WRKY transcription factors constitute a large family in plants and play important roles in many different physiological processes. Many members of WRKY genes have been studied in various processes including abiotic and biotic stress tolerance in Arabidopsis thaliana. However, the functions and molecular mechanisms of many WRKY genes are still unknown. In this report, the WRKY72 gene in Arabidopsis, which is a member of group Ⅱb is studied. In order to explore the characteristics of WRKY72 gene and its role in abiotic stress tolerance, various cellular and molecular biology approaches were employed. Subcellular localization using green fluorescence protein reporter gene (GFP) in leaves of Nicotiana benthamiana showed that the WRKY72 transcription factor was localized in the nucleus of leaf epidermal cells. qRT-PCR was used to examine its response to multiple stress and phytohormone treatments at 3 time-points. The results demonstrated that WRKY72 transcription was inhibited by cold, heat, salt, abscisic acid (ABA), methyl viologen (MV), low nitrogen (LN) and glucose (Glc) treatments, which showed that WRKY72 may play a role in these stress signaling pathways. The transcriptional activity of WRKY72 was assessed through in vivo yeast assay, revealing that WRKY72 was a transcriptional repressor. Through a dual luciferase reporter system, WRKY72 was identified to significantly repress the transcription of firefly luciferase gene under the control of tandem repeats of W-box cis-elements, compared to the control. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 and the results showed that WRKY72 interacted with homologous WRKY9, WRKY36, WRKY47, WRKY61 and itself. These indicated that WRKY72 was able to form both heterodimers and homodimers in vivo. Finally, 2 T-DNA insertion mutants of WRKY72 gene were identified through PCR, and semi qRT-PCR assay indicated loss-of-function mutation. Phenotypic assay was done to compare the response of the knock-out mutant to wild-type Col-0 under different abiotic stress conditions at the post-germination level. Root elongation assay showed that the elongation of wrky72 mutant roots were significantly shorter under MV and LN treatments whereas no significant difference was observed under any other stress treatment, suggesting that WRKY72 might participate in the signaling pathways of oxidative stress tolerance and nitrogen absorption and/or utilization. In summary, the experimental evidences provided here support that WRKY72 was a transcriptional repressor and played a role in abiotic stress signaling by forming a complex with homologous WRKY9, WRKY36, WRKY47 and WRKY61. These data have laid a foundation for in-depth dissection of function and molecular mechanism of WRKY72 transcription factor gene in the context of abiotic stress signaling.

The Rabies virus (RV) G protein is the only structural protein that stimulates the body to produce cellular immunity and induces the production of neutralizing antibodies. In this study, the optimized rabies G gene sequence was inserted into the intermediate vector pMP3, and the intermediate vector pMP3-G was successfully constructed. Then the intermediate vector pMP3-G and the plant vector pCAMBIA1300 were double-digested and ligated, and the plant expression vector pCAMBIA1300-G was successfully constructed. The blank non-transgenic rice (Oryza sativa) seed TP309 was selected and disinfected. The callus was grown on the callus induction medium for 3 d at 31 °C, and the callus was inoculated with Agrobacterium tumefaciens EHA105 which had been transferred to the rabies G gene. The callus was co-cultured for 3 d, and the rabies G gene was introduced into rice callus. The callus was further screened, differentiated, germinated, rooted, and 313 plants were obtained, and the leaves of 313 plants were extracted by hexadecyl trimethyl ammonium bromide (CTAB) method. A total of 79 strains were positively identified by leaf genomic DNA amplification PCR. A total of 50 positively expressed plants were obtained after refining and planting. The seeds of 50 positive plants were randomly selected, cut in half, labeled, and half of the endosperm-free seeds were ground. The extract was extracted from the protein and detected by antigen test strips and western blot. The results showed that G protein was in the transgenic rice seeds. Successfully expressed and has good reactogenicity. Four strains with high expression and best reactivity were selected from 50 strains, and the other half of the seeds containing the endosperm corresponding to the positive test results in the T₁ generation of the 4 strains were planted and planted in the plant. Before and after planting the field, the genomic DNA of T₁ plants was extracted by CTAB method, and the endogenous gene of rice (rice starch branching enzyme, RBE4) was used as an internal reference. The homozygous plants in positive plants were screened by relative fluorescence quantitative PCR. Absolute real-time PCR was used to calculate the copy number of homozygous plants. As a result, 35 out of 132 plants were homozygous plants. The copy number of the homozygous plants were 1, 2, 3, 5, 7, 11, and 16, respectively. This study is dedicated to the expression of rabies G gene in rice and its genetic stability, laying the foundation for the preparation of new rabies subunit vaccines in the future.

