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ABSTRACT: Previously, a suppression subtractive hybridization library was constructed to identify differentially expressed genes in
peel pitting of navel orange fruit and a cDNA fragment sharing high similarities to cysteine protease genes was identified.
In this study, we cloned its full-length cDNA sequence, designated CsCP, using the Rapid amplification of cDNA ends approach. It consists of 1,409 nucleotides and its ORF encodes 361 amino acids
predicted to have an N-terminal signal peptide. Phylogenetic analysis revealed that CsCP belonged to the aleurain group in
papain family of cysteine proteases. According to quantitative RT-PCR, the expression of CsCP was enhanced during the development of postharvest peel pitting concomitant with senescence, although it was detectable in
all tested tissues including root, leaf, flower and peel of fruit. RNA gel blot analysis showed that the CsCP expression was induced by hypoxia (3% O2), but repressed by anoxia (0% O2), wounding, ethylene and high temperature (40°C). Conclusively, the CsCP is a senescence-associated gene and up-regulated during the development of citrus postharvest peel pitting, which provides
a basis to understand its role in citrus peel pitting.
Plant Cell Tissue and Organ Culture 04/2012; 98(3):281-289. · 3.09 Impact Factor
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ABSTRACT: In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified
as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816bp. The
calculated molecular weight of the mature protein was 27.05kDa and theoretical isoelectric point was 7.93. The deduced protein
contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues
in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C),
and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since
low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results
provided us a clue that CsEXP may play a role in response to peel pitting related stresses.
Plant Growth Regulation 04/2012; 59(1):13-19. · 1.60 Impact Factor
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ABSTRACT: A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70 degrees C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28 degrees C (pH 6.0) in flasks stirred at 150 x g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.
Indian journal of biochemistry & biophysics 12/2010; 47(6):348-52. · 1.14 Impact Factor
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ABSTRACT: This study was carried out to determine the changes of volatile compounds composition during pickled mustard tuber production. The samples were collected at different pickling stages from the traditional industry production way in Chongqing, China. Gas chromatography–mass spectrometry were performed after simultaneous distillation and extraction. The results showed that ITC (isothiocyanates) possessed the highest relative percentage area (RPA) 78.87%, followed by sulphide compounds with RPA of 14.48%. Main volatile compounds that determined were allyl ITC, dimethyl trisulphide, (2-isothiocyanatoethyl)-benzene, n-Hexadecanoic acid and linolenic acid ethyl ester, which were responsible for the hot and pungent flavour of pickled mustard tuber. The sulphides, acids, aldehydes, alcohols, phenols, esters, nitriles and heterocyclic compounds increase with pickling time but ITC increase at the first and second pickling stage, then start to decrease in the last pickling stage. It was also demonstrated that more than 60-day pickling time was necessary for the formation of the typical aroma notes.
International Journal of Food Science & Technology 10/2009; 44(11):2278 - 2286. · 1.26 Impact Factor
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ABSTRACT: Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.
Bioscience Biotechnology and Biochemistry 08/2009; 73(7):1541-9. · 1.28 Impact Factor
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ABSTRACT: A cDNA subtraction library had been constructed to identify differentially expressed genes in peel pitting of citrus fruit.
Based on the sequence of a cDNA fragment homologous to NAC gene family, the full-length cDNA of 1,203 nucleotides was cloned from “navel” orange by rapid amplification of cDNA ends.
It was designated as CsNAC, encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2kDa, and theoretical
isoelectric point was 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at the N terminus,
which is considered as the characteristic of the NAC protein family. CsNAC protein was orthologous to Arabidopsis
thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to
stress stimuli. RNA gel blot analysis showed that the expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4°C) and exposure to ethylene
also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40°C) but not affected by low oxygen (3%). Our results
may provide the basis for future research of NAC-like gene’s role in stress-induced citrus peel pitting.
Plant Molecular Biology Reporter 11/2007; 25(3):145-153. · 2.45 Impact Factor
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ABSTRACT: In this study, the pitted peel and non-pitted peel of 'Fengjie' navel orange fruits were used as experimental materials to construct and screen the peel pitting related genes by suppression subtractive hybridization (SSH). The results showed that suppression subtractive hybridization was very effective. A cDNA library of differentially expressed genes was constructed. The library included about 200 clones with an average insert size of around 300 bp. Part of the positive clones were picked up randomly and sequenced. Six of the 50 clones had no homologous sequences being found and three had unknown functions in GenBank. According to the analysis of the homology, four homologous (Ca2+ binding protein, cysteine proteinase, NAC-domain protein and expansin) genes were chosen to examine their expressions through semi-quantitative RT-PCR analysis in pitted and non-pitted navel orange fruits. The expression of four genes were all higher in pitted peel than that in non-pitted peel. It suggests that these genes in the SSH cDNA library may be involved with peel pitting and can be subject of future investigation to explore the molecular biological mechanism of the pitting of citrus fruit.
Zhi wu sheng li yu fen zi sheng wu xue xue bao = Journal of plant physiology and molecular biology 03/2007; 33(1):71-6.
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ABSTRACT: Citrus fruit is prone to develop peel pitting during development and storage, which greatly decreases its fresh market value because of the deterioration of the peel. In the present study, we have examined the effect of different temperatures (15 degrees C and 4 degrees C), waxing and mechanical damage on the changes in the activity of phenylalanine ammonia-lyase (PAL) and the incidence of peel pitting in 'Fengjie' navel orange (Citrus sinensis Osbeck) fruits. The expression levels of PAL2, PAL6 genes in the peel during the development of peel pitting have been investigated through semi-quantitative PCR method. The incidence of peel pitting was greatly enhanced by waxing and mechanical damage and was decreased in lower temperature storage (4 degrees C) (Fig.1). Waxing and mechanical damage might be the important factors inducing peel pitting and suitable low temperature could decrease the incidence of this disease. The PAL activity increased during the whole storage period in accordance with the development of this pitting (Fig.2). The expression levels of PAL2 and PAL6 genes in damaged peel were higher than those in healthy peel and the expression of PAL2 is much more higher than that of PAL6 (Figs.4 and 5). The results suggested that the enzyme activity of PAL, along with the expression of PAL2 gene is highly related to this peel pitting occurred on 'Fengjie' navel orange fruits.
Zhi wu sheng li yu fen zi sheng wu xue xue bao = Journal of plant physiology and molecular biology 07/2006; 32(3):381-6.
