Cloning and characterization of a gene encoding phage-related tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.     

氯化钇对黑腹果蝇生育率的影响

探讨稀土元素钇（Y）对黑腹果蝇生育率的影响。在不同浓度（1、4、16 mg/L）氯化钇（YCl₃）培养基中饲养野生型黑腹果蝇（红眼Canton-S）。并设置普通培养基作为对照，收集8 h内羽化的果蝇，雌雄果蝇分别放置在0、1、4、16 mg/L的YCl₃培养基中喂养10 d，后分别与异性果蝇按照1:3的比例在普通培养基中进行杂交，7 d后去除异性果蝇，第10 d开始统计羽化的果蝇总数量（F1），连续统计5 d后，计算新生成果蝇的数目。结果表明YCl₃浓度为1 mg/L时，雄性果蝇繁殖能力未见明显异常；浓度增加到4 mg/L时，实验组雄果蝇的生育率与对照组相比有所下降；随着浓度的不断增加，雄性果蝇的生育率下降80%。然而以雌性果蝇作为实验对象，无论是在低浓度还是高浓度的YCl₃喂养，其生育率与对照组相比没有明显下降，统计学没有显著差异（P＞0.05）。YCl₃超过一定浓度会降低黑腹果蝇的繁殖能力，对果蝇的生殖系统产生不利影响。并且我们此次实验发现，随着YCl₃浓度的升高，对雄性果蝇生殖系统的危害要远大于对雌性果蝇的影响，存在显著的性别差异且呈现明显的剂量效应关系。     

红芪加工废弃物活性多糖对果蝇生育力和寿命及抗氧化能力的影响

研究红芪废弃物中提取的生物活性多糖对黑腹果蝇的生殖能力、寿命和抗氧化能力的影响.通过不同浓度的红芪多糖对黑腹果蝇的存活试验,研究了一代至四代(F1-F4)的生殖能力和寿命、雌雄个体数、抗氧化酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性和丙二醛(MDA)含量的变化.结果表明,与对照组相比,添加多糖可提高F1-F...     

Reporter-based screen for <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants compromised in nonhost resistance

Plants are exposed to many potentially pathogenic microbes in the environment, but each species is only susceptible to a limited number of pathogens. The broad resistance is referred to as nonhost resistance. To date, little is known about the underlying mechanism of nonhost resistance and the signaling transduction process. Here we describe a simple method for isolating Arabidopsis nonhost resistance mutants against a nonadapted bacterial pathogen. A RAP2.6 promoter-driven LUC reporter system was developed to replace the tedious bacterial growth assay during the primary screening. The RAP2.6-LUC reporter gene is normally induced by the virulent bacterium Pseudomonas syringae pv tomato but not the nonadapted bacterium P. syringae pv phaseolicola. By using this method we iso- lated 4 mutants displaying strong reporter activity in response to P. syringae pv phaseolicola, which were characterized in some details, ebsl, ebs2, ebs3, and ebs4 （enhanced bacterial susceptibility） were compromised in resistance against P. syringae pv phaseolicola and/or P. syringae pv tomato. In addition, ebs4 showed enhanced hypersensitive response to the incompatible bacterium P. syringae pv tomato （avrB）. These results demonstrated that the method is suited for large scale screening for nonhost resistance mutants.     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSDl-like zinc finger domains regulate disease resistance and programmed cell death （PCD）. We cloned a rice OsLOL2 gene, orthologous to LSD1 of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci （Pst）. RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related （PR） protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR proteins and hypersensitive response-like reaction.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

The evolutionary foundation of genomic imprinting in lower vertebrates

In mammals,genomic imprinting confers developmental asymmetry and complementation on the parental genomes and makes both parental genomes essential for complete development.Genomic imprinting is,therefore,the first regulatory step of genome-wide gene expression of embryogenesis and thought to be the epigenetic foundation of bisexual reproduction.However,how the genomic imprinting is originated,established and maintained during vertebrate evolution remains unknown.Because no endogenous imprinting gene has be...     

