Abstract Flow cytometry is a prevailing method to measure nuclear DNA content and confirm plant ploidy level for its convenience and reliable results, the suspension with highly purified and complete nuclear is the key to the accurate observation of plant ploidy level by flow cytometry and the buffer heavily affects the state of the nucleus. Although many researches have studied the ploidy of pear (Pyrus), but little is known about the screening of the optimal nuclear isolation buffer for pear. In this study, Xinjiang 'koral fragrant' pear (Pyrus sinkiangensis) plantlets were selected as material for extraction of nucleus with direct shear method, through systematical comparison to the nucleus morphology, DNA relative content and nuclear extraction efficiency, this study filtered the optimal nuclear isolation buffer from the five buffers (general purpose buffer (GPB), OTTO, Galbraith's, woody plant buffer (WPB) and N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)(HEPES)). Galbraith's was finally screened as the most suitable lysis for producing pear nuclear suspensions. The results suggested that nucleus suspension in OTTO and GPB can't completely reduced the redundant ingredients and ensured the integrity of the nucleus. There were rarely complete nuclei, in addition, a large number of flocculent substances were scattering a strong fluorescence. What's more, the nuclear suspensions prepared by OTTO and GPB formed an artifactual bands. The results also showed that the suspensions produced by WPB, Galbraith's and HEPES contain a little of bits and pieces, but they had round or oval cell nuclei uniformly distributing in the view. Additionally, the histogram of the relative DNA contents from cell nuclei showed that WPB, Galbraith's and HEPES provided an acceptable and normally distributed peak at G0/G1 phase, the variation coefficients were less than 5%, 4.85%, 4.53%, 4.57%, respectively. Moreover, the suspensions prepared by WPB and Galbraith's formed a DNA peak in the G2/M phase, but the suspension produced by HEPES did not form the peak. So the HEPES buffer may lead to some errors in the analysis of DNA ploidy, cell cycle and the number of cells at mitosis. The nuclear fluorescence scatter diagram indicated that the WPB, Galbraith's and HEPES formed a cell population at G0/G1 phase (PE-A=4.7×104~4.8×104), obviously distincting from the surrounding particles in the 10 000 events. Furthermore, an investigation of both WPB and Galbraith's also formed cell colony at G2/M phase (PE-A=9.4×104~9.6×104). Therefore, it was ultimately confirmed that Galbraith's should be the most suitable performing buffer for the preparation of pear nuclear suspension. This result would lay the foundation for further plant cytological observation and molecular study.

Abstract N-acetylneuraminic acid (Neu5Gc) is an important non-galactose antigen which can cause xenograft rejection, and it is catalyzed synthesis by cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, the CMAH gene knockout Bama mini pigs (CMAH-/-) were generated by CRISPR/Cas9 based on the alpha-1, 3-galactosyltransferase (GGTA1) gene knockout pigs. The health and reproductive condition of the CMAH-/- pigs were evaluated. Single guide RNA (sgRNAs) were designed to target the porcine CMAH gene and the efficiency of the sgRNAs was verified. High-efficiency CMAH-gRNA1 expression vector was transfected into the ear fibroblasts of Bama mini pigs (BMEF). Cells identified as positive were used as nuclear donors, and somatic cell nuclear transfer were used to prepare CMAH-/- pigs. CMAH gene mutations in piglets were identified by PCR and sequencing; Immunofluorescence detection of CMAH-/-porcine organs with anti-Neu5Gc antibody to determine the expression of Neu5Gc in piglets; The 4-month-old CMAH-/- pigs' blood was collected for the detection of its physiological indexes, the 6 months old CMAH-/-boars mating with wide type sows, the litter size of sows was computed to evaluate the CMAH-/- pigs'breeding capacity. The CMAH-/- piglets had two homozygous genotypes: -2 bp/-2 bp and -1bp/-1bp. Immunofluorescence detection results showed that the Neu5Gc was not detected on the surface of CMAH-/-pigs'organs, indicating that the CMAH gene was knockout. Compared with the wild type pigs (WT), there was no significant difference in blood routine and blood biochemical parameters, and the number of sows mating with CMAH-/- boars was normal. In this study, CRISPR/Cas9 technology was used to generate GGTA1/CMAH knockout pigs, the health and reproductive condition of CMAH-/- pigs were normal. The pigs could provide a low immune antigen donor for further reducing the acute immune rejection of xenotransplantation.