Seed shattering is one of the major agronomic traits closely related to rice (Oryza sativa)production. Easy shattering is not good for seeds harvest, nor suitable for mechanized production of rice. qSH1 (QTL of seed shattering in chromosome 1; LOC_Os01g62920) is one of the major genes to control rice seed shattering. Higher expression of qSH1 in the abscission zone (AZ) would promote the formation of AZ, and lead to easy shattering. The CRISPR/Cas9 system was used to edit qSH1 in this study. Three CRISPR/Cas9 vectors, which contain 5 targets for qSH1 gene or the key SNP site at its 5' regulatory region, were constructed and transformed to Oryza sativa ssp. indica HR1128 respectively. 31 T₀ transgenic plants were obtained and confirmed by T-DNA specific PCR. The mutant efficiency for 3 vectors amounted to 85.71%, 76.92%, 63.64%. Through sequencing analysis of 10 T₀ mutant plants, it was be found that they would mutate in a way of deletion, insertion or substitution, etc. As a consequence of variation in qSH1, it would either terminate translation in advance for a frameshift in CDS, or change its transcriptional level for an influenced binding ability for ABI3 transcription. In this study, the qSH1 gene or its regulatory region were targeted-edited successfully and obtained mutant plants with various types. These results will be used for analysing new functional or key regulated sites of qSH1, and rice suitable seed-shattering breeding.

E3 ubiquitin ligases, a set of crucial component of ubiquitination pathway, are involved in many aspects of biological process. In order to seek more genes which have key function in antiviral response, this study based on the previous research achievements amplified the Oryza sativa ubiquitin fusion protein (OsUF) cDNA sequence (GenBank No. XM_015757177.2) by using reverse transcription-PCR (RT-PCR) method. The bioinformatics analysis showed that OsUF contained 5 ubiquitin motifs (Ubiquitin, Rad60-SLD, Ubiquitin-2, Rad60-SLD-2 and DUF2407), and 2 non-ubiquitin motif (Ribosomal_L4F01020 and zf-C4pol). Then the full-length of OsUF cDNA was reconstituted into pEarleyGate103, a transient expression vector fused expression with green fluorescent protein (GFP). Agrobacterium-mediated transient expression of OsUF was performed in the epidermal cells of Nicotiana benthamina leaves. Subcellular localization and 4',6-diamidino-2-phenylindole (DAPI) dyeing showed that OsUF protein was localized in the nucleus. Western blotting showed that the GFP-fused OsUF protein was expressed. This analysis of motif and subcellular localization pattern of OsUF may provide key insights into the elucidation of biological function of OsUF and gives more information to learn the mechanism of antiviral response in rice.

DREB (dehydration responsive element binding) in plant plays important roles in resisting drought, high salinity and low temperature, etc. However, the further study on its family showed that the resistance of this gene to different crops was complicated and limited. TaDREB1 was cloned by hexaploid wheat (Triticum aestivum) and its diploid and tetraploid wild relative species as materials (GenBank No. DQ195070.1). It was found that there were 3 DNA sequences in the hexaploid wheat, corresponding to the 3 genomes A, B and D, and were named as TaDREB1-a/b/d, respectively. Develop functional markers according to polymorphic sites of TaDREB1-b between different materials, TaDREB1-b was mapped using a doubled haploid (DH) and recombinant inbred lines (RIL) population. TaDREB1-b was mapped in the intervals between markers P2449-185 and WMC231 on chromosome 3B, with 19.2 and 16.0 cM from the flanking markers using DH population, and mapped in the intervals between markers Xfbb117 and Xgwm566 on chromosome 3B, with 3.4 and 6.4 cM from the flanking markers using RIL population; At the same time, the specific primers of TaDREB1-a/b/d were designed by using wheat variety Hanxuan 10 as the material, and the expression pattern of TaDREB1-a/b/d under 4 stresses was analyzed by qRT-PCR. The expression peak and degree of TaDREB1-a/b/d were different in leaves and roots. TaDREB1-a/b expression were up-regulated in leaves under PEG, NaCl and ABA condition, while the expression of TaDREB1-d were down-regulated. TaDREB1-a/b/d was up-regulated under low temperature in leaves. In roots, the expression of TaDREB1-a/b/d were up-regulated in all condition. TaDREB1-a/b/d were all involved in responding to PEG, NaCl, ABA and 4 ℃ stresses, but different in expression patterns. The functional markers developed in this study provides the tools for molecular marker-assisted selective breeding. Meanwhile, the relationship between TaDREB1-a/b/d and abiotic stress response is preliminarily explained at the genome level, laying a foundation for in-depth exploration of the molecular mechanism of wheat stress resistance.