Zn2+对果蝇生育力和寿命及抗氧化能力的影响

为了研究Zn~(2+)对果蝇生育力和寿命及抗氧化能力的影响。以黑腹果蝇(Drosophila melanogaster)为研究动物,用生存实验来考查不同浓度的Zn~(2+)处理果蝇,研究果蝇F1-F4代生育力和寿命的变化,雌、雄果蝇数量的变化,抗氧化酶SOD、CAT的活力及MDA含量的变化。结果显示:添加Zn~(2+)浓度为1.0 g/mL可以使F1-F4代雌、雄果蝇世代生育力增强,寿命延长,而在Zn~(2+)浓度为2.0×10-3g/mL时果蝇体内MDA含量大量积累,SOD和CAT活性急剧降低,使果蝇提前衰老、死亡并且降低或丧失生殖能力,而Zn~(2+)对果蝇性别分化影响不大,总体接近于1:1。因此,适当浓度的Zn~(2+)可以延长果蝇寿命,增强其生殖力,提高抗氧化能力。     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

井冈山地区铁皮石斛的繁殖生物学研究

通过野外实验观察和人工控制套袋等方法,从铁皮石斛的花部基本特征、开花动态、花粉活力、柱头可授性、杂交指数、套袋实验等方面对其繁殖生物学进行了研究。结果显示:(1)井冈山地区铁皮石斛4～6月开花,种群花期近60 d,多数花(50%)在约30 d内开放,果实一般需要180 d左右才能成熟。(2)铁皮石斛花粉活力开花后24 h即可达到(62.67±6.43)%,至开花第三天,活力可达到90%,柱头可授性达到最高,至第5天柱头仍保持高可授性。(3)套袋实验表明,部分自交亲和,异交,需要传粉者。自然条件下未观察到井冈山铁皮石斛居群结实,人工自花授粉率为22.2%。该研究结果为保护和利用井冈山铁皮石斛种质资源提供了科学依据。     

Expression ofbcl-2 gene increases in apoptosis of vascular endothelial cells induced by rattlesnake venom

Apoptosis of vascular endothelial cells (VEC) has been induced by deprivation of survival factors (aFGF and serum) and by rattlesnake venom. The expression ofbcl-2 gene has been examined by Northern blotting in the two apoptosis inducing systems. Our results show that the expression ofbcl-2 has not been detected in normal culture cells and in apoptotic cells induced by deprivation of survival factors. But in apoptotic cells induced by rattlesnake venom (10 ng/mL), the expression ofbcl-2 increases, and its mRNA exhibits two bands. The data first suggest that increasing expression and splitting ofbcl-2 mRNA may play an important role in apoptosis of VEC induced by rattlesnake venom, and this finding is helpful to understanding the role ofbcl-2 in regulation of apoptosis.     

具有反秀丽隐杆线虫捕食能力的根际细菌的筛选及其反捕食机理的研究

虽然生物菌肥具有多效、环保、可持续等多种优点,但经常被生活在土壤中的其他生物捕食而减少其实际应用效果.细菌为应对捕食演化出多种机制,排斥或杀死线虫.对具有反捕食能力的菌株进行筛选,能够得到具有高竞争力的菌株.以秀丽线虫为样本,对细菌进行对峙试验,得到两株具有较高反捕食能力的植物促生细菌:橘黄假单胞菌(Pseudomonas aurantiaca)JD37、荧光假单胞菌(Pseudomonas fluorescens)P13.以大肠杆菌(Escherichia coli)OP50为对照,线虫对3株细菌的偏好性由强至弱依次为P13、OP50、JD37.缓慢杀线试验中,JD37、P13对秀丽线虫的致死率分别达26.12%和18.66%.在单培养下,JD37能使线虫活力下降,产卵数量减少.化学和分子生物学研究显示:JD37不能产生任何已知的杀线次级代谢产物,而P13能够产生氰化氢(HCN).以上结果表明JD37能够抵御秀丽线虫捕食,在捕食压力下有较强竞争力,作为生物菌肥具有广泛的应用前景.     

