GDSL gene family is featured with a conserved GDSL motif at N' terminus and encode a variety of lipases that exist in various species from prokaryotic to eukaryotic. GDSL gene family in plant consists of a wide range of members that exhibit structural and functional divergences, playing important roles in plant physiological processes including development, stress response and lipid metabolism. In this review, we summarize the previous literatures about plant GDSL genes, with respects of their structural divergences, classification, evolutional changes, expression and functions. In particular, here we report our recent findings regarding to the role of some GDSL genes in seed fatty acid accumulation of oilseeds.

As a well-known medicinal fungus, Cordyceps militaris has been widely used as a crude drug and folk tonic food in East Asia. It has been proved that C. militaris has the therapeutic actions against various kinds of cancer cells, while studies concerning the leukemia cells are not common, especially with fermentation products of this fungus. In this study, the effects of three aqueous extracts of C. militaris on human leukemia Jurkat and K562 cells were investigated. All aqueous extracts from C. militaris mycelium (CMME), fermentation broth (CMBE) and whole fermentation product (CMPE, extracts from both mycelium and fermentation broth) significantly inhibited the growth of Jurkat and K562 cells in a concentration- and time-dependent manner and the cytotoxic effects were clearly evident even from the early time point (24 h) using methylthiazoletetrazolium (MTT) assay. In comparison with CMBE, both CMME and CMPE showed a remarkably higher inhibitory effect on human leukemia cells, while no significant difference was observed between the two. Additionally, for the three extracts from liquid culture of C. militaris, IC50 value (inhibitory concentration for 50% growth inhibition) followed the ascending sequence: CMME＜CMPE＜CMBE. Further, the Annexin V-FITC/PI staining and flow cytometric analysis showed that the cytotoxic effects observed in response to the CMME and CMPE were associated with the induction of apoptotic cell death, and both early and late apoptosis rates were increased in a dose dependent manner in both Jurkat and K562 cells after the treatments. Both CMME and CMPE showed similar effects on the Jurkat cell apoptosis induction, although the former induced more K562 cells to undergo apoptosis than the latter. Generally, among the three extracts, CMME showed a stronger effect on cell viability and apoptosis than both CMBE and CMPE. In response to CMME, the level of anti-apoptosis B-cell lymphoma-extra large(Bcl-xL) expression was significantly inhibited in Jurkat cells after a relatively long time (48 h) at the concentration of 4.00 mg/mL, but slightly decreased in K562 cells (up to 60 h of treatment) at a higher concentration of 8.00 mg/mL. Taken together, the results here suggested that the anti-proliferative properties of C. militaris aqueous extracts were associated with the apoptotic induction and that CMME might have a great therapeutic potential in human leukemia treatment than the other two extracts. Antileukemia activities of the three aqueous extracts from C. militaris reported here indicated that the effective compounds were produced intracellularly rather than extracellularly. Further investigation may focus more on the mycelium production rather than fermentation broth. The results provide references for future study on application of C. militaris products in leukemia treatments and other related research.

Constitutive photomorphogenesis and dwarf gene (CPD) is a key rate-limiting enzyme in brassinosteroids(BRs) synthesis, gene expression patterns and functions of which in Arabidopsis, rice (Oryza sativa) and tomato (Lycopersicon esculentum) have been researched, and not in cotton(Gossypium arboreum L.). To clone and verify CPD gene function in cotton, a cytochrome P450 monooxygenase gene, GaCPD2 (GenBank accession: KJ183066) was cloned from cotton by RT-PCR. Expression pattern and functional bioinformatics analysis showed that GaCPD2 was 1 413 bp, which encoded a deduced protein including 470 amino acid residues with theoretical relative molecular weight of 53.98 kD and pI 9.40, mainly composed of alpha helix, beta turn, extended strand and randon coil. Multiple sequence alignment revealed that the most similarity was 84.0% between the amino acid sequence of GaCPD2 and that of other reported species, which had conserved domain of cytochrome P450. Real-time PCR revealed that GaCPD2 expressed in root, stem, leaf, flower and fiber, and had the highest expression level in 10 days post-anthesis (DPA)fiber, which was about 10 times that of 0DPA. Cloned promoter sequence of GaCPD2 contained many cis-elements related to light response by bioinformatic analysis, which implied that GaCPD2 was light regulated. Overexpression vector 35S :: GaCPD2 for GaCPD2 transformation restored the growth of cpd91, which is a brassinosteroids deficient dwarf mutant of Arabidopsis (Columbia background). The transgenic lines showed flattened leaves and restored fertility. These results revealed that GaCPD2 played an important role in brassinosteroids biosynthese. These results lay the base for the further reseach of BR regulation in cotton development, and will help to reveal the BR regulation of plant molecular mechanism of plant type and yield traits.

