Northern Blot and Immunohistochemistry Analysis of HMOX1 Expression in Blue- and Brown-shelled Chickens

HMOX1 is an important functional candidate gene for chicken blue egg in view of its role in biosynthesis of biliverdin for blue egg coloration. To elucidate molecular mechanism of blue egg formation, this study detected expression of HMOX1 in blue-shelled chickens and brown-shelled chickens. Expression and alternative splicing of HMOX1 were detected by Northern blot, expression traits of HO-1 protein in shell glands of blue- （n=4） and brown-shelled （n=4） chickens were analyzed by immunohistochemistry. 3＇ UTR of HMOX1 was cloned using 3＇RACE. Results showed that the expression of HMOX1 at mRNA level had no significant difference between two groups of chickens, but at protein level HO-1 protein was highly expressed in blue-shelled chickens. Immunohistochemistry analysis showed that HO-1 protein expression was predominately located in villus epithelial cell of shell gland. Length of HMOX1 3＇ UTR were 586 bp. In 3＇ UTR we found a SNP of rs13866562 showing significant association with blue egg phenotype. Further miRNA prediction showed that it might influence interaction of some miRNAs and target sequences. The data suggested that blue egg is relevant to high expression of HO-1 in villus epithelial cell of shell gland. Further experimental validation for biological relevance of miRNAs is dispensable to elucidate reason for differential expressions of HO-1 protein.     

Studies on Immunoregulation of Cytokine of Chickens after Inoculation with Marek''''s Disease Vaccines

The spleenic and thymus T lymphocyte proliferaitivc reactions (TLPR) were enhanced, the intcrlcukin 2(IL-2) inductive activity was raised, and the IL-2 receptor (IL-2R) inductive expression was increased significantly in chickens after inoculation with trivalcnt Marck's disease vaccine in comparison with control chickens and those after vaccination with herpcsvirus of turkey (HVT) vaccine. In chickens after inoculation with HVT vaccine, in contrast with control chickens, the splcenic TLPR was strengthened and IL-2R inductive expression was hoisted significantly, the thymus TLPR, splccnic and thymus T lymphocyte IL-2 inductive activity, the thymus T lymphocyte IL-2R inductive expression were not significantly increased.     

Effect of in ovo zinc injection on the embryonic development,tissue zinc contents,antioxidation, and related gene expressions of broiler breeder eggs

Two experiments were conducted to investigate the effect of in ovo zinc(Zn) injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders. Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection. A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design. The eggs were injected with 0.1 m L sterilized water at 3, 6 and 9 embryonic days of incubation(E3, E6 and E9) or non-injection(the control), respectively. The results from experiment 1 showed that E3 and E6 injections increased(P0.05) the embryonic mortalities, and decreased(P0.05) hatchabilities compared to the non-injected control, but no differences(P0.05) between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability. The findings suggest that the E9 is the optimal embryonic age for early in ovo injection. In experiment 2, a total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design. The eggs were injected with 0(the negative control), 50, 100, 150, 200, or 250 μg Zn/egg as reagent grade Zn SO4·7 H2 O in a 0.1-m L solution, or non-injection(the positive control), respectively at E9–10. The results from the experiment 2 demonstrated that no differences(P0.05) among 50 and 100 μg Zn/egg groups and the negative control were observed in the embryonic mortality and hatchability, however, the injection of 200 μg Zn/egg increased(P0.05) the embryonic mortality, and injections of 150 and 200 μg Zn/egg decreased(P0.05) hatchabilities compared with the controls. The embryonic tibia Zn contents at E20 were increased(P0.05) by injections of 150, 200 and 250 μg Zn/egg. Zinc injection did not affect(P0.05) malonaldehyde(MDA) contents, copper-and Zncontaining superoxide dismutase(Cu Zn SOD) activities and m RNA expression levels in the liver and heart of chick embryos at E15 and E20. Compared with the negative control, injections of 50, 150 and 200 μg Zn/egg up-regulated(P0.05) the metallothionein(MT) mR NA expression levels in the embryonic liver at E20. These results indicated that in ovo Zn injections increased Zn contents in the embryonic tibia and MT m RNA expression levels in the embryonic liver at E20, however, injections of 150–200 μg Zn/egg were harmful to the embryonic development.     

Studies on lmmunoregulation of Cytokine of Chickens after Inoculation with Marek‘s Disease Vaccines

The spleenic and thymus T lymphocyte proliferaitive reactions(TLPR) were enhanced,the interleukin 2(IL-2)inductive activity was raised,and the IL-2 receptor (IL-2R)iductive expression was increased significantly in chickens after inoculation with trivalent Marek‘s discase vaccine in comparison with control chickens and those after vaccination with herpesvirus of turkey (HVT) vaccine.In chickens after inoculation with HVT vaccine,in contrast with control chickens,the spleenic TLPR was strengthened and IL-2R inductive expression was hoisted significantly,the thymus TLPR,spleeinc and thymus T lymphocyte IL-2 inductive activity,the thymus T lymphocyte IL-2R inductive expression were not significantly increased.     

