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Zhao D.,Guizhou University | Qin L.,Guizhou University | Sun H.,Guizhou University | Zhao J.,Guizhou Academy of Tobacco Science
Tobacco Science and Technology | Year: 2014

For revealing the function of CYP82E4v1 gene involving in nornicotine biosynthesis in Nicotiana tabacum L., an Agrobacterium tumefaciens-mediated transformation method was used to transform tobacco cv. K326 with two plant expression vectors, pLF-35S-CYP, which contained CYP82E4v1-overexpressing fragment, and pLF-35S-CYPi containing interfered expression fragment, respectively. Sixty-one transgenic plants were acquired, including forty-two overexpressing plants and nineteen interfered ones. The results of HPLC showed that the nornicotine content in CYP82E4v1-overexpressing plants was 88.9% higher than, that in interfered expression plants was 61.1% lower than that in the plants of cv. K326 (CK). It indicated that the overexpression of CYP82E4v1 increased nornicotine content in tobacco, whereas the interfered expression of CYP82E4v1 gene inhibited nornicotine biosynthesis. The results of Real-Time PCR analysis indicated that the expression level of CYP82E4v1 gene in overexpressing plants was over 20 times the level in the CK, while that in interfered expression plants was only 6% of that in the CK. Meanwhile, the other nornicotine biosynthesis genes, such as CYP82E5v2 and CYP82E10, were suppressed to different extents in interfered expression plants. In addition, the "Gene-deletor" system contained recombinase gene FLP driven by the promoter of Arabidopsis thaliana senescent gene SAG12, which deleted the exogenous gene GUS ∷ NPT II during the late stage of leaf development, was introduced into the mentioned expression fragments for the reason of acquisition of selectable-free transgenic tobacco plants with low nornicotine content. Source

Hai-Ming T.,Hunan Soil and Fertilizer Institute | Xiao-Ping X.,Hunan Soil and Fertilizer Institute | Wen-Guang T.,Hunan Soil and Fertilizer Institute | Ye-Chun L.,Guizhou Academy of Tobacco Science | And 2 more authors.
PloS one | Year: 2014

Residue management in cropping systems is useful to improve soil quality. However, the studies on the effects of residue management on the enzyme activities and microbial community of soils in South China are few. Therefore, the effects of incorporating winter cover crop residue with a double-cropping rice (Oryza sativa L.) system on soil enzyme activities and microbial community in Southern China fields were studied. The experiment has conducted at the experimental station of the Institute of Soil and Fertilizer Research, Hunan Academy of Agricultural Science, China since winter 2004. Four winter cropping systems were used: rice-rice-ryegrass (Lolium multiflorum L.) (R-R-Ry), rice-rice-Chinese milk vetch (Astragalus sinicus L.) (R-R-Mv), rice-rice-rape (Brassica napus L.) (R-R-Ra) and rice-rice with winter fallow (R-R-Fa). The result indicated that the enzyme activities in the R-R-Ry, R-R-Mv and R-R-Ra systems were significantly higher (P<0.05) than in the R-R-Fa system during the early and late rice season. The β-glucosidase activities reached peak values at the tillering stage after residue application, and alkaline phosphatase activities reached peak values at the booting stage after residue application, respectively, the activities of β-glucosidase and alkaline phosphatase gradually decreased after this. Arylsulfatase activities reached peak values at the maturity stage. Arylamidase activities reached peak values at the maturity stage. The numbers of aerobic bacteria, actinomycete and fungus of residue treatments were significantly higher (P<0.05) than that the R-R-Ra system. However, the number of anaerobic bacteria under the R-R-Ry and R-R-Mv systems was significantly lower (P<0.05) than that under the R-R-Fa system during early rice and late rice growth stage. Thus, incorporation of winter cover crops into rotations may increase enzyme activities and microbial community in soil and therefore improve soil quality. Source

Zhou X.-M.,Wuhan University | Zhao P.,Wuhan University | Wang W.,Wuhan University | Zou J.,Guizhou Academy of Tobacco Science | And 3 more authors.
DNA Research | Year: 2015

Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses. © 2015 The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. Source

Zhao P.,Wuhan University | Zhou X.-M.,Wuhan University | Zou J.,Guizhou Academy of Tobacco Science | Wang W.,Wuhan University | And 3 more authors.
Journal of Experimental Botany | Year: 2014

Cystatins are tightly bound and reversible inhibitors of cysteine proteases in C1A and C13 peptidase families, which have been identified in several species and shown to function in vegetative development and response to biotic/abiotic stresses in plants. Recent work revealed their critical role in regulating programmed cell death during embryogenesis in tobacco and suggested their more comprehensive roles in the process of sexual plant reproduction, although little is known about cystatin family genes in the processes. Here, 10 cystatin family genes in Nicotiana tabacum were identified using an expressed sequence tag (EST)-based gene clone strategy. Analysis of their biochemical properties showed that nine of them have the potency to inhibit the activities of both commercial cathepsin L-like proteases and extracted cysteine proteases from seeds, but with different Ki values depending on the types of proteases and the developmental stages of the seed tested. This suggests that cystatin-dependent cathepsin L-like proteolytic pathways are probably important for early seed development. Comprehensive expression profile analysis revealed that cystatin family genes showed manifold variations in their transcription levels in different plant cell types, including the sperm, egg, and zygote, especially in the embryo and seed at different developmental stages. More interestingly, intracellular localization analysis of each cystatin revealed that most members of cystatin families are recognized as secretory proteins with signal peptides that direct them to the endoplasmic reticulum. These results suggest their widespread roles in cell fate determination and cell-cell communication in the process of sexual reproduction, especially in gamete and embryo development, as well as in seed formation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. Source

Cao Y.,Yunnan University | Cao Y.,Guizhou Academy of Tobacco Science | Tian B.,Fujian Normal University | Ji X.,Yunnan University | And 3 more authors.
Journal of Basic Microbiology | Year: 2015

The root knot nematode (RKN), Meloidogyne incognita, belongs to the most damaging plant pathogens worldwide, and is able to infect almost all cultivated plants, like tomato. Recent research supports the hypothesis that bacteria often associated with plant-parasitic nematodes, function as nematode parasites, symbionts, or commensal organisms etc. In this study, we explored the bacterial consortia associated with M. incognita at different developmental stages, including egg mass, adult female and second-stage juvenile using the pyrosequencing approach. The results showed that Proteobacteria, with a proportion of 71-84%, is the most abundant phylum associated with M. incognita in infected tomato roots, followed by Actinobacteria, Bacteroidetes, Firmicutes etc. Egg mass, female and second-stage juvenile of M. incognita harbored a core microbiome with minor difference in communities and diversities. Several bacteria genera identified in M. incognita are recognized cellulosic microorganisms, pathogenic bacteria, nitrogen-fixing bacteria and antagonists to M. incognita. Some genera previously identified in other plant-parasitic nematodes were also found in tomato RKNs. The potential biological control microorganisms, including the known bacterial pathogens and nematode antagonists, such as Actinomycetes and Pseudomonas, showed the largest diversity and proportion in egg mass, and dramatically decreased in second-stage juvenile and female of M. incognita. This is the first comprehensive report of bacterial flora associated with the RKN identified by pyrosequencing-based analysis. The results provide valuable information for understanding nematode-microbiota interactions and may be helpful in the development of novel nematode-control strategies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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