Abstract Efficient expression of human complement regulatory gene human cluster of differentiation 55(hCD55) is particularly important in xenotransplantation. Human CD55 gene was knocked-in porcine Rosa26 (pRosa26) locus via CRISPR/Cas9 on the basis of GGTA1 knockout (α-1,3-galactosyltransferase knockout, GTKO) swine ear fibroblasts, to establish the cell lines with efficient expression of hCD55 gene. Generating a Cas9 expression vector targeted the intron 1 (intron 1) of the reverse orientation splice acceptor β-geo26 (Rosaβgeo26) site in swine. A expression vector homologous-recombinating hCD55 at the pRosa26 site were constructed, which contained the the hCD55 gene under the control of polypeptide chain elongation factor 1α(EF1α) in the heterogeneous Loxps. The hygromycin B phosphotransferase (HPH) gene was the positive screening gene. The thymidine kinase (TK) was the negative gene. The Cas9-Rosa26 expression vector and the pEF1α-hCD55 expression vector were co-electroporated to the ear fibroblasts of pig. Ganciclovir (GCV) and hygromycin (Hyg) were added into the culture medium for screening cells, and then cultured the cell to single cell clone. The integration of hCD55 was detected by PCR with two pairs of primers across the 5' or 3' homology arms, respectively. The expression of hCD55 in the cells was detected by reverse transcription PCR(RT-PCR) and Western blot. Ninety-four clones were obtained after the drug screening. Two clones were confirmed that hCD55 site-specific integration by PCR. The site-specific integration efficiency was 2.1%. The results of RT-PCR showed that the positive clones expressed hCD55 gene, and Western blot results further confirmed that hCD55 was expressed in the cells. This study successfully constructed a cell line expressing hCD55 on the basis of GTKO Bama mini pig's ear fibroblast line, which would provide data foundation for the generation of xenotransplanted donor pigs.

Abstract Exogenous gene stably integrated into the host genome, and stably inherited to the offspring, producing transgenic animals is a basic requirement. Analysis of transgenic animals for inheritance and expression stability of transgene is an important part of establishing a transgenic animal strain. The T cell-activated costimulatory pathway can be blocked by LEA29Y expressed in islet cells of transgenic pig (Sus scrofa), and the T cell-mediated immune rejection in humans after islet transplantation surgery is reduced. A transgenic pig prepared by this research group that specifically expressed LEA29Y in islets, but its inherited genetic condition remains to be determined. In order to obtain islet-specific expression of LEA29Y transgenic pig strain for islet xenograft, the inheritance and expression of two-generation LEA29Y transgenic pigs was continuously traced. 1 male (coded 81 ) and 2 females (coded 77, 83)，a total of 3 LEA29Y founder pigs (F0) were bred each other or bred with wild-type pigs to get F1 generation pigs. Positive individuals in the F1 generation pigs and wild-type pigs were bred to get F2 generation pigs. The transgenic piglet was detected by PCR. The LEA29Y expression of transgenic pig's heart, liver, spleen, lung, kidney, pancreas, muscle, and other tissues were tested by reverse transcription-PCR(RT-PCR). Especially the specific expression of LEA29Y in islet was analyzed by immunohistochemistry and immunofluorescence staining. Three founder pigs coded 77, 81, 83 can all be bred, and a total of 95 progenies were obtained, PCR tested that 53 progenies were positive. RT-PCR results showed that individuals coded 77 and 83 were non-specific expression LEA29Y in 77 and 83 lines. The results of RT-PCR, immunohistochemistry, and immunofluorescence showed that the islet-specific expression of LEA29Y was both in the F0 generation coded 81 and F1 generation coded 271 in 81 lines. And the inheritance of genetic was stable. All above, this study successfully screened for a genetically stable line of LEA29Y transgenic pigs, which can serve as a reliable donor for the study of islet xenotransplantation.

