infrared spectroscopy technique with One-way ANOVA and Tukey’s HSD and hierarchical cluster analysis model were promising for the rapid, accurate and reliable classification for citrus as well as studying citrus phylogenetic relationship. © 2015, © 2015 TSI® Press.


Zhu S.,Chinese Academy of Agricultural Sciences | Zhu S.,National Citrus Engineering Research Center | Wang F.,Chinese Academy of Agricultural Sciences | Shen W.,Chinese Academy of Agricultural Sciences | And 6 more authors.
Acta Physiologiae Plantarum | Year: 2015

The polymorphic markers of nuclear SSR (simple sequence repeat), SNP (single nucleotide polymorphism)/InDel (insertion–deletion length polymorphism), and cpSSR (chloroplast SSR) were used to evaluate the genetic diversity of 52 trifoliate oranges and their relatives. The two genomic DNA-based markers produced high values of PIC (polymorphic information content) at 0.45 for SNP/InDel and 0.32 for nuclear SSR, indicating that there is a large diversity of genetic variation within the trifoliate oranges. Three UPGMA (unweighted pair-group method with arithmetic means) dendrograms were generated based on the results of three marker systems. At 0.76 of coefficient, all of Poncirus accessions were clustered into one group, except the 74-1 Early Poncirus, one of the three precocious trifoliate oranges, which was placed separately into another group by nuclear SSR. At a higher coefficient, the trifoliate oranges were discriminated into 4, 6 and 6 groups by nuclear SSR (0.86), SNP/InDel (0.95), and cpSSR (0.95), respectively. The results revealed that three precocious trifoliate oranges were originated from different sources. Hubei Early Poncirus and Beibei Early Poncirus were the asexual mutants of different trifoliate oranges, and 74-1 was probably a sexual hybrid. The present study also indicated that ‘Zhiwenzhou,’ a diploid chimera of trifoliate orange and Satsuma is more closely related to Satsuma rather than to trifoliate orange, which is in concordance with the results of morphological observation. © 2015, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.

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