Tripterygium wilfordii is an important medicinal plant that has anti-cancer, anti-inflammatory and immune system regulation effect, and also has strong insecticidal activity. Celastrol is the most representative triterpenoid in T. wilfordii which has high pharmacological activity for medical application and potential development value. Oxidosqualene cyclase (OSC) is considered to be the key rate-limiting enzyme in the triterpenoid biosynthesis pathway. The present study cloned 4 OSC genes, TwOSC1 (GenBank No. MH412928), TwOSC2 (GenBank No. MH412929), TwOSC3 (GenBank No. MH412930) and TwOSC4 (GenBank No. MH412931), from T. wilfordii. The ORFs of TwOSC1, TwOSC2 and TwOSC3 were all 2 289 bp and encoded 762 amino acids. The TwOSC4 ORF was 2 280 bp and encoded 759 amino acids. Bioinformatics analysis showed that the amino acid sequence similarity of the 4 TwOSCs was high, and the similarity of multiple sequence alignments between each 2 sequences was 53% to 77%. Phylogenetic analysis showed that TwOSC1 clustered with β-amyrin synthase, TwOSC2 and TwOSC4 clustered with cycloartenol synthase, and TwOSC3 clustered with lupeol synthase. The expression patterns of 4 TwOSC genes in different tissues and the expression level in T. wilfordii hairy roots treated by methyl jasmonate (MeJA) were detected by qRT-PCR. TwOSC1 had a very strong tissue-specificity and was only expressed in the root. However, TwOSC2, TwOSC3, and TwOSC4 were expressed in all tissues. The expression level of TwOSC1, TwOSC2 and TwOSC4 increased by different degrees in T. wilfordii hairy roots after MeJA treatment. But MeJA treatment inhibited the expression of TwOSC3. The content of celastrol that was detected by high-performance liquid chromatography (HPLC) increased after MeJA treatment in T. wilfordii hairy roots. The change of TwOSC1 expression was consistent with the change of celastrol content after MeJA treatment. Above results indicate that TwOSC1 may be involved in the synthesis of celastrol in T. wilfordii hairy roots. The present study provides some basic data for the synthesis and regulation of triterpenoid in T. wilfordii.

The large outbreak of canker disease has posed a serious threat to the sustainable development of hickory (Carya cathayensis) industry. For the purpose of studying the effect of bacterial diversity and function of C. cathayensis on canker disease, this research adopted 16S rRNA gene high-throughput sequencing, compared the differences of pH values, nutrients and bacterial diversity in sick parts and healthy parts, bark and xylem, respectively of healthy hickory trees and diseased trees during diseased period of canker disease, and the correlation were also analyzed. The results indicated that the pH values of the bark and xylem of healthy hickory trees respectively were 7.03 and 7.63. The neutral pH was significantly higher than that of acidic hickory trees (P<0.05). The tannin and soluble sugar contents of healthy hickory bark were 0.317% and 3.348% respectively, while that of the xylem were 0.079% and 0.626% respectively, of which all the values were remarkably higher the values of diseased trees (P<0.05). The reducing sugar contents in tree bark and xylem of healthy hickory trees were 0.865% and 0.420%, respectively, of which both were lower than that of diseased hickory trees (P<0.05). The amount of bark bacterial operational taxonomic units (OTU) in healthy hickory trees was significantly higher than that in diseased trees, while the amount of xylem bacterial OTU was significantly lower than that in diseased trees. The amount of the main bacterial genus found in healthy hickory barks, sick parts and free-diseased parts of diseased hickory barks were 7, 8, and 8 respectively, while the amount of that in xylem respectively were 8, 4, and 8. The species and abundance of the main bacterial genus in the barks and xylem of healthy and diseased hickory trees presented remarkable differences (P<0.05). The dominant bacteria genus lived in healthy hickory bark were Bdellovibrio, Acidocella, and Sphingomonas, and their abundance were 0.13%, 0.01%, and 15.32%, respectively. While the dominant bacterial genus founded in xylem were Massilia and Sphingomonas, and their abundance were 1.2% and 4.41%, respectively, of which both were remarkably higher than that of diseased hickory trees (P<0.05). In the diseased hickory trees, the dominant bacterial genus of sick and free-diseased bark both were Byssovorax and Methylobacterium. In diseased parts, the abundance of these 2 genera were 7.56% and 6.46% respectively, both higher than that in free-diseased parts. Both of the abundance of these 2 bacteria genera in 2 parts, tree bark, of the diseased C. cathayensis trees were obviously higher than that of the healthy hickory trees (P<0.05). In diseased parts xylem and free-diseased parts xylem of diseased hickory trees, Methylobacterium, Luedemannella, and Byssovorax performed as the dominant genera, and their abundance were 2.36%, 0.35%, 2.35% and 1.76%, 1.81%, and 0.55% respectively. All these figures were significantly higher than that of healthy hickory trees (P<0.05). The index of Ace, Chao, Shannon, and Simpson in the bark and xylem of healthy hickory trees were 599, 603, 6.59, 0.98 and 440, 454, 6.35, 0.95. All values were significantly higher than that of diseased hickory trees (P<0.05). The analysis of redundancy analysis (RDA) and monte carlo testing indicated that the impact of pH value, soluble sugar, and reducing sugar on the dominant bacteria genera in the bark and xylem of C. cathayensis trees were remarkable and significant (P<0.05). This research result could be considered as a reference for a sustaining and healthy operation run by the hickory forest.

