To clarify the relationship between brassinosteroid(BR) and Ca2+ signal transduction and their regulating mechanism in plant growth and development, 2D-DIGE technology combined with mass spectrometry analysis was used to identify proteins differentially expressed in d61-4(BR insensitive mutant in rice(Oryza sativa L. ssp. Japanica)) and brd1-3(BR synthesis mutant in rice) by comparing with the wild type plant. A protein significantly upregulated both in d61-4 and brd1-3 was identified. The protein was related to Ca2+ signal transduction network(putative calmodulin, PCAM). This gene was overexpressed in Arabidopsis thaliana. Transgenic Arabidopsis lines overexpressing PCAM were smaller in size, and plant growth period was prolonged. The expression of SaurAC(a key gene in BR signal transduction pathway) in transgenic strains was obviously down-regulated. These results suggest a role of PCAM in the signal transduction of BR is as a negative regulator of the plant growth and development.

Sheep mannose-binding lectin(MBL) is an important part of the body's natural immune system. Nine pairs of primers were designed according to human(Homo spiens) and bovine(Bos taurus) MBL gene sequence, combined with PCR technique to amplify the MBL gene of sheep(Ovis aries). Cloning, sequencing and splicing to analyze bioinformations with the DNAMAN software and online tools. The results showed that sequence of DNA of the sheep MBL gene was 4 462 bp containing promoter, four exons, three introns, which encoded 249 animo acids, submitted it to GenBank (Accession No. FJ977629). Its amino acid sequence had an overall similarity with a comparable region of bovine MBL-C gene(96.39%), phylogenetic tree of amino acid analysis indicated that the sheep MBL gene was MBL-C. Expasy were employed to predict specific structure and function of sheep MBL gene, 1~19 amino acids were signal peptide region, exon 1 encoded N-terminus cysteine-rich domain and 8 Gly-X-Y repetitive sequences of collagen-like domain, and exon 4 encoded carbohydrate recognition domain(CRD) which had common features of C-type lectin. This study is the first report about complete genomic DNA sequence data of sheep MBL gene, and it provides basal data for further study on the structure, function and defects in the molecular mechanism of MBL.

In order to study the yak (Bos grunniens) populations' genetic diversity and genetic structure and promote the rational use and protection of the yak populations genetic resources in Gansu Province, 15 microsatellite markers were used to detect six yak populations (240 individuals). The results showed that146 alleles were found in 15 microsatellite loci, in which allele number (106) in Maqu yak population was highest, and that in Tianzhu white yak was at least (74). The analysis of polymorphism information content(PIC), expected heterozygosity(He) and observe heterozygosity(Ho) indicated that the genetic diversity of 3 yak populations from Hezuo city were rich, but the genetic diversity of Tianzhu white yak, Tianzhu yak and Sunan yak population were relatively low. DA genetic distanceand Nei's standard genetic distance (DS) and the phylogenetic tree constructed by DA and DS genetic distance and NJ method showed that the relationship of Maqu yak, Luqu yak and Xiahe yak were very near, which indicated that the 3 populations might have the same ancestors. But the genetic distance of Tianzhu While yak, Tianzhu yak and Sunan yak populations were near, they might have the same ancestors. The results of classification in principal component analysis and genetic structure analysis were same with the phylogenetic tree, the 6 yak populations were divided into two clusters, and consisted with the geographic distribution of the 6 populations.

