[Identification of bear bile powder and its adulterants by using DNA barcoding technology].

To identify the precious bile powder and its adulterants by DNA barcoding, and establish its standard experimental process to ensure the safe and effective utilization. Total twelve sequences from samples of bear bile powder which come from Ursus thibetanus for DNA extraction, PCR(polymerase chain reaction) and sequence, then using CodonCode Aligner V 7.0.1 shear primer region to obtain COI sequence. The COI sequences of U. arctos and their adulterants were obtained from GenBank. MEGA7.0 software was applied for analyzing mutation, calculating intraspecific and interspecific K2P(Kimura 2-Parameter) genetic distance and constructing the Neighbor-joining tree(NJ). The results showed that the maximum K2P genetic distance of bear bile powder of U. thibetanus and U. arctos are far less than minimum K2P genetic distance within its adulterants species, and the results of NJ tree demonstrated that each species could be distinguished from the counterfeits obviously. DNA barcoding is a safe, convenient and reliable technique for species identification, and it is important to establish the standard sequence of COI sequences for animal medicines.

Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis.

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.).

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.

Unravelling mitochondrial retrograde regulation in the abiotic stress induction of rice ALTERNATIVE OXIDASE 1 genes.

Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2·(-) )-generating chemical methyl viologen was stronger than that of hydrogen peroxide (H2 O2 ). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O2·(-) . Furthermore, the stress induction did not rely on chloroplast- or cytosol-derived O2·(-) . Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O2·(-) is involved in the process.