Class Ⅲ Homeodomain Leucine Zipper (HD-Zip Ⅲ) protein is mainly involved in the regulation of vascular tissue formation, apical meristem differentiation, embryonic morphogenesis and the polarity establishment of the lateral organs. In this study, the HD-Zip Ⅲ genes of Chinese fir (Cunninghamia lanceolata) were cloned by RACE based on the transcriptomic sequencing. Bioinformatic characteristics of the cloned ClHDZs were analyzed using online service. To understand genic roles in wood formation of Chinese fir, the expression patterns were analyzed in different organs and tissues. The results showed that four HD-Zip Ⅲ genes in Chinese fir were cloned and named ClHDZ1, ClHDZ2, ClHDZ3 and ClHDZ4, respectively. The corresponding encoding proteins were composed of 857, 841, 851 and 842 amino acid residues, respectively. And they contained 4 conserved domains of HD(homeodomain), LZ(leucine zipper), START(steroidogenic acute regulatory protein-related lipid transfer domain), SAD (START adjacent domain) and MEKHLA(Met, Glu, Lys, His, Leu, Ala). Phylogenetic tree suggested that HD-Zip Ⅲ proteins from gymnosperm and angiosperm were clustered in different branches. ClHDZ1 and ClHDZ3 appeared early in the evolution of eukaryotes, and ClHDZ2 and ClHDZ4 were grouped into the C8-subclass of gymnosperms. Four ClHDZs were expressed at different levels among the organs, and the expression levels in roots were the lowest. ClHDZ1 was mainly expressed in coniferous leaves and ClHDZ4 was preferentially expressed in female cones. ClHDZ2 and ClHDZ3 presented the similar expression patterns and were predominantly expressed in the stem, and their expression levels increased as the lignification progressed. This suggested that these 2 genes might participate in xylem development of Chinese fir. Further quantitative PCR analysis showed that ClHDZ2 was mainly expressed in the phloem and xylem, and the corresponding values were 1.0 and 1.2 times higher than that of the cambium. The expression level of ClHDZ3 in xylem was more than 8 times that of phloem and cambium. These 2 genes exhibited different expression patterns in response to compression wood induction. ClHDZ2 was predominantly expressed in the opposite wood. And transcripts of ClHDZ3 showed the same pattern as lignin changes, which were most abundant in compression wood. It further suggested that ClHDZ2 and ClHDZ3 might involve in regulation of Chinese fir wood formation. The molecular biology of the HD-Zip III gene of Chinese fir has provided a new scientific basis for revealing the formation of Chinese fir wood and has laid a foundation for further study on the molecular mechanism of Chinese fir wood formation.

Influence of epigenetic variation on phenotypic diversity has become a research hotspot recently. However, it remains largely unknown how epigenetic variation regulates plant phenotypic diversity. In this research, the leaf length diversity of 27 Arabidopsis thaliana genotypes and its correlation with DNA methylation of related genes to leaf development were studied. The results showed that a great leaf length diversity existed among different A. thaliana genotypes. After treatment with demethylation agent 5-azacytidine (5-azaC), leaf length variation of different genotypes decreased significantly. The leaf length of long-leaved A. thaliana genotypes decreased after 5-azaC treatment while short-leaved populations tend to increase. Leaf development related genes of different A. thaliana genotypes showed various DNA methylation levels via bioinformatics analysis. Meanwhile, leaf length of different A. thaliana genotypes were positively and significantly correlated with DNA methylation of 4 genes struwwelpeter (SWP), auxin response factor 2(ARF2), growth-regulating factor 1(GRF1), erbb-3 epidermal growth factor receptor binding proteinebp1(EBP1)which involved in A. thaliana leaf development. Three typical A. thaliana genotypes with different leaf lengths, long-leaved Gr-1, mediate leaved Ty-0 and short-leaved Br-0, were selected and used for gene expression analysis. The results showed that long-leaved Gr-1 had highest methylation levels of SWP, ARF2, GRF1 and EBP1 genes among the three selected populations while short-leaved Br-0 had lowest methylation levels of SWP, ARF2 and GRF1 and mediate methylated EBP1 gene. Q-PCR analysis showed that expression levels of these 4 genes were positively correlated with DNA methylation level and leaf length of A. thaliana genotypes, respectively. Gr-1 had the highest expression levels of SWP, ARF2, GRF1 and EBP1 genes compared with the other two populations. Br-0 had the lowest expression of SWP, ARF2 and GRF1 genes and the mediate expression level of EBP1 gene. However, relative expression level differences of these 4 genes among the 3 genotypes decreased significantly after 5-azaC treatment. The relative expression levels of SWP, ARF2, GRF1 and EBP1 in Gr-1 decreased significantly after 5-azaC treatment while relative expression levels of those four genes in Br-0 increased. In conclusion, demethylation of these 4 genes led to smaller relative expression level differences among the 3 populations, which contributed to the less leaf length diversity. These results indicated that DNA methylation of SWP, ARF2, GRF1 and EBP1 genes play a role in regulating leaf length diversity of A. thaliana. This may provide a basis for further study of the role of epigenetic variation on plants phenotypic diversity.