Dehydration responsive element binding protein (DREB) belongs to the APETALA2/ethylene response factor (AP2/ERF) family in plants, which is a large group of plant-specific transcription factors. The DREBs widely participate in various abiotic stresses, such as drought, salt, heat, and cold. In order to explore the function of DREB in Malus zumi, a new gene named MzDREB2A (GenBank No. MH992511) was isolated and cloned using the method of rapid amplification of cDNA ends (RACE). The open reading frame of MzDREB2 was 1 197 bp without intron structure, encoding 398 amino acids and containing an AP2 domain with the molecular weight 43.9 kD and the isoelectric point 4.93. Bioinformatic analysis showed that MzDREB2A was a hydrophilic protein and had no transmembrane region, and the secondary structure contained 7.54% α-helix, 4.52% β-sheet, and 87.94% random coils. Homology analysis and phylogenetic analysis showed that MzDREB2A protein had the highest sequence similarity (about 99%) and closest genetic relationship to Malus sieversii. qRT-PCR analysis showed that MzDREB2A exhibited the highest expression level in roots followed by blades, and the lowest expression level in stems. The expression level of MzDREB2A sharply increased under NaCl or PEG-6000 treatment, and MzDREB2A expression reached the maximum when NaCl concentration was 150 mmol/L and PEG-6000 concentration was 20%, respectively. This study successfully cloned the MzDREB2A of AP2/ERF family in Malus zumi, which might provide a reference for further study on the function of MzDREB2A and the molecular mechanism of stress resistance in Malus zumi.

Carotene hydroxylases are key enzymes that catalyze the synthesis of carotenoids in metabolic pathway of carotenoids. It consists of the β-carotenoid hydroxylase (chyb) and the cytochrome P450 (cyp97) family. In order to investigate the bioinformatic characteristics of the chyb and the cyp97 gene family and the expression of chyb and cyp97 under different stress conditions in Dunaliella viridis, cDNA sequences of chyb1, chyb2 and cyp97c of Dunaliella viridis were obtained from the transcriptome data. Bioinformatic analysis showed that the proteins encoded by chyb1, chyb2 and cyp97c genes of D. viridis were hydrophilic proteins, of which CHYB1 and CHYB2 had four transmembrane structures respectively, and CYP97C protein had no cross-membrane structure. Multiple sequence alignment showed that both CHYB1 and CHYB2 contained conservative histidine residues (HXXXXH and HXXHH) while CYP97C contained a heme site (FXXGXRXCXG) and a conservative site (EXXR). Phylogenetic tree analysis revealed that the chyb1 and chyb2, belonging to chyb gene family, while cyp97c was a member of cyp97 gene family and most closely related to the Chlamydomonas reinhardtii. Moreover, qRT-PCR results revealed that all of the 3 tested genes were up-regulated (P<0.01) during high light stress. Besides the content of lutein, zeaxanthin, and the active oxygen level in D. viridis increased significantly, indicated that carotenoid hydroxylase gene family was involved in high light stress and regulated reactive oxygen level to protect algal from oxygen stress. In addition, excess salt or low temperature stress or plant growth regulators (e.g. methyl jasmonate, acetylsalicylic acid, arachidonic acid, ammonium cerous sulfate) could not induce these two family genes simultaneously, revealed that different family genes had different preference to stress response. The present study initially revealed the metabolic control mechanism of carotenoid of D. viridis, which provides excellent genetic resources for increasing the content of lutein and zeaxanthin, and a new sight for improving plant stress tolerance.