To improve the utilization of goat oocytes and reduce the decline in oocytes quality caused by aging, the senescence of goat(Capra hircus) oocytes cultured in vitro were studied by comet assay. The results showed that, with the prolonged culture time, especially more than 24 h, the first polar body(PBⅠ) degenerated significantly and the DNA damage increased gradually. These results suggested that apoptosis increased seriously during oocytes culture in vitro. Before 24 h of culture, the percentage of oocytes with PBⅠ increased visibly with the extended culture(20 h, (45.74±2.67)%; 22 h, (66.11±1.12)%). All the mean length of comet tail was less than 100 μm, however, among oocytes with PBⅠ, the percentage of oocytes with comet tail length distributed in 0~50 μm region (20 h, (77.27±2.27)%; 22 h, (40.12±1.55)%) was less than oocytes without PBⅠ(20 h, (83.86±1.34)%; 22 h, (51.39±8.45)%). At 24 h of culture, oocytes with PBⅠ increased to the peak rate(80.19±2.07)%. And there was no difference in the mean comet tail length for both of oocytes with PBⅠor not ((96.08±4.52) μm vs (95.25±9.28) μm). Moreover, the distribution of mean comet tail length was major in 50~100 μm((45.71±0.50)%) and 100~150 μm ((35.45±2.92)%) for oocytes with PBⅠ, while 0~50 μm ((30.92±4.02)%) and 50~100 μm ((33.02±3.23)%) were the major distribution for oocytes without PBⅠ. When the culture time extended to 26 h later, the rate of oocytes with PBⅠ decreased dramatically (26 h, (72.01±2.88)%; 28 h, (59.77±3.59)%). And the mean comet tail length increased continuely for both of oocytes with PBⅠor not, and the percentage of oocytes with more than 150 μm comet tail length increased rapidly. At 30 h of culture, only (46.34±2.07)% of PBⅠ could be observed in oocytes, and the mean comet tail length was (180.11±10.33) μm, (58.33±4.81)% of them with a more than 150 μm comet tail; but for oocytes without PBⅠ, the mean comet tail length was (270±17.72) μm, and (80.95±2.38)% of them with a more than 150 μm comet tail. This study showed that goat oocytes matured after in vitro muturation became increasingly aged along with the extended culture time, and the proportion of aging oocytes increased dramatically when cultured more than 24 h. Through this study, we can get a further more understanding on the mechanism of oocyte aging. It contributed to improve the oocyte quality by reducing the oocyte aging.

Rice stripe disease, caused by Rice stripe virus (RSV), has led to severe losses in many rice-cultured countries and regions. RSV is transmitted by the small brown planthopper (SBPHs), Laodelphax striatellus Fallén, in a persistent-propagative, and it also can be transmitted transovarially (vertically) by the small brown planthoppers to their offspring. In order to study the transovarial transmission characteristics of RSV by SBPH vectors, we tested viral infections in internal organs and in ovaries of SBPHs with RT-PCR assay and immunofluorescence for virus antigens. Our results showed that only 5.3% of SBPHs contained virus antigens in the ovaries with immunofluorescence for virus antigens, although the virus was detected by RT-PCR in 28.6% of SBPHs that had access to infected plants. Thus, the ovaries would form the transovarial transmission barrier for RSV, which might determine the efficiency of the transovarial transmission of RSV by SBPH vectors. We further investigated the sequential infection of RSV in the ovaries of SBPH vectors with immunofluorescence for virus antigens. Immunofluorescence studies revealed that RSV initially infected the oviduct, and then progressed to the follicular cells of ovarioles. RSV also could accumulate in the germarium and the nutritive cord of the ovary and in the blastodermal cells of the eggs after oviposition. All these results suggested that RSV might spread into the oocyte together with the nutrition transport from the follicular cells of the ovarioles and germarium to the developing oocytes. Based on the fact that the germarium would develop to oocytes and follicular cells, we deduced that RSV might initially infect germarium, which finally formed viruliferous oocytes, although RSV might also spread into oocytes when accompanied by the nutrition transport. Taken together, our results provide a possible mechanism that RSV can exploit the pathway for the nutrition transport into the oocytes, and finally transport into the blastodermal cells of the eggs.

The Chinese mitten crab is an indigenous species in China, its hatchery production and larval farming have been performed almost exclusively in aquaculture farms for many years. It is worthy to know whether the genetic diversity of cultured crab has been changed in the closed off or half-closed off farm. The genetic diversity of Chinese mitten crab (Eriocheir sisensis) was investigated in three cultured populations (Jiangsu, Anhui and Liaoning Province, China) and wild population in Yangtze River by using 10 microsatellite markers. The results showed that the mean effective number of allele (Ne) of four populations were 8.77, 6.94, 8.71 and 9.03 respectively, the mean observed heterozygosity (Ho) was from 0.70 to 0.75, the mean expected heterozygosity (He) was from 0.87 to 0.90, the mean polymorphism information content (PIC) was from 0.84 to 0.87, the level of genetic diversity of the wild population was higher than that of the cultured population. Coefficient of genetic ddifferentiation F-statistic(FST) among populations ranged from 0.0123 to 0.0207. Analysis of molecular variance (AMOVA) revealed that a higher portion of variations existed within individuals, while lower portion existed among populations. FST and AMOVA analysis across all populations indicated the low level of divergence among the populations. The NJ clustering tree based on DA genetic distance demonstrated that there were two different groups: one being composed of cultured population in Jiangsu and wild population in Yangtze River, and the other of cultured populations in Anhui and Liaoning. A hundred and seven individuals obtained from different place could be grouped into two potential populations based on the genetic structure, the crabs cultured in Jiangsu and the wild crabs in Yangtze River were grouped into one population; the crabs cultured in Anhui and Liaoning were grouped into another population. These data demonstrated that the cultured mitten crab is in high levels genetic diversity and exhibite low levels of genetic variation as compared with wild population. There is potentiality for breeding selection.

