cDNA cloning, characterization, and pharmacologic evaluation of anticancer activity of a lectin gene in Pinellia integrifolia.

Plant lectins are proteins that possess at least one non-catalytic domain, which could reversibly bind to specific monosaccharides or oligosaccharides. The important roles played by plant lectins in immune regulation, signaling pathways, and plant defense could be attributed to their specific binding activities with carbohydrates. In this study, a Pinellia integrifolia lectin gene, designated pia, was cloned using rapid amplification of cDNA ends. The open reading frame (ORF) of pia was constructed into the pET-28a vector, and a 33-kDa recombinant protein was induced in Escherichia coli BL21. The hemagglutination and anticancer properties of the purified recombinant protein were assayed in vitro. The results indicated that the full-length cDNA of pia was 1210 bp long, containing an 807-bp ORF encoding a 268-amino acid peptide. The putative P. integrifolia lectin protein (PIA) contained three mannose-binding sites. The agglutinating activity exhibited by PIA was inhibited by D-mannose. PIA was also shown to exert an anti-proliferative activity against nasopharyngeal carcinoma, human cervical carcinoma, and human breast cancer cell lines in vitro. These results could be applied to determine the function of PIA in the future.

Identification of novel and useful EST-SSR markers from de novo transcriptome sequence of wheat (Triticum aestivum L.).

Simple sequence repeats (SSRs) are highly informative, polymorphic, and co-dominant Mendelian markers that provide an important genomic resource for genetic research. Recently, the use of large-scale transcriptome sequence has become a reliable and efficient approach for the identification and development of new EST-SSR markers. In this study, 8389 potential SSRs with a minimum of five repetitions for all motifs were identified from 121,210 unigenes. Gene ontology analysis indicated that the unigenes containing SSR loci participate in various biological processes of regulation, growth, development, metabolism, and apoptosis in wheat. As in many other plants, trinucleotide repeats were found to be the most abundant repeat units with a frequency of 62.33%. A subset of 300 EST-SSRs was randomly selected for the applicability of EST-SSRs to be evaluated. Of the 300 primer pairs tested, 177 (59%) yielded unambiguous PCR products among five wheat cultivars. Using the Chinese Spring nulli-tetrasomic line, 131 of the 177 EST-SSR primer pairs yielded products and 178 loci were found to be located on all the 21 wheat chromosomes. These findings suggest that the novel EST-SSR markers, as a basis for future genetic linkage and gene tagging analysis, are a valuable tool for genetic mapping, marker assisted selection, and comparative genome analysis.

Identification of differentially expressed genes in a pistillody common wheat mutant using an annealing control primer system.

HTS-1 is a new kind of pistillody wheat. All or parts of its stamen are transformed into pistils or pistil-like structures, and it has more seed sets per floret than normal wheat under normal cultivation conditions. To investigate the expression divergence in this mutant, an annealing control primer system was used to identify differentially expressed genes (DEGs) in the young spikelets. As a result, three DEGs, including HDB2, HGF2, and HCG4, were detected, with variable expression in HTS-1 and the control. After further confirmation using real-time reverse transcription polymerase chain reaction analysis, these genes were overexpressed in HTS-1 wheat. NGF2 was identified in the double ridge to floret differentiation stages; HDB2 and HCG4 were identified in the stage of pistil and stamen-differentiating. Therefore, we inferred that the homeotic transformation of stamens into pistil-like structures occurred during the early stage of stamen development. Sequence alignment analysis revealed that HDB2 encodes a putative protein of 189 amino acids, with high homology to the DEAD-box ATP-dependent RNA helicase, and HCG4 was identical to the Chinese spring wheat cDNA clone predicted protein according to GenBank. However, NGF2 was not found to have significant similarity to any reported proteins, suggesting it is a new functional gene in wheat. The results suggest that HDB2, HCG4, and HGF2 are minor genes contributing to pistillody trait formation in HTS-1.

Molecular cloning, characterization and expression analysis of WAG-1 in the pistillody line of common wheat.

Wheat WAG-1 is a C-class MADS-box gene, which is orthologous to AGAMOUS in Arabidopsis. In this study, we report the cloning, characterization, and expression patterns of WAG-1 in the pistillody mutant HTS-1 and its sib-line CSTP. The cDNA of WAG-1 was found to be 765 bp in length, which was equal to the length of its open reading frame, encoding 254 amino acids. The location of WAG-1 revealed that it has three homologous genes from the short arm of chromosome 1A, 1B, and 1D. Their genomic sequences were determined to be 5864, 6454, and 6447 bp long, respectively, and possessed seven exons and six introns. Young spikes from HTS-1 contained higher levels of WAG-1 transcript than did those from CSTP, and the transcript levels in the young spikes (7-10 mm in length) of HTS-1 increased 3.3-fold relative to those of the CSTP line. The transcript level in the pistil and pistil-like stamens of HTS-1 was over 2-fold higher than that in the stamens of CSTP, and expression in the pistil-like stamens of HTS-1 was slightly higher than that in its pistils. These data provide a basis for future research into the function of WAG-1, and offer further insight into the molecular mechanism of the pistillody mutation in common wheat.

