Abstract The fatty acyl-acyl carrier protein thioesterase (FATA) is a key enzyme in the accumulation of fatty acids, which directly regulates the content and composition of fatty acids. The function of the acyl-ACP thioesterase A gene (GenBank accession number: EU267122.2) in Jatropha curcas has not been reported before. In this study, the JcFATA gene RNAi expression vector had been constructed and down-regulation of JcFATA gene expression was accomplished, the positive fragment FATA1 (439 bp) of JcFATA gene was cloned from Jatropha curcas leaf cDNA , which was then inserted into pYL RNAi.2-35S contained intron to construct the intermediate vector pYLRNAi.2-35S-FATA1; the reverse fragment FATA2 (439 bp) was amplified by PCR used the intermediate vector as a template, and then inserted into the intermediate vector to construct the JcFATA gene RNAi expression vector pYLRNAi.2-35S-FATA1-FATA2; it was transformed into Agrobacterium tumefaciens EHA105 competent cells by electroporation, and then transformed into Arabidopsis thaliana by inflorescence infection method. Finally, the transformation was confirmed by PCR and Southern blot. In addition, preliminary phenotypic observation, expression analysis and determination of fatty acid content of transgenic Arabidopsis plants was carried out in this study, the results indicated that the growth and development of transgenic plants might be inhibited by RNAi expression of JcFATA gene. The results of this study can provide a theoretical basis for further discussion of the function of JcFATA gene and its application in the genetic improvement of Jatropha curcas.

Abstract Virus disease caused by Turnip mosaic virus (TuMV) is one of the most serious problems during Chinese cabbage (Brassica rapa ssp. pekinensis) cultivation. Initial infection of TuMV in Chinese cabbage has been demonstrated to be related to wild type BraA.eIF(iso)4E.a (A) and BraA.eIF(iso)4E.c (C). Loss-of-function mutation in one or two genes hinders TuMV infection and results in virus-resistant phynotype in Chinese cabbage. Two kind of allelic variations, miss splicing mutation (a1) and psedogene mutation (a2) in eIF(iso)4E.a, frame-shift mutation (c1) and transposon-insertion mutation (c2). in eIF(iso)4E.c, has been identified respectively from Chinese cabbage. In this study, the differences among three kinds of eIF(iso)4E.a genotype were compared again. Simple and convenient markers were developed. The precise genotyping of eIF(iso)4E.a and eIF(iso)4E.c was detected among 38 hybrids bought from seed market and 96 germplasms created by our breeding team. The results showed that the allele frequencies for A, a1 and a2 were 36.8%, 40.8%, 22.4% among hybrids and 26.1%, 28.1%, 45.8% among germplasms respectively. Total genotype frequency for two mutant alleles of a1 and a2 was 70.1%. Comparatively, only one germplasm carried c1c1 genotype and 13 germplasms carried c2c2 genotype. The total genotype frequency for c1 and c2 was only 10.4%. The results provide molecular markers for utilizing the genes in germplasms detection and new vriaties breeding. They also provide molecular evidence for continuing expanding of those virus-resistance hybrids.