Abstract As an important signaling molecular, branch chain amino acids (BCAA) participate the synthesis and catabolism of protein of amino acids, and regulate food intake et al. in animal body. These processes include two conservative signaling pathway, one is the mammals target of rapamycin (mTOR) which sense of amino acid abundance, and another is the general amino acid control non-derepressible 2 (GCN2) which sense the absence of one or more amino acids by virtue of direct binding to uncharged tRNAs. This review mainly focused on the effect of BCAA on mTOR and GCN2 signaling pathway to further elucidate how BCAAs are involved in protein synthesis and catabolism. This review provides a reference for the study of protein turnover regulation in mammalian and gives a theoretical basis for BCAA application in feed additives.

Abstract Translationally controlled tumor protein (TCTP) exists widely in eukaryotic cells. TCTP was involved in a variety of cellular processes including mitotic regulation, DNA damage repair, and plant resistance to pathogen infection. Thaumatin-like protein (TLP) is a pathogenesis related protein in plants and is thought to participate in the various processes of plants in response to pathogens infection. Previous work in this laboratory has demonstrated that TaTCTP was involved in the response of wheat (Triticum aestivum) to Puccinia triticina infection. This study focused on the interaction between TCTP and TLP. Vectors were constructed for yeast two-hybrid and Bi-molecular fluorescence complementation (BiFC), respectively. Through yeast two-hybrid test, it was found that yeast AH109 carried both TCTP and TLP could grow on SD-Leu/-Trp/-His and SD-Leu/-Trp/-His/-Ade medium and have detectable α- galactosidase gene (MEL1) reporter activity. This result indicated that there is a physical interaction between TaTCTP and TaTLP. Through the BiFC test, it was found that a strong yellow fluorescence signal could be observed in tobacco epidermis cytoplasm when co-transforming Agrobacterium with TCTP and TLP. These results indicated that TaTCTP and TaTLP can interact with each other in cytoplasm. This study lay a foundation for further exploring the function and mechanism of TCTP in wheat resistance to Puccinia triticina infection.

Abstract The growth and development of stem could be induced and changed by the infection of Ustilago esculenta in Zizania latifolia, but the regulatory of swollen stem is still unclear. In this research, Z. latifolia WRKY transcription factor 72 (ZlWRKY72) (GenBank number: MG365889) was cloned with full DNA length of 2 831 bp with one intron. The full length of its cDNA was 1 085 bp with a 738 bp of ORF coding frame encoding 245 amino acids, the typical structure domain of WRKYGQK and a zinc finger structure of Cys2.His2 were found in ZlWRKY72. The phylogenetic analysis showed that the ZlWRKY72 has the highest homology with Oryza sativa subsp. japonica OsWRKY72. Expression analysis showed that the higher expressions of ZlWRKY72 during the development of swollen stem of Z. latifolia, liking the higher expression in leaves than stems at tillering stage seedling (P＜0.05), while the higher expression in stems than leaves at stage of initiation swollen using qRT-PCR (P＜0.05). The expression of ZlWRKY72 in stem was related to the successfully infection and reproduction of U. esculenta. After the initiation of swollen stem, the higher expression of ZlWRKY72 were detected in the stem of Jiaobai than grey Jiaobai (P＜0.05), which was reduced significantly during the development of swollen stem in Jiaobai (P＜0.05). The expression of ZlWRKY72 were higher in male Z. latifolia than Jiaobai during the initiation of swollen. It was found that the reduction expression of ZlWRKY72 occurred following with the increasing hypha reproduction of U. esculenta in stem. The expression responses and regulation of ZlWRKY72 are investigated and discussed during the development of swollen stem in Z. latifolia, which might be used in the research of swollen stem. It provides a theoretical basis for studying the mechanism of swelling development of Zizania latifolia.