Aquaporins(AQPs) are ancient channel proteins localized in biological membranes and facilitating water transport through cell membranes rapidly. Aquaporins play an important role in plant development. A gene encoded aquaporin was isolated from cotton (Gossypium hirsutum) fiber and named GhAQP2 (GenBank accession No.: KJ920936). The full length of GhAQP2 composed three introns and four exons, and contained a open reading frame of 837 bp encoding 279 amino acids. Analysis of multiple alignment and phylogenetic tree showed that GhAQP2 shared the highest identity with NtPIP2; 1(86%) and belonged to PIP2 protein family. Semi-quantitative RT-PCR was used to investigate the expression profile of GhAQP2 in different tissues and fibers from different days post-anthesis. The results showed that GhAQP2 was preferentially expressed in cotton fiber, and the transcript level increased along with fiber development and maintained at high abundance in fiber cells at 6 to 18 days post anthesis, after which time it gradually decreased. The GhAQP2 was transformed into tobacco (Nicotiana tobacum) suspension BY-2 cells to preliminary analyze the gene function. Overexpression of GhAQP2 in BY-2 cells led to cell shape significantly changed from round to long strip, and the cell state changed from aggregating to loose. All our results manifested that GhAQP2 may have important effect on the elongation of cotton fiber cell.

Sugar transporters (SUT) are proteins which are responsible for mediating sucrose transmembrane transport, and play a vital role in plant growth and developing. At present, 5 members are identified in rice(Oryza sativa L.), which are named as OsSUT 1~5, respectively, and the most functions of OsSUT3, OsSUT4 and OsSUT5 are not documented. In order to understand the role of OsSUT5 in rice callus induction and regeneration, experiments were conducted using antisense OsSUT5 transgenic rice and wild type rice MingHui86. The results showed that scutellum of antisense OsSUT5 transgenic rice became enlargement, and the induction and increase rate of callus was significantly declined(P＜0.05). The dry weight of the first and second generation callus of transgenic rice were 0.028 80 and 0.119 93 g/peridish, respectively, significantly lower than that of the wild type(0.058 70 and 0.174 55 g/peridish)(P＜0.05). After subculturing five generations, proliferation rate of callus flesh weight of transgenic rice was not significently different compared with wild type rice callus, however, the callus vitality of transgenic rice was significantly lower than that of wild type rice, and the callus of transgenic rice had obviously hygrophanous shape, and the green seedling differentiation rate was 65%. The callus differentiation rate was significantly decreased in the transgenic rice compared to the wild type(82.5%)(P＜0.05). Real-time PCR analysis of the OsSUTs expression of the two generations of callus showed that the OsSUT5 expression decreased nearly one time of transgenic rice compared to the wild type, and OsSUT1 gene expression exhibited the same trend with the OsSUT5, while the other three OsSUTs genes were not influenced by OsSUT5 down expression. Our results suggested that sucrose transporter OsSUT5 can play an important role in regulating the rice callus induction and regeneration. This study provide the reference data for further analysis of OsSUT5 gene function.

Wheat(Triticum aestivum)-Thinopyrum intermedium introgression line CH5383 is high resistance to wheat stripe rust and powdery mildew. It was characterized by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and molecular marker analysis. GISH using Th.intermedium DNA as probe couldn't detect the alien signals. Strong hybridization signal in the distal region of wheat chromosome 3BL was observed when FISH using pHvG38 containing the simple GAA repeat as a probe. A total of 135 PLUG primer pairs were used to test in wheat Chinese Spring (CS), CH5383 and wheat related species. The results showed that TNAC1383 displayed unique 1 300 bp bands of CH5383 and Th. intermedium. Experimment also verified that CH5383 was a small wheat-alien introgression line and the alien Th. intermedium DNA segments were possibly located in wheat bin 3BL-0.81~1.00 region. Due to its good diseases resistance, CH5383 can be useful in breeding programs.