Abstract The formation of skeletal muscle affects the growth and development of animals. Through screening SNPs of MyoD1 gene promoter in different breeds of cattle (Bos taurus), and studying its effect on growth body size traits and promoter function, to further provide research basis for the molecular genetic markers of growth related traits and MyoD1 gene promoter revealed promoter functional components. 200 Guizhou native cattle (including Guanling cattle, Sinan cattle, Liping cattle, Weining cattle, 50 heads , respectively) as the research material, the experiment amplified MyoD1 promoter and used direct sequencing method to detect SNPs. The experiment found 5 SNPs in four groups, g.-1141T>C、g.-871C>T in Guanling cattle, Sinan cattle, Liping cattle and g.-1778T>C、g.-871C>T、g.-797T>C、g.-761T>C in Weining cattle; Correlation analysis results showed that in Guanling cattle -1141 T>C, g.-871C>T sites had significant influence on abdominal extension girth, body length and chest girth(P＜0.05), respectively; in Liping cattle -1141T>C, g.-871C>T sites had significant influence on cannon circumference(P＜0.05); in Sinan cattle g.-1141T>C site had significant influence on hucklebone width, while g.-871C>T had no significant influence on growth traits; in Weining cattle g.-1778T>C、g.-871C>T、g.-797T>C、g.-761T>C sites had significant influence on body length, hucklebone width and wasitband height(P＜0.05). Using the yeast one-hybrid (Y1H) system, the results found the SF1 transcription factor could be combined with the MyoD1 promoter; using dual luciferase report system, SF1 transcription factor increased the MyoD1 promoter activity. When the SF1 transcription site produced g.-1141T>C mutation, it had significant effect on abdominal girth, cannon circumference and hucklebone width and so on growth traits in three cattle(Guanling cattle, Liping cattle, Sinan cattle). SF1 has important influence for the activation and regulation of the MyoD1 gene.

Deeply studied of the genetic mechanisms for ear-related traits in maize (Zea mays) can provide references for using ear-related traits to breed high-yielding maize varieties. Thus, the 2 F1 hybrids and their derived 202 (POP1) and 218 (POP2) F2 populations with the common male parent TS141 were developed to identify quantitative trait loci (QTLs) for ear number per plant (EN), ear weight (EW), cob weight (CW), 100-kernel weight (KW), ear length (EL) and kernel ratio (KR) via composite interval mapping (CIM) in this study. It showed that EN, EW, KW and EL of the 2 F1 showed positive over-parent heterosis significantly, CW showed positive over-parent heterosis weakly, and KR showed mid-parent heterosis significantly. The F1 heterosis index and relative heterosis simultaneously displayed that EW>EN>EL>KW>CW>KR. However, the F2 advantage reduction rate showed that EW>EN>EL>KW>CW>KR. Totally 49 QTLs were identified in the 2 F2 populations, these QTLs were located on the 10 chromosomes and explained 4.10%~15.73% of phenotypic variation in single QTL. QTLs for EW and KR mainly showed additive effects, QTLs for EN, KW and EL mainly showed non-additive effects, and QTLs for CW showed both half of additive and non-additive effects. Eight stable QTLs (sQTLs) were simultaneously identified across POP1 and POP2, namely, the sQTL controlled EW, CW and KR in Bin1.03-1.04, the sQTL controlled KR in Bin1.06-1.07, the sQTL controlled KW in Bin4.04, the sQTL controlled EN in Bin6.05, the sQTL controlled KR in Bin7.02 and the sQTL controlled CW in Bin9.04, respectively. These results can lay a foundation for further revealing genetic mechanism for maize ear-related traits, and these sQTLs are stably expressed under 2 genetic backgrounds may play an important role in controlling maize ear-related traits and can be used as the candidate loci for marker-assisted selection (MAS), fine mapping and positional cloning.

