2A peptide is a novel ribosomal skipping based technique, which takes advantage of a cis-acting hydrolase element of a length of 18~22 amino acids with profiles of short structure and high protein segmentation efficiency. Therefore, it is increasingly used in polycistronic expression vectors. At present, it is widely used in yeast (Saccharomyces cerevisiae) and mammal cells, less in plants. In order to explore 2A peptide application in plants, the polycistronic co-expression vector pX6-2A with Porcine teschovirus-1 2A (P2A) and Thosea asigna virus 2A (T2A) linking 3 ORFs (open reading fragments) including a chimeric transcription activator gene XVE (X, the DNA-binding domain of the bacterial repressor LexA; V, the acidic trans-activating domain of VP16; E, the regulatory region of the human estrogen receptor.), GFP (green fluorescent protein) and Bar (bialaphos resistance) in series under the control of 35S promoter was constructed, and transformed into Arabidopsis thaliana, to obtain successful transgenic plants. Western blot results showed that these 3 proteins XVE, GFP and Bar expressed separately, indicated that both P2A and T2A peptide could transcribe, express and translate normally, and there was no interference with each other. It suggested that it could be an ideal way to express polycistronic in plants. These results indicated that P2A and T2A could do 'ribosome skipping' and separate proteins completely in plants. P2A and T2A were suitable for application in plants, which provides an important theoretical and referential significance for the popularization and application of 2A peptide in transgenic plants.

Calcineurin B-like proteins (CBLs) represent a family of newly emerging plant-specific Ca²⁺ sensors, which could selectively interplay with their respective kinase effectors (CBL-interacting protein kinases, CIPKs), thus playing critical roles in response to signal transduction of various abiotic stresses. To identify the functional characteristics of the CBLs gene involved in stress response in Vitis plants, the cold-tolerant species of Vitis amurensis 'Zuoshan-1' was used as the material to isolate one calcineurin B protein family member VaCBL01 by homologous cloning. Analysis of its sequence and structure indicated that the ORF of VaCBL01 gene (GenBank No. MH921999) was 642 bp, encoding 213 amino acids, with four EF-conserved domains, and a myristoylation site at the N-terminus. Chromosome location prediction indicated that VaCBL01 could be mapped to locus of 5 571 989~5 581 623 in chromosome 2. Sequence alignment revealed that VaCBL01 shared high sequence homology with Vitis vinifera VvCBL01 (100%), followed by tomato (Solanum lycopersicum) SlCBL01 (95.3%), Arabidopsis AtCBL01 (95.3%) and PtCBL01 (94.8%). Phylogenetic analysis of CBL01 gene families across a variety of species suggested VaCBL01 protein had the closest evolutionary relationship with proteins clustered in GroupⅡ, including V. vinifera, tomato, Arabidopsis and Populus trichocarpa. Furthermore, the expression levels of VaCBL01 genes either in different tissue and organs or in response to abscisic acid (ABA) and cold stress were examined by qRT-PCR. Expression of VaCBL01 was found to be ubiquitous in all tissues in V. amurensis, but its expression was relatively high in tendril. VaCBL01 gene was found to respond to ABA and cold stress, suggesting that the grapevine CBL-CIPK network may be a point of convergence for several different signaling pathways. Also, a comparison of interaction patterns of VaCBL01 and 19 VaCIPKs was performed using yeast two-hybrid system. The results showed that none of the 19 VaCIPKs had autotoxicity and self-activation in yeast cells. VaCBL01 exhibited a significant interaction only with a subset of 12 CIPKs (VaCIPK01, 02, 04, 06, 09, 10, 11, 12, 13,16, 17 and 18). For 5 additional CIPKs (VaCIPK03, 07, 15, 19 and 20), a weak tendency of complex formation was observed, whereas 2 CIPKs (VaCIPK08 and 14) did not show any affinity toward AtCBL01. The results would provide an important foundation for further functional dissection of the CBL-CIPK signaling network in grapevine.