The swollen stem of zizania latifolia was induced by the interaction between Ustilago esculenta and Zizania latifolia, following with a series of immune defense in zizania latifolia. Study on activation of resistance gene (activated disease resistance 1, ADR1) in the development expression pattern of zizania latifolia will assist researching the formation mechanism of swollen stem of zizania latifolia. In this study, ZlADR1 gene(GenBank number: KP 729625.1) was cloned with full length of 1164 bp and encoding 387 amino acids, which belonged to the resistance gene with CC-NBS-LRR domain. There were two introns in the genome sequences and the amino acid sequence of ZlADR1 contained the domains of Kinase2a, Kinase3a, RNBS-C and HD. Moreover, ZlADR1 had the highest homologous gene sequence similarity with Oryza brachyantha. Meanwhile, expressions analysis of the ZlADR1 gene were proceed in different development stages, swollen types and tissues in zizania latifolia. Expression of ZlADR1 gene raised before the growth of swollen stem, it may related to the large growth of U. esculenta in the stem of zizania latifolia. And ZlADR1 gene was expressed both in the stem and in the leaves, but the expression in stem where U. esculent highly grew and distributed was significantly higher than in the leaves (P＜0.05). Besides, in the early wollen stem, expression of ZlADR1 gene in white zizania latifolia stem was significantly higher than that of grey zizania latifolia and male zizania latifolia (P＜0.05). While no significant difference was found between grey zizania latifolia and male zizania latifolia. Expression of ZlADR1 gene was induced by the growth and distribution of mycelium of U. esculenta in zizania latifolia. The vegetative growth state and distribution of U. esculenta in the stem was closely related to the expression of ZlADR1 gene. In this study, the expression responses of ZlADR1 gene have been preliminarily investigated during the swolling of zizania latifolia, which would benefit the mechanism of the swollen research in zizania latifolia.

Flagella is the major organelles for motility, is also an important pathogenic factor. Chemotaxis is the movement of an organism in response to a chemical stimulus, bacteria direct their movements according to certain chemicals in their environment. The chemotaxis gene (cheA) mutants caused significant reduction in virulence. In order to study the roles of cheA and flagellin gene (fliC) double mutant in Acidovorax citrulli, The double mutant strain ΔcheAΔfliC and his complemented strain were constructed by homologous recombination approach. Meanwhile, ΔfliC for control and his complemented strain were constructed. The influence of ΔcheAΔfliC illustrated by testing the biological characteristics. The results showed that the pathogenicity, motility, growth ability and seed adhesion of ΔcheAΔfliC were significantly decreased and the biofilm formation ability was significantly enhanced. Compared with ΔfliC and ΔcheA, the virulence of ΔcheAΔfliC was dropped significantly, biofilm formation ability significantly increased. The effect of fliC and cheA on the ability of the pathogenic and biofilm formation of watermelon were even more significant. The movement ability of ΔcheAΔfliC, ΔcheA, ΔfliC were basically consistent, indicating that fliC deletion and cheA deficiency also affect the athletic ability of A. citrulli, and further explain the chemotaxis core gene cheA of A. citrulli by regulating the movement of flagellum and thus affect the movement of A. citrulli ability. The adhesion ability of ΔfliC and ΔcheAΔfliC were significantly lower than that of ΔcheA, indicating that fliC played an important role in adhesion, and it would provide a basis for the prevention and control of the disease and its effective control agents' development.