RNA interference (RNAi) technique has been used on gene function research frequently. In order to construct a kind of simple RNAi expression vector which is adapted to rice(Oryza sativa ssp. japonica) rapidly and efficiently, a pair of forward and reverse primers oligonucleotides with 9 bp of complementary nucleosides at 3' end was designed, and a small interfering segment with inverted repeated sequence of target gene by polymerase extending was formed, which was digested and ligased into the basic expression vector pCAMBIA1301 to construct RNAi vector with an inverted repeated sequence. After Agribacterium tumefaciens transformation, both wild type and transgenic lines of rice were validated by leaf GUS staining and qRT-PCR of target genes in grains. The GUS staining result showed that the cut edges of transgenic rice leaves were blue, and there was none in the wild type, suggesting that the genes within T-DNA region of RNAi vector had already been integrated into the rice genome and expressed. qRT-PCR result showed that the mRNA relative expression quantity of the target gene decreased to 18%~55%(P<0.01), showing that the small interfering segment within rice genome could decrease the expression level of target gene. Comparing with wild type rice, the single kernel weight of 10 transgenic rice plants decreased significantly (P<0.05), and the size of transgenic rice kernel was smaller, both length and width changed significantly(P<0.05). This showed that simplified RNAi vector could effectively interfer target genes expression. This research provides a simple RNAi construction for quick and efficient rice gene function research.

Flower time is important trait in Brassica, FLCs are key genes to flowering. Forward primers were designed on polymorphic region of exon 4, reverse primers were designed on conserved region of exon 7. Four Specific primers were developed for Genotyping of BrFLC1, BrFLC2, BrFLC3, BrFLC5, BoFLC1, BoFLC2, BoFLC3 and BoFLC5. FLCs were identified by above mentioned specific primers in the Chinese cabbage monosomic alien addition line which introduced chromosome 2 from cabbage, the results showed that besides the 4 BrFLCs, BoFLC3 was existing in the monosomic alien addition line. Which provide new way to identification of BoFLCs in the background of Chinese cabbage, and supply generation of Chinese cabbage translocation lines added BoFLC3 with special materials.

Phytochrome B (PHYB) is one of key factors regulating photoperiod-dependent flowering in plant. At present study, whole genomic sequences of PHYB, which were isolated from extremely late- and early-bolting Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred lines 06-247 and He102, were analyzed bioinformatically. The results revealed that the total length of PHYB in both lines were 5 986 and 5 528 bp respectively. And the sequences of both were not completely identical to that of Chiifu-401 from BRAD (Brassica Database), thus they were all mutants of wild type PHYB and named as BraphyB1(GenBank No. KJ866947) (from 06-247) and BraphyB2 (GenBank No. KJ866948)(from He102). There were 476 Inserts/Deletions(InDels) and 57 SNPs, among which 473 InDels and 31 SNPs located in promotor and 3 InDels and 26 SNPs located in coding region. InDels appeared 445, 18 and 3 bp deletions in phyB2 promoter, 5'UTR and coding region, respectively, among which 445 bp deletion in promotor containing two TATABOX4, two MYB-like transcription factors biding sites and one GA-responsive element; 18 bp deletion in 5'UTR might influence secondary structure of mature mRNA and then the scanning of pre-initiation complex and translation efficiency; 3 bp deletion in coding region of phyB2 caused an amino acid deletion. Furtermore, among 26 SNPs in coding region, 3 were nonsynonymous mutations which caused amino acid subtitutions between phyB1 and phyB2. A set of codorminant markers relating to large fragment InDel in promotor were developed and named as phyB1-762/phyB2-317. A segregating F2 population derived from 06-247 and He102 was detected with the markers. Association analysis between markers and flowering time revealed that phyB1-762 and phyB2-317 were significantly (P<0.05) related to late and early flowering respectively. Real-time quantitative RT-PCR analysis revealed that the relative mRNA level in tissues of He102 were evidantly lower than that in 06-247, the difference was statistically the most significant (P<0.01) at all development stages tested. Thus the large fragment deletion mutation in promotor was one of the most important factors causing low level phyB2 expresson and early flowering in He102. The results provide clues for research on molecular mechanism which influences Chinese cabbage flowering time by PHYB mutation at the level of trancription, translation and protein structure.

