Work had been done with the purpose to illustrate the regulation mechanism and function of auxin receptors and their signaling pathway in cucumber(Cucumis sativus L.), and also with the wish to find new approaches for enhancing production. Compared with the sequences of auxin receptor gene TIR1/AFBs in Arabidopsis, two highly conserved sequences were selected by screening the cucumber genome database, which were named CsTIR and CsAFB. cDNAs of both CsTIR and CsAFB genes were cloned and verified by sequencing. Overexpression vectors were constructed for further reverse genetic research in cucumber. Their sequences were submitted to GenBank and recruited with the accession of KC414935.1 and KC414934.1, respectively. The basic physical and chemical properties, as well as functional protein domains, of CsTIR and CsAFB were predicted by bioinformatics analysis. Phylogenetic and conservation analysis were taken between sequences from different plants, which made these two genes putative auxin receptors in cucumber. The expression patterns of the two genes were analyzed by semi-q RT-PCR. CsAFB and CsTIR both accumulated more in roots and leaves than in stems in 10-day-old plants while CsTIR was undetectable in stems. Expressions of both genes were detected in every tissue from 70-day-old flowering plants and showed no tissue specificity. This work provides new evidence of auxin receptors in cucumber, and will contribute to the further study of their functions and regulation mechanisms.

Gibberellins (GAs) are endogenous hormones that play important roles in the regulation of plant growth and development. Their regulation functions also have a great impact on the plant's ornamental performance. Petunia is a model system for comparative research, and a model plant for investigating the traits that influence ornamental performance. Although most of the genes involved in the biosynthesis and catabolism of the bioactive GAs have been identified in Arabidopsis thaliana and rice(Oryza sativa), no GA metabolism genes in petunia (Petunia hybrida) had been cloned and analysed previously. In this study, three full-length GA 2-oxidase cDNAs were isolated from petunia using a RACE PCR method and we named them PhGA2ox1~3(GenBank accession numbers：GU059939, JQ323102 and JQ323101). Evolutionary analysis suggested that proteins encoded by the three cDNAs all belong to C19 CGA 2-oxidase clade, closing to SlGA2ox2, -4, and -5 of Solanum lycopersicum and NtGA2ox1~3 of Nicotiana tabacum. qRT-PCR analysis indicated that their expression profiles were similar. A relatively higher expression level was observed in stems, leaves, stamens and pistils on the first day of anthesis, and in wilting petals, compared to a very low expression level in sepals and petal on the first day of anthesis and in fruits seven days after pollination. However, PhGA2ox3 transcripts were much less abundant than those of PhGA2ox1 and -2 as the cycle threshold (Ct) values of PhGA2ox3 were four cycles lower than those of PhGA2ox1 and -2 for the same cDNA template (Ct PhGA2ox1~26, Ct PhGA2ox2~25 and Ct PhGA2ox3~30), using primers with similar efficiency. 2×35S::PhGA2ox1 transgenic petunia plants were created by Agrobacterium tumefaciens-mediated transformation. The transgenic plants exhibited various degrees of GA-deficient phenotypes such as shortened internodes, smaller and dark-green leaves, which were reported and well described by other researchers, suggesting the PhGA2ox1 cDNA encodes a functional gene.

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine. Genetic and pharmacological evidences suggest that eIF5A is essential for cell survival and proliferation. However, the precise function of eIF5A is still unclear. In the present study, We isolated the full length PtoeIF5A4 (GenBank Accession number: HQ529482) CDS from white poplar based on bio-information analysis. Sequence analysis showed that the sequence of PtoeIF5A4 shared 99% identity with the sequence of Populus trichocarpa. The completed ORF of PtoeIF5A4 was 483 bp and encoding 160 amino acid residues with a calculated molecular mass of 17.5 kD and a pI of 5.76. Real-time PCR results showed that PtoeIF5A4 expressed in various organs in poplar，especially in the stem. The expression of PtoeIF5A4 increased with growth time until one year. The expression of PtoeIF5A4 in 1 year P. tomentosa tree stem secondary xylem was 4 times than that of in 1 month. These results demonstrate that PtoeIF5A4 may play an important role in growth and development of trees secondary growth