Radiation therapy has become an important method of treating tumors, but at the same time, high doses of ionizing radiation can cause damage to the tissues and organs. To study the effects of high-dose ionizing radiation on the pathological injury and related cytokine expression in the heart, liver, spleen, lung, kidney and intestinal tract of rhesus monkey (Macaca mulatta), three male rhesus monkeys were irradiated with 9.5 Gy of 60Co γ for 4 h, and the organs were sacrificed 15 days after irradiation. The pathological changes of heart, liver, spleen, lung, kidney and intestinal tissues were observed by hematoxylin-eosin staining (HE). The expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, IL-2 and transforming growth factor β1 (TGF-β1) in each tissue were detected by immunohistochemistry and image analysis. The results showed that the heart, liver, spleen, lung and kidney tissues of treated rhesus monkeys had obvious pathological changes compared with the control, and the expression of factors was significantly higher than that of the control group (P＜0.05) at 15 days after irradiation. This indicates that radioactive injury caused by ionizing radiation has an effect on the expression of related cytokines. The results of the study provide a supplementary reference for the diagnosis of radioactive injury.

Grapevines (Vitis vinifera) are widely planted in China. Grape gray mold caused by Botrytis cinerea, is one of the most common diseases threating grape production. The aim of this work is to estimate the phenotypes and virulence variability of B. cinerea isolates from grapes of different viticulture climate zones in China. In this study, characters of 143 B. cinerea isolates, containing phenotype, growth rate and virulences to Red globe grape (V. vinifera cv. 'Redglobe'), Mare nipple grape (V. vinifera cv. 'Mare Nipple') and rape (Brassica campestris) leaves, were tested by applying wound inoculation with mycelial blocks. The results showed that there were two phenotypes, mycelial type and sclerotial type, accounting for 40.56% and 59.44% respectively. Six sub-mycelial-types containing M1, M2, M3, M4, M5, and M6, were found, and the number of M6 type isolates was more than that of other sub-mycelial-types. There were 5 sub-sclerotial-types containing S1, S2, S3, S4, and S6, and the number of S3 type isolates was more than that of other sub-sclerotial-types. The growth rate variation of 143 isolates was very high and ranged from 4.76 mm/d to 12.91 mm/d, and 3 levels were found by clustering analysis. All 143 isolates could successfully infect red globe grape and mare nipple grape, while virulence differentiation was very serious. The sizes of lesion area were 46.03~258.55 mm2 and 14.80~385.34 mm2 respectively, which were divided into 4 levels by clustering analysis. 62 of 65 isolates could successfully infect rape leaves, and the radius of the lesions was between 2.88 mm and 16.63 mm. The correlation analysis between test data containing phenotype and virulence, and isolates' information containing collection regions, viticulture climate zones and grape hosts was performed. The results indicated that there was no significantly correlation between the two indexes and geographical distance and host grapes, while there was a certain correlation between phenotype and viticulture climate zone. The virulence also had some correlation with viticulture climate zone. The correlational analysis among phenotype, growth rate, and virulence were performed. The results showed that there was the slightly positive correlation among isolates' virulences to red globe grape, mare nipple grape and rape. While no significant negative correlation between growth rate and virulence to Red globe grape, and significant positive correlation between growth rate and virulence to Mare nipple grape and rape were found. In addition, phenotype was not obviously related to growth rate and virulence. This study lays the foundation for analysis of molecular phylogeny to the new phenotype and researching the mechanism of virulence differentiation. The results of this study also provide some helpful basis for studying B. cinerea diversity and controlling B. cinerea effectively.