Acetyl­CoA carboxylases alpha (ACACA) and fatty acid synthase (FASN), as crucial rate-limiting enzymes, exert significant roles in the biosynthesis of fatty acid and were regulated by multiple hormones in mammals. However, it is still unclear about the expression and regulation characteristics about ACACA and FASN genes in hen (Gallus gallus). The experiment was designed to explore the expression and regulation characteristics of ACACA and FASN genes related to lipid metabolism in hen. A total of 10 tissues including heart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary were obtained from 20- and 30-week-old Lushi green eggshell hens.In addition, more liver tissues were obtained from hens at different developmental stages including 5-, 15-, 20-, 30- and 35-week-old Lushi green eggshell hens. These tissues were used to detect the temporal and spatial expression pattern of ACACA and FASN genes via qRT-PCR. The hens and chicken embryonic primary hepatocytes (CEPHs) were treated with different concentrations of estrogen, and the regulatory mechanism of ACACA and FASN genes mediated by estrogen in vivo and in vitro were analyzed using qRT-PCR. The hens were fed with the basal diet supplemented with 4% or 8% soybean for 30 days, respectively, to study the effect of different doses of fat feed on ACACA and FASN genes expression. The results demonstrated that ACACA and FASN genes were expressed in all detected tissues including heart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary and shared a highest expression in livers. In addition, the expression levels of ACACA and FASN genes were highly significantly higher in the liver of peak-laying hens than that in the liver of pre-laying hens (P≤0.01). Moreover, compared to the control group, ACACA and FASN genes showed significantly or extremely significantly higher expression abundance in livers of hens treated with the 0.5, 1.0, 2.0 mg/kg dose of 17β-estradiol dissolved in olive oil or primary hepatocytes treated with either 25, 50 or 100 nmol/L dose of 17β-estradiol dissolved in ethyl alcohol (P≤0.05 or P≤0.01). Compared to the control group, ACACA and FASN genes were significantly (P≤0.05) and extremely significantly high (P≤0.01) in 8% soybean feed group compared to the control group, but no difference in 4% soybean feed group. Taken together, ACACA and FASN genes were likely to participate in the lipid metabolism in laying hen and their transcriptional expression were regulated via estrogen, and 8% soybean supplement in the basal diet contributes to an significant increase in their expression levels. This study paved the way for the further understanding the regulatory mechanism of ACACA and FASN genes on laying hen lipid metabolism.

The adenylsuccinate lyase (ADSL) belongs to a member of the aspartase /fumarate superfamily family, and plays an important role in the initial synthesis of purine nucleotides and purine nucleotide cycle as well as improving muscle flavor. This study was to clone the cDNA full length of ADSL gene (GenBank No. MK161097) of Xupu goose (Anser cygnoides) by rapid amplification of cDNA ends (RACE), and analyze the ADSL mRNA level in different tissues/organs at sexual age (80 d), the nucleic acid and protein sequences by ADSL conducted by bioinformatics analysis. The result showed that the full length of ADSL cDNA sequence was 1 496 bp, containing a 202 bp 5'UTR, a 31 bp 3' UTR and a 1 263 bp ORF, encoding a protein of 420 amino acid, no signal peptide sites, non-secreted protein. The gene mainly found in cytoplasm by subcellular localization, and was an unstable hydrophilic protein. Sequence analysis revealed that the ADSL amino acid sequence of the Xupu goose was similar with those of the eastern Zhejiang White goose (99.68%), duck (Anas platyrhnchos, 88.35%), chicken (Gallus gallus, 85.64%), pigceon (Columba livia, 82.24%). Based on the relationship of ADSL to build construction system and showed that Xupu goose had the closest phylogenetic relationship to the eastern Zhejiang White goose, followed by the close relationship between duck and chicken, and the distant relationship with mammals. The qRT-PCR results showed that ADSL gene was expressed in different tissues, and the mRNA level of ADSL from high to low were breast muscles, leg muscle, liver, sebum and abdominal fat. The expression of ADSL in breast muscle was significantly higher than that of sebum and abdominal fat (P<0.05). These results provide a reference for further study on the mechanism of the ADSL gene in the goose meat quality.