MicroRNA(miRNA) is a class of small RNA, it is involved in the intracellular complicated and precise regulatory networks. In order to study the effect of castration on the expression of miR-122 and miR-378 and the regulation effect of miR-122 and miR-378 on androstenone and skatole metabolism, we detected the skatole content in subcutaneous fat of boars and barrows with the help of high performance liquid chromatography (HPLC) and the expression of miR-122 and miR-378 in various growth stages in liver of Jinhua Pig (Sus scrofa) by quantitative Real-time PCR (qRT-PCR) and analysed the expression variation between boars and barrows. The results showed that the skatole content in adipose tissue was higher (P<0.01) in boars (mean 87.21 μg/kg, n=9) than that in barrows (79.87 μg/kg, n=9), and there was a significance change of the expression of miR-122 and miR-378 between embryonic and growth period. In the embryonic period the expression of miR-122 increased firstly and lasted to the 80th day, and then decreased. In the growth period the expression of miR-122 was decreased gradually. With the development of embryonic liver the expression of miR-378 continued to increase, while in the growth period miR-378 increased firstly and then decreased, and at 60 d reached the maximum. After 60 d the expression of both miR-122 and miR-378 of boars and barrows decreased significantly (P<0.01) with the increase of day age. At 60 and 120 d the expression of miR-122 of barrows were significantly decreased (P<0.01) by comparison to boars, and at 90th day it was also decreased significantly (P<0.05). After castration, the expression of miR-378 was decreased significantly (P<0.01) at 60, 90 and 120 d. We also predicted the transcription factors which may affect the transcription of pre-miR-122 and pre-miR-378, and the target genes which may affect the androstenone and skatole metabolism. We found that miR-122 and miR-378 had target relationship with androstenone and skatole metabolism genes SULT1A1(sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), SULT2A1(sulfotransferase family, cytosolic, 2A, dehydroepiandrosterone (DHEA)-preferring, member 1) and CYP2E1(cytochrome P450, family 2, subfamily E, polupeptide 1). We forecasted that the upstream sequence of 5.2~4.9 kb of pre-miR-122 and the upstream sequence of 2.2~1.8 kb of pre-miR-378 were the promoter region, and there were related transcription factor binding sites. In the upstream sequence of pre-miR-378, there was a CpG island close to the promoter region, which indicated that the transcription of pre-miR-378 might be affected by methylation. In conclusion, we suggested that the changes of hormone level after castration may affect the expression of microRNA through the relevant transcription factors, and then affect the boar taint by regulating the androstenone and skatole metabolism directly or indirectly. And microRNAs may play an important role in the regulatory networks.All in all ,we provide a new idea for further research on boar taint.

Space mutation breeding with the characteristics of high efficiency, large variation rang and short breeding period , is an important way of improving germplasm, broadening germplasm foundation and enriching germplasm resources. In order to understand the mutagenic effect on the combining ability for different inbred lines after spaceflight, three maize(Zea mays L.) inbred lines 08-641, RP125 and 18-599 were used as basic materials for space mutation, some mutation lines obtained from these three inbred lines were used as female parent for three sets of diallel cross design with Mo17, 478, Huangzaosi and so on as male parent according to NCⅡdesign. The combinations were evaluated at two different locations both in Sichuan and Yunnan，and the general combining ability (GCA) for plant yield was analysed . The results showed that GCA of yield per plant in these mutant lines from 08-641 and RP125 decreased compared with their original materials but GCA of yield per plant in the mutant line Y2709 and Y3235 from 18-599 increased significantly. Space mutation had an effect on the combining ability of plant yield traits in three maize inbred lines with different degrees, the overall performance showed that the variation range in the RP125 mutant line was the biggest, the 18-599 mutation line was the second, and the 08-641 mutation line was the smallest. Hybrid heterosis in mutant lines S1449, Y2803, Y3165, and Y3235 performed significantly higher compared with their original material and might have great potential in breeding. These results not only enrich the material basis of corn breeding, but enrich the theoretical content for mutation breeding.