g authors, xhmei0791@hotmail.com Abstract Antitrypsin is a major trypsin inhibitor in the human body. The lack of antitrypsin can lead to various diseases such as emphysema and respiratory distress syndrome, and the demand quantity for this kind of drugs is large, so the recombinant anti-trypsin has become a hot spot, the recombinant anti-trypsin has been expressed from prokaryotes such as Escherichia coli, eukaryotes such as yeast, plants such as rice(Oryza sativa) and animals such as goats(Capra hircus). But the safety of the recombinant antitrypsin should be concerned. To evaluate the safety of transgenic antitrypsin rice, the transgenic rice and its non-transgenic counterpart were fed to Sprague-Dawley(SD) rats (Rattus norvegicus) during 90 d. This study added the genetically modified (GM) rice (JY150248) and its non-transgenic control rice (JY150250) in a mass ratio of 17.5%, 35.0% and 70.0% respectively to the basal feed. The influence and safety of GM rice were evaluated by monitoring SD rats' nutrition, physiological status and biochemical indicators. During the 90 d feeding for SD rats, the body weight and food intake were monitored. At the end of the study, we surveyed the effects of physiological indexes, such as body weight, hematology and serum biochemistry indexes, and dissected the animals for pathological observation and calculated viscera coefficient. After 90 days, animals of each group were in good condition. There were several significant differences between GM and its corresponding non-GM group in indexes of the serum biochemistry, hematology and relative organ weight (P＜0.05), but these differences were not dose-relative or gender-relative. For which it meant no biological significance. The study does not find genetically modified rice has subchronic toxicity in experimental animals. The results also lay a scientific basis for the development of antitrypsin drugs using transgenic means.

As the major structural polysaccharide of plant cell walls, cellulose is the most abundant organic material on earth. Cellulose biodegradation offers the potential to produce fuels and other chemicals from renewable substrates. In cellulase biodegradation system, endogluconase attacks cellulose at random places, to break the long-chain polyer of glucose into short chains, exoglucanase attacks the ends of the cellulose, to release cellobiose, and β-glucosidase cuts cellobiose and cello-oligosaccharide into glucose. Endoglucanases are classified by the Carbohydrate-active Enzyme data into 13 glycosyl hydrolase families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 74, 124 and 131) based on protein sequence similarity and catalytic domain structure. Glycosyl hydrolase family 5 (GH5) is one of the largest GH families, historically known as “cellulase family A” as it was the first cellulase family described. GH5 members are commonly found to be encoded as parts of multi-modular polypeptide chains mainly containing a catalytic domain and a cellulose-binding domain and play an important role in the cellulase biodegradation enzyme system. In this study, an endoglucanase (EGII) was isolated from thermophilic fungus Scytalidium thermophilum. Based on sequence alignment analysis, two catalytic residues of EGⅡ were identified by homology to the other GH5 structures as Glu 199 (acid-base) and Glu 306 (nucleophile), and 6 residues conserved among the glycoside hydrolase family 5 were also found. The 6 residues include Arg 115, His 159, Asn 198, His 264, Yyr 266, Trp 399. The His159 of the EGⅡ is highly conserved in the family 5 of glycoside hydrolases, but its function is unclear so far. To determine His159 function of catalysis, we cloned EGⅡ gene from S. thermophilum and constructed pPIC9K/egII recombinant plasmid and then mutated the His159 into Ala, Asp, Phe, Trp, Arg and Tyr by site-directed mutation, respectively. The mutant enzymes H159A, H159D, H159F, H159R, H159W, H159Y and wild-type enzyme WT (EGⅡ) were expressed in Pichia pastoris and purified using nickel affinity chromatography and characterized. Activity measurements indicated that the specific activity of all mutant enzymes was lower than that of WT. The specific activity of WT, H159A, H159D, H159F, H159R, H159W, H159Y is (11.02±0.41), (6.42±0.10), (2.09±0.07), (0.32±0.02), (0.66±0.01), (1.89±0.13), (0.19±0.02) U/mg, respectively. Further kinetic measurements indicated that Km values of all the mutant enzymes were higher than that of WT, indicating that the affinity of the mutant enzymes to the substrate is reduced. Compared with WT, kcat values of H159D, H159F, H159R, H159W and H159Y decreased but kcat value of H159A was significantly increased, indicating that His159 is involved in the binding of enzymes to substrates. The optimum reaction temperature was 70 ℃ for WT, 60 ℃ for H159A, H159D and H159F, and 55 ℃ for H159R, H159W and H159Y. After incubation at 60 ℃ for 10 minutes, H159A still retained more than 80% activity, H159D and H159W had a significant decrease, H159F, H159R and H159Y were substantially inactivated, indicating that His159 is also involved in EGⅡ thermostability. Our data provide insight into understanding of the function of His159 in the family 5 of glycoside hydrolases.