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine;and we haven't found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰlocus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.     

The Influence of Selection for Plasma very Low Density Lipoprotein Concentration upon Production Performance in Layer Type Chickens

The chickens studied were from a pure line of brown shell egg-type(YAFA) female grandparents of 17-week-old.Plasima very low denstiy lipoprotein(VLDL) concentration was measured with turbidimetric assay.The experiment results showed that the phenotypic correla-tions between 29-week or 50-week plasma VLDL concentration and egg production (EP) were positive in the early stage of laying period,but those in the latter stage were negative,Selection for low plasma VLD concentration will decrease the EP in the early stage of laying period but in-crease the one in the latter stage,There was a significant negative phenotypic correlaiton between the atge at first egg (AFE) and 18-week body weight(BW),The phenotypic correlation between 29-week plasma VLDL concentration and the AFE was negative,AFE was influenced by BW and body fatness as well.There was significant positive phenotypic correlation between plasima VLDL concentration and body weights(BWs) at the same stage in laying period and the phenotypic correlation was aslo positive between 29-week plasma VLDL concentration and the BW at middle stage of laying period,indicating that selection for low plasma VLDL concentration would reduce BWs at various stages to different degree.     

Analysis of Developmental Proteome at Egg Stage of Drone Honeybees （A. m. ligustica）

An investigation on the proteome of drone egg development of native Italian bee （Apis mellifera ligustica Spinola,1806） was carried out in order to prove up the characteristics in protein expression and regulation at egg stage and open out the molecular mechanism of the development. The experiment was carried out by two-dimensional gel electrophoresis. The results showed that there were 200, 242 and 233 proteins in a wide rang of molecular weight （12.42-169.60 kDa） and in a relatively narrow scope of pI （4.50-9.00） detected on day 1, day 2 and day 3, respectively, during the developmental process of the drone egg. Meanwhile, 164 protein spots were resolved at all the images （i.e., the protein was consistently expressed） along with the egg development, among which 7 were significantly up-expressed （P 〈 0.05） and 4 were significantly down-expressed （P 〈 0.05） while 79 had no significant differences （P 〉 0.05）. In addition, the specific proteins expressing proteins on day 1, day 2 and day 3 were 11, 18 and 18, respectively. Besides, 17 proteins expressed both on day 1 and day 2 but silenced on day 3, and 43 proteins expressed both on day 2 and day 3 but silenced on day 1, while only 8 proteins expressed both on day 1 and day 3 but silenced on day 2. The results indicate that 2-d-old eggs are at the most active expressional stage in the development of drone egg. The protein expressing at all images suggests that it should be indispensable for drone egg development, but their expression pattern is different. The proteins expressing at a specific age of egg suggest that specific proteins are needed in different developmental stages to regulate. And there are more house-keeping proteins in the developmental process of the drone egg than that of worker egg, and it will provide more targets for gene improvement.     

Associations of Gonadotropin-Releasing Hormone Receptor （GnRHR） and Neuropeptide Y （NPY） Genes＇ Polymorphisms with Egg-Laying Traits in Wenchang Chicken

Single nucleotide polymorphisms （SNP） of chicken gonadotropin-releasing hormone receptor （GnRHR） and neuropeptide Y （NPY） were selected to identify the genotypes of Wenchang （Chinese indigenous breed） chicken with restricton fragment length polymorphisms. The associations of the SNPs with the total egg production （NE）, average days of continual laying （ADCL）, and number of double-yolked eggs （DYE） traits were analyzed. The frequency of restriction enzyme A/a alleles in the population was for GnRHR 0.69 （Bpu1102 Ⅰ A） and 0.31 （Bpu1102 Ⅰ a） and for NPY 0.46 （Dra Ⅰ B） and 0.54 （Dra Ⅰ b）. Trait data from a total of 120 hens, which were purebred introduced from Hainan Province, China from one generation were recorded. Two significant effects of genes＇ marker were found： for GnRHR and number of eggs （dominant; t= 2.67, df= 116） and NPY and number of eggs （additive; t= 1.97, df= 116）. The current research supports the effects of GnRHR and NPY genes on egg-laying traits of chickens.     

Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

Toll-like receptors （TLRs） are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors （ChTLRs） and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum ofE. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection （hpi）, ChTLR3, ChTLRI 5 and MyD88 mRNA expression in the spleen ofE. tenella infected chickens were significantly higher （P〈0.05） than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregnlated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.     