Expression ofBcl-2 in cells with different telomerase activities

Both telomerase andBcl-2 are important genes in controlling apoptosis. The activation of telomerase and the abnormal regulation ofBcl-2 are also closely related to carcinogenesis. However, little is known about the linkage between telomerase andBcl-2. The effect of activated telomerase on the expression ofBcl-2 has been investigated. It is demonstrated that in tumor and transformed cells with higher telomerase activity,Bcl-2 expression is significantly lower than that in telomerase negative or less telomerose activity cells. Further study showed that in the telomerase gene-transformed 2BS-fibroblasts,Bcl-2 expression is inhibited significantly while the exogenous telomerase catalytic subunit gene is re-expressed in fibroblasts. Results indicated that there might be a certain linkage between the expression of telomerase andBcl-2, and overexpression of exogenous telomerase gene might down regulate the expression ofBcl-2. They contributed equally to this work.     

Functional characterization and mapping of two MADS box genes from peach （Prunus persica）

Previously an AGAMOUS gene homologue PpMADS4 and a FRUITFULL gene homologue PpMADS6 were isolated from peach （Prunus persica）, and both genes were shown to express in the developing floral and fruits. To gain insight into their function, the two genes were constitutively expressed in Arabidopsis thaliana and their effects on plant growth and floral organ development were studied in this work. The transgenic plants all displayed early flowering and conversion of inflorescence to floral meristem. However, the two genes had different effects on the floral organ structures in A. thaliana. The transgenic plants overexpressing PpMADS4 displayed homeotic conversion of floral organs, and particularly the perianth abscission was inhibited. The plants overexpressing PpMADS6 showed early flowering, produced higher number of carpels, petals, and stamens than nontransgenic plants, and pod shatter was prevented; significantly, the transgenic plants yielded more than one siliques from a single flower. A SSR molecular marker was developed for PpMADS4, and it was then assigned into the G5 linkage group of Prunus sp. Both PpMADS4 and PpMADS6 genes were located at the same region in the G5 linkage group. Our results showed the potential application of these two MADS box genes for crop and fruit tree improvement.     

S 100A8 mediates the activation of P65/HLA- B/S 100A8/BCL-2/Caspase-9 （-3） pathway in laryngeal carcinogenesis

S100 calcium binding protein A8 （S100A8）, a possible novel member of NF-kappa B signal pathway in laryngeal squamous cell carcinoma （LSCC）, interacts with human leukocyte antigen B （HLA-B） which carries an NF-kappa B binding site within the enhancer A. The objective of this study was to explore the molecular mechanism of S100A8 in laryngeal carcinogenesis. RT-PCR, Western blotting and immunohistochemistry staining were applied to evaluate the expression levels of IKKα, P65, REL-B, S100A8, APAF-1 and BCL-2 genes. The signal transduction passway in which S100A8 might participate was explored by RNA interference. Flow cytometry, TUNEL assay and cell invasion in vitro were used to detect the biological behavior of Hep2 cells induced by S100A8 gene. Our results showed that high expression of S100A8 was related to tumorigenesis in LSCC and negatively correlated with the degree of differentiation, indicating that S100A8 gene could inhibit apoptosis and promote metastasis in LSCC. Additionally, the suppression of S100A8 by RNA interference down-regulated BCL-2 but not APAF-1, P65 and IKKα, while, the suppression of P65 could significantly down-regulate the expression of S100A8 gene. In conclusion, S100A8 plays an important role in P65/HLA-B/S100A8/BCL-2/Caspase-9 （-3） pathway in laryngeal carcinoma.     

Fine mapping of the red plant gene R1 in upland cotton(Gossypium hirsutum)

Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chromosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R ₁ gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R ₁ by using the F₂ segregating population containing 1259 plants derived from the cross of Sub 16 and T586 and the backbone genetic linkage map from G. hirsutum×G. barbadense BC₁ newly updated by our laboratory. Genetic analysis suggested that the segregation ratio of red plants in the F₂ population fit Mendelian 1:2:1 inheritance, confirming that the red plant trait was controlled by an incomplete dominance gene. Preliminary mapping of R ₁ was conducted using 237 randomLy selected F₂ individuals and JoinMap v3.0 software. Then, a fine map of R1 was constructed using the F₂ segregating population containing 1259 plants, and R ₁ was located between NAU4956 and NAU6752, with only 0.49 cM to the nearest maker loci (NAU6752). These results provided a foundation for map-based cloning of R ₁ and further development of cotton cultivars with red fibers by transgenic technology. Supported by National Natural Science Foundation of China (Grant No. 30730067) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025)