Homeotic gene family with MADS-box is widely involved in the growth and development of plant and plant morphology construction. MADS-box genes can be classified into types I and type Ⅱ, type Ⅱ genes are also named as MIKC type. In this study, the MIKC type genes, from wheat(Triticum aestivum), rice(Oryza sativa) and arabidopsis (Arabidopsis thaliana) from genome databases published in plant transcription factor family databases, were carried out with multialignment and phylogenetic analysis. Total 51 MIKC family genes were screened out from Chinese Spring genomic sequence data. These MIKC type genes were classified into agamous(AG), suppressor of overexpression of constans(SOC), agamous-like gene 6(AGL6), sepallata/agamous-like gene 2(SEP/AGL2), short vegetative phase(SVP), pistillata(PI), apetala3(AP3), gnetum gnemon mads13(GGM13), agamous-like gene 12(AGL12), agamous-like gene 17(AGL17) and squamosa/apetala 1(SQUA/AP1) subfamilies And the number of genes in these subfamilies were 8, 2, 3, 6, 2, 3, 4, 8, 5, 5 and 5 respectively. Moreover. total 105 MIKC type genes were identified from the wheat amino acid sequences in NCBI database. Among them, 12 genes belonged to classA (SQUA/AP1 subfamily), 10 genes belonged to classB (AP3, PI and GGM13 subfamily), 7 genes belonged to classC (AG subfamily), 7 genes belonged to classD (AG subfamily), 28 genes belonged to classE (SEP/AGL2subfamily) according to ABCDE homeotic gene model. The remaining 41 genes belong to SOC, AGL6, SVP, AGL12, AGL17, and OsMADS32 subfamilies, the number of genes in these subfamilies were 11, 5, 12, 2, 8, and 3 respectively. Local blast analysis was carried out using wheat MIKC type full length transcripts identified above, total 46 MIKC type unigenes were screened out from the transcriptome data of the dwarf, multi-pistil and male sterility (dms) mutant. Cluster, phylogenetic and expression pattern analysis were carried out for the MIKC type unigenes. Five genes in class A, TaAP1-1 (T1_60629), TaAP1-2 (T4_56463), TaAP1-3 (T1_42731), TaAP1-3 (T4_52821) and TaAP1-3 (T2_46748), expressed highly in wheat young spikes than that in the young stem tips. Compared with the normal plants, genes TaAP1-2 (T4_56463) and TaSEP-5A (T2_44069) were down-regulated, genes TaPI-1 (T4_15141), TaMADS82 (T3_871), TaAG-2B (T2_43881), TaSEP-2B (T4_38414), Tam7 (T2_37491), TaSEP-4 (T4_8204) and TaMADS12 (T4_56460) were up-regulated in dms mutant. These genes may be associated with abnormal differentiation of wheat flower organs. These results provide useful information for further studies on MIKC type genes in wheat development.

Green leaf volatiles (GLV), including C6 and C9 aldehydes, alcohols and lipids, are important aroma components in grapes(Vitis vinifera L). and wines. Lipoxygenase (LOX) is a key enzyme for the synthesis of GLVs. In this study, full-length cDNAs of VvLOX1 (KF033130) and VvLOX2 (KF033131) were cloned from Cabernet Sauvignon(V. vinifera) berries. By bioinformatics analysis, these two genes belonged to type Ⅰ 9-LOX and type Ⅱ 13-LOX, respectively. The expression levels of VvLOX1 and VvLOX2 were higher in leaves and roots than those in stems and tendrils. During the development of grape berries, the expression levels of VvLOX1 and VvLOX2 were related to GLV synthesis, but the peaks of two genes expression appeared 2 weeks earlier than those of C6 and C9 contents. After wounding treatment, there was a significant positive correlation between VvLOX2 expression level and C6 content, which indicated that this gene played an important role in plant stress resistance. The results of this study provide the basic data for exploring the function mechanism of LOXs in aroma formation of grape berries.

