ABSTRACT
OBJECTIVE: To investigate the gene differential expression in Pesudostellariae Radix from different habitats, isolate and evaluate associated differential genes. METHODS: cDNA-AFLP technique was applied to analyze the differential expression genes of Pesudostellariae Radix from four different habitats. RESULTS: Six primer pairs were selected and amplified. Thirty-four differentially expressed trivially distributed file system (TDFs) were obtained from Pesudostellariae Radix samples from different habitats. Then these TDFs were cloned and sequenced, and the nucleotide sequences of 21 TDFs were obtained. By BLASTX analysis, 15 of them were found to have homologous sequences in the databases, among which seven TDFs had known function. These proteins were mainly involved in the growth and development of plants and played a role in the metabolisms of defending diseases and insect pests, and improving the ability of plants to resist abiotic stress. CONCLUSION: This research evaluates differential expression genes in Pesudostellariae Radix from different habitats, providing the basic information for revealing the molecular mechanism of the property formation of Pesudostellariae Radix.
ABSTRACT
The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.
Subject(s)
Humans , Amino Acid Transport Systems/genetics , Antimony/pharmacology , Antipruritics/pharmacology , Drug Resistance , Leishmania tropica/drug effects , Leishmaniasis, Cutaneous/parasitology , Protozoan Proteins/genetics , Ubiquitin/geneticsABSTRACT
In the present paper, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to examine and identify differentially expressed genes in Capsicum annuum exposed to UV-B irradiation. Around 4000 transcript derived fragments (TDFs) were visualized and in total 183 TDFs were isolated, sequenced and analyzed by Blast 2 go. Among these TDFs, 84 of them showed homology to known genes. There were 43 TDFs showing up-regulated expression, 24 TDFs showing down-regulated expression and 27 TDFs showing both up-regulated and down-regulated expression, respectively. Some of these TDFs were found to be in response/related to UV-B stress, including carbonic anhydrase, calcium-dependent protein, thionin-like protein, bzip protein and so on. In particular, chlorophyll a/b binding protein (Capcab) responding to UV-B stress was cloned. It was concluded that Capcab could play a protective role in plant anti-UV-B and maintaining photosynthetic rate under UV-B stress.
ABSTRACT
Angelica sinensis (Oliv.) Diels (Umbelliferae) is a well-known medicinal plant mainly distributed in Gansu Province of China. Its local and global demand is significant because of its food and medicinal applications. However, the early bolting rate of Angelica sinensis (Oliv.) Diels reaches 20 percent-60 percent, which seriously affects its food and medicinal qualities. Thus, differences in gene expression between the flower bud and sprout-shoot apical meristem underwent analysis, by means of cDNA-amplified restriction fragment length polymorphism, to better understand the flowering mechanism. 64 primer sets, each of which amplified to 60 transcript-derived fragments (TDFs), were used. Among these TDFs, 26 were expressed specifically in the flower bud. After cloning and sequencing, 32 distinct sequences were obtained from these 26 TDFs, and 25 were found with homologous sequences in databases. Confirmation of differential expression of 13 sequences was obtained by semi-quantitative RT-PCR, their showing higher expression levels in flower buds. These homologous sequences encode transposable elements, pentatricopeptide repeat-containing proteins, DNA-binding transcription factors, zinc finger (B-box type) family proteins, NADP-dependent sorbitol 6-phosphate dehydrogenase (S6PDH), amongst others.
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By means of cDNA amplified fragment length polymorphism(cDNA-AFLP) technique, a fragment P1708 was amplified from Polima cytoplasmic male sterile Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinenesis Makino, syn, B. rapa L. ssp. chinenesis) 'Bpol97-05A'. RT-PCR showed that this fragment was specifically expressed in male sterile material. Sequencing and BLAST search in GenBank database indicated that P1708 had 100% homolog with chloroplast ndhJ-trnF gene region except a 54 bp insertion. Gene specific primer pairs were synthesized according to ndhJ-trnF gene region and two fragments about 1 900 bp were amplified respectively using genomic DNA templates of Polima cabbage and male fertile oilseed rape. The sequencing results showed that the gene region ndhJ-trnF of Polima cabbage contained two 54 bp repeats and some variation sites. The repeat part shared the same sequence as trnF gene except three bases at 5′ ends. For the insertion of 108 bp sequence, a new open reading frame was created.
ABSTRACT
Objective To construct for the heredity linkage map and to study the functional gene research of Carthamus tinctorius,the factors affecting cDNA amplified fragment length polymorphism(cDNA-AFLP) of C.tinctorius were investigated with developing and optimizing the cDNA-AFLP reaction system.Methods Improved Trizol method was used to extract RNA from compounds in new petals specific to safflower.With the help of M-MLV RTase without RNase activity combined with replacement synthesis method,double-stranded cDNA was synthesized from total RNA;cDNA was digested by restriction enzyme MseI/EcoRI and ligated by two steps.Then the products were provided for pre-amplification and selected amplification of different concentration gradients.After tiny modifications of system concentration,finally PAGE electrophoresis and silver-staining were performed.Results High purity and integrated total RNA for later cDNA synthesis were obtained and high quality cDNA was synthesized with the help of M-MLV RTase without RNase activity combined with replacement synthesis method.The cDNA-AFLP reaction system in C.tinctorius was as follows: 250 ng integrity cDNA was digested thoroughly at 37 ℃ for 6 h,and ligated 12 h at 16 ℃.Furthermore,the sample dilution multiplication was 10 fold for pre-amplification and 150 fold for selected amplification under the proper system concentration.According to the above reaction system,the polymorphous strips with high resolution power in PAGE electrophoresis were clear and stable.Conclusion The cDNA-AFLP reaction system established and optimized in this experiment is suitable for the functional gene analysis of C.tinctorius.
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Objective To study the relationship during exogenous hormone-endogenous hormone-idiogene, and establish the foundation to illuminate the molecule mechanism during process of somatic embryogenesis in Panax ginseng. Methods AFLP Analysis on the period of different culture in development pathway of P. ginseng was carried out simultaneously, the endogenous IAA, ABA, and the saponin content in the period of different cultures during process of somatic embryogenesis in P. ginseng were determined. Results It indicated the content of IAA was the highest in early embryo period and ABA was the highest in the mature embryo period. The ratio of ABA/IAA was higher at the stage of mature embryo than that of others. The gene expression was different in the different period cultures during process of somatic embryogenesis in P. ginseng. The total saponin content of test-tube plantlet is four fold higher than that in the period of cotylen embryo. Conclusion The condition that maintains normal development of somatic embryogenesis in P. ginseng is the presence of IAA. ABA correlates with the embryo priming, somatic embryogenesis and development. Especially, it influences the later period of somatic embryogenesis. It produces the different types of gene regulation in different physiology condition or different culture conditions. There is idiogene expression in the different development processes, which induces the different cell differentiation. With gradually completion of morphogenesis, it is the accumulative process of secondary metabolite.