The gene functional identification in roses is limited by the lack of efficient stable transformation protocols. As an alternative, Agrobacterium-mediated transient gene expression and RNAi-based gene silencing can be used conveniently and effectively in plants. To optimize a transient gene expression system in rose petals, we investigated the effects of rose cultivars, flowering stages, petal positions, Agrobacterium strains, bacterial density and infiltration solution on transient gene expression in rose(Rosa hybrida) petals. The agro-infiltration method was used and GUS was selected as a marker gene in this study. We found that the cultivar Honey could have strongest GUS expression among all the 5 cultivars tested, while the other 4 cultivars Hollywood, Samantha, Honeymoon and Xiushanhong showed weak or no GUS expression. Petals of the middle whirl at flowering stage 3 displayed the highest expression level of GUS in Honey, suggesting that vigorous and fully developed petals were more suitable for transient gene expression. Compared to Agrobacterium strains of C58C1 and EHA105, GV3101 was a more effective laboratory strain and the optimal bacterial density was OD600=0.9. The infiltration solution is better to be supplemented with 10 mmol/L MgCl2 and 10 mmol/L 2-(N-Morpholino) ethanesulfonic acid (MES). The optimized transient system could result in around 100% strong GUS expression. By using the optimized transient expression system, we transiently expressed ihpRNA constructs to silence the reporter gene GUS and RhSAG. Compared to control, GUS-silencing showed less strongly stained petals and a lower average staining density, while RhSAG-silenced petals wilted more slowly, indicating senescence delay. In addition, both silencing treatments led to decreased gene transcripts. Together, the results suggested the system could be used in gene silencing and provide an effective tool for gene function analysis in rose petals.

DNA methylation is one of the most important epigenetic markers, which has been well characterized and demonstrated to be involved in many biological processes, including transposable element silencing, genomic imprinting and X chromosome inactivation etc. Yamanaka reprogrammed the mouse(Mus musculus) fibroblasts back to a pluripotent state via ectopically expressing four embryonically special transcription factors Oct4, Sox2, Klf4 and c-Myc, and named the cells as induced pluripotent stem cells (iPSCs). During reprogramming, there was accompanied with a series of epigenetic changes, so iPSCs can be used as a useful tool to study the interplay of epigenetic modifications and dynamics. In this study, porcine(Sus scrofa) fetal fibroblasts were isolated from 35-day-old fetus, and were reprogrammed into iPSCs using classical four factors recombination, namely Oct4, Sox2, Klf4 and c-Myc. The pluripotent markers of colonies were identified by immunofluorescence staining. With the method of bisulphate treatment, methylation status of promoters in endogenous pluripotent genes Oct4, Sox2, Klf4 and c-Myc were detected. It was found that the methylation status were very low which indicated that it was accompanied by decreased methylation status in promoters during the reprogramming of somatic cells into iPSCs. Therefore, this study is helpful to understand the DNA demethylation of pluripotency gene, and provides further insight into epigenetic regulations that control the pluripotency genes expression during reprogramming.

Glucagon-like peptide-2(GLP-2) can promote the growth of intestine, enhance nutrient absorption and repair intestinal injures. In this study, porcine glucagon-like peptide-2 (pGLP-2) microspheres were prepaired by Water in oil in water(W/O/W)method and solid in oil in water(S/O/W)method. A better one was chosen to investigate the potential therapeutic effects on intestinal injures of colitis in mice(Mus musculus) induced by dextran sulfate sodium (DSS). The results showed that microspheres prepared by W/O/W method could encapsulate 60% pGLP-2 with (54.19±17.28) μm in diameter while the drug burst was 65.1% and the surface was porous; Microspheres prepared by S/O/W method could encapsulate 72% pGLP-2 with (31.17±8.17) μm in diameter and 15.58% in drug burst. BALB/c mice were inoculated with 3% DSS solution for 7 d to induce colitis successfully. DSS inoculated mice were treated with pGLP-2 microspheres (10 mg per mice) prepared by S/O/W method in the third day. DSS+pGLP-2 microspheres group significantly decreased body weight loss and colonic damage and increased colon length (P＜0.05). Villus height and villus height /crypt depth ratio in mice of DSS+microspheres group significantly increased compared with that of DSS-only inoculated group and water group (P＜0.05). This study demonstrates pGLP-2 microspheres prepared by S/O/W method had higher encapsulation efficiency, lower drug burst and more stable than that of microspheres prepared by W/O/W method. Administration pGLP-2 microspheres once reduced the severity of colonic injury in murine colitis. The potencies of this product highlight its potential as therapeutic agent for intestinal diseases in piglet.