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. However, little information is available on the genetics and functional genomics of the fungal pathogen. In order to develope a polyethylene glycol (PEG)-mediated transformation system of Didymella bryoniae, it was transformed by PEG-induced fusion of protoplasts. The plasmid pSGate1 carrying hygromycin B phosphotransferase gene (hph) gene was used and D. bryoniae ZJDB32 isolate was used as the host strain. After 1010 conidia were incubated for 20 h in PDB medium (200 g/L potato extract, 20 g/L dextrose) by shaking at 150 r/min, the mycelia were collected and enzymatically hydrolyzed for 3 h at 28℃ by shaking at 90 r/min at 40 mL of enzyme solution (NaCl 2.34 g, 1 mol/L MgCl2 0.4 mL, 100 mmol/L K3PO4 4 mL, 400 mg lysing enzyme, 200 mg driselase, and dH2O), the most protoplasts (3.8×107 /g fresh mycelia) were generated. The pelleted protoplasts were suspended in a 4∶1 mixture of STC (sorbitol, 1.2 mol/L; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L)∶PTC (PEG moleculor weight 3 350, 50 g/100 mL; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L) and adjusted to a concentration of 2×108 /mL. Twenty micrograms of the plasmid in less than 20 μL STC∶PTC (4∶1) were added to 100 μL of the above protoplast suspension, mixed, and incubated on ice for 20 min. The protoplast: plasmid suspensions were amended with 100, 300, or 600 μL PEG∶STC solution (25 g PEG molecular weight 3 350 with STC in a total volume of 50 mL) and incubated for 20 min at 25℃. Finally, the mixtures were amended 1, 3, and 4 mL of STC, respectively, and mixed gently. Protoplasts were pelleted by centrifugation at 3500 g for 10 min, re-suspended in 1.6 mL recovery medium (RM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%) and incubated at 25℃ for 2~4 h with gentle shaking at 75 r/min. Each protoplast suspension was then mixed gently with 20 mL of recovery agar medium (RAM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%; agar, 0.8%) containing hygromycin B at 100 μg/mL at 50℃. Each mixture was poured into a Petri plate and incubated at 28℃. After 3~5 d, transformants were transferred to fresh potato dextrose agar (PDA) (200 g/L potato extract, 20 g/L dextrose, 15 g/L agar) with hygromycin B at 100 μg/mL and incubated at 25℃. The transformants were purified by single spore isolation. About 1230 hygromycin resistant transformants were generated per mg plasmid DNA. All tested twenty transformants had stable resistance to hygromycin B after four serial passages. The hph gene was confirmed by PCR with hph-specific primers and integrated into the genomes of all 20 tested transformants. Transformants had similar colony morphology, growth rate, sporulation, and pathogenicity compared to the wild type. The transformants can be used in the subsequent functional research of D. bryoniae strain ZJDB32. In conclusion, we have demonstrated and optimized a PEG-mediated protoplast transformation system for D. bryoniae strain ZJDB32 with pSGate1. Therefore, this paper establishes an alternative genetic transformation system for D. bryoniae for functional genomics studies.