Abstract Hybridization is a critical pathway to obtain excellent germplasm, however, little publications are referred to hybridization of Pinellia ternata that is an important medicinal plant in China. In this study, different staining methods (iodine-potassium iodide (I-KI), peroxidase, triphenyl tetrazolium chloride (TTC), or acetocarmine method) and in vitro pollen germination were used to detect pollen viability; And benzidine-hydrogen peroxide method and artificial pollination were carried out to test stigma viability; Artificial pollination for different parental combinations that are originated 4 populations of P. ternata were executed to obtain hybrid seeds and traditional chromosome tabletting technique was employed to determine chromosome numbers for parents or hybrids. Results showed that the TTC staining was the most appropriate method to detect pollen viability; Pollen viability was maximum at 9:00~10:00 am in 2~3 day after flowering, Stigma possess high receptivity during 1 d before flowering to 2 d after flowering. The optimal temperature for pollen storage is 4 degree Celsius. Rates of hybrid seed setting (individuals or flower) for Hezhang×Baoding, Hezhang×Pingliang and Xiangyang×Hezhang were 43.75%, 11.11% and 33.33%, respectively whereas rates of hybrid seed setting (ovules) for these 3 combinations were 66.14%, 48.89% and 38.29% respectively. The chromosome number of all hybrids were consistent with its the female parent. These results indicated that new germpalsm of P. ternata could be obtained through hybridization between populations that possess different features. The present study further perfected technical link of hybridization breeding operation of P. ternata, which can be used to guide the practical operation of hybridization breeding of P. ternata.

Abstract 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyse the reaction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid (MVA) which is the first committed enzyme in the MVA pathway in the cytosol, and the reaction of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) which is the first committed enzyme in the MEP pathway in the plastid, respectively. For the study of the effect of over-expressing TwHMGR(KU246037.1) and TwDXR(KJ174341.1) genes on the production of secondary metabolites in Tripterygium wilfordfii, the expressing vector of TwHMGR and TwDXR was constructed and integrated into the genome of T. wilfordfii via Agrobacterium-mediated transformation methods. Three transgenic hairy roots were screened and confirmed respectively. The results of qRT-PCR showed that the expressing level of target genes in transgenic hairy roots were significantly higher than those in non-transgenic control, with highest levels at 4.73 times and 5.81 times, respectively. According to the results of high-performance liquid chromatography (HPLC), the contents of wilforgine, wilforine and triptolide of transgenic hairy roots significantly higher than controls. The results showed that the increase of TwHMGR gene and TwDXR gene expression could improve the yield of terpenoids secondary metabolites of T. wilfordii. This study provides the basis for the further use of metabolic engineering methods to improve the content of secondary metabolites and to elucidate of terpenoid biosynthetic pathway in T. wilfordii.

Abstract The miR-124a-3p has a potential interaction relationship with the acetyl-CoA acyltransferase 2 gene (ACAA2) in chicken (Gallus gallus). The research around biological function of chicken miR-124a-3p and its targeting effect on ACAA2 gene have not been reported. This study analyzed the potential biological function of miR-124a-3p by integrating the results of tissue expression profile and target gene prediction, and verified its relationship targeting with ACAA2 in chicken. The results of qRT-PCR analysis showed that miR-124a-3p was expressed both in all the 13 detected tissues at 4 developmental stages, especially high abundance and specific expression in the hypothalamus. Bioinformatics analyses indicated that the predicted target genes of miR-124a-3p were enriched in different biological processes and pathways, such as lipid transport, regulation of neuron migration, adipocytokine signaling pathway and axon guidance. The relevance analysis indicated that the expression of miR-124a-3p and ACAA2 appeared a negative relevance in pectorales, liver and subcutaneous adipose tissue. When miR-124a-3p mimics were transfected in chicken fibroblasts cells, the luciferase activity and expressions of ACAA2 gene were remarkably inhibited. These results demonstrated that miR-124a-3p as a generalized miRNA exhibited striking temporal expression characteristics in chicken, and regulate a variety of biological process related to lipid metabolism by targeting the ACAA2 gene. The data in this study provide a useful clue for further studying the function of miR-124a-3p and uncovering the formation mechanism of traits involved in fat deposition in chicken.