The transcription factor myf6 is a member of the MyoD family that plays a key role in myogenic lineage determination. This study was carried out to clone and analyze sequence of goose (Anser anser) myf6 gene, and to reveal its expression during embryonic stage and after overfeeding. In order to obtain full-length of myf6 cDNA, RT-PCR and rapid-amplification cDNA ends (RACE) were used. The structure and function of myf6 gene was analyzed by bioinformatics methods. Real-time fluorescence quantitative RT-PCR was performed to detect myf6 mRNA expression levels in various embryonic stages and after overfeeding status. A full-length of 1 196 bp myf6 cDNA contains an open reading frame, the 90 bp 5'UTR and the 383 bp 3'UTR (GenBank Accession: JQ905627). The open reading frame was 723 bp, which encodes polypeptides of 240 amino acids, with a calculated relative molecular mass of 26.214 4 kD and pI of 5.68. The myf6 was a kind of hydrophilic and unstable protein, with average of hydropathicity -0.595. The myf6 protein was predicted to distinctly had Basic and HLH protein domains, and the bHLH domain of goose myf6 was forming α-helix dimer that was like a scissors, and HLH sequence was highly homologous among different species. Multiple sequence comparisons showed Anser anser myf6 had high homology with Anas platyrhynchoss at 98.62%, about 81% with mammals, and the lowest with fish at only 62%~66%. Phylogenetic relationship analysis indicated that all mammals, birds, bony fish each was formed a large branch, and amphibians was formed an independent branch. Anser anser was firstly clustered with Anas platyrhynchoss and also closely related with Gallus gallus, which was similar to the results of the biological classification. Real-time PCR was performed to analyze the expression profiling of myf6, results showed that goose myf6 was expressed in early embryos at E7, then the expression level gradually increased, the expression level of myf6 in muscle reached the peak at E18, then decreased and stabilized before hatch. The expression level of myf6 at E21 was nearly four times as E25, and after E25 the expression of myf6 was gradually stabilized. And the expression level at E18 was significantly different from those at E14, E15, E25 and E28 (P＜0.01). The similar results were also seen between E21 and E25, E28 (P＜0.01), and E14, E15 (P＜0.05). In addition to these, the expression of myf6 in the breast muscle and thigh muscle of overfeeding group was much higher than that in the control group (P＜0.05). The results showed the expression of myf6 is tissues-specific, and also provide theoretical foundation for the further study of the structure and function of myf6 in goose myogenesis and development during embryonic stage.