Agouti-signaling protein (ASIP), encoded by Agouti and acted as an antagonist of Melanocortin 1-receptor (MC1R), plays a key role in inhibiting deposition of eumelanin and forming the coat color of tan striped character. Mink (Neovison vison) has a colorful coat and is an important economic animal for fur. However, the role of Agouti in mink coat color is unclear. To get the Agouti gene sequence of mink and its structure character, the distribution of 601 bp insertion mutation and the relationship between transcription of fragment with insertion and Agouti gene was explored. The genome of black mink, white mink and coffee mink and the RNA from skin of black mink and coffee mink were used to amplify Agouti gene sequence and transcriptome sequencing, respectively. PCR cloning and sequencing, and bioinformatics analysis were used to get Agouti gene sequence and mutations, respectively. And the spliceosome types were predicted. Population analysis was done for the 601 bp insertion in the three-color-type mink, and the luciferase promoter activity was detected for fragments in both directions with and without the 601 bp insertion. The transcripts of Agouti gene were analyzed based on the transcriptome of mink skin. The mink Agouti gene sequence for 25 478 bp was submitted to NCBI (GenBank No. KP981640.1) and 7 spliceosomes were predicted. 60 SNPs and Short interspersed nuclear element (SINE), a 601 bp insertion mutation were found in Agouti gene. The wild genotype frequency in black mink was significantly higher than white mink and coffee mink (P<0.05). The promoter activity of the 1 605 bp fragment in forward direction, which included the SINE and the up and down stream sequence of the insertion locus, was significantly higher than that of the fragments in inverse direction and the deletion fragments in both directions (P<0.01). A 973 bp transcript contained the 601 bp SINE was 100% covered and similar with the intron 3 sequence of Agouti gene. However, none of the Agouti transcripts was detected. It was concluded that the Agouti transcription was silenced by the 601 bp SINE insertion in the intron 3 of Agouti gene, and the insertion fragments had promoter activity and the transcript. The research discovered that Agouti gene might not take part in the coat color formation of mink, which provides some data for further studying relationship between expression regulation of transposable elements and gene function.

Bordetella bronchiseptica (Bb) is the main pathogen of respiratory infectious in rabbits (Oryctolagus cuniculus). To investigate the effect of cold stress on the infection of Bb in rabbits, 90 New Zealand laboratory rabbits (45-day-old healthy) were randomly divided into 5 groups, injected intranasally with 0.5 mL 4×10⁹ CFU/mL Bb (group: Bb/RT, Bb/cold) or phosphate buffer saline (PBS) alone (group: PBS/RT, control /RT, PBS/cold). Rabbits in Bb/RT group and PBS/RT group (20±2 ℃) were kept in different cages in the same room. Bb/cold group and PBS/cold group (6±2 ℃) were kept in different cages in the same room, while control/RT group (20±2 ℃) were kept, separately. Three rabbits of each group were randomly sampled at 7 h, 10 h, 1 d, 3 d, 7 d and 12 d post-infection. The concentration of Bb in the respiratory tract (nose, trachea, lungs) , heart, liver, and kidney were tested by TaqMan fluorescence quantitative PCR. The changes of rabbit interleukin (IL-10), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) mRNA were also determined by SYBR-Green reverse transcription (RT-PCR). The results indicated that Bb can spread through the air to rabbits, Bb was detected in nasal cavity of rabbits in both PBS/RT group and PBS/cold group at 7 h. Cold stress had no significant effect on Bb proliferation in the nasal cavity of infected rabbit. The Bb concentration in the nasal cavity of Bb/RT group did not differ significantly from Bb/cold group, PBS/RT group did not differ significantly from PBS/cold group at any time point examined. Bb proliferation in trachea of infected rabbits which exposed to cold stress was faster in the first 3 days. The concentration of Bb in Bb/RT and Bb/cold group was close but significantly higher than PBS/RT group and PBS/cold group (P<0.01) at 12 h, Bb/RT group higher than PBS/RT group (P<0.05) at 1 d and 3 d; there was no significant difference for all infected groups after the 7 d. None of Bb was detected in lungs, livers, kidneys, and hearts of all rabbits. Based on 2^-ΔΔCt transformation, IL-10, IFN-γ and TNF-α were found to increase in expression in the blood of infected rabbits. The expression of IL-10 was up-regulated, but there was no significant change. The expression levels of IFN-γ and TNF-α changed among sampling time and peaked at 3 d. However, there was no significant difference in the expression change of IL-10, IFN-γ and TNF-α between different infected groups at the same sampling time. Cold stress could promote the propagation of Bb in trachea of rabbits within 3 d after exposure. Nasal cavity and trachea were the main organs that Bb invades and colonizes. Cold stress did not significantly effect the cytokines expression in Bb infected rabbits. This study provides a technical basis for the prevention and control of Bb infection.