Tamba Black Soybean (Glycine max 'Tamba') is an aluminum-resistant plant whose aluminum-resistant mechanism is that their root tips secrete the citric complex aluminum ions. In order to explore its excellent resistance to aluminum genetic resources, this study, with the foundation of transcriptome data about Tamba black soybean's aluminum stress, cloned one of Tamba black soybean's gene of citrate transporter by using reverse transcription PCR (RT-PCR) and this gene was named GmMATE2 (GenBank No. MF497433.1). CDS of GmMATE2 was 1 674 bp in length and GmMATE2 encoded 557 amino acids with a relative molecular mass of 60.38 kD, the isoelectric point of 9.54. GmMATE2, whose instability index was 28.38, was a stable protein. Amino acids sequence of GmMATE2 analysis showed that it contained nine transmembrane structures, one Polysacc_synt_C domain and two family-specific MatE structures of multidrug and toxic compound extrusion (MATE). Evolutionary tree analysis showed that GmMATE2 was closely related to MATE proteins of Medicago truncatula, Trifolium repens and Cicer arietinum. When Tamba black soybean subjected to aluminum stress (pH 4.5, 0.5 mmol/L CaCl₂, 100 μmol/L AlCl₃), the semi-quantitative RT-PCR analysis showed that GmMATE2 was up-regulated in the root tips, with the highest expression at 24 h and citric acid secretion in the root tips increased with time, which suggested that GmMATE2 was involved in response to the aluminum stress. The plant overexpression vector pBI121-GmMATE2 was constructed and 3 transgenic tobaccos (Nicotiana tabacun) were obtained by agrobacterium-mediated leaf disc method. When 3 transgenic tobaccos subjected to aluminum stress (pH 4.5, 0.5 mmol/L CaCl₂, 50 μmol/L AlCl₃), qRT-PCR analysis indicated that GmMATE2 was up-regulated in transgenic tobaccos. The citric acid secretion of transgenic tobacco root tips were 2.56~3.79 times that of wild types. The relative elongation of transgenic tobacco roots were 1.91~3.45 times that of wild types. The root tips of transgenic tobaccos were more stained lightly with chrome azurine S than that of the wild types. All the results above indicated that GmMATE2 of Tamba black soybean was a citrate transporter gene, which could improve citric acid secretion and aluminum resistance of transgenic tobaccos. This study provides genetic resources for improving aluminum resistance of aluminum sensitive plants.

The genetic mechanism of oleic acid in rapeseed (Brassica napus) is complex. The main gene FAD2 (fatty acid desaturase 2) is studied mostly, but little about minor genes now. In order to discover new related genes and promote the research of fatty acid metabolism in high oleic acid rapeseed, 20 high oleic acid rapeseed strains were used as materials in this study. Not only were their fatty acid composition tested by gas chromatography (GC), but also the expressions of OPR (1,2-oxo-phytodienoic acid reductase) and AT(acetyltransferase) gene, which screened from microRNA sequencing and related to fatty acid metabolism, were determined by qRT-PCR. Meanwhile, the relationship between the expression of the 2 genes and the fatty acid composition were analyzed. The results showed that the content of oleic acid was negatively correlated with linoleic acid and α-linolenic acid. Linoleic acid, α-linolenic acid and palmitic acid were positively correlated with each other. The expression patterns of OPR and AT genes were similar during the whole growth period. Their expression increased gradually at seedling stage, the highest expression was in blooming flowers at flowering stage, and decreased gradually at pod stage, while the expression level of OPR gene was much lower than that of internal reference gene, and it was opposite with AT. The analysis between expression of 2 genes and fatty acid composition was found that AT gene was significantly or extremely significantly correlated with oleic acid, linoleic acid and α-linolenic acid in leaves of 5~6 leaf stage, budding stage and selfed seeds of 35 d; OPR was positively correlated with oleic acid and negatively correlated with linoleic acid and linolenic acid during the whole pod stage. The 2 genes might be related with oleic acid synthesis as the minor genes. Besides, AT could be used for early screening of high oleic acid materials. The results of this study could promote the research of molecular mechanism in high oleic acid rapeseed.

JAZ (jasmonate ZIM-domain) is a major regulator of jasmonic acid signaling pathway and plays an important role in regulating plant development and responding to biotic and abiotic stresses. In the present study, GhJAZ10 (GenBank No. XM_016834302.1) was cloned from upland cotton (Gossypium hirsutum) 'zhongmiansuo 24' (CRI24), which was 723 bp in full length and encoded 240 amino acids which contained TIFY and Jas conserved domains. Subcellular localization by fusion of GFP reporter gene and transient transformation of tobacco (Nicotiana tabacum) showed that GhJAZ10 localized in the nucleus. qRT-PCR was used to analyze JAZ10 expression pattern in different tissues and different cultivars treated with PEG6000. The results showed that JAZ10 was highly expressed in flower tissues, roots and stems, and PEG6000 treatment up-regulated JAZ10 expression in tetraploid upland cotton (CRI24 and upland cotton genetic standard line TM-1), diploid cotton (Gossypium arboretum) 'Shixiya1' and wild-type diploid cotton (Gossypium raimondii). The gene was transformed into Arabidopsis thaliana by the floral dip method, and the homozygous transgenic line was obtained by resistance screening. The results showed that the stomata number of the transgenic line was significantly reduced compared with the wild type, root length and fresh weight increased significantly under drought conditions in transgenic lines, and stress response genes such as DREB2A (dehydration-responsive element binding protein 2A), RD22 (responsive to desiccation 22), DREB1A and DREB1B were up-regulated. This study preliminarily demonstrates the relationship between GhJAZ10 and drought stress response at the molecular level, and provides a theoretical reference for further exploration of its molecular mechanism in drought resistance.