The plant-type of maize(Zea mays L.) is characterized by the leaf angle and leaf spacing. And the yield is associated with the distribution of light within canopy and utilization of solar energy within groups, which are affected by the plant-types of maize. To analyse the genetic mechanism of leaf angle and leaf space above ear position in maize, a genetic linkage map composing of 212 simple sequence repeat markers(SSR) was constructed based on the F2 population derived from the cross between compact inbred line CY5 and horizontal-leaf type inbred line YL106. The map covered 1 153.39 cM with an average interval of 5.44 cM. By using the inclusive composite interval mapping method, QTLs for leaf angle and leaf space above ear position of maize were identified based on a field mapping population consisting of 144 F2∶3 families in three environments(XM, short for Xiema test site; BB, short for Beibei test site; HC, short for Hechuan test site). Twenty-two QTLs were detected in single-environment analysis, ten for leaf angle and twelve for leaf space, including nine in XM, eight in BB and five in HC, respectively. Totally eleven QTLs were detected in joint-environment analysis, which were distributed on chromosomes 1, 3, 5 and 7. Five QTLs for leaf angle were detected, which were distributed on chromosomes 1, 3 and 5. Six QTLs for leaf space were detected, which were distributed on chromosomes 1 and 7. Three consistent QTLs including qSecLA1a, qThiLA1a and qThiLS7 were all detected in single-environment and joint-environment analysis. qSecLA1a between bnlg1803 and bnlg1007 explained 26.99% and 18.51% of the phenotypic variation, qThiLA1a between bnlg1803 and bnlg1007 explained 24.14% and 22.00% of the phenotypic variation, qThiLS7 between bnlg1305 and umc1787 explained 13.77% and 9.96% of the phenotypic variation, in single-environment analysis; while in joint-environment analysis explained 29.10%、31.86% and 11.20% of the phenotypic variation, respectively. Therefore it can be concluded that the three loci qSecLA1a, qThiLA1a and qThiLS7 stably expressed in different environments. The results of this study may provide references for genetic modification of leaf angle and leaf space and molecular marker-assisted selection.

In order to construct the fingerprinting of the main seedless watermelon(Citrullus lanatus) varieties to achieve fast and accurate identification and evaluate the genetic diversity, core simple sequence repeat (SSR) markers were used to study the DNA fingerprinting and the genetic diversity of 54 seedless watermelon major varieties in China. Twenty three polymorphic primer pairs could amplify 63 genotypes, and 2.74 genotypes were detected by each primer pair with the range from 2 to 5. The polymorphic information content(PIC) values ranged from 0.04 to 0.67 with the mean of 0.39. Five varieties had unique bands. The genetic similarity coefficients of the 54 varieties ranged from 0.643 9 to 1.0 with the mean of 0.859 3, which indicated that the tested varieties had higher genetic similarity. Besides Xuefenghuapiwuzi and Zhengkangwuzi No.1, Zhengkangxin No.1 and Guangxi No.3、Heibaowuzi and Guiguan No.1, the other varieties could be separated clearly from each other. The standard fingerprinting of 5 main tested varieties was constructed using 10 pairs primer with PIC>0.4. Furthermore, all the varieties were classified into 7 groups, at the level of genetic similarity coefficient 0.82 according to cluster analysis by an un-weighted pair-group average method with arithmetic mean. This study established the standard DNA fingerprinting for seedless watermelon major varieties in China, which provids a technical foundation of varieties' authenticity identification and intellectual property protection.

The root endophytic fungi Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. In order to study the influence of P. indica on salt tolerance in Nicotiana tobacum, we compared the malondialdehyde (MDA) content, Proline (Pro) content, the relative conductance and salt-related genes expression level of P. indica-colonized and un-colonized N. tobacum plants after treated with 300 mmol/L NaCl. The results showed that the MDA concentration and relative conductance in N. tobacum leaves colonized by P. indica were significantly lower compared with the un-colonized ones (P＜0.05) and the Pro content in P. indica colonized N. tobacum leaves was significantly higher than that in un-colonized ones (P＜0.05). RT-PCR analysis showed that the expression of salt tolerance gene(osmotin promoter binding protein) OPBP1 and pathogenesis-related (PR)protein genes PR-1a, PR2, PR3 and PR5 were upregulated in both P. indica colonized and un-colonized N. tobacum leaves, and the expression of these genes in P. indica colonized N. tobacum was significantly higher than the un-colonized ones (P＜0.05), which showed that P. indica can enhance the expression level of salt-related genes of N. tobacum. This study indicated that the salt resistance improvement in P. indica colonized N. tobacum was associated with malondialdehyde and proline contents, plasmamembrane permeability, the expression level of salt tolerance genes and pathogenesis-related protein genes. P. indica confer N. tobacum salt tolerance via maintaining cell biomembrane system stability, balancing cell osmotic pressure, and reducing the level of membrane lipid peroxidation of plants. This study preliminarily confirmes the role and partly mechanism of salt tolerance of N. tobacum conferred by P. indica, and conforred the possibility of better understanding of the role and related mechanisms in P. indica colonized N. tobacum during stresses resistance.