Abstract The high-efficiency expression and stable inheritance of Bacillus thuringiensis gene (Bt) are crucial to transgenic rice breeding and commercial production. A transgenic line B2A68 (Oryza sativa) was obtained by introducing biplaphos resistance gene (Bar) and insecticidal crystal proteins gene (Cry2Aa) into the early season rice restorer line D68 via Agrobacterium mediated method. In this study, the temporal and spatial expression profile of Cry2Aa protein between homozygous and heterozygous genotype, different tissues and various developmental stages were studied by enzyme linked immunosorbent assay (ELISA), and the relationship between expression level, stage and tissue of insecticidal protein and insect resistance was revealed by referring bioassay results, in order to provide technical parameters for developing excellent lepidopteran resistant rice. The results showed that the expression level of Cry2Aa protein in homozygous plant leaf was significantly higher than that in heterozygous plant leaf (P＜0.01). At milk stage, the contents of Cry2Aa protein in each organ were: leaf ＞ glume and hulled grain ＞ stem and sheath (P＜0.05). At hard dough stage, the contents of Cry2Aa protein in each organ were: leaf ＞ hulled grain ＞ stem and glume (P＜0.05), but there were no significant differences in the contents of Cry2Aa protein between hulled grain and sheath, sheath and stem, and stem and glume, respectively. From seedling to ripening phase, the Cry2Aa protein content in leaf increased first, then decreased, and later increased again, fluctuating in a same pattern in T3 and T4 generation. The mortality of striped rice borer larvae was highly correlated with the Cry2Aa protein content in different organs at milk stage (r=0.837), and the mortality of rice leaf roller larvae was highly correlated with the Cry2Aa protein content in leaves at different stages (r=0.988). For the heterozygous plant leaf with low Cry2Aa protein content, the lethal time of the rice leaf roller feeding with heterozygous plant leaf was slightly longer than that of feeding with homozygous plant leaf, but shorter than 5 days. This study will contribute to controlling Lepidopteran pests in field and developing transgenic rice in the future.

Abstract NAC (NAM-ATAF-CUC)transcription factors play important roles in various stress response. This study currently, NAC4 gene were cloned from the Nicotiana tabacum, Its the coding region of the cDNA was 1 422 bp and encoded 473 amino acids. The phylogenetic tree analysis showed that the amino acid sequence encoded by the tobacco NAC4 gene has extremely high similarity with Capsicum baccatum and Durian zibethinus. The plant expression vector pSH737-35S-NAC4 was constructed and transformed into tobacco using Agrobacterium tumefaciens-mediated leaf disc. Six different transgenic lines, i.e. TP4, TP6, TP7, TP8, TP9 and TP10 as well as the wild-type lines, were subjected selected to simulate the drought tolerance of seed germination rate and seedling stage with different concentrations of mannitol simulated drought stress. The results showed that the seed germination rate was more than 40% higher than that of wild type when treated with 300 mmol/L mannitol for 15 d. The growth of wild type plant roots was obviously inhibited by observing the growth of seedling roots. The transgenic plants and wild plants 7 d were treated with 20% polyethylene glycol 6000. The results showed that under drought stress, the superoxide dismutase, The activities of enzymes and peroxidase were significantly higher than those of wild type, and the content of malondialdehyde was significantly lower than that of wild type plants. The results showed that the expression of NAC4, pyrroline-5-carboxylate synthetase (P5CS) and ornithine-oxo-acid transaminase (δ-OAT) were up-regulated under drought stress by qRT-PCR. Studies have shown that the expression of NAC4 gene may increase the drought resistance of plants. This study provides the basis for further study of the function of NAC4 gene and basic data for the creation of transgenic drought-tolerant plants.

Abstract MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor plays an important role in plant development and their response to various environmental stresses. In this paper, a MYB gene named BnMYB1 was isolated from ramie (Boehmeria nivea) by rapid amplification of cDNA ends (RACE) based on the analysis of expression profiling of cadmium response genes in ramie. The full length of BnMYB1 (GenBank No.: MF741318) coding sequence was 1 029 bp, which encoded 342 amino acid. BnMYB1 protein belonged to 1RMYB subfamily which had only one conserved SANT (switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear receptor co-repressor (N-CoR), transcription factor (TFIIB)) domain. The similarity comparison revealed that BnMYB1 shared 86%, 77%, 79%, 80% and 76% of similarity with SANT/MYB proteins of Humulus lupulus (CBY88798.1), citrus sinensis (XP_006477144.1), Vigna radiate (XP_014509497.1), Prunus mume (XP_008238436.1) and Cicer arietinum (XP_004508939.1). Phylogenetic analysis revealed that BnMYB1 was closely to HlMYB4 of Humulus lupulus. qRT-PCR analysis indicated that BnMYB1 was a constitutive gene, but the expression of BnMYB1 in leaf was significantly higher than that of in root (P＜0.05). Furthermore, BnMYB1 gene was up-regulated by Cd stress, and the expression of BnMYB1 changed along with the stress time and Cd concentration. Collectively, our data suggest that BnMYB1 is a cadmium-responsive factor and may play potential roles in the plant adaption to cadmium stress.