Cloning and characterization of thioredoxin h in the three-pistil line of common wheat.

Thioredoxin h (Trxh) is a ubiquitous protein that reduces disulfides in target proteins, and is itself reduced by NADPH-thioredoxin reductase. In the current study, the complementary DNA sequence and the genomic sequence of the three-pistil (TP) line of common wheat (Triticum aestivum L.) were obtained from spikes through reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR. Sequence alignment of amino acids of TPTrxh then allowed for predictions of its physicochemical properties, secondary structures, tertiary structures, and functional domains. Furthermore, the TPTrxh gene was overexpressed in Escherichia coli and its activity was demonstrated using a dithiothreitol-dependent insulin assay. The expression patterns of TPTrxh were analyzed through real-time RT-PCR in different tissues and across different developmental stages of young spikes. The complementary DNA of TPTrxh was found to be 411 bp in length, encoding 118 amino acids. Its genomic sequence was determined to be 2632 bp, possessing 3 exons and 2 introns. Functional domain analysis indicated that TPTrxh contained a WCGPC motif located at the end of the second ß-fold and on the initial side of the second α-helix. The TPTrxh protein reduces intramolecular and intermolecular disulfide bridges in target proteins. Young spikes contain higher levels of TPTrxh transcripts than do stems and leaves. The transcript levels in the young spikes (2-5 mm in length) of the Chinese Spring TP line increased 2.84-fold relative to those of young spikes (2-5 mm in length) of the Chinese Spring line. These data provide a basis for future research into the function of Trxh, and offer further insight into the molecular mechanism of the TP mutation in wheat.

Morphological variation of mutant sunflowers (Helianthus annuus) induced by space flight and their genetic background detection by SSR primers.

After sunflower seeds were exposed to space conditions, various mutant plants were screened from the descendent plants. The morphological characters of plants changed in flower color from golden to yellow, light yellow, or even to yellowish green. The ligulate petals of the unisexual floret broadened, or became thin, while the short tubular petals of bisexual floret elongated to some extent, or even turned into semi-ligulate petals or ligulate petals, making the phenotype of the whole inflorescence like a chrysanthemum. The shape and thickness of leaves varied in some of these plants. Absolute sterile plants in mutant plants were found to possess neither normal bisexual florets nor unisexual florets, but the "pseudo-floret" only consisted of pieces of shield-like bracts on protuberant floral disc. Thirty-five pairs of simple sequence of repeat primers were used to detect the genetic variation of the plants, and the results showed that only a variation was tested in the mutant plants from 4 primers. The different PCR products obtained were extracted for sequencing and alignment analysis, and the aligned results showed that the DNA sequence changed by deletion, insertion and replacement that occurred at some sites. The results proved the high mutagenic efficacy of space flight, and ways of DNA transformation due to space conditions.

A new reduced height gene found in the tetraploid semi-dwarf wheat landrace Aiganfanmai.

Aiganfanmai is a dwarf tetraploid wheat landrace (Triticum turgidum var. turgidum) that stably produces the semi-dwarf trait. Plant height varies from 80-105 cm under cultivation. Compared with tall durum wheat (T. turgidum var. durum) variety Langdon, we found it to have short spikes and seeds, besides a semi-dwarf character. We crossed Aiganfanmai with Langdon to analyze the genetic basis of the semi-dwarf trait. The F(2) population segregated at a 1:3 ratio for the short trait to the normal, which demonstrates that Aiganfanmai carries a recessive reduced height (Rht) gene. This gene was found to be located between the molecular markers Xgwm471 and Xgwm350 on chromosome arm 7AS by microsatellite analysis. No Rht gene had been reported from this chromosome; we designated it as Rht22. Rht 22, unlike other previously reported Rht genes, does not reduce internodal cell length. Reduced cell numbers might explain the short stem trait.

[Clinical effects of perfusing drugs into hepatic artery to promote blood circulation in late stage of hepatocarcinoma].

The sophisticated technology of hepatic artery catheterization was used since 1990 in treating exacerbated patient after chemotherapy, 4-8 ml Salvia miltiorrhizae composita injection with 20 ml of 10% glucose was used. The same Chinese herbal drug based on Syndrome Differentiation as well as Western drug to relieve the symptoms was used in both treated and control groups. After two treatment courses, the symptom of treated group was relieved, the tumor mass shrunk and the biochemical parameters improved, the total effective rate was 65%, while that of control group was 25%, the difference between two groups was very significant, P < 0.01. This result suggests that is one of effective methods which worth further investigation.