Study on the Relationship between Plasma very Low Density Lipoprotein Concentration and Egg Composition Traits in Layer Type Chickens

The chickens used in this study were from a pure line of brown shell egg-type(YAFA) female grandparents of 17-week-old.Plasma very low density lipoprotein(VLDL) concentration was measured with turbidimetrie assay ,The experimental results showed that there was a close rela-tionship between plasma VLDL concentration and egg composition traits,and a positive phenotypic correlation trend was found betewwn 29-week plasma VLDL concentration and 44-week yolk weght,and the genetic correlation coefficient was 0.234, Both the phenotypic and genetic correlations betewwn 29-week plasma VLD concentration and albumen weight were neg-ative,Significant positive phcnotypie correlations ,were,however,observed between yolk ratio or the yolk/albumen ratio and 29-week plasma VLD concentration and their genetic correlation coeffieients were 0.250 and 0.223,respectively.     

Genetic diversity center of cultivated soybean(Glycine max) in China——New insight and evidence for the diversity center of Chinese cultivated soybean

Information on the center of genetic diversity of soybean(Glycine max) will be helpful not only for designing efficient strategies for breeding programs, but also for understanding the domestication and origin of this species. Here, we describe an analysis of genetic diversity based on simple-sequence repeat(SSR) variations within a core collection of 2 111 accessions of Chinese soybean landraces. Prior to the diversity assessment, the geographic origin of each accession was mapped. The map was then divided into grids each 2.5° in latitude and 5° in longitude. We found two regions that had higher number of alleles(NA) and greater polymorphic information content(PIC) values than the others. These regions are adjacently located within grid position of 30°–35°N×105°–110°E, which includes the valley of the middle and lower reaches of the Wei River, and the valley of the upper reaches of the Hanjiang River. It was also observed that in many regions, genetic diversity decreased with the increase in distance from the center. Another region, in northern Hebei Province(115°–120°E×40°–42.5°N), was observed having higher diversity than any surrounding regions, indicating that this is a sub-center of soybean diversity. Based on the presented results, the domestication and origin of soybean are also discussed.     

The Relationship of Color Formation with Related Enzymes and Protein Contents in the Seedcoat of Oilseed Rape （Brassica napus）

Three pairs of near-isogenic lines with different genetic backgrounds of yellow-seeded and black-seeded rape (Brassica napus L.) were used as experiment materials to study the relationship of color formation in the seedcoat with enzyme activity and protein content in it. The results showed that with similar genetic backgrounds, phenylalanine ammonia-lyase (PAL) and polyphenol oxidase(PPO) activities in the black-seeded lines were much higher than in their yellow-seeded counterparts and maximum PAL activity in the seedcoat occurred comparatively late while no significant difference was present in glutamine synthetase (GS) between the two types of rape. The plants were treated with red light,blue light, p-hydroxybenzoic acid (a PAL inhibitor), polyvinylpyridoxal (a PPO inhibitor), urea (a protein synthesis promoter) or chloramphenicol (CM, a plastid protein synthesis inhibitor) during seed development. It is speculated that PAL may be primarily responsible for coloration in the yellow seed; PPO may be the main factor contributing to the darkness of the testa of the black genotypes; and nitrogen assimilation is, probably, not directly related to the difference in protein content observed between yellow- and black-seeded genotypes, which may be induced mainly by PAL.     

Effects of Immunization Against Inhibin on Egg-Laying Performance in Magang and Landaise Geese

This study aimed to improve egg-laying performance in incubating Magang geese of Guangdong origin and Landaise geese of French origin. In experiment 1, 50 adults, egg-laying Magang geese were inoculated intramuscularly （i.m.） on days 0, 22, and 45 with 1 mL of immunogen containing 1 mg of recombinant chicken inhibin fusion protein. Immunization significantly increased blood antibody titers against inhibin fusion protein, but did not affect the egg-laying performance within 10 days after the first inoculation. From day 15, the egg-laying rate in inhibin-immunized group increased and was significantly higher than the values of control geese from day 40 to 55. However, the reverse was true from day 55 to 75 when more immunized geese developed incubation. In the entire 120 days of the experiment, the immunized geese laid 17.3 eggs in contrast to 16.4 eggs laid by the control geese. From day 30 till the end of the experiment, weight of eggs in the control geese was significantly greater than that in inhibin-immunized birds. In experiment 2, 40 Landaise geese were immunized against inhibin, as described in experiment 1. These geese laid 9.0 eggs on average in contrast to 7.3 eggs laid by nonimmunized control geese over 90 days of egg laying. The above results demonstrated that immunization against recombinant chicken inhibin fusion protein improved egg-laying performance in geese, and the increment was higher in nonincubating geese.     