Abstract Siraitia Grosvenorii is an dioecious plant with medicinal and editable fruits as harvest, native to north of Guangxi Province, China. To explore the molecular mechanism of ethylene signal pathway, based on the unigene information from transcriptome data and combined with the rapid amplification of cDNA ends (RACE) technique and high-efficiency thermal asymmetric interlaced polymerase chain reaction (hi-TAIL PCR) technique, the full length mRNA and DNA of the key enzyme of ethylene synthesis was obtained from S. grosvenorii. The relative expression of genes on the male plant, female and Ag+treated female plants at different stages of different tissue parts were detected by real-time fluorescence quantitative technique. The results showed that the total length of the mRNA was 1 954 bp, named as SgACS3 (GenBank accession number: KY705404), and the full length DNA of the gene was 2 530 bp and contains three introns. The expressions of SgACS3 gene of the female leaves and buds before pollination were particularly high. The relative expressions of buds and leaves in the female plants were significantly higher than those in Ag+treated plants, suggesting that SgACS3 gene was widely involved in the growth and development of S. grosvenorii, stamen development in the buds, and it was closely related to the formation of female flowers and Ag+ by impact negatively regulate expression. This study provides an important base for revealing the molecular mechanism of SgACS3 in regulating the sex express and organ morphogenesis of flower and its application in breeding.

Abstract Festuca arundinacea is a widely cultivated forage in the world with excellent stress and disease resistance. Qiancao No.2 was cultivated by Guizhou Institute of Prataculture, which has two different strains (broad-leaved and fine-leaved) with obvious differences in the leaf shape. In order to recover the mechanism of leaf formation and resistance of Qiancao No.2, in this study, a differential display reverse transcription PCR (DDRT-PCR) was performed to screen the differential expression genes between the broad-leaved and fine-leaved strains. Finally, 50 differential bands were separated and recovered. The DNA fragments were cloned and sequenced. Most of different expressed genes were enriched into photosynthesis, growth regulation and stress resistance. After further verification by qRT-PCR, it was found that only the genes of 18S ribosome were up-regulated in broad-leaved, and the rest of them were up-regulated in fine-leaved. These results suggested that plants with large leaves usually allocated more biomass to the cell wall while to enhance the toughness of leaves, and accumulated more photosynthetic products to prepare for the growth and development, this situation would lead to lower photosynthetic efficiency in unit area of leaves. However, plants with narrow leaves allocated more organic in chloroplast which made them had higher photosynthetic capacity to accelerate growth, and with strong competition ability in natural environment. In particular, the growth of aboveground parts made them with strong competition ability in natural environment. This study provides a theoretical foundation for the further research on the functional genes of leaf morphogenesis, gene expression and the stress resistance.