β-glucosidase is one of the three members of the cellulase system and is considered to be the rate-limiting enzyme of cellulose saccharification. The objective of the study was to clone the β-glucosidase gene (bgl1) from Asperjillus niger, and analyze its expression in Chinese hamster ovary (CHO) cells. The DNA fragment was amplified by PCR from Asperjillus niger, and then cloned into pMD18-T vector. A DNA fragment containing the coding sequence of pig parotid secretory protein signal peptide (sp) and bgl1 was assembled by SOE-PCR and subcloned into the expression vector pcDNA6/His A using BamHⅠand XhoⅠrestriction sites to generate pc6-sp-bgl1. To evaluate transgene expression, plasmid DNA was purified and transiently transfected into CHO cells, then RT-PCR and Western blot analyses were used and the enzymatic activity was determined by DNS(dinitrosalicylic acid). Sequence analysis showed that the DNA sequence and the putative amino acid sequence shared 91% and 99% identity with the reported sequences, respectively. RT-PCR and Western blot analyses showed that the exogenous gene bgl1 was expressed and recombinant protein was secreted into cell culture medium. The enzymatic activity of culture supernatants from 48 h-transfected cells to D-(-)-salicin was 1.74 U/mL, indicating that the recombinant protein with the activity of β-glucosidase. We cloned the β-glucosidase gene bgl1 from Asperjillus niger and expressed the gene in CHO cells. The study paves the way for further research of β-glucosidase in monogastric animals.

To search and identify the gene regions which control yield and quality in maize, 64 pairs of SSR (simple sequence repeats) markers for the core 257 groups composed of maize(Zea mays L.) inbred lines were genotyped, linkage disequilibrium(LD) of groups of loci and population structure were analysed on this basis, using the GLM(general linear mode) procedure in TASSEL software to make regression analysis for phenotypic data of plant height, ear height, growth duration, ear length, row number of ear, 100-seed weight, fat content, protein content, starch content and maker data, and determined the interpretation of phenotypic markers of the rate in this study. The results showed that: (1) SSR loci combinations had a certain degree of linkage disequilibrium in the public genetic map. LD pairs of loci of the total sites combined was 20.29% when P<0.01, and the proportion of sites was 12.65% when D'>0.5. (2)SSR analysis of genetic structure of the data showed that groups could be divided into five subgroups.(3) A total of 26 marker loci associated with the 9 traits was identified, mostly concentrated in the 4,6,7 and 10 chromosom. The most markers was chromosome 7, which was possessed of five markers. Eight markers were significantly related with six traits such as plant height, ear height, growth duration, ear length, ear number of rows, protein content at the level of 0.01 (P<0.01).Umc194 was associated with plant height and the phenotype variation explained was 7.2%; umc1741 and phi116 acted on the ear and the phenotype variation explained were11.37% and 8.57% respectively; phi328175 and phi260485 played a vital role in reproductive age and the phenotype variation explained were 4.74% and 5.6% respectively ; umc1741 has effected on ear length and the phenotype variation explained was 5.77%; umc1309 has effected on rows per ear and the phenotype variation explained was 6.68%; bnlg1450 and bnlg1185 have effected on protein content and the phenotype variation explained were 9.41% and 9.81% respectively. The other 18 markers were associated with 9 traits at the level of 0.05(P<0.05). The number of markers which associated with a single trait ranged from one to seven, and the phenotype variation explained varied from 4.74% to 14.31%. There were 30 and 7 markers associated with yield and quality traits respectively. The former number was far more than the later number. One of those markers was simultaneously associated with multiple traits, which may be explained by the traits relationship and the gene pleiotropic effect. Some traits were associated with a marker at the same time, and much of which were according with the result of QTL mapping. The study suggests that those makers and many QTLs associating with yield and quality traits may play important roles to improve those traits in maize.

