黑籽南瓜NBS类基因CfRFN2的克隆及VIGS验证

黑籽南瓜(Cucurbita ficifolia)是云南特有的具有高抗枯萎病遗传性状的瓜类种质资源。为鉴定黑籽南瓜中NBS-LRR类基因的抗病功能,该研究从其叶片中克隆了NBS类基因CfRFN2 (GenBank ID:MK618462),测序全长为4 303 bp,完整的编码框长度为4 092 bp,编码1 363个氨基酸残基,该基因注释为拟南芥抗病蛋白At4g27190类转录体X1的同源基因,含有1个NB-ARC和2个LRR结构域,属于具有信号肽的可溶性蛋白。核苷酸相似性分析显示,CfRFN2与其他瓜类NBS类基因相似性在87%～98%之间;系统进化树分析表明,CfRFN2蛋白和瓜类的其他NBS类抗病蛋白聚为一个分支,其中CfRFN2蛋白与中国南瓜和美洲南瓜的RPS2、印度南瓜的RPS2-like亲缘关系最近,其次是黄瓜的At4g27190和苦瓜的At4g27220,与甜瓜的Atg27190亲缘关系相对较远;组织表达特性分析表明,CfRFN2基因在黑籽南瓜叶片中表达量最高,其次是茎,而在果皮和根中表达量较低。该研究采用烟草脆裂病毒载体系统,构建了黑籽南瓜VIGS沉默载体pTRV2-CfRFN2,含沉默载体的农杆菌侵染黑籽南瓜幼苗后接种枯