Abstract Litter size is one of the most important economic traits in sheep (Ovis aries) which is controlled by minorgenes. While genes that have been found to be related to litter size are numbered, and it is still a hotspot to mine them. To investigate the tissue expression level and polymorphisms of follistatin-like protein 1 gene (FSTL1) which is found by whole genome re-sequencing (WGS) and its association with litter size in sheep, semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) and quantitative real-time PCR (qRT-PCR) were performed to investigate the expression of FSTL1 gene in sheep with different lambing abilities. Multiparous (380 Small Tail Han sheep) and uniparous (total 380 for Tan, Sunite, Suffolk, Dorper and Prairie Tibetan sheep) sheep breeds were selected and Sequenom MassARRAY?SNP assay was applied to genotype four single nucleotide polymorphism sites (SNPs) of FSTL1 gene. Then the association was analyzed between FSTL1 and litter size in Small Tail Han sheep. The results showed that FSTL1 expressed in all tissues selected. qRT-PCR indicated that the expression of FSTL1 had no significant difference between uniparous and multiparous sheep (P＞0.05), but the expression in pituitary, ovary, oviduct, uterus body and uterine horn of uniparous Sunite sheep was higher than that in multiparous Small Tail Han sheep, and the expression in pituitary, oviduct and uterus body of ++ was significantly higher than those in BB and B+ (P＜0.05). From genotyping, this study found the genotype frequencies and allele frequencies of the four SNPs were significantly different between uniparous and multiparous sheep (P＜0.05). Population genetic analysis showed that four SNPs were at moderate polymorphism in Small Tail Han sheep (0.25＜PIC＜0.5) and partial at moderate (0.25＜PIC＜0.5) and partial at low polymorphism (PIC＜0.25) in other sheep breeds. The χ2 test indicated that, the four SNPs were under Hardy-Weinberg equilibrium (P＞0.05) in most of the sheep breeds that chose. Association analysis indicated that the polymorphisms of the four SNPs in FSTL1 had no significant correlation with the litter size of each parity in Small Tail Han sheep (P＞0.05), and the litter size of mutation homozygotes was lower than that of wild homozygotes for almost all SNPs. Therefore, the results concluded that there may be a certain negative correlation between the expression level of FSTL1 and the litter size in sheep, but the four SNPs have no significant association with the litter size of different parities of Small Tail Han sheep (P＞0.05), which indicated they may not be the key loci that affect the expression or function of FSTL1. This study might provide a basis for litter size trait selection in sheep.

Abstract Clinical mastitis is a kind of the most serious disease with high incidence in dairy industry, and has been plagued in the development of dairy industry. It has significant effects on milk performance and lifetime which will cause huge economic losses in milk production process. It's very important to study the related genes about mastitis for the improvement of milking traits and elongation of lifetime of cows with molecular techniques, and scientific basis would be provided for the breeding of anti-disease and improvement of economic benefits of dairy farm. In order to verify the effects of lactoferrin gene polymorphism on the production traits of Chinese Holstein (Bos taurus), SNPs of LF gene were selected by PCR and direct sequencing for 20 cows with low SCS and 20 cows with high SCS according to the sequence of 5' untranslating region (UTR) of LF gene. Finally, LF-131C>T and LF-28A>C for 866 Chinese Holstein cows were detected by flight mass spectrometry. The dairy herd improvement production recordings information including the number of test cow, ID number of test cow's father, date of test, related genealogical information, test-day milk yield, fat content, protein content, somatic cell score, lactose, total solid, milk urea nitrogen, clinical mastitis and productive life of tested cows were collected from the management system of dairy farm. The effects of LF-131C>T and LF-28A>C on milk performances and lifetime were analyzed by multi factor variance analysis and Cox regression. The results showed that the dominant genotype of LF-131C>T and LF-28A>C loci were TT and AA with the frequency of 0.813 and 0.580, respectively. The dominant genes were T and A, respectively, and their frequencies were 0.903 and 0.762, respectively. Two loci were in the state of Hardy-Weinberg Equilibrium. LF-131C>T had extremely significant effects on SCS, lactose content and 305 days milk yield (P＜0.01), the effects on test-day milk yield and protein content were close to significant level (0.05＜P＜0.1), but LF-131C>T did not have significant effects on fat content total solid and milk urea nitrogen (P>0.05). Multiple comparisons showed that individuals with genotype of LF-131CC had significantly higher 305 days milk yield and lower SCS and lactose content than that of LF-131 CT and TT. LF-28A>C extremely significantly affected test-day milk yield, fat content, protein content, lactose content, total solid and 305 days milk yield (P＜0.01), but had no significant effects on SCS and MUN (P>0.05). Individual with LF-28 CC genotype had significantly higher fat content, protein content, total solid and 305 days milk yield and lower lactose content than that of LF-131 AA and AC, they also had significantly higher test-day milk yield than that of LF-131 AA. LF-28A>C had significant effects on production lifetime (P＜0.05)，individuals with CC genotype had shorter lifetime than that of AA genotype. LF -131C>T had effects on lifetime, but the effects were not significant. Relative researches about the association between LF-131C>T and LF-28A>C and clinical had important significance for improving milk performance and prolonging production life of dairy cows. It could provide scientific basis for disease resistance breeding and economic benefit improvement of dairy cows.