Abstract As a key enzyme involved in the formation and regulation of melanin, tyrosinase (TYR) has an important role in animal hair color phenotype. Tyrosinase dysfunction can block the pathway of melanin formation. The mutation of tyrosinase causes white phenotype of skin, hair, and feathers. Transcriptional regulation of gene promoter region plays an important role in gene expression. The research was designed to study the activity and the structure of TYR promoter. Dual-luciferase expression vectors were constructed and transfected to chicken embryo fibroblasts DF1 cells. The dual-luciferase detection kit was used to measure the relative luciferase activity. The length of the fragment in 5′ flanking region of TYR gene in chicken was 1 813 bp. Seven fragments with different length of promoter regions were amplified and cloned into the vector pGL3-Basic. Two mutant vectors of the promoter region were constructed. The core promoter region from -810 bp to +65 bp was identified in chicken TYR gene. The region from -810 bp to -213 bp may positively regulate TYR gene during the transcription process. The transcription factor (SP1, Oct-1, GATA-1, C/EBP, SRF, NF-1, YY1, NF-κB, TBP, and AP-1) may play a pivotal role in the gene transcription. The polymorphic sites of -963~-957, -955, -932, -692, -391, and -321 were found and the function of these sites in regulating promoter activity was critical but not all. Eight haplotypes (Hap1~Hap8) were predicted by the SHEsis software. The difference of genotype and allele frequency between white feather and black feather chicken was significant (P＜0.05). The dominant genotypes of white feather chicken were BB, DD, FF, II, KK, and MM respectively, the frequency of Hap1 was 100%. The dominant genotypes of black feather chicken were AA, CD, EF, HH, JJ, and LL respectively, the frequency of Hap2 had the highest proportion of haplotype (40%). The promoter activity value of mut-2 was significantly lower than P2, and the promoter activity value of mut-1 was significantly lower than P6 (P＜0.05). The decrease of promoter activity may affect TYR gene transcription, thus affecting the synthesis of melanin. The present study defines the activity and the structure of TYR promoter and provides the basis for studying the expression of this gene in hair follicle tissue.

Abstract Slow skeletal muscle troponin I 1 (TNNI1) located in slow-twitch myofibers that provides a calcium-sensitive switch for the contraction of striated muscle and plays an important role in muscle growth. The aim of this study was to clone the cDNA full length of TNNI1 gene of Gaoyou duck (Anas platyrhynchos domestica), analyze its sequence characteristics, mRNA expression profile in different tissues/organs, which might provide basis to further study the function of TNNI1 gene in muscle development in duck. Gaoyou duck was selected as materials, the full length of TNNI1 cDNA was cloned from breast muscle by 5' and 3' rapid-amplification of cDNA ends (RACE) and its homologies with TNNI1 gene of other animals was analyzed. The TNNI1 mRNA levels in different tissues/organs at sexual age (70 d), as well as in the breast and leg muscles during the developmental stages were measured using quantitative real-time PCR (qRT-PCR). The results showed that the full length of TNNI1 cDNA sequence was 965 bp, containing a 56 bp 5′UTR, a 345 bp 3′UTR and a 564 bp open reading frame (ORF), encoding a protein of 187 amino acids (GenBank accession number: KY926794). Sequence analysis revealed that the TNNI1 amino acid sequence of the Gaoyou duck is similar with those of Beijing duck (Anas platyrhynchos, 97.9%), quail (Coturnix japonica, 97.9%), chicken (Gallus gallus, 97.3%) and mammals (87.7%). Phylogenetic tree analysis showed that Gaoyou duck has the closest phylogenetic relationship to the Peking duck, quail and chicken, but were far away with mammals TNNI1 in amino acid sequences. The qRT-PCR results showed that TNNI1 were expressed in different organizations. And the mRNA level of TNNI1 in leg muscle were significantly higher than that of other tissues (P＜0.01), and the heart were the second. There was no significant difference between the mRNA level of breast muscle and glandular stomach (P＞0.05), while the lowest level was in brain, liver, spleen, and hypothalamus. During embryogenesis and post-hatching development, the expression pattern of TNNI1 in breast muscle and leg muscle showed an extreme significant difference as time went on. The mRNA level peak of TNNI1 in breast muscle was at embryonic age of 21 days, and in leg muscle was at embryonic age of 27 days. The mRNA level of TNNI1 gene in breast muscle was significantly lower than that of leg muscle at the embryonic days 27 and the neonatal age of 5 days, but was significantly higher than that of leg muscle at the embryonic age of 21 days. The results of these experiments showed that the amino acid sequence of TNNI1 belonged to the same branch as that of poultry, and was far away from mammals. qRT-PCR results showed that TNNI1 could be expressed in different tissue of Gaoyou duck, and existed some difference. The present study also found that there were significant differences in mRNA level of TNNI1 gene between the breast muscle and the leg muscle at different developmental stages. Therefore, structural characterization and tissue expression of TNNI1 gene in Gaoyou duck lay the foundation for further research on the function of TNNI1 gene in duck muscle development.