Synchronization of somatic embryogenesis helps accelerate the process of crop molecular breeding. The specific expression of key genes such as Agamous-like15 (AGL15) and Aintegumenta-like5 (AIL5) plays a role similar to switch during somatic embryogenesis. To further explore the molecular mechanism of hypothermia induced somatic embryo synchronization, in this study, GbAGL15 (GenBank No. MK580460) and GbAIL5 (GenBank No. MK591943) were cloned by homologous sequences methods from the embryonic callus of Gossypium barbadense XH16, and the expression pattern of GbAGL15 and GbAIL5 genes in low temperature treated embryogenic cells were checked by qRT-PCR. The results showed that the coding region of GbAGL15 gene was 753 bp and encoded 250 amino acids, which contained the MADS-MEF2-like domain and the K-box conserved region. Phylogenetic tree analysis showed that there was a close relationship among GbAGL15 together with GrAGL15 (G. raimondii) and GaAGL15 (G. arboretum). The coding region of GbAIL5 gene was 1 626 bp and encoded 541 amino acid in the coding segment, and it contained two AP2 domains. Phylogenetic tree analysis showed that GbAIL5 was located in the same evolutionary branch with GhAIL5 (G. hirsutum). qRT-PCR results showed that the expression levels of GbAGL15 and GbAIL5 in embryogenic callus were significantly lower than those of the control group after low temperature treatment. It was speculated that the GbAGL15 and GbAIL5 genes might be involved in somatic embryogenesis and played regulatory roles. Under low temperature conditions, the two gene expression might be inhibited, resulting in the development and differentiation of hypocotyl callus tissue at low temperature, which might promotes the synchronization of somatic embryos. And after the stress conditions were removed, the embryo development could be swithced on in synchronization pattern. The study partially elucidates the mechanism of hypothermia induced somatic embryo synchronization, promoting the foundation of high efficient system of somatic embryogenesis-dependent genetic transformation in G. barbadense.

At present, cotton transgenic technology highly depends on somatic embryogenesis,and preservation of the cultured tissues needs subculture which might cause genetic variation or species degradation of preserved materials so that it is not conducive to the preservation and utilization of resources. Ultra-low temperature preservation can maximize the genetic stability of preserved materials. To establish the method of cryopreservation of Gossypium barbadensee XH33 embryonic cells by droplet vitrification, the present study optimized the system from preculture, vitrification solution and processing time, defrost method, and recovery cultivation, respectively. And the genetic stability of embryonic cell after cryopreservation was checked through the methods of flow cytometry, SSR molecular markers and physiological index detection. The results showed that XH 33 embryogenic cells were pre-cultured in 0.3、0.5、0.7 mol/L sucrose medium in sequence at 4 ℃ for 3 d, and then loaded and dehydrated for 20 min in the modified PVS2 (plant vitrification solutions 2). After stored in liquid nitrogen for 12 h, the cells were thawed in 40 ℃ water bath and washed with 0.5, 0.7, 1.0 mol/L gradient concentration of sucrose solution and then cultured in darkness for 3 d. The cell survival rate was as high as 55.55%. The results of flow cytometry and SSR molecular markers showed that cryopreservation did not affect the genetic stability of cells, and there were no significant changes in the physiological activities of cells after cryopreservation. This study established the cryopreservation system for embryogenic cells of G.barbadensehave by vitrification which provides a reference for long-term preservation of embryogenic cells in island cotton.