Transposon is a DNA sequence that can change its position within a genome and sometimes creat or reverse mutations. During the cloning of Chinese cabbage (Brassica rapa ssp. pekinensis) isoform of eukaryotic translation initiation factor 4E (eIF(iso)4E.c), a large DNA fragment with 3 195 bp was found to insert into its third intron of 2 accession (DaQinBai and 826) of 12 Chinese cabbage inbred lines. Bioinformatics analysis revealed that a set of typical 8 bp target site duplications and 17 bp short terminal inverted repeats were found in two side of the fragment, in which the structure was more like that of hAT (after hobo from Drosophila melanogaster, Ac from Zea mays, and Tam3 from Antirrhinum majus) superfamily transponson. The large inserted fragment contained 2 exons which consisted of an intact ORF coding for a product with DUF 659 domain and transposase-like function. The transponson was named BraD8 after Chinese cabbage lines DaQinBai and 826. Further analysis showed that BraD8-like elements were ubiquitous in B. rapa genome and mostly located on chromosome A04 and A09. Its organization in B. rapa genome was like that of activator/dissociation (Ac/Ds) in maize (Zea mays). The copy number of 5'-and 3'-termini of BraD8 varied greatly in different Chinese cabbage lines. DaQinBai contained only a few copies of both 5'-and 3'-terminus, while line 826 contained about 19 and 45 copies respectively. Both termini varied greatly in lines 94 610 and 05-46; 73 and 06-247 only contained the 3'-termini, and neither terminus was detected in lines 322, 8 407, 71-3-62 and T03. PCR amplification was performed by using 17 bp TIR sequence as single primer and genomic DNA from DaQinBai and 826 as templates. The intact BraD8 sequence was only amplified from DaQinBai, while small fragments forming three main band ranging from 1 000~2 000 bp were observed in 826. Sequencing analysis revealed that the small fragments shared higher than 80% identity with the 3'-termini of BraD8 and were named BraD8-like elements. To explore the distribution of BraD8 in B.rapa genome, BLAST search was performed in B. database with the intact BraD8 sequence as query. The search results didn’t find complete identical BraD8 sequence but a lot of fragments ranged from 200 to 600 bp. They were mostly scattered on chromosome A04, A09. The pattern of BraD8/BraD8-like elements in B. rapa genome was line Ac/Ds transponson in maize. Considering the theoretical research and practical applications of Ac/Ds in maize, the characterization of BraD8 will potentially facilitate molecular genetic studies and functional genes identification through transposon tagged protocol in Chinese cabbage .

The APETALA2/ethylene-responsive element binding protein (AP2/EREBP) superfamily is one of the largest and most conserved gene families in plant. It has great contributions in plant growth, development and response to diverse stresses such as extreme temperature (freezing damage, and heat stress), drought, high salinity and pathogen infection. It has also been involved in various hormone-related signal transduction pathways. The AP2/EREBP superfamily could be classified as four subfamilies, i.e., ERF (ethylene-responsive factor), DREB (dehydration-responsive element binding protein), RAV (related to ABI3/VP) and AP2. DREB subfamily genes play an important role in response to drought, high salt and low temperature stress in higher plants. In our previous analysis of Brachypodium distachyon AP2/EREBP gene superfamily, we found that B. distachyon DREB38 gene (BdDREB38) of DREB subfamily was significantly induced by cold, while its expression profile under other stress conditions and the activity of its promoter was still unclear. In this study, the expression profile of BdDREB38 gene under several abiotic stress conditions including cold, drought, NaCl, abscisic acid (ABA), salicylic acid (SA) and H2O2 was detected by qRT-PCR. The result showed that the expression of BdDREB38 gene had no obvious change at each time point of NaCl and H2O2 treatments in contrast with the control (without treatment), but increased gradually with the extension of drought treatment time. Moreover, the expression of BdDREB38 gene was significantly higher than that of control at 1 h point after cold treatment and at 2 h point after ABA treatment, whereas the expression of BdDREB38 gene decreased at 5 h point after SA treatment. These results indicated that the promoter of BdDREB38 gene might be a stress-inducible promoter. To further investigate the structure and function of BdDREB38 promoter in B. distachyon, a 1 510 bp fragment (named as PBdDREB38) at the upstream of this gene was cloned. Plant CARE analysis reveals that this promoter not only consists of the basic cis-elements such as TATA box and CAAT box, but also includes some cis-elements involved in adversity stress and light response, such as LTR (lower temperature response element)、HSE (Heat shock response element), TC-rich repeats (defense and stress response element), SP1(light response element), TCA-element (SA response element) and so on. To investigate the expression profiles of this promoter, it was fused with the β-glucuronidase (GUS) reporter gene in the recombinant expression vector pCAMBIA1381-GUS, and then the constructed expression vector was transformed into tobacco (Nicotiana tabacum) through Agrobacterium-mediated method. GUS staining result showed that BdDREB38 promoter could be significantly induced by drought stress, but was not induced by cold treatment, although BdDREB38 gene enhanced its expression after 2 h cold treatment. This case may be the reason that one regulatory element in the promoter region is not enough to drive the expression of its downstream target genes and needs other remote cis-acting element such as enhancer in the promoter region to promote the binding of transcription factors and finally activates their downstream target genes. This study would provide a theoretical basis for further functional study of BdDREB38 gene and its promoter in B. distachyon.