Abstract Colaphellus bowringi is a common vegetable pest. As the current Colaphellus bowringi full genome has not yet been announced, the various related gene information can not be known, leading to the Colaphellus bowringi gene function research is slow. The complete transcriptome data of Colaphellus bowringi was obtained by a new generation of high-throughput sequencing. Transcriptome data was analyzed with bioinformatics analysis. A complete transcriptomic database of Colaphellus bowringi was obtained, and the obtained original data was uploaded to National Center for Biotechnology Information (NCBI), and accession number is of SRP125298. Blast X tool was used to search the unigenes against NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), KEGG (Kyoto Encyclopedia of Genes and Genomes), Swiss-Prot(Manually Annotated and Reviewed Protein Sequence Database), Pfam (Protein Family), GO (Gene Ontology) and KOG (enKaryotic Ortholog Groups) databases to perform gene function and pathway annotation. In this study, 40 489 unigenes were de novo assembled. 19 955 unigenes were annotated in the public protein databases. A total of 17 437, 4 158, 6 817, 12 129, 15 890, 8 641, 11 416 unigenes had NR, NT, KEGG, Swiss-Prot, Pfam, GO, KOG database classification. The microsatellite (SSR) analysis, which found a total of 2 992 SSR exist in 2 692 unigenes, the average length of 14.61 bp. The most abundant type of repeat motif was mononucleotide (65.51%) and trinucleotide (21.76%). The most frequent motifs in mononucleotide and trinucleotide were A/T (94.95%) and TTA/TAA (8.45%). A total of 115 repetitive primers were found, of which 12, 60 and 38 of the dinucleotide, trinucleotide and tetranucleotide were found, respectively. The repetition frequency of the motif was between 5 and 50 times, and the length of the motif was mainly in the range of 10 ~ 20 bp. This study provides basic data for species identification and genetic diversity analysis through the acquisition of the primary data of the transcriptome of the Colaphellus bowringi.

Abstract Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-1,3,5-triazine) is widely used in many countries, and has great threat to the environment and human beings. In order to obtain a highly efficient atrazine-degrading bacteria, the present study isolated a bacterial strain (LY-2) which could use atrazine as a sole nitrogen source to grow by enrichment culture. The source was from the surface soil of maize field where atrazine had been used for many years. LY-2 was identified as Enterobacter sp. strain. It was the first reported Enterobacter sp. strain in detail that could degrade atrazine. The results of PCR test showed that LY-2 contained atrazine-degrading gene atzA, atzB, and atzC, which were involved in the degradation of atrazine to cyanuric acid. The strain LY-2 could degrade 98.7% atrazine (100 mg/L) within 48 h. The optimum temperature range of the strain was 25~35 ℃ and the optimum pH range was 6~9. Additional nitrogen sources did not affect the degradation of atrazine. The experiment of soil remediation showed that the highest degradation rate of the strain was 86.7% after incubation for 7 d, and the highest degradation rate of strain was 90.1% after incubation for 14 d, the concentration of atrazine in contaminated soil was 100 mg/kg. The strain LY-2 shows good bioremediation effect in a relatively short period of time and has a better potential for bioremediation of atrazine-contaminated soil.