Xylanase VmXyl1 plays an important role in the pathogenesis of Valsa mali. In order to reveal the action mechanism of VmXyl1 in the interaction of V. mali and host, a recombinant plasmid pET-32a-VmXyl1 was constructed in this study. The fusion protein VmXyl1 was expressed successfully in Escherichia coli Rosetta (DE3) strain when induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The solubility and purification effects of the fusion protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, New Zealand white rabbits (Oryctolagus cuniculus) were immunized with purified VmXyl1 protein and polyclonal antibody was prepared. The titer and specificity of the antibody were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the fusion protein VmXyl1 mainly existed in the form of inclusion body, and the protein was expressed abundantly under induction by 0.5 mmol/L IPTG for 8 h at 30 ℃. The target protein with high purity was obtained by the Ni-NTA affinity chromatography. ELISA showed that the titer of prepared antibody was up to 1∶102 400. Western blot analysis demonstrated the polyclonal antibody could specifically recognize both the fusion protein VmXyl1 and xylanase protein in the apple (Malus pumila) bark tissue infected by V. mali, which indicated that the prepared antibody had high efficiency and specificity. The results in this study provide basic data for further exploring the pathogenesis of V. mali xylanase and its interaction mechanism with the host.

： In September of 2017, a serious virus disease of tomato (Solanum lycopersicum) broke out in Dongyuan town, Zhangzhou city, Fujian province, which caused severe yield loss and accompanied with the occurrence of a large number of whiteflies (Bemisia tabaci). In order to identify what type of the pathogenic virus, molecular diagnosis of the virus strain isolates was done in this research, and then, the biotype and the virus carrying rate of B. tabaci were surveyed. At the same time, the acquisition, transmission and retention of gemini viruses by B. tabaci were studied in laboratory. The results revealed that the virus sample was Tomato yellow leaf curl virus (TYLCV), the B. tabaci was B-biotype and had a very strong ability to acquire and transmit the TYLCV. The virus-carrying rate could reach up to 100%. All this indicated that whitefly-transmitted gemini viruses (WTG) was a main reason to cause the outbreak of TYLCV disease on tomato in Zhangzhou city. This will provide the basis and help for the prevention and control of TYLCV in the future.

Salmonella enteritidis (SE) always colonized in the intestinal and reproductive tracts of the poultry, causing direct contamination and vertical transmission of eggs that were difficult to eradicate and prevent. It not only affected the egg production, but also caused egg pollution as well as environmental pollution and imperiled human health. The effect proteins of the type secretion system (T3SS) played a critical role when SE attached to the host cell. In order to study the role of the T3SS1 major effect protein SipC during SE infection, the sipC deletion mutant strain of SE MY1 were constructed by using λRed homologous recombination system, and a complementary strain MY1ΔsipC/psipC was constructed by transformation of the a plasmid pBR322 carrying the sipC gene cloned from MY1. The growth curve, the ability of adhesion and invasive to human (Homo sapiens) colorectaladeno carcinoma cell line (Caco-2) and duck (Anas platyrhynchos) granulosa cell (dGC) of the wild-type strain MY1, mutants MY1ΔsipC and MY1ΔsipC/psipC in vitro were measured. And the strains were orally inoculated to 7-day-old shaoxing ducklings separately to study the pathogenicity of sipC in vivo. The results showed that the mutants MY1ΔsipC and MY1ΔsipC/psipC were successfully constructed and SE MY1 contained a sipC gene with 100% identity to the S. typhimurium. No significant difference was observed between the parental strain MY1 and sipC mutant strains in growth curve. However, after 2 h of infection, the ability of SE MY1ΔsipC adhering to Caco-2 and dGCs were decreased about 44.9% and 34.5%, respectively, as compared with the wild-type adherence, and the invasive ability of SE MY1ΔsipC to Caco-2 and dGCs were also reduced 37.9% and 30.4%, respectively. The virulence of the sipC mutant was significantly reduced in a 7-day-old duckling model of SE disease (P<0.05), as determined by quantifying the lethal dose 50% of the bacterial strains. Collectively, a novel function of sipC in contributes to SE virulence was covered and experimental reference of the interaction between sipC and host genes were offered for further study.

MicroRNA (miRNA) are a class of non-coding small RNAs of about 20~24 nucleotides in length that are produced in vivo, and play an important role in plant growth and development, biotic and abiotic stress responses. As a sessile growth plant, tobacco (Nicotiana tabacum) cannot be moved like an animal to obtain the energy needed for growth and development and to avoid adverse environmental factors. Therefore, it is necessary to have its own special physiological mechanisms such as: Initiation of control of key gene expression related to chlorophyll to promote growth and development, initiation of defense-related key gene expression in response to stress, and many miRNA participate in related processes. Recent studies have shown that many biological processes in plants are regulated by miRNA, including cell maintenance and differentiation, growth and development, signal transduction, and response to environmental stress. The expression level of plant miRNA changes with environmental factors. By regulating the expression of their corresponding target genes, miRNA can make plants adapt to the environment both physiologically and morphologically. In this paper, recent advances in miRNA-mediated tobacco stress response to drought, salt, nutrients, temperature and disease stress, as well as tobacco growth and development, The mechanism and prospects for future research on tobacco miRNA.