Mitochondrial DNA (mtDNA), as an extranuclear genetic material, carries important genetic information, while its D-loop region sequence reflects the genetic diversity of the species.To assess the genetic diversity of Guizhou pig (Sus scrofa domesticus) species,10 local pig breeds in Guizhou Province and 3 foreign varietes were chosen as the researching object in this study, they were Gaopo pig, Guanling pig, Congjiang xiang pig, Qiannan black pig, Qianbei black pig, Kele pig, Jiangkou Luobo pig, Zongdi hua pig, Baixi pig, Nuogu pig and Meishan pig, Rongchang pig, Yorkshire.The full-length sequence of the D-loop region of mitochondrial DNA was amplified and analyzed by PCR amplification combined with direct sequencing.The research results showed that the full sequence of the D-loop region of the mtDNA of the experimental group was 1 117~1 148 bp, and there were 12 sequences of length. Except for the difference in the base composition of the Zongdi hua pig, there was no difference in the base content of other varieties, which showed the highest content of T, the lowest content of C, and the base A+T content was significantly higher than C+G. Then, 66 polymorphic loci were found in the 149 splicing sequences of the valid sequencing results, and 16 haplotypes were defined, with a haplotype diversity of 0.421 5. The genetic relationship between the Gaopo pig population and other pig populations was randomly selected, and it was found that there was less genetic communication among the populations, and the gene exchange value (Nm) was less than 1. The genetic differentiation index between Gaopo pig and Zongdi hua pig was the highest, and it was 0.412 78, and the genetic differentiation index of Gaopo pig and Qiannan black pig and Guanling pig were lower, indicating that the genetic relationship among the three pigs was relatively close; The genetic diversity analysis showed that the Gaopo pig were closely related to the Qianbei black pig, while the Zongdi hua pig was far related to other 12 varieties. Based on the results of this experiment, it could be inferred that the genetic diversity of 10 local pig breeds in Guizhou was threatened to a certain extent, and it was imperative to properly carry out the conservation of local pig breeds. This study provides theoretical support for further understanding of the origin differentiation and genetic diversity of Guizhou pig breeds, and provides a reference for local pig breed protection.

In China, seasonal reproduction limits both diversification and intensification of sheep (Ovis aries) production, thus it is necessary to explore the key genes that affect the seasonal reproduction of sheep at the molecular genetic level. In order to explore the association between tissue expression levels and polymorphisms of period 1 gene (PER1) with seasonal reproduction in sheep, semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) and quantitative real-time PCR (qPCR) were performed to investigate the expression of PER1 gene in different tissues including pineal gland, hypothalamus, pituitary, ovary, oviduct and uterus in Small Tail Han sheep and Sunite sheep respectively. Seasonal estrous breeds (Prairie Tibetan sheep, Sunite sheep and Tan sheep) and year-round estrous breeds (Small Tail Han sheep, Hu and Celeblack sheep) were selected, and Sequenom MassARRAY^®SNP assay was applied to genotype 2 single nucleotide polymorphism sites (SNPs) of PER1 gene. Gene and genotype frequencies of those 2 sites were analyzed in sheep with different breeding traits. The results showed that PER1 expressed in all tissues selected. Expression levels of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in all tested tissues, and there were significant differences in pineal gland, ovary, and uterus (P<0.05). There were three genotypes in both year-round estrous breeds and seasonal estrous breeds. The allele frequencies of the PER1 g.27342699G>A was significantly different between year-round estrous and seasonal estrous sheep (P<0.05). Association analysis indicated that there were no significant correlation between the polymorphisms of the 2 SNPs in PER1 gene and the litter size of each parity in Small Tail Han sheep. In conclusion, the expression of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in the tissues relating with seasonal reproduction, which showed significant differences in pineal, ovary and uterus. Therefore, the higher level of PER1 gene was probably related to the seasonal estrus regulation, which will provide a theoretical basis for studying the seasonal reproduction mechanism of sheep.