The study was conducted to establish the DNA fingerprints and throw light on the genetic diversity of the japonica rice(Oryza sativa L.) in Henan Province. The simple sequence repeat (SSR) fingerprints map and genetic diversity of 37 major cultivars in Henan during 1963~2010 were studied by 37 pairs of primers equally distributed on 12 chromosomes of rice. Thirty-three SSR primers showed polymorphism among 37 varieties, and 114 polymorphic bands were detected, with an average of 3.53 bands per pair of primers. The frequency of polymorphism of the primers ranged from 0.054 to 0.739. The range of genetic similarity coefficient of the 37 varieties was from 0.627 to 0.992, with the average of 0.812. Seventeen cultivars could be identified by their single characteristic SSR marker, while the other 20 varieties could be identified by combination of different primers. Nineteen pairs of core primers were used to construct the SSR fingerprint map, and all the 36 varieties could be identified using the fingerprint map. Un-weighted pair-group of arithmetic mean(UPGMA) cluster analysis indicated that 37 varieties were clustered into two groups at the level of genetic similarity coefficient 0.710. Most of the breeding varieties of Henan were classified into the same group. It is demonstrate that the genetic basis of the japonica rice in Henan is narrow.

This paper was to obtain the fusion proteins of betaine aldehyde dehydrogenases(OsBADH and SpBADH) from rice (Oryza sativa subs. japonica) and spinach (Spinacia oleracea) by means of gene cloning, prokaryotic expression and protein purification for measuring the enzyme activities of the two aldehyde dehydrogenases in vitro, proved that rice existed OsBADH with the function of aldehyde dehydrogenase. In this study, OsBADH and SpBADH full-length gene were cloned by RT-PCR from japonica rice and spinach, respectively. The amino acid sequences alignment showed 70.8% similarity. Recombinant plasmids of both OsBADH and SpBADH could be expressed in Escherichia. coli strain BL21 (DE3). SDS-PAGE analysis revealed that the fusion protein bands were observed at approximately 70 kD after inducing by 0.4 mmol/L IPTG. The proteins from E. coli strains BL21 were purified by TALON metal chelating resin and then the enzyme activities were determined. The result indicated that the purified recombination proteins exhibited the enzymatic activities of BADH. The catalytic activity of OsBADH was 74.87 nmol/min/mg with SpBADH (86.84 nmol/min/mg) as control, suggesting that OsBADH could catalyze the reaction effectively but no glycinebetaine(GB) production was detected by HPLC. These results image that little level of glycinebetaine in rice may not due to the activity deficiency of BADH. The study may provide basic information for further research of the molecular mechanisms of glycinebetaine synthesis in rice.

In order to analyze the SSR distribution in genomic sequence of Brassica rapa and develop new SSR markers, the DNA sequences of Chinese cabbage A10(16899818~17299817) were screened using SSRHunter software and 394 Simple sequence repeats(SSRs) were mined with an average distance of 1.02 kb. Dinucleotide and trinucleotide repeat SSRs were the dominant types, accounting for 79.44% and 18.78%, respectively, of the SSR obtained. In order to improve the accuracy and to test the transferability of the developed SSRs, the mined SSRs were blasted and 15 SSR sequences were selected and primers were designed. Morever, some differences of Insertion/Deletion(InDels) rather than SSRs were found in some SSRs sequences and 19 InDels primers were designed. The 34 resultant primer pairs were used for polymorphism analysis in 6 genotypes of Chinese cabbage(Brassica rapa ssp. pekinesis). Twenty eight primer pairs had expected PCR products, accounting for 82.35% of designed primers, and 27 primer pairs showed polymorphisms, accounting for 79.41% of the designed primers. PCR products from 4 random SSR primer pairs were used for sequence analysis. The results showed that 100% of the fragments contained target SSRs. The percentage of available SSRs and InDels amplified in cabbage(B. oleracea), rapeseed(B. napus) and radish(Raphanus sativus) samples were 85.71%, 100% and 77.78%, respectively. It showed that these SSRs and InDels had good polymorphisms and transferability among Brassicaceae. A subset of 6 validated primers was also used to assess genetic diversity in a collection of 48 elite Brassicaceae germplasms. The dendrogram revealed that all germplasms were obviously classified into 3 groups, including Brassica rapa and Brassica oleracea, Raphanus sativus and Brassica napus, and the result accorded with the conventional taxonomy. The SSR and InDel markers can be used in genetic analysis of Brassica rapa.