Zeaxanthin is a derivative of β-carotene found in nature and it is the most curcial portion of the macula and retina ,helping protect the macular region of the eye from harmful form of light can cause photoxidative damage to the eye. The biosynthesis of zeaxanthin is regulated by a series if enzymes, β-carotene hydroxylase(HYB) is the rate-limiting enzyme during the synthesis. To investigate the changes of Dschyb expression and content of zeaxanthin in Dunaliella salina under some stress, the full-length cDNA of chyb (GenBank accession No. JN118489) had been obtained from D.salina based on RACE technology, and four factors influencing Dschyb and zeaxanthin were analyzed. The cDNA was comprised of 1 433 bp containing a 969 bp open reading fragment, which encoded a polypeptide of 322 amio acids with a predicted molecular mass of 35.5 kDa and theoretical pI of 9.01. It had four conserved histidine residue motifs and was homologous with Volvox carteri f. nagariensis (64%) and Chlamydomonas reinhardtii(58%). Sequence analysis showed that D. salina CHYB had four transmembrane domains and chloroplast transit peptide, which further proved that β-carotene hydroxylase was located in the thylakoid membrane, no signal peptide was predicted in DsCHYB. Phylogenetic tree demostrated CHYB in D.salina and CHYBs from other green algae like Volvox carteri f. nagariensis and Chlamydomonas reinhardtii were grouped into one clade. Upon the observation of chyb regulation expression, the Dschyb expression level had little fluctuation when treated with intensive light at the treatment groups of 6 and 12 h, but with a significant rise from 24 h (P＜0.01), and at the peak of 48 h (P＜0.01). When darked-cultured cells were exposed to high light, sodium acetate and ferrous sulfate, the Dschyb transcription obviously increased within the initial 6 h (P＜0.01), but then declined after 12 h of the treatment. When D. sallina was exposed to glucose, the expression level of Dschyb reached a peak after 1.5 h. In contrast to the control group, there was no change observed in the group of 6 h. Most strikingly, glucose induction had not been observed after treatment with RNA polymerase inhibitor actinomycin D, indicating that actinomycin D might have a strong inhibitory effect on glucose. Dschyb mRNA was expressed significantly less than that in the control group (P＜0.01), especially in groups of 1.5 and 3 h. Zeaxanthin was identified according to their chromatograms on HPLC and UV-vis absorption spectra. The retention time of the standard was 26.742 min while the sample was 29.008 min. The content of zeaxanthin increased due to sodium acetate, ferrous sulfate, high light and glucose treatment. The content of zeaxanthin had increased 16%(P＜0.05), 28%(P＜0.05) and 53%(P＜0.01) compared with control group, respectively. These results support that high light and glucose play critical roles in Dschyb expression and provide a helpful message for production of zeaxanthin.

The increase of membrane permeability in plant cell results to imbalance in cellular homeostasis and expression of heat shock gene. As fatty acids are the basic elements of the cell membrane, the content and the saturation of lipid are related to the cell membrane stability under high temperature. Two genotypes of Triticum aestivum L. tolerant TMA107 and susceptible Chinese spring(CS), were chosen according to their genetic background diversity in thermotolerance. Membrane permeability, the fluorescence ratios Fv/Fm，fatty acids composition (C14:0, C16:0, C16:1c, C16:1t, C18:0, C18:1, C18:2 and C18:3) and transcript of fatty acid desaturase gene (FAD7) under high temperature were quantified. Increase of membrane permeability and reduction of fluorescence ratios Fv/Fm and trienoic fatty acids content were observed in both genotypes. However, changes in unsaturated fatty acids content in Chinese spring were significantly higher than that of TAM107. These indicated that the membrane system of heat resistant genotype wheat is much more stable under heat stress than heat sensitive genotype. The expression of FAD7 was more sensitive to heat stress in CS than in TAM107, and as a result, the content of trienoic fatty acids in membrane changed much more in CS after heat stress . This indicated that the susceptible genotype CS is more sensitive to high temperature than tolerant genotype TAM107 on transcript level. The observed changes on trienoic fatty acid content under high temperature may be a positive trait for heat tolerance qualification.