Bacillus subtilis is a probiotics that can be added directly to the diet to improve the uptake of nutrients in animals. With the rapid development of gene technology, the expression system of B. subtilis is widely used in the livestock industry. In order to ameliorate the transformation efficiency of B. subtilis competent cells, different types and concentrations organic solvents were added in the process of competent cells preparation to optimize the transformation efficiency. In this study, 1.0%, 2.0%, 3.0%, 4.0% and 5.0% of the Tween-80, methanol and acetone were added in preparation of wild-type Bacillus subtilis LN competent cell. The number of viable bacteria was calculated by dilution plate counting method to determine the best addition reagent and its concentration. The plasmid pGEM-kpgt and pGEM-kmpgt with the length of 500 and 2 680 bp was transformed into individual competent cells. The transformation results were determined by PCR using P4326F/P4326R and KgF/KgR as primers and the transformation efficiency was calculated. The results showed that the count of viable competent cell is 546±13 cell/μL and the highest transformation efficiency is 52±4 transformants/μg DNA when adding 4% methanol in the preparation process. When the length of the target gene fragment was extended to 2~3 kb, the transformation efficiency was 29±2 transformants/μg. Through the optimization of the preparation method of the competent cell, the transformation efficiency of the competent cell of Bacillus subtilis was improved, which provides a potential tool for genetic engineering of Bacillus subtilis.

Coronatine (COR) is a chlorosis-inducing non-host-specific phytotoxin that is produced by several pathovars of Pseudomonas syringae including P. syringae pv. tomato, P. syringae pv. glycinea, P. syringae pv. maculicola and P. syringae pv. atropurpurea. COR is a structural and functional mimic of jasmonates but it is more active than jasmonates. COR is involved in a wide array of effects on plant development and defence responses including inhibition of root elongation, hypertrophy, senescence, accumulation of defense-related protease inhibitors, secondary metabolite production, ethylene emission and resistance to abiotic stresses, and can function as a novel plant growth regulator. The objective of this study is to establish a rapid method for screening coronatine producing strains, which plays an important role in enriching its strain resources and promoting the research and application of COR. According to the key genes in COR biosynthesis gene clusters, corS and corR, two sets of PCR primers were designed. Polymerase chain reaction (PCR) and agarose gel electrophoresis gel electrophoresis were conducted for initial screening of wild COR-producing strains. The yield of COR for wild strains was determined by high performance liquid chromatography (HPLC). As a result, the target bands corS (850 bp) and corR (450 bp) were amplified when we used the genomic DNA of a typical COR-producing strain P. syringae pv. glycinea MW123 as a template. And it was consistent with the result of colony PCR of P. syringae pv. glycinea MW123. The PCR method was verified with the typical COR-producing strains and non-COR-producing strains. The results showed that the corS (850 bp) and corR (450 bp) bands were amplified only from the typical COR-producing strains, including P. syringae pv. glycinea MW123, P. syringae pv. glycinea MFB1 and P. syringae pv. tomato DC3000. The 2 target bands were not be obtained when using the non-COR-producing strains as templates. This showed that the designed primers had a fairly good specificity for COR-producing strains. The above PCR method was applied to preliminary screening of 32 wild stains isolated from diseased plants in the field. The specific PCR assay displayed that a 850 bp (corS) band could be obtained from the strains numbered 7, 18, 19 and a 450 bp (corR) band could be obtained only from the strains of 7 and 18. The 2 target bands, corS (850 bp) and corR (450 bp), weren't be observed in the PCR products of the rest wild strains. Among 32 wild strains, 3 wild strains (7, 18 and 19) were picked out as potential COR-producing strains. The selected strains were inoculated and cultivated in HSC culture medium for COR fermentation. Subsequent HPLC analysis showed that the strains of 7 and 18 could synthesize COR and the yields of COR were 6.5 mg/L and 2.1 mg/L, respectively. Two novel producing-COR strains were successfully screened out from 32 wild strains by specific PCR and HPLC. In comparison, the specific PCR is more efficient for COR-producing strains and it is suitable for screening rapidly COR-producing strains which could help to found higher yield of COR-producing strain and provide support for the industrial production and large-scale agricultural applications of COR.