Pig induced pluripotene stem cells(iPSCs) are ideal materials for regenerative medicine and cell biology research because of their self-renewal and multilineage differentiation potential. In order to explore the embryo chimeric ability of pig iPSCs, the piggyBac transposon vectors, PB[Act-RFP] DS and Act-PBase, were used to cotransfect pig iPSCs. The PS24-R, a pig iPS cell line, was successfully obtained which expressed red fluorescent protein (RFP). We have got chimeric embryos through microinjecting the PS24-R cells into the 4~8 cell parthenogenetic embryos in vitro. Then we collected the statistics of blastocyst rate and chimeric rate as the chimeric embryos developed to the blastocyst stage. The result showed that the PS24-R cell using piggyBac vectors to transfect the iPS cells could stably express RFP. The pig iPS cells could be effectively observed in chimeric embryos. We also tried to microinject one, five, ten PS24-R cells and one PS24-R clone respectively to pig embryos to produce chimeric embryos. Along with increasing of the number of donor cells, the chimeric rate of embryos gradually increased, whereas, blastocyst rate declined. Compared with the parthenogenetic embryos, the expression lever of the stem cell pluripotent factors, OCT4(octamer binding transcription factor 4), SOX2(sry related HMG box-2) and NANOG, had significant upregulated in chimeric embryos. In summary, the pig iPS marked by PiggyBac vector can achieve chimerism in the pig early embryonic development stage. This work lays a foundation for the production of pig iPS chimeric animals.

To investigate the stability of reference molecules with different insertion segments, there standard plasmids were developed with different insert segments: 186 bp (Cry1Ab), 273 bp(Cry1Ab and SPS) and 880 bp(Cry1Ab, SPS, fsACP, 35S, NOS and NPTII). The plasmids were stored for different time (1, 3, 7, 14, 21, 28 days or 3, 6, 12 months) under different temperature (-70, -20 and 4℃). The stability of Cry1Ab of 186, 273 and 880 bp were compared. The results showed, the Cq(quantification cycle) values of Cry1Ab in 186 and 273 bp were stable in 6 months, but increased rapidly and significantly differed (P<0.05) under -70℃ and 4℃ storing for 12 month. The Cq values of Cry1Ab in 880 bp were increased and significantly differed (P<0.05) at the 6 month. The stability of SPS in 273 bp and SPS, fsACP, 35S, NOS, NPTII in 880 bp were also studied, and we found that the Cq values of SPS in 273 bp were stable in 6 months, and the Cq values of the other five segments in 880 could maintain stability only in 3 months. It was showed that plasmid with shorter insert segments were more stable than that with the longer one, and the stability varied with different insertion. This study provides a scientific basis of the stability of reference molecules.

Sex determination is a plastic biological developmental process, which has always the intriguing aspect in evolutionary biology and developmental biology. In vertebrates, the sex determination including genetic sex determination (GSD) and environmental sex determination (ESD) acts to differentiate an initially bipotential gonad primordium into either testes or ovaries. GSD is governed by a series of sex-related genes involving in genetic pathway that initiate by sex-determining gene during critical periods of gonadal development. Though many downstream genes in sex determination pathways are conserved, even among vertebrates and invertebrates, the upstream sex determination gene can vary even between closely related species. Up to now, five sex-determining genes (SRY, DMRT1, DMY, DMW and AMHY) had been identified in vertebrates. This paper reviews the recent progress of sex-determining genes in vertebrate and analysis of the differences on the conservation of sex-determining genes between the higher vertebrates and lower vertebrates. We found that the sex-determining genes (SRY and DMRT1) are conserved in therian and aves, respectively, comparing to the sex-determining genes DMW, DMY and AMHY, which are unstable in their respective taxonomic systems. Correspondingly, the sex chromosomes in higher vertebrate attained high differentiation during their evolution, whereas no obvious (or even no) differentiation was observed in the sex chromosome of most extant lower vertebrates. Generally, the appearance of sex-determining gene in certain organism was always accompanied with the evolution of their sex chromosomes, which was defined by coevolution. We thus concluded that the difference of conservation on sex-determining genes between higher vertebrates and lower vertebrates may be caused by the sex chromosome differentiation or not. Following these conclusions, we then put forward two models on the relationship between sex-determining gene and sex chromosome evolution. One is the model of differentiation on sex chromosome that developed through diversification of one region of the progenitor chromosomes. In this model, transposons and repetitive elements were accumulated around a sex-determining gene and its homolog evolved into a pseudo-gene because the recombination was ceased in the sex-determining region. We thus expect a dosage dependent sex-determining gene in homogametic sex or a heteromorphic chromosome-linked (usually Y and W) determining gene in heterogametic sex such as DMRT1 in chicken and SRY in human, respectively. The other model is undifferentiation of sex chromosome where sex-determining gene derived from a duplicated region from elsewhere in the genome that was inserted on it. The large region of sex chromosomes in such case can still recombine, though the duplicated region could accumulate few repetitive elements. In this case, the sex-determining genes would reside on the heterogametic chromosome (usually Y and W) such as DMY, DMW and AMHY. In all, sex determination is a complex biological process; these two models cannot represent the types of sex determination in all species, but above models proposed would make for the isolation of new sex-determining gene.