Cell lines as a immortalized cell materials play an important role in exploring gene function,producing transgenic animals and discovering new drugs. However, because of difficulties in immortalizing primary cells, until now only a few types of cells were reported to be immortalized. In order to overcome this problem, we aimed to construct a recombination lentivirus expressing human telomerase reverse transcriptase to improve the efficiency of immortalization so as to establish cell lines. So, we cloned ORF of hTERT gene from the retrovirus vector of pBABE-neo-hTERT and inserted into pLV-puro vector to construct pLV-puro-hTERT. After that, lentivirus particals were successfully packaged and used to infect pig preadipocytes.Then, positive cell clones were selected by puromycin. Finally, we examined the expression of telomerase reverse transcriptase by Real-time PCR, and evaluated the ability of proliferation and differetiation by DAPI and Oil red O staining. The results showed that porcine preadipocytes infected with pLV-puro-hTERT lentivirus displayed a 12-fold increase in telomerase mRNA level compared to control(P<0.01) with significant enhancement in cell proliferative activity, whilst no effects of lentivirus on porcine preadipocyte differentiation were observed. Taken together, these results indicate that the lentivirus vector can be used to promote primary cells proliferation, meanwhile it doesn't change primary cells' original biological characters, so it provides us a powerful tool to immortalize more primary cells in the future.

Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol. We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector, and packed and amplified the virus for a high titer, as a antecedent work for the further study of cellular level function of SERBP2 gene. Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA. A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No. NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR). The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid. The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease PmeⅠ and subsequently transformed into Escherichia coli BJ5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV. And then, the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with PacⅠand transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV, and to collect virus of high titer. The viral titer of Ad-SREBP2 and Ad-CMV was 7×108 and 1.3×109 GFU/mL respectively, measured by green fluorescent protein (GFP) labelled method. Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues. The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h, determined by quantitative Real-time PCR. The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular level.

Tocochromanols (Vitamin E) are important natural fat-soluble antioxidants and dietary nutrients in seed oils, protecting the polyunsaturated fatty acids from lipid peroxidation. In the past decade, along with tremendous progresses in the understandings of the mechanisms of vitamin E biosynthesis, the manipulations of Vitamin E qualities in oilseeds were remarkable. In this review, the components and biosynthetic pathway of Vitamin E were briefly described. The relationships between vitamin E and fatty acid components in oilseeds and the molecular breeding progresses in the modification of oilseeds with desired vitamin E qualitative traits were summarized. Finally, attempts targeting on the integrated qualitative traits breeding with desired fatty acid composition and vitamin E components were proposed.

Aim to current situation of urgent need to develop a simple, rapid, sensitive and specific technique for White spot syndrome virus (WSSV) detection in shrimp (Penacus orientalis) culture industry. Six specific primers including one set of outer primeres F3 and B3, one set of inner primers FIP and BIP, and one set of loop primers LF and LB were targeted on the conserved region in ORF120 of WSSV genome. These primers were designed and synthesized for amplifying the target gene ORF120 of WSSV in a loop mediated isothermal amplification (LAMP) system. A visual WSSV-LAMP was developed based on the optimized reaction conditions in which calcein was applied to be detection indicator. As none amplification taken place in the detection, calcein was cancellation by chelated with manganese chloride. When WSSV target gene was existed in the detected sample, a large amount of DNA was synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combined with metallic ion competitively to form an insoluble pyrophosphate salt. Calcein was emitted visual Kelly fluorescence. It took about 60 min to finish the detection during the one-step amplification reaction. Testing results showed that this WSSV-LAMP assay was able to detect the target gene contained in WSSV positive samples by nake-eye observation through the Kelly fluorescence emited by the amplification production. The lowest detected limitation was 1fg of target gene DNA. The preliminary tested results of clinical application showed that fifteen positive samples with same labels were figured out from 250 shrimp clinical samples by WSSV-LAMP and WSSV-PCR recommened by Office International Des Epizooties, 2012, respectively. The diagnose accordance rate between WSSV-LAMP and WSSV-PCR was hundred percent.WSSV-LAMP could also detected another three samples as positive, which were confirmed negative by WSSV-PCR. The comparison between these two methods, WSSV-LAMP was higher positive detection rate than that of WSSV-PCR , in which WSSV-LAMP was 7.2%(18/250) and WSSV-PCR was 6.0%(15/250). Results suggest that WSSV-LAMP is with higher technical advantage than WSSV-PCR in the detection of clinical samples with lower virus quantities. It can be used as an on-the-spot detection tool for WSSV in clinical diagnosis, breeding and port quarantine inspection,as well as sea ecological monitoring.