Abstract SSR primers with dinucleotide repeat sequences are prone to continuous multi-peak, which influences the accurate collection of fingerprints. In order to improve the accuracy and automation of fingerprint analysis of such primers, an algorithm for the collection of fingerprints with continuous multi-peak was developed, and interactions between the continuous multi-peak algorithm and algorithms for other peaks including adjacent peak filtration, N+1 peak recognition, high-low peak filtration and diploid filtration were tested. Results demonstrated that the continuous multi-peak algorithm improved the accuracy and efficiency of fingerprint reading for SSR primers with dinucleotide repeat sequences, increased the accuracy of peak identification and location, and facilitated the estimation of peak height to truly reflect primer amplification. The error recognition probability of the continuous multi-peak peak was reduced from 50% to less than 1%, the recognition accuracy was increased from 2~4 bp to less than 1 bp, and the overall analysis time was shortened by more than 30%. By adjusting parameters, the intercoordination between the continuous multi-peak algorithm and algorithms for other peaks was guaranteed. The continuous multi-peak algorithm solves the identification and location of continuous multi-peak and the estimation of peak height, improves the efficacy of fingerprint collection of SSR primers with dinucleotide repeat sequences, eliminates the restriction in the application of SSR primers with dinucleotide repeat sequences that have the highest polymorphism in genomes and the most widely application, and provides important technical support for the better application of SSR primers in practice.

Abstract Phoebe sheareri is one species of "Nanmu with golden tint", which is recognized as a vulnerable species in China. The existing natural populations of P. sheareri is only scattered in the southern region of the Yangtze River, and the wild resources are gradually reducing now. In order to further study genetic diversity and genetic structure of natural populations in P. sheareri, it was imminent to develop numerous EST-SSR markers. In the present study, transcriptome data of P.sheareri was obtained by high-throughput sequencing technology. The SSR loci were screened and the EST-SSR markers were developed, as well as the preliminary validation and the transferability within Phoebe species was conducted. The results showed that a total of 43 781 unigenes were obtained using de novo assembly, and 6 958 SSR loci were identified using MISA, at a frequency of 15.89%, with one SSR locus occurred in per 5.31 kb. The length and number of SSR repeats were largely different, the most abundant SSR length was 15~19 bp, accounting for 49.05% of the total repeat types，and the most abundant repeat number of SSR motif was 4~8 times, accounting for 60.25%. A total of 340 repeat types of SSRs were identified, dinucleotide and trinucleotide were the dominant repeat types, representing as AG/CT and AAG/CCT, accounting for 23.99% and 12.81%, respectively. Ninety-four primer pairs from typical types were synthesized, which were verified by PCR amplification, with genomic DNA from 10 P. sheareri samples. The transferability of SSR markers within Phoebe species was also conducted, using the genomic DNA from four Phoebe species. The results showed that 41 primer pairs could yield the expected PCR products, of which 32 primer pairs were polymorphic. The number of alleles was averagely 2.75 per locus. The average value of observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information content (PIC) were 0.369, 0.563 and 0.541, respectively. These markers also showed high transferability within Phoebe species. In conclusion, the unigenes generated from transcriptome data of P.sheareri can be used as an effective source to development EST-SSR markers. The EST-SSR markers could be used in genetic diversity analysis, genetic map construction and germplasm resources protection for P.sheareri and other Phoebe species.