Abstract Microsatellite marker (SSR) was an effective method of analysis of genetic diversity of fish. The genetic variation in Epinephelus lanceolatus (♂), E. moara (♀) and their hybrid progenies Yunlong grouper were studied by using 27 pairs of microsatellite primers from the relative species of Epinephelus. Polymorphism was detected in Yunlong grouper by using 25 of 27 SSR markers. The genetic diversities of 3 grouper species were analyzed by calculating the effective allele number(Ne), observed heterozygosity`(Ho), expected heterozygosity(He), Shannon index(I), genetic similarity Nei's genetic distance, polymorphism information content (PIC), Overall inbreeding coefficient (Fit), inbreeding coefficient in population(Fis), genetic differentiation coefficient (Fst). The results showed that the average Ne were most in E. moara (2.8926), followed by Yunlong grouper (2.8010), and the smallest in E. lanceolatus (2.0564). The average Ho of E. moara, E. lanceolatus and Yunlong grouper were 0.604 6, 0.566 1 and 0.501 0, the average polymorphic information content were 0.577 8, 0.544 6 and 0.342 0. The inbreeding coefficient of the 3 populations was 0.150 8, and the genetic differentiation coefficient among populations was 0.067 7, which indicated that 6.77% of the genetic variation was from intergroup, and 93.23% of the genetic variation came from the differences among individuals in the population. The genetic differentiation coefficient was -0.072 2 among the 3 grouper species, and the intergroup population gene flow (Nm) was 2.396 3. The cluster analysis diagram constructed by using the UPGMA method showed that the hybrid Yunlong grouper and the female E. moara first clustered together. The results could provide the genetic basis for the formation of heterosis traits of Yunlong grouper and the identification of hybrid germplasm at the molecular level.

Abstract The cluster of differentiation 63 (CD63) belongs to the tetraspanin superfamily, which plays an important role in immuno-physiological functions including cell adhesion, signal transduction and immune cell activation. In recent years, there are serious disease problems in the aquaculture of Lateolabrax japonicus, in order to strengthen the basic research for fish diseases and immune. In this study, CD63 cDNA sequence of Lateolabrax japonicus was cloned using rapid-amplification of cDNA ends (RACE) method. The protein molecular weight, isoelectric point and structure prediction using online software. To expression analysis of CD63 using qRT-PCR. The experimental results showed that the amplified sequence was 1 219 bp, including 114 bp 5'-UTR, 382 bp 3'-UTR and 723 bp ORF which encoding 240 amino acids. It was predicted that the molecular weight of protein was 26.26 kD and the isoelectric point is 8.43. The results of online software analysis showed that the protein was a 4 transmembrane protein, including 4 transmembrane regions (13~35, 50~72, 85~107 and 207~229 aa), three intracellular domains (1~12, 73~84 and 230~240 aa) and 2 extracellular loops (36~49, 108~206 aa). A conserved Cys-Cys-Gly motif and 3 potential N-glycosylation sites (Asn128, Asn150 and Asn172) were found in the large extracellular loop region. The carboxy terminus of the protein had a tyrosine lysosome target sequence (GYEVM, Gly-Tyr-Glu-Val-Met). Bioinformatics analysis indicated that Lateolabrax japonicus CD63 peptide share 61%~84% similarity with other fish counterparts. Quantitative real-time PCR showed that the mRNA of CD63 was expressed in all tissues examined, including liver, spleen, head kidney, intestines, gill, fat, muscle, brain and blood. The higher expression of CD63 was detected in the liver, muscle and head kidney. The expression level of CD63 in head kidney didn't show any difference at 3 h after intraperitoneal injection with Vibrio harveyi, but up-regulated significantly at 6 h (P＜0.05) and down-regulation at 12 h. In vitro experiment, the mRNA expression of CD63 showed significantly up-regulation in head kidney macrophages after stimulation with LPS 12 h (P＜0.05). The CD63 expression were also significantly up-regulated in macrophages at 24 h after stimulating by Poly I:C (P＜0.05). The results indicated that CD63 may play important roles in pathogen invasion and inflammation reaction. This provides the theoretical basis for the further study of fish immune response molecules regulatory networks.