Sec14p, one of the phosphatidylinositol transfer proteins (PITPs), is originally found in Saccharomyces cerevisiae that has the functions of transporting phosphatidylinositol (phosphatidylinositol, PI) and phosphatidylcholine (phosphatidylcholine, PC) and is widely present in eukaryotes. Plant Sec14-like phosphatidylinositol transfer protein has high sequence homology with yeast Sec14p. In recent years, with the development of molecular biology and lipid genomics, more and more plant phosphatidylinositol transfer proteins have been excavated. In plants, the structure of those proteins has evolved from the original Sec14p domain to integrate with the membrane-localized Nlj16 domain and the vesicle transport Golgi dynamics (GOLD) domain, resulting in multi-functions, such as osmotic regulation, cell polarity growth, nodule development, protein transport, plant immune regulation and virus interactions. This paper reviews the differences and associations of structures and functions among Sec14-only proteins, Sec14-nodulin proteins and Sec14-GOLD proteins in plants, and highlights their multiple biological functions.

Synthesis and accumulation of cyanine nucleoside in Red pear (Pyrus pyrifolia) peel are key factors influencing red pear fruit coloring and biosynthesis regulated by enzymatic reaction genes, screening of stable and reliable reference genes has important significance to research the key genes and their regulatory mechanism in red pear fruit coloring. In order to select reference genes for qRT-PCR analysis in different growth period (young fruit period,slow growth period, fast growth period, mature period) and different shading conditions (natural light, partial shading, complete shading) in different varieties (Zaobaimi, Zhongshu32, Yunhongli No.1) of red pear fruit, 6 common house-keeping genes were chosen as candidate for internal gene which included β-actin (ACT), 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), histone (His), elongation factor 1α (EF-1α), and α-tubulin (TUB). The qRT-PCR amplification efficiency, the stability parameters and the relative expression in different samples of internal reference genes were analyzed. The BIO-RAD CFX Manager v2.0 software was used to analyze and compare the stability parameter index such as cycle threshold (Ct), average expression coefficient (M) and variation coefficient (CV). The results showed different expression stability in different candidate genes, and EF-1α and His showed stable expression trend in different samples from different varieties, growth stages, and processing conditions. Analysis of relative expression of reference genes in different samples showed that the expression of EF-1α and His in different samples were relatively stable. As a result, EF-1α and His could be used as reference gene for qRT-PCR analysis in red pear peel tissues which might standardize genetic selection for relative quantitation about coloring related gene expression analysis in red pear.

Excessive use of fertilizers leads to serious degradation of cultivated lands which greatly affects crop yield and quality. Through the screening of acid-protease strains, combined with modern fermentation techniques, the present study attempted to develop new amino acid organic fertilizers. An acid-protease strain named Zju-c was screened from the hydrolytic liquid of fish meal using the transparent hydrolysis circle method and the Folin-Phenol method. Morphological observation and 26S rRNA molecular identification were performed, and the fermentation condition was optimized. Results of physiological and biochemical reactions showed that most sugars and a small portion of alcohols could be utilized directly, and casein, starch, gelatin, and tartrates could be hydrolyzed, and the growth rate was fast. The concentration of NaCl can be tolerated upto 13%. The BLAST of 26S rRNA sequence in GenBank indicated that the strain belonged to Pichia pastoris and the phylogenic tree of the strain, which was generated by Vector NTI Suite 7 software based on 26S rRNA sequence, showed that it was most close to Pichia galeiformis (GenBank No. JF781422.1) strain with 100% of identity. In addition, the optimum fermentation conditions were 300 g/L fish meal, 50 g/L glucose, 30 g/L soy peptone, 30 g/L NaCl and 0.9 mg/mL Mg²⁺ with 3% inoculation quantity, with the culture condition of 30 ℃, pH 5.0, and 180 r/min for 7 d.The contents of free amino acids and enzymic activity were the fermentation index, total amino acids content in the fermentation broth eventually increased from initial 29.4±2.1 mg/mL to 88.4±1.4 mg/mL, and the enzymic activity was optimized from 190.2±2.9 U/mL to 400.1±3.8 U/mL. Therefore, the strain has good application potential for the development of liquid bio-organic fertilizer.