Mycoplasma mycoides subsp. capri (Mmc) causes Mycoplasma pneumonia of goats and sheep (MPGS). The existing methods for Mmc detection are time-consuming and unquantifiable, but not suitable for accurate and rapid detection of Mmc. In order to establish a SYBR Green I real-time PCR assay for detecting Mmc. A pair of primers was designed based on the hypothetical protein gene (MLC_1770) gene of Mmc. The recombinant plasmid of Mmc-1770 was constructed as positive control for standard curve development, and the specificity, sensitivity and reproducibility of the assay were evaluated. The result showed that the correlation coefficient (R2) of standard curve was 0.999 and the amplification efficiency (E) was 101.3%. There was no cross reactions with Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma ovipenumoniae (Mo), Pasteurella multocida (Pm), Escherichia coli (Ec), Staphylococcus aureus (SA) and Orf virus (ORFV), indicating a good specificity. The sensitivity of this assay was 226 copies/μL which was 100 times higher than that of the conventional PCR. The coefficients of variation between the intra-groups assay were 0.54%~0.79%, and the inter-groups assay was 0.70%~0.96%. The 95 clinical samples were tested with this assay, the Mmc positive rate was 11.6% (11/95). The assay could be used to detect Mmc rapidly in clinical samples with strong specificity, high sensitivity and reliable reproducibility.

Crown rot caused by Fusarium pseudograminearum is one of the main devastating diseases in wheat (Triticum aestivum) production. Studying the interaction mechanism between F. pseudograminearum and wheat varieties is the most economical and effective measures to control of wheat crown rot. The objectives of this study are to optimize the Agrobacterium tumefaciens-mediated transformation (ATMT) technology system of F. pseudograminearum, and obtain the transformant strains of F. pseudograminearum (WZ-8A) successfully transformed with with the green fluorescent protein (GFP). Firstly, the transformation conditions were optimized, and then the GFP gene was transformed into the conidia of F. pseudograminearum strain WZ-8A using the A. tumefaciens strain carrying plasmid pCAM-GFP-Hyg. After transformation, transformants randomly collected were screened and identified through the analysis of the hygromycin B (hyg) resistance, as well as using the fluorescence microscopy techniques. A pathogenicity detection was subsequently conducted. The results showed that a satisfactory transformation with a efficiency of 42 transformants per 1×106 spores could be achieved in a sytem of 1×106 spores per milliliter of F. pseudograminearum spore suspension which were co-cultured with Agrobacterium cells under the culture in the presence of co-culture medium containing CaCl2 at 2.6×10-2 g/L at 28 ℃ for 2 d. The transformants was stable when grown on hygromycin B-free PDA medium for 5 generations. The transformants were found to be hyg-positive by PCR amplification and Southern blot analysis, and to be GFP-positive through the detection using fluorescence microscopy, compared with the wild-type strain of WZ-8A. The subsequent test showed that the transformants really did not lose the pathogenicity. It thus be concluded that the T-DNA bearing GFP gene is successfully inserted into the genome of F. pseudograminearum with the optimized system mediated by A. tumefaciens. The optimized genetic transformation sysytem mediated by A. tumefaciens in F. pseudograminearum could be used for the studies on the pathogenic mechanisms as well as the resistance mechanisms in wheat cultivars.

The APETALA2/ethylene responsive factor (AP2/ERF) family transcription factor is plant-specific. It has been reported that the AP2/ERF family members involve in plant stress response and govern plant growth and development. In this article, we reviewed the classification, structure characteristics, and the distribution in rice of the AP2/ERF family, summarized the biological functions of the AP2/ERF in rice and gave a perspective on the application of the AP2/ERF transcription factor in rice breeding.