Periploca forrestii Schltr. is one of the national medicine in the Yunnan-Guizhou plateau of China.In order to assess the genetic diversity of Periploca forrestii Schltr. from different geographic population at molecular levels, sequence-related amplified polymorphism (SRAP) molecular markers were used to detect the genetic diversity of 11 P.forrestii Schltr. accessions and 4 P.calophylla (Wight) Falc. accessions for the first time mainly from Southwest China (Yunnan, Guizhou, Chongqing and Sichuan). Forty primer pairs had been selected to identify the polymorphism among the 15 test materials and 420 distinguishable DNA fragments were amplified, of which 333 bands were polymorphic. The percentage of polymorphic bands in average was 79.29%. The range of genetic distance of the test accessions was 0.040~1.613 with an average of 0.840, and the genetic similarity coefficient(GS) of them was 0.391~0.973 with an average of 0.718. All of these suggested that the genetic diversity was comparatively rich among the test materials. The 15 accessions were classified into 2 cluster groups of P.forrestii Schltr. and P.calophylla (Wight) Falc. by cluster analysis using UPGMA(unweighted pair-group method with arithmetic mean) at the similarity coefficient of 0.59. At the similarity coefficient of 0.80, the 11 accessions of P.forrestii Schltr. could be furtherly classified into 3 sub-groups which reflected the 3 geographical origins of Yunnan, Guizhou, Chongqing-Sichuan. Moreover, accessions from the same origin frequently clustered into one group, showing a certain degree of eco-geographical distribution. Results of this study will provide references for the collection, breeding and conservation of P.forrestii Schltr..

Using plants bioreactor to produce pharmaceutical proteins has special development advantages, however, foreign gene silencing usually affecte gene expression. Since gene silencing suppressor can effectively inhibit the gene silencing，we focused on using gene silencing suppressor helper component-proteinase (HC-Pro) to improve gene expression. The results showed that green fluorescent protein (GFP) expressed in Nicotiana benthamiana plants by leaf infection of Agrobacterium suspension containing p35S-30B-GFP vector. Compared with the wild type tobacco Nb, the expression level of GFP significantly increased in HC-Pro transgenic tobacco analyzed by protein electrophoresis and Western blot. The results provide the basis for gene silencing suppressor applied in plant bioreactor.

G protein-coupled receptor 43(GPR43) is the member of G protein-coupled receptors(GPCRs) family and has a potential role of regulating lipid metabolism. The aim of this study was to clone GPR43 and analyze its mRNA expression level in different tissues and lactation periods of diary goats(Capra hircus). Based on the published nucleotide sequence of bovine(Bos taurus) GPR43 gene(NM_001163784) in GenBank, a pair of specificity primers was designed. Coding sequence(CDS) of GPR43 was amplified (GenBank accession number: HM623658) by RT-PCR. The results showed that the CDS length of GPR43 gene was 987 bp, coding 329 amino acids. The homology analysis of dairy goat GPR43 gene indicated that the nucleotides and amino acids had high similarity with bovine, human(Homo sapiens) and mice (Mus musculus) and reached up to more than 90% with bovine. The protein structure analysis showed that GPR43 protein had 7 transmembrane helices and the C-terminal was high hydrophobic but the N-terminal showed high hydrophilism. Real-time quantitative PCR was used to detect the expression of GPR43 gene in different tissues. The results suggested that GPR43 mRNA expressed in all 10 tissues examined. GPR43 mRNA expressed abundantly in spleen, followed by the small intestine, liver, adipose tissues but the lowest in lung, muscle and kidney, and the relative low expression was also detected in mammary gland tissue. Two lactation stages expression analysis showed that expression of GPR43 gene was significantly higher in the lactation stage than that of dry stage (P＜0.05). Taken together, these results demonstrated that GPR43 had a different range of tissue-speci?c expression, and might play an important role during lactation stages in dairy goat. In conclusion, this work serves as the basis for further study on GPR43 in the dairy goat mammary gland.