Abstract The brown planthopper (BPH)(Nilaparvata lugens) is one of the insect pests significantly damage the rice production in China. Transgenic crops, carrying Bacillus thuringsis (Bt) genes, is widely utilized in the control of agricultural insect pests, including lepidopteran and coleopteran. However, BPH is not susceptible to most of Bt toxins. Thus, management of BPH will become an urgent problem. Binding of Cry toxins with aminopeptidase N (APN) anchored on midgut brush boarder membrane has been reported as a key step in the intoxication-mechanism of Cry toxins. In this study, full-length sequence of a putative membrane-bound APN, which was previously identified from midgut transcriptome of BPH, was cloned into pDEST8 vector, and then transformed into DH10Bac competent cells to produce recombinant Bacmid-bphAPN plasmid. Through baculovirus expression vector system, bphAPN was eukaryotically expressed in a Spodoptera frugiperda (Sf9) cell line. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that compared to control Sf9 cells, a specific circa 110 kD band was detected in the Sf9 cells transfected by Bacmid-bphAPN plasmid. The successfully preparation of bphAPN in Sf9 was then verified by western blot analysis using anti-bphAPN primary antibody and LC-MS/MS analysis. Immunofluorescent localization showed that strong fluorescence was observed in the cells expressed bphAPN, while weak fluorescence was observed from the control cells (without over expression of bphAPN). Cytotoxicity assays showed that compared to the control cells, over expression of bphAPN in Sf9 cells did not significantly increase the cell mortality after exposure to Cry1Ac toxin. Results of this work indicated that the bphAPN, which was highly expressed in the midgut of BPH nymphs could be successfully prepared in vitro by eukaryotic expression. These results will be helpful to analyze the characters of BPH midgut APN and may provide information for innovation of novel pore-forming toxins, such as Bt Cry toxins, against BPH.

Abstract Saccharopolyspora spinosa (S. spinosa) is an important industrial microorganism known for spinosad production, which is a kind of biological insecticides with high efficiency, broad spectrum and low toxicity. At present, the yield of spinosad is low for industrial production in China. In order to improve the yield of spinosad, it is necessary to gain insight into the regulatory mechanisms and networks of spinosad biosynthesis in S. spinosa. Studies have shown that quorum sensing affects morphological differentiation and/or secondary metabolism in microorganism through cascade regulation. However, no report on the quorum sensing regulation mechanism of S. spinosa has been published yet. Present bioassay found that fermentation broth extract of S. spinosa could induce γ-butyrolactone (GBL)/receptor indicator strain Streptomyces coelicolor M145 to produce pigment, and the spinosad yield increased by 22.6% through adding water extract of its fermentation broth, which indicates that there is GBL/receptor cascade regulation of quorum sensing in S. spinosa. Bioinformatics analysis showed that S. spinosa had one GBL receptor homologous protein SsbR(Saccharopolyspora spinosa γ-butyrolactone receptor) and 3 central transcription regulators homologous proteins SstA1, SstA2, SstA3 (Saccharopolyspora spinosa central transcription activator). Real-time quantitative PCR (qRT-PCR) was used to detect the expression of key homologous genes related to cascade regulation. The expression levels of ssbR and sstAs during the fermentation conformed to model that the expression of sstAs was repressed by SsbR. In addition, the expression levels of 3 target proteins homologous genes griR (grixazone regulator), sprA (serine protease A) and ssgA (sporulation of Streptomyces griseus A) in S. spinosa were the same as that of sstAs, which suggests that these 3 genes may be activated by SstAs. This study provides a reference for the further research of the quorum sensing regulation mechanism of S. spinosa.