Pituitary specific transcription factor 1 (POU1F1) is a member of the POU family (pituitary specific transcription factor, Pit-1; octameric binding protein, Oct-1 and Oct-2; caenorhabedit Ⅰ nematode nerve Unc-86), who is a positive transcription factor regulating pituitary hormone-related genes and plays an important role in the growth and development of mammals. Thus, POU1F1 gene is extensively considered as a candidate gene for growth performance in goats (Capra hircus). In order to verify the genetic effects of flanking region of POU1F1 gene on the growth traits in Shaanbei white cashmere (SBWC) goats, the genetic polymorphisms of 3'-untranslated region (3'-UTR) within POU1F1 gene was detected in all adult SBWC goats (n=621) by direct DNA sequencing method, and the associations of different genotypes of polymorphisms and 9 kinds of growth traits in SBWC goats were analyzed. Sequencing results showed that a single nucleotide polymorphism (SNP), named NC_030808.1:g.34235967T>C and located at position 164 of the 3'-UTR within POU1F1 gene (c.876+164A>G), was identified in these SBWC population. The frequencies of alleles A and G of this locus were 0.894 and 0.106, respectively; the number of effective alleles (Ne) was 1.235; the homozygosity (Ho) was 0.810; the heterozygosity (He) was 0.190; and the polymorphism information content (PIC) was 0.172 and the loci was low polymorphisms. The values of these genetic polymorphism indexes reflect the inferior degree of genetic variation in this population, and the value of PIC belongs to the low PIC (PIC<0.25). In addition, the c.876+164A>G locus conformed to Hardy-Weinberg equilibrium (P>0.05) in the SBWC goat population. The further association analysis results demonstrated that the c.876+164A>G locus was significantly associated with the body length and cannon circumference in SBWC goats (P<0.05). However, there were no significant effects from the SNP for other growth traits, including body weight, body height, height at hip cross, chest circumference, chest depth, chest width and hip width. The individuals with AG genotype were significantly superior to other genotypes in both body length (P=0.047) and cannon circumference (P=0.035) in this population. Thus, the AG genotype could be considered as a positive genotype for this SNP in SBWC goats. In addition, the mutation in 3'-UTR could not only affect its binding to target miRNA, but also change transcription factor binding sites. Here, bioinformatics prediction of transcription factors on this SNP was performed by Genomatix MatInspector software v3.11, and analysis result indicated that the binding of the transcription factor MEL1 (myelodysplasia syndrome 1/ecotropic viral integration site1-like gene 1) with POU1F1 gene was affected when this locus mutated. MEL1, also called PRDM16, could simultaneously activate and inhibit gene transcription, which might be related to multiple transcriptional domains in gene structure. Therefore, when the A allele was replaced by G allele at the c.876+164A>G locus, the binding of the MEL1 with POU1F1 gene was hampered and the inhibition of POU1F1 gene was discharged, which might affect growth and development of individuals by facilitating the expression level of POU1F1 gene. These research results unveiled that the c.876+164A>G had strong effects on body length and cannon circumference of SBWC goats, which could be used as a candidate marker for marker assisted selection of greater growth performance and would provide scientific information for the genetic breeding and improvement in goat industry.

Agrilus zanthoxylumi is a significant and destructive branch-borers pest in the Zanthoxylum bungeanum planting areas of northern China. This insect mainly recognizes volatile odor molecules through sensitivity and specificity via antenna, which plays an important role in its survival and reproduction. To select Agrilus zanthoxylumi adult male and female antenna of differentially expressed genes, preliminary parsing and Agrilus zanthoxylumi candidate differences related to the behavior and the biological prevention and control in terms of seeking mate genes, this study selected adult female and male antenna transcriptome sequencing as the research object, using bioinformatics software to analyze the transcriptome sequencing data obtained and screening of differentially expressed genes between the 2 samples of male and female, further analysis of differentially expressed genes. The results showed that 36 209 unigene were obtained from the antenna transcriptome data of the male and female adults of Agrilus zanthoxylumi. A total of 726 genes were detected with significant differences in expression, among which 459 genes were up-expressed and 267 genes were down-expressed. Through GO (Gene Ontology) enrichment analysis, the differentially expressed genes were found in binding function, catalytic activity, cell process, metabolic process, cell components and organelles which were significantly enriched. A total of 472 differentially expressed genes were annotated in the KEGG database, and Pathway enrichment analysis showed that they were involved in 165 metabolic pathways, with 394 up-expressed and 78 down-expressed genes. This study obtained Agrilus zanthoxylumi adult antenna of the genetic basis of enrichment of the active ingredient based on the transcriptome sequencing technologies, discussed the adult male and female antenna differentially expressed genes, revealed the gene function, understood the relationship between genes. It doesn't only further improve the mechanism of tentacles feelings outside odor material plays an important role, but also provide the basic data for pest prevention and control technology.