Metallothionein-1(MT-1) is an ubiquitously dispersed metal-binding protein in animals, which serves in maintaining metal homeostasis and heavy metal detoxification, and also scavenges free radicals, resists ionize radiation and anti-stress. For further research on the molecular mechanism of MT-1 gene involved in stress resistance, full length cDNA sequence of MT-1 gene in Tianzhu white yak(Bos grunneins) was cloned by RT-PCR and RACE (rapid amplification of cDNA ends). Physicochemical property, structure and homology of MT-1 encoded protein were analyzed by bioinformatic methods. Real-time PCR was used to analyze MT-1 mRNA expression profile in different tissues of Tianzhu white yak. The result showed that the full length of MT-1 cDNA was 408 bp (GenBank accession No. KF770836), with an ORF (open reading frame) of 182 bp in length, which encoded 61 amino acids with a predicted molecular mass of 5.981 kD. The predicted amino acid sequences showed 100% similarity with Bos taurus, and was highly homologous with most mammalians. The mRNA expression of MT-1 gene was found in 8 different tissues of Tianzhu white yak by Real-time PCR, the highest expression level was found in heart. Therefore, the full length cDNA of MT-1 gene was successfully obtained, which provides basic data for further study on its function.

The performance of competing binding reactions between metal-ion group and human serum albumin(HSA) was investigated using affinity capillary electrophoresis (ACE) method. Based on the site binding model, a model about the interaction between [Zn2+, Cu2+] as the ligand and HSA as the receptor was established, a theoretical equation of competing binding reactions between multimetal-ions and macromolecule had been suggested, the binding parameters and dynamic mechanism were also measured and analyzed. The result showed that, the competitive combining reactions of metal-ion group [Zn2+, Cu2+ ] with HSA were reacted to form compounds Zn2+-HSA and Cu2+-HSA. Based on changes of the effective mobility, the mean apparent competition binding constant was determined through established theoretical equation by using non-linear fitting method, and the average apparent binding constant K[Zn2+-HSA]=(4.67±0.13)×104 L/mol, K[Cu2+-HSA]= (7.69±0.11)×104 L/mol. The binding reactions of Zn2+/Cu2+-HSA were equilibrium reactions, and that Cu2+ had antagonistic effect on Zn2+. Dosage-dependent effect existed among the peak height of compounds with the ligand strength of binding reactions and stability of compounds. This work has referential meaning for understanding deeply the transport mechanism of multimetal-ions with protein.

Avian primordial germ cells (PGCs), as precursor of sperm and eggs, can develop to sperms and eggs in the sexually matured avian. Based on this property, PGCs can be used in the fields of PGCs preservation, transgenic avian preparation and developmental biology and will play very important role in the fields of scientific research and practical application. In this study, the PGCs from blood of chicken (Gallus gallus)embryos which had been hatched to Hamburger-Hamilton stage 13~15 (HH13~15) were separated and purified by Nycodenz density gradient centrifugation. The purity of the PGCs was to 99% and the diameter of the purified PGCs was about 13~15 μm after manually selected by microinjection glass needle. The purified PGCs were identified by alkaline phosphatase(AKP) staining, periodic acid-schiff(PAS) staining and stage-specific embryonic antigens-1(SSEA-1) immunohistochemical staining to show the specific reaction of PGCs. The purified PGCs were labeled with fluorescent dye (PKH 26) and successfully labeled PGCs showed red fluorescence under fluorescent microscope. About 200 labeled PGCs were microinjected into the dorsal aorta of recipient chicken embryos which had hatched to HH14~16. After 8 days of incubation, the gonads of recipient chicken embryos were then separated under a stereoscope and embedded in Neg-50 frozen section medium and frozen in liquid nitrogen. Frozen sections were prepared and observed under fluorescence microscope, the labeled donor PGCs could be observed in the gonad of the recipient chicken embryos, which indicated that the purified PGCs were capable of migrating and settling in gonads of the recipient embryos. This study will lay a foundation for the study of PGCs based germplasm preservation and transgenic technologies.