Abstract Rabbit haemorrhagic disease virus (RHDV) belongs to the genus Lagovirus of Caliciviridae. VP60 protein is one of the capsid proteins of RHDV, and also is main immunogenic protein which the major antigenic epitopes mainly locate in the N-terminal region. In this study, the VP60 truncated gene was cloned by employing the method RT-PCR and then subcloned to pMD20-T. After complete sequence analysis, the prokaryotic expression plasmid pET-RHDV-VP60 was successfully constructed and then inserted into the expression Escherichia coli Rosetta(DE3). The target protein was highly expressed under the induction with 1 mmol/L isopropyl β-D-1 -thiogalactopyranoside (IPTG), and the Balb/C mice were immunized with purified expression products, then the polyclonal antibody was prepared. Subsequently, the immunogenicity of recombinant protein was analyzed using indirect enzyme linked immunosorbent assay (iELISA) and Western-blot. Twenty clean rabbits were randomly divided into 4 groups and subcutaneously injected once, using commercial vaccine (5 rabbits), the RHDV GS/YZ inactivated by 0.4% formaldehyde (5 rabbits), recombinant protein mixed with adjuvant (5 rabbits) and PBS as negative controls (5 rabbits)，respectively . The dosage of recombinant protein was 500 μg mixed with equal amounts of freund's complete adjuvant per injection, with 14 days interval, the recombinant protein was 250 μg mixed with equal amounts of freund's incomplete adjuvant per injection, After 21 days, all the rabbits attacked by the RHDV GS/YZ that HA titer was 210 and observed the results after 14 days. The results showed that the homology of nucleotide sequences of VP60 truncated gene from RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 91.9% to 98.6%, and had 98.6% nucleotide sequence identity with the RHDV HB strain(accession number: KU207100.1) and RHDV WX strain (accession number: AF402614.1). The homology of amino acid sequence of RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 96.7% to 99.1%, and had 99.1% amino sequence identity with the RHDV WX strain (accession number：AF402614.1) and RHDV Mexico 89 strain (accession number：AF295785.1). The target gene was successfully expressed in Escherichia coli, and the recombinant protein expression in the form of inclusion body. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD. The titer of polyclonal antibody was 1∶32 000 by iELISA and Western-blot demonstrated that the recombinant protein could bind with antibody induced by RHDV GS/YZ and the polyclonal antibody induced by recombinant protein could also combine with vaccine of RHDV - Pasteurella multocida and wild viruses of RHDV GS/YZ. So it had good immunogenicity. The immune protection experiment showed that the rabbits in the RHDV GS/YZ inactivated by 0.4% formaldehyde group, commercial vaccine group and recombinant protein mixed with adjuvant survived facing the challenge without any clinical signs of RHD while those in the negative control group died within 96 h post-infection and exhibited typical clinical symptoms. The titer of hemagglutination inhibition of commercial vaccine was 25~28, the self-made inactivated vaccine was 25~26, and the recombinant protein mixed with equal amounts of freund's adjuvant was 24~25. The results showed that the proteins expressed by Rosetta (DE3) could be ideal candidate for RHDV vaccines.

Abstract Bamboo is the fastest high growing plant and has attracted much attention of researchers. In recent years, the molecular mechanism of rapid growth of bamboo plants has been the hot research focus, and has achieved some research results. Based on the previous studies on the height growth of bamboo plants, the dynamic changes of height growth rhythm, cell structure, physiology and biochemistry and endogenous hormones in bamboo shoots were discussed in this paper. At the same time, we combined with analysis of transcriptome miRNA and proteome data. We systematically summarized the research progress of the mechanism of rapid growth of bamboo plants, and pointed out the future research direction of bamboo plants.