Bothrogonia ferruginea is an important agricultural and economic pest in China. Sequencing and analyzing the complete mitochondrial genome of B. ferruginea and exploring its phylogenetic status in the suborder Auchenorrhyncha at the mitochondrial level are of great significance to its systematic taxonomy. The complete mitochondrial genome sequence of B. ferruginea was obtained by PCR amplification and sequencing, and its general features and base composition were analyzed. The plyogenetic tree of mitochondrial protein-coding genes of 22 species of Auchenorrhyncha and 2 outgroups was constructed by using the Bayesian inference (BI) and maximum likelihood method (ML). The complete mitochondrial genome of B. ferruginea was 15 262 bp (GenBank No. KU167550), including 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and one control region. The nucleotide composition and gene distribution sequence of B. ferruginea showed the same as insect of Auchenorrhyncha. The A + T content of mitochondrial genome is 76.48%, and the AT-skew is 0.170 2. There were 14 overlapping genes and 10 intergenic regions. All protein-coding genes use the ATN as initiation codon. Majority of PCGs had a complete termination codon (TAA or TAG), except that COⅡ gene terminated with the incomplete stop codon TA. All the predicted tRNAs showed the classic clover-leaf structure, except for the absence of DHU (dihydrouracil) arm of tRNA^Ser(AGN). The A+T rich region contained some conserved sequence (ATTTA) and a typical microsatellite structure (TA)₂₅. Two special structures, ATCTA and CCCTCT, were found in the tandem repeats in the control region of Cicadellidae, but not in the family Membracidae and other Auchenorrhyncha. The phylogenetic relationships of 22 species of Auchenorrhyncha based on mitochondrial genome were identified as Cercopoidea+(Fulgoroidea + Membracoidea). B. ferruginea, Nephotettix cincticeps, Idioscopus nitidulus, Empoasca vitis and Homalodisca vitripennis belonged to Auchenorrhyncha, Membracoidea and Cicadellidae. The gene arrangement pattern of the mitochondrial genome of B. ferruginea was identical to those of other Cicadellidae insects. The phylogenetic relationship of Auchenorrhyncha constructed based on the mitochondrial genome was consistent with those in traditional morphological taxonomy. B. ferruginea belonged to Auchenorrhyncha, Membracoidea and Cicadellidae. Our research provides data for the further study of auchenorrhyncha classification system.

Compared with the insecticidal crystal proteins, vegetative insecticidal proteins (Vips) have no homology of amino acid sequences and have no competitive relationship with the sites of insecticidal action. As the protein structure of Vips has not yet been elucidated, the mechanistic study on insecticidal action has lagged behind. In order to identify the key amino acids affecting insecticidal activity of Bacillus thuringiensis (Bt) Vip3Aa11 protein, site-directed mutagenesis was performed on 3 amino acid sites in Vip3Aa11, and 3 mutant proteins G200S, F442S and S726T were successfully constructed, and their insecticidal activities against Spodoptera exigua and Helicoverpa armigera were determined. The results showed that the insecticidal activity of mutant protein S726T against S. exigua was 4 times of that of Vip3Aa11, while the mutant proteins G200S and F442S showed no significant change in insecticidal activity against S. exigua and H. Armigera. To find out the reason of activity change in mutants, 3 mutants and the wild type Vip3Aa11 were treated with trypsin. The results indicated that both variants and wild type Vip3Aa11 could yield a 62~66 kD fragment by trypsin digestion in vitro and the effects of the 3 mutants on trypsin sensitivity were basically consistent. Through secondary structure prediction, it was found that the conformation of mutant protein S726T was shifted backward on α-helix which indicated that the increase of insecticidal activity caused by S726T mutant might be related to the slight change of protein structure space. In this study, the difference in insecticidal activity between Vip3Aa11 protein and each mutant protein for different insects was compared, and the reason for the difference was preliminarily analyzed, the results may provide a guideline for further study on the structure and function of Vip3Aa proteins.