To investigate the effects of various physical and chemical factors on the growth and the hyoscyamine production in hairy root cultures of Anisodus acutangulus, the influence of different minimal mediums, carbon sources, growth regulators, pH, temperature and photoperiod were studied by liquid culture. The results showed that B5 medium was most suitable to the growth of hairy roots and the formation of hyoscyamine in hairy roots. Hairy roots growth and hyoscyamine formation were promoted when B5 was used as the basic medium, at pH 6.5, temperature of 25 ℃ and photoperiod of 18 h/d. The effect of 5% sucrose was better to hairy roots growth within carbon source concentrations of 1% to 7%, but hyoscyamine contents in hairy roots were decreased with the increase of carbon source concentration. 0.05 mg/L of NAA, IBA and 2,4-D all could promote the growth of hairy roots, but such growth regulator substances inhibited strongly scopolamine formation in hairy roots regardless of the concentration of high or low. This research on the effects of these physical and chemical factors can provide a scientific reference for the optimization of the culture conditions of Anisodus acutangulus hairy roots and the mass production of hyoscyamine.

Wild-abortive (WA) and dwarf wild abortive (DA) are two important cytoplasmic male sterility (CMS) types in rice(Oryza sativa). CMS can be restored by nuclear restorer gene (Rf). One or two dominant restorer alleles (Rf3 and/or Rf4) are usually suggested be responsible for the fertility of WA-CMS and DA-CMS. To develop the strong restorer lines for WA-CMS and DA-CMS and analyse the pyramiding effect of the Rf3 and Rf4 genes, seven indica chromosome double segments pyramiding lines (DSPLs), carrying the genotype Rf3Rf3/Rf4Rf4, and five second single segment substitution lines (SSSSLs) with the Rf3 on chromosomes 1 (or Rf4 on chromosomes 10) gene were selected by marker-assisted selection (MAS) in the F2 and F3 populations generated from the ten crosses among the seven SSSLs which possess the Rf3 (or Rf4) gene on fertility restoration in rice with WA-CMS and DA-CMS in this study. There were wide differences in restoring abilities for WA-CMS and DA-CMS among the six DSPLs. Of which the restoration ability of the DSPL11-01/14-10P with the genotype Rf3-4Rf3-4/Rf4-4Rf4-4 was the strongest, the DSPL07-01/14-10P carrying the genotype Rf3-1Rf3-1/Rf4-1Rf4-1 appeared to be weakest restoring ability, and the restoration abilities of the other DSPLs ranged from DSPL11-01/14-10P to DSPL07-01/14-10P. The length of substituted chromosome segments in the five SSSSLs ranged from 0.1 cM to 45.5 cM with an average of 18.7 cM, and the total length of substituted chromosome segments from them was 93.5 cM. To reveal the pyramiding effect of the Rf genes, the DSPL11-01/14-10P and its corresponding SSSLs were crossed with a typical WA-CMS lines of Bobai A (BBA) and a typical DA-CMS line of XieqingzaoA (XQA), respectively. In the F1 populations, the plants carrying the Rf3rf3/Rf4rf4 genotype were selected and their phenotyping for pollen was evaluated. The result showed that even if the restoration ability of the DSPL11-01/14-10P showed stronger than that of corresponding SSSLs, the difference between them was not significant, which indicated that there was not interaction effect between Rf3 and Rf4 genes in the DSPL11-01/14-10P. These studies have led to the development of three-line in rice and the transfer of Rf3 and Rf4 genes into adapted cultivars through backcrossing in an active hybrid rice breeding program.