Abstract Lily (Lilium brownii var. viridulum) is one of the main fresh cut flowers in the world which possesses both high ornamental and economic value. However, the yield will be greatly reduced because the low temperature inhibits the growth of the lily. To improve the antifreeze function of Lily and breed the new characters, Antifreeze protein gene (AFPⅢ) originates from Anarhichas minor which was codon optimized according to the codon usage bias in plants and the antifreeze gene (AFⅠ) was synthesized by the company. Then AFⅠ was inserted into Pcambia1390 vector by enzyme digestion and ligation with the Peanut chlorotic streak caulimovirus (PCISV) promoter. Thus the plants expression vector carrying AFⅠ gene was constructed. And then the P1390-PCISV-AFⅠ plasmid was transformed into Agrobacterium EHA105 by electric shock. Subsequently, AFⅠ gene was introduced into lily callus mediated by Agroinfection, and the results of PCR confirmed that the AFⅠ gene had been integrated into Oriental Lily cultivar "Conca D or"．Restoring growth test was carried out to examine the antifreezing function of the positive transgenic plants, and the results of the test revealed that experimental group had a great antifreezing function in -2 ℃ compared with the control group, and the Semi-quantitative reverse transcription PCR demonstrated that the expression of AFⅠ gene leads to the antifreezing function of transgenic plants. This study provides an important reference for the cultivation of new varieties of antifreeze plants.

Abstract Bacillus strains are widely used as animal probiotics. Cellulase produced by Bacillus strains could be used to increase efficiency of feed utilization and improve the intestinal microflora. The aim of this study was to screen the Bacillus strains with high celluloytic activities from the rhizosphere soil of plants (sugarcane and hemp, and so on) to provide strain resources for development of animal probiotics. Firstly, culturable Bacillus strains were isolated from the soil samples through dilution seperation method on LB plate. Eight celluloytic Bacillus strains were obtained by screening with the sodium carboxymethyl cellulose (CMC-Na) medium. Among them, the strain FJAT-29941 exhibited the strongest cellulase activities and was identified as Bacillus amyloliquefaciens according to the morphology, physical and biochemical characteristics and 16S rRNA gene sequences analysis. The cellulase production conditions, gastrointestinal tolerance, filter paper degradation and hemolytic activity of strain FJAT-29941 were studied furtherly. The fermentative conditions for cellulase-producing of the strain FJAT-29941 were as follows: pH 4.0~9.0 (optimum pH 9.0), 20~50 ℃ (optimum 30 ℃ ), 18~72 h (optimum 42 h) and the optical fermentative medium was NA (nutrient agar) containing 0.5% NaCl. Moreover, the cellulases displayed excellent tolerance to pH and temperature. The survival rate of the strain FJAT-29941 in the artificial intestinal fluid containing 1% typsin and the artificial gastric fluid containing 1% pepsin was 27.98% and 64.91%, respectively, suggesting a great tolerance to gastrointestinal environments. It was showed that the cultures could rapidly degrade intact long filter paper scrips into fragments within 48 hours of incubation, showing strong ability of degradation on cellulose substrates. Finally, no hemolysis of the strain FJAT-29941 was observed on the goat blood agar plates, indicating its safety toward use of this isolate as a potential probiotics. Taken together, the strain B. amyloliquefaciens FJAT-29941 exhibited both excellent cellulose degrading ability and probiotic potential, which would provide a strain resource and scientific basis for the probiotics development.

Abstract Listonella anguillarum has emerged as a serious pathogen causing signi?cant mortalities in various ?sh and shell?sh species over a wide geographical area. With the large-scale application of intensive farming model in aquaculture, the speed of the transmission of aquatic pathogens has been greatly improved. Cultivation of Listonella anguillarum is seen as the gold standard for detection, although it is very time consuming and labour intensive. Accordingly, the establishment of practical and effective diagnostic methods has long been desired. In this study, several pairs of primers and an exo-probe targeting the zinc metalloprotease gene (empA) of Listonella anguillarum was designed and a rapid real-time detection method for Listonella anguillarum based on recombinase polymerase amplification (RPA) was established by optimizing the reaction system and conditions. The results showed that the established Real-time RPA technique could detect Listonella anguillarum directly within 20 min and did not cross-react with other seven common aquatic pathogenic bacteria. The detection limit of Real-time RPA for gDNA of Listonella anguillarum was 1 pg/μL, and reproducibility was good. However, the lowest detection limit of artificial pollution sample was 3.4×102 CFU/mL. It is expected to be the routine which can provide scientific basis and technical reference for the development of rapid diagnostic kit and commercial aquaculture Listonella anguillarum.