Entomopathogenic nematode symbiotic bacteria and insecticidal toxins have been a research hotspot in the field of pest control. PirAB (photorhabdus insect related toxin AB) toxin is a special binary toxin from symbiotic bacteria of entomopathogenic nematodes with high insecticidal activity, and the insecticidal characteristics of the toxin and its effect on the enzyme activity of host insects were carried out in present study. Firstly, the pirAB gene of Xenorhabdus nematophila HB310 was expressed in prokaryotic expression system, and the biological activity of the recombinant PirAB protein was determinated on the 4th instar larvae of Helicoverpa armigera by intracavity injection method, and the influence of PirAB against related enzymes (acetylcholinesterase, carboxylesterase, polyphenol oxidase, tyrosinase and peroxidase) was further detected. The results showed that recombinant PirAB protein demonstrated a high intravenous injection activity (LD₅₀=2.003 μg/larva). After injection of PirAB toxin with LD₅₀ dose, there was no significant difference in acetylcholinesterase activity between the treatment and the control group; Carboxylesterase activity showed a dramatic change of activation, inhibition and re-activation, and tended to be stable after 36 h treatment; Polyphenol oxidase activity also showed a trend of first activation and then inhibition, and the change of activity tended to be stable after 24 h, and was significantly lower than that of the control group (P<0.05). Tyrosinase activity was basically inhibited after toxin treatment, and there was a significant change between 12 and 36 h which decreased sharply and increased again, and the tyrosinase activity was the same as that of the control; The overall change trend of peroxidase activity was not obvious and continued to be lower than that of the control. This study provides basic information for revealing the immune mechanism of host insects against PirAB toxin.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger molecule widely present in bacteria and can participate in innate immune response through stimulator of interferon genes (STING) signaling pathway and is a potential vaccine adjuvant. In order to clarify its innate immune regulation mechanism and evaluate the effect of immunoadjuvant, the efficient synthesis of c-di-GMP needs to be solved urgently. In order to establish a relatively simple and efficient c-di-GMP biosynthesis method to prepare c-di-GMP in vitro, this study constructed Escherichia coli dinucleotide cyclase (DncV) recombinant expression vector, and the target protein DncV was obtained by IPTG (isopropy-β-D-thiogalactoside)-induced expression and His affinity purification; Detection of c-di-GMP from GTP catalyzed by DncV recombinant protein by ultra performance liquid chromatography (UPLC) in vitro. The results showed that the IPTG-inducible expression vector pET-28a-His-DncV was successfully constructed, and the purity of the recombinant protein obtained by affinity purification was 92%. The recombinant protein could generate c-di-GMP in one step by enzymatic reaction; Metal ions had a significant effect on the activity of the enzyme. Three kinds of metal ions (Mg²⁺, Mn²⁺, Co²⁺) could improve the catalytic efficiency, and the other 7 metal ions (Cu²⁺, Zn²⁺, Ni²⁺, Mo²⁺, Bo²⁺, Fe²⁺, Ca²⁺) inhibited the enzymatic reaction. This study established a method for the synthesis of c-di-GMP by E. coli DncV recombinant protein, which provides basic data for the large-scale preparation and functional study of c-di-GMP.

In the previous study, the laboratory isolated and screened a cold-tolerant aerobic denitrification strain Y-12 from long-term flooded winter paddy field. In this study, the characteristics of nitrification, denitrification characteristics and simultaneous nitrification and denitrification characteristics of strain Y-12 at 15 ℃ were studied using single nitrogen source (ammonium (NH₄⁺-N), nitrate (NO₃^--N) or nitrite (NO₂^--N)) and different concentrations of mixed nitrogen sources (ammonium and nitrate, or ammonium and nitrite). The results showed that at 15 ℃, when the nitrogen source was ammonium nitrogen, the removal rates of NH₄⁺-N and total nitrogen (TN) were 100% and 46.2%, respectively. When the nitrogen source was nitrate nitrogen, the removal rates of NO₃^--N and TN were 99.7% and 53.6%, respectively. When the nitrogen source was nitrite nitrogen, the removal rates of NO₂^--N and TN were 99.9% and 57.7%, respectively. The total nitrogen removal rates of the low concentration mixed nitrogen source (5 mg/L NH₄⁺-N+5 mg/L NO₃^--N, 5 mg/L NH₄⁺-N+5 mg/L NO₂^--N) was 50.0% and 52.4%; the total nitrogen removal rates of high-concentration mixed nitrogen (100 mg/L NH₄⁺-N+100 mg/L NO₃^--N, 100 mg/L NH₄⁺-N+100 mg/L NO₂^--N) were 31.8% and 24.7%, and both of them could effectively remove ammonium nitrogen, nitrate nitrogen and nitrite nitrogen. The strain could still grow when the pH raised to about 9.0 under mixed nitrogen source conditions. The research proves that strain Y-12 is a cold-tolerant aerobic denitrifying bacteria with certain alkali resistance, which has good advantages and application potential for the treatment of low temperature and alkaline wastewater (especially ammonium-containing nitrogen), provides theoretical basis and alternative strains for the simultaneous treatment of wastewater containing various nitrogen source in the late autumn to early spring.

Seedless fruits are especially appreciated by consumers because of their high edible rate, good taste and their convenience. Seedless is a desirable economic trait for fruits and an important index in breeding work. Seedless fruits can be obtained through parthenocarpy which the ovary develops without fertilization and by stenospermocarpy which the ovule is fertilized but the seed is aborted for lots of reasons. In this paper, we reviewed the potential roles and underlying mechanisms of auxin, gibberellin, cytokinin and ethylene, and analyzed the crosstalk of different hormones during the set and development of fruits.