The objective of current study is to identify the genetic resource and phylogenetic relationship of indigenous horses in Gansu based on control region of mitochondrial DNA (D-loop), which was re-sequenced from Equus przewalskii, Mongolian horse , Qilian horse and Maqu horse (Maqu population, Hezuo population and Luqu population). The results revealed that higher proportion of A+T content than that of G+C in all breeds. Fifty-six haplotypes were reconstructed base on 50 polymorphic sites in all populations of Gansu horses. In addition, the nucleotide diversity (π) and haplotyple diversity (Hd) was 0.967±0.004 and 0.01782±0.0076, respectively, which suggested extensive divergence among Gansu horses. Six clusters (A, C, E, F and G) and fifteen sub-clusters(A1-A4, A6, C2, D3-D6, G and F1-F3) were distributed in Gansu horses. Cluster E was identified from Hequ horses merely; Most frequency of Cluster C and Cluster D were filled with Hequ and Qilian, respectively; Cluster A was found from all Gansu horses breeds. Notably, phylogenetic analyses showed Qilian horse share the same haplotype with other population in this study, which implied Qilian horse have the same maternal origin with them. To reveal the relationship between gansu indigenous horses and other breeds in other places of china and all over the world. NJ (Neighbor-Joining) phylogeny tree obtained from phylogenetic analyses compared with other places horses' NJ (Neighbor-Joining) phylogeny tree made based on Vilà C. and Janse T. identified standard cluster. The result suggested E.przewalskii had the same maternal origin, otherwise, E. przewalskii got mutation during their evolution. Mongolian horse, Qilian horse and Hequ horse not only had the same maternal origin with domestic horse (Guanzhong horse, Tibtan horse, Jinjian horse, Datong horse, Debao pony, Lichuan horse, Baise horse) but also had the same maternal origin with horses in East Asia, west Asia , central Europe, Western Europe, North America and Middle East. In addition, Qilian horse and Hequ horse also had the same maternal origin with E. przewalskii, potentially. The study results will be a reference for genetic resources conservation and development and utilization of indigenous horses in Gansu.

To compare the effects of three different doses of CpG oligonucleotide (CpG-ODN) adjuvants combined with subgroup J Avian leukosis virus (ALV-J) gp85 recombinant protein on serum and egg yolk antibodies in breeding hens(Gallus domesticus), the recombinant protein containing ALV-J gp85 gene was prepared using the prokaryotic expression system and vaccinated combining with three different doses of CpG-ODN adjuvant into adult Hy-line Brown parent breeding hens, the booster immunization was conducted with two weeks interval. The serum antibodies and the egg yolk antibodies against ALV-J were detected weekly using ALV-J antibody kits from 1 day post inoculation to 49 day post inoculation and the data were statistically analyzed with ANOVA software. The results of protein gel electrophoresis showed that the recombinant protein containing gp85 gene of ALV-J was successfully prepared with prokaryotic expression system and the size of the recombinant protein was 45 kD; the Western blotting analysis indicated that the purified recombinant protein could specifically bind with the monoclonal antibody JE9 of ALV-J gp85 protein; the serum antibodies in breeding hens were successfully induced by three different doses of CpG combining with the recombinant gp85 protein, the serum antibody titers were increasing from the first post inoculation and reached a peak at the 28th day post first inoculation and then decreased slightly. Between three vaccinated groups, the antibody titers in the 50 μg CpG dose group were higher than those in the other two vaccinated groups. The ratio of positive antibodies in the 50 μg CpG dose group was up to 92%, but it was up to 80% in the 100 μg CpG dose group and only 67% in the 200 μg CpG dose group, respectively. The egg yolk antibodies against ALV-J in three vaccinated groups were successfully detected from the 3rd week to the 7th week post first inoculation; the antibody titers in the 50 μg CpG dose group were optimized at the 4th, the 6th and the 7th week post the first inoculation, which also were better than those in the other two vaccinated groups. Based on these results, it can be concluded that three does of CpG combining the recombinant protein containing gp85 gene of ALV-J could induce the vaccinated breeding hens to produce serum antibody and maternal antibody against ALV-J and the dose of 50 μg per hen was better than that of the other two doses. These results can provide a scientific basis for the application of CpG adjuvants combing with ALV-J gp85 subunits vaccine in blocking ALV-J early infection with maternal antibodies in breeding hens.