Studies on the Developmental Genetics of Tiller Number in Three-line Indica Hybrid Rice

Following NC Ⅱ design, the developmental genetic behavior of tiller number (TN) in three-line indica hybrid rice was studied using additive-dominance developmental genetic models and the corresponding statistical methods. The results showed that dominance effects were predominant for TN. The expression of those additive effects were affected by environment and genotype interaction, but the expression of dominance effects were not affected. Heterosis was the strongest in the middle developmental periods of TN. Additive effects and dominance effects were selectively expressed throughout in the entire tillering developmental stage.Analysis of genetic correlation between TN at different stages and the productive panicles indicated that a close correlation appeared earlier in the populations with higher heterosis than in those with less heterosis. Utilization of heterosis at the middle tillering stage might enhance the final biomass but reduce the percentage of productive panicles.     

Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection

The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects. In the construction of CRISPR/Cas9-mediated dsLmRNase2~(–/–) mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newlylaid eggs that were less tanned. However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G_1 progeny was similar between adults derived from the tanned or less tanned engineered eggs. Further, the similar effective mutant ratios in the normally developed eggs and G_0 adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9. Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs. We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection. Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure. In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.     

Comparative Analysis of Population Genetic Structure in Bemisia tabaci （Gennadius） Biotypes B and Q Based on ISSR Marker

Bemisia tabaci （Gennadius） biotypes B and Q are two invasive biotypes in the species complex. The comparison of the population genetic structure of the two biotypes is of significance to show their invasive mechanism and to their control. The intersimple sequence repeats （ISSR） marker was used to analyze the 16 B-biotype populations and 4 Q-biotype populations worldwide with a Trialeurodes vaporariorum population in Shanxi Province, China, and a B. tabaci non-B/Qbiotype population in Zhejiang Province, China, was used as control populations. The analysis of genetic diversity showed that the diversity indexes of biotype Q including Nei＇s gene diversity index, Shannon informative index, and the percentage of polymorphic loci were higher than those of biotype B. The high genetic diversity of biotype Q might provide the genetic basis for the excellent ecological adaptation. Cluster analysis suggested that the ISSR could not be used in the phylogenetic analysis though it could easily distinguish the biotypes of B. tabaci. The difference of the population genetic structure between the biotype B and the biotype Q exists based on the ISSR marker. Meanwhile, the results suggested that the molecular marker has its limitation in the phylogenetic analysis among the biotypes of B. tabaci.     

Characterization of Embryonic Stages through Variations in the Egg＇s Contents in Spodoptera fitura

This paper describes the embryonic development of the common cutworm Spodoptera litura （Fabricius） （Lepidoptera： Noctuidae） which is divided into eight embryonic stages, through variations in the egg＇s contents. Experiments were conducted on the eggs ofS. litura under laboratory conditions. The highest concentration of the protein （min.-max.： 32-! 40 lag/egg） and the lowest of the carbohydrate （min.-max.： 3.0-44.0 lag/egg） were obtained; similarly, the DNA content was smaller （min.-max.： 1.0-38.2 lag/egg） than the RNA （min.-max.： 9.2-360.0 lag/egg） throughout the embryogenesis. Three peaks of ecdysteroid titre were observed which were responsible for appearance of particular events during embryogenesis. The last highest peak of 400_＋33.4 lag/egg was appeared at 84 h after oviposition when larva was ready to hatch. Therefore, it is concluded that eight embryonic stages in S. litura were characterized by fluctuation in the egg＇s contents, which were responsible for gradual-progressive embryonic development. The egg-larval development did not show a normal distribution of embryonic developmental-time.     

Effects of Exogenous Jasmonic Acid on Concentrations of Direct-Defense Chemicals and Expression of Related Genes in Bt （Bacillus thuringiensis） Corn （Zea mays）

Bt corn is one of the top three large-scale commercialized transgenic crops around the world.It is increasingly clear that the complementary durable approaches for pest control,which combine the endogenous defense of the crop with the introduced foreign genes,are promising alternative strategies for pest resistance management and the next generation of insect-resistant transgenic crops.In the present study,we tested the inducible effects of exogenous jasmonic acid(JA) on direct-defense chemical content,Bt protein concentration,and related gene expression in the leaves of Bt corn cultivar 34B24 and non-Bt cultivar 34B23 by chemical analysis,ELISA,and RT-PCR.The results show that the expression of LOX,PR-2αMPI,and PR-1 genes in the treated leaf(the first leaf)was promoted by exogenous JA both in 34B24 and 34B23.As compared with the control,the concentration of DIMBOA in the treated leaf was significantly increased by 63 and 18% for 34B24 and 34B23,respectively.The total phenolic acid was also increased by 24 and 12% for both 34B24 and 34B23.The Bt protein content of 34B24 in the treated leaf was increased by 13% but decreased significantly by 27% in the second leaf.The induced response of 34B24 was in a systemic way and was much stronger than that of 34B23.Those findings indicated that there is a synergistic interaction between Bt gene and internally induced chemical defense system triggered by externally applied JA in Bt corn.