Transgenic soybean (Glycine max) MON89788 is approved for commercial planting earlier. It has a wide range of cultivation with a large circulation of products. In this study, a series of primers combining with a special probe were designed for recombinase polymerase amplification (RPA) based on the event-specific sequence of MON89788. Then a strategy that forward primers and reverse primers mutually amplifying was employed to obtain the optimal primers combination with the highest amplification efficiency. In addition, the RPA reaction conditions, including reaction temperature, the concentration of primers and probe, were selected and optimized. The results indicated that RPA had a wide range of amplification temperature: Among 29.5~43.1 ℃, and the high concentration of probe in the reaction system would affect the amplification efficiency of RPA. Further, the specificity, sensitivity and applicability of the RPA detection system were tested. Then the MON89788 real-time fluorescent RPA (RT-RPA) detection method was established. The detection method was specific, and the absolute limit of detection (aLOD) of MON89788 could reach 40 copies, the relative limit of detection (rLOD) was 0.05%. Furthermore, for real samples detection, the RT-RPA detection was completed within 10 min at 39 ℃, which was 0.07~0.13 times of quantitative real-time PCR (qRT-PCR) detection time. This isothermal and rapid detection method provides new technical support for the rapid detection of genetically modified components, and is expected to be used for rapid on-site detection of genetically modified components.

Porcine epidemic diarrhea virus (PEDV) is one of the important pathogens that affect severe diarrhea in 3~10 day-old piglets (Sus scrofa domestica). Now the variant strain (GⅡtype) causes serious loss in farms. In order to differentiate the genotypes of PEDV, a rapid and accurate diagnostic method is established and used to detect PEDV from clinical diarrhea samples, those are advantageous to the prevention and control of these diseases. In this study, a pair of primers based the N-terminal domain of the PEDV S gene were designed, and 2 probes were designed in the light of the different regions of PEDV spike gene (S) from the variant strains (GⅡ) and the classical strains (GⅠ). The 5' end of those probes were individually marked by 5-carboxyfluorescein (FAM) (GⅡ) and 5-hexachloro-fluorescein hosphoramidite (HEX) (GⅠ) fluorescent signals. One single-step duplex qRT-PCR based on specific probes was established to distinguish different genotypes of PEDV by experiments on amplification conditions, specificity, sensitivity, repeatability, and so on. Firstly, the standard curves were established. Within the range of 10^-1~10⁸ copies/μL, this method showed good amplification efficiency for GⅡstrain (R²=0.9892) and GⅠstrain (R²=0.9914), and there was a good linear relation-ship between Ct value and concentration as well. Secondly, the sensitivity was 7.34 copies/μL or 2.3×10² TCID₅₀ (tissue culture infective dose)/0.1 mL for GⅡstrain, 4.32 copies/μL or 4.3×10³ TCID₅₀/0.1 mL for GⅠstrain, respectively. Thirdly, there was no cross-reaction between GⅡand GⅠstrains, in addition between PEDV and other pathogens about Porcine deltacoronavirus (PDCoV), Classic swine fever virus (CSFV), Transmissible gastroenteritis virus(TGEV), Porcine rotavirus (RV), Japanese encephalitis virus (JEV), Porcine parvovirus (PPV). Therefore, the specificity of this method was good. In the end, the coefficient of variation between and within groups was low, so the reproducibility was good. In detecting clinical samples by this method, the results were 100% consistent with virus isolation. This study are useful tools for quantifying viral load, detecting PEDV, and differentiating PEDV genotypes, and providing one technique for the prevention and control of PEDV.

Avilamycin is widely used as a new type of metabolic regulator and digestive enhancer in livestock feeding. The low production capacity of the strain and the immature fermentation process limit the industrial of avilamycin in China. Ribosome engineering technique was used to carry out resistance breeding of the Streptomyces viridochromogenes gs 77 by streptomycin to obtain a high-yield mutant strain of avilamycin, . Through the drug sensitive test and high performance liquid chromatography, a high-yield avilamycin mutant S.viridochromogenes gs 77-54 was obtained. The yield of avilamycin reached the maximum at 8th day of shake flask, and increased to 1.80 fold than that of the original strain. Passage experiments showed that the high-yield performance of mutant strains was genetically stable. rpsl gene (encoding the ribosomal protein S12) and the morphological characteristics of the strains parent and after the mutation were analyzed. The results showed that the aerial hyphae of the mutant strain were long and straight, and the spores were elliptical with a diameter of about 1.1 µm× 0.6 µm, which was significantly different from the starting strain. Sequence analysis of the rpsl gene revealed a point mutation in the gene fragment, the G mutation at position 356 was mutated A, which was mutated to glutamine by arginine. The avilamycin high-yield mutant strain was constructed successfully, which would provide a new idea for the breeding of S. viridochromogenes.