Characteristics of livestock myofiber not only determine meat production but also closely relate to meat quality. The present study was firstly designed to research the characteristics of myofiber during late embryonic and post-hatch development and their associations with skeletal growth and development in two duck breeds(Anas platyrhynchos domestica) (Gaoyou ducks and Jinding ducks) with different growth rate. Gastrocnemius muscles collected from two duck breeds of 21, 25 and 27 embryonic development and 7 day post-hatching were cut at -20℃ using a cryotome and then stained with the method of myosin-ATPase. The results showed that there was no significant difference between two breeds in leg weight, body weight, myofiber diameter, cross-sectional area and density during late embryonic development, but the age effect was significant. The myofiber diameter and cross-sectional area of the two duck breeds increased while the myofiber density decreased throughout the embryonic development. At 7 day post-hatching, leg weight and body weightof Gaoyou duck was significantly higher than that of Jinding duck (P＜0.01), but there was still no significant difference in myofiber diameter, cross-sectional area and density between two breeds (P＞0.05). The myofiber cross-sectional area of two duck breeds decreased slightly (P＞0.05) at 7 day post-hatching while the myofiber density increased significantly (P＜0.05). Gastrocnemius myofibers of two duck breeds could be divided into three types (typeⅠ, typeⅡa and typeⅡb ), and the proportion of type IIb was highest. There existed a trend in duck gastrocnemius muscles that a few of type IIb was switching to type I during late embryo to early hatching period. The correlation analysis showed that in both two duck breeds leg weight and body weight were positively correlated with myofiber diameter and cross-sectional area and negative correlated with myofiber density. These results provide an important reference for studying the development and regulation mechanism of skeletal muscle and meat quality in ducks.

High density lipoprotein binding protein (HBP) plays an important role in fat metabolism. Lipid is indispensable to fish energy material, and it is very important for carnivorous fish which has low efficiency to use carbohydrate, especially. The single nucleotide polymorphism(SNP) in HBP gene may affect the fat metabolism, and is correlated with growth traits. Studying the association between the SNP and growth traits can provide candidate markers for marker-assisted selection. In this study, partial genomic fragments of HBP gene were amplified based on the sequences of one available contig in the EST-SNP database of largemouth bass (Micropterus salmoides). Three single nucleotide polymorphism(SNP) mutations were identified after sequencing the PCR products of twenty individuals (H1: G+2782T, H2: T+2817C, H3: G+2857A), which were located in 3'non-coding region. For the three SNPs, the genotype and gene frequency of 165 largemouth bass were further assayed with the method of SnaPshot. Genetic structure was analyzed by POPGENE32 software. A general linear model(GLM) was used in the correlation analysis between SNPs and growth traits. The results showed that three locus were in Hardy-Weinberg equilibrium. The sites of H1 and H2 formed two haplotypes (A and B) and three genotypes (AA, AB, and BB). The sites of H1, H2 and H3 formed six diplotypes(D1, D2, D3, D4, D5 and D6). Association analysis showed that each SNP was not significantly associated with growth traits. The body weight and total length of genotype BB and AB were significantly different (P＜0.05). The body weight and total length of diplotypes D2 and D6 were significantly different (P＜0.05). Our data suggested a significant association between genetic variations in the largemouth bass HBP gene and growth traits. These results indicated that the SNP markers founded in HBP gene 3'non-coding region were candidate genetic markers, which can be used in future breeding programs of largemouth bass.

To establish two-dimensional gel electrophoresis methods(2-DE) for cashmere goat skin, the experimental conditions of 2-DE were optimized, and various extraction and purification methods, different loading quantities and pH IPG strips, different isoelectric focusing were studied. The results showed that the process of grinding in liquid nitrogen and following by ultrasonication, the effect of sample purification with 2-D clean up kit, pH 4~7 17 cm pH IPG strip, the 450 μg loading quantity, isoelectric focusing process repeated several times in addition to salt step, maps were of good quality. Meanwhile, the common problems occurring in 2-DE experience were analyzed. The results indicated that the 2-DE maps of cashmere goat skin were obtained with high resolution and reproducibility, and can be used for the further proteome analysis. This study got a good 2-DE maps for cashmere goat skin protein, proved that it was feasible to conduct proteomics research for cashmere goat, and provided important technical information for researching development cycles and revealing origin and development law of skin hair follicle.

The Real-time quantitative PCR (qRT-PCR) is a common approach to analyse the gene expression level, in order to obtain reliable experimental data, it is critical to select an appropriate reference gene. However, there has been little research on the insect reference genes. This research was conducted to determine stable referece gene(s) for normalization in future expression studies on the red flour beetle (Tribolium castaneum). The expressions of candidate reference genes β-actin, GAPDH, α-Tubulin, SYN1, SYN6, RPS3, RPS18 and RPL13a in Tribolium castaneum after exposure to the phosphine were detected by qRT-PCR using relative quantification. According to the qRT-PCR reaction result, these eight candidate reference genes were all specifically amplified by the primers pairs with high efficiency. Then the expression stability of these genes was analysed by geNorm and NormFinder software. Based on the results of geNorm analysis, the stability of the eight candidate reference genes from high to low was RPS18 =RPL13a (0.007)＞SYN6(0.015)＞RPS3(0.038)＞β-actin (0.044)＞α-Tubulin(0.105)＞SYN1(0.154)＞GAPDH (0.205), and V2/3 value of pairwise variances analysis of eight candidate reference genes was 0.006. The results showed that RPS18 and RPL13a were the most suitable reference genes in T. castaneum after exposure to the phosphine and the appropriate number of reference genes was 2, whereas GAPDH, α-Tubulin and β-actin which have a wide range of application were poorly ranked. On the other hand, based on the results of the NormFinder analysis, the stability of eight candidate reference genes from high to low was RPS18 (0.002)＞RPL13a(0.016)＞β-actin (0.042)＞RPS3 (0.044)＞SYN6(0.055)＞α-Tubulin (0.066)＞SYN1(0.077)＞GAPDH (0.126). The results showed that RPS18 was the most suitable reference gene, and the best combination of two genes were RPS18 and RPL13a. All above results suggest that RPS18 and RPL13a are suitable reference genes and can be used to normalize mRNA levels in different strains of T. castaneum after exposure to phosphine . This work is contributable to lay a foundation for future gene expression studies in the T. castaneum, and may also act as a resource to screen reference genes for expression studies in other insects.

Ferritins are key proteins of iron metabolism that serves the dual function of iron detoxification and iron storage. The importance of their dual function is underscored by their ubiquitous distribution throughout all organisms. Not only iron in phytoferritin from legume seeds is required for seedling germination and early growth, but also phytoferritin is considered to be a new iron supplementation strategy for the 21st century. Therefore, it is important to elucidate its mechanisms of iron oxidative deposition and iron reductive release for its physiological research and its successful development as the iron supplement in future. Despite common ancestry, plant and animal ferritins are remarkably different in the structure of protein. For example, the mature phytoferritin contains an own EP(extension peptide) at its N-terminal, and only H-type subunit has been identified in phytoferritin which usually consists of H-1 and H-2 subunits. The different properties and function have been identified due to the specific structure of phytoferritin. This review focused on recent research progress in structure and function of phytoferritin and its mechanisms of iron oxidative deposition and iron reductive release, aimed at providing the reference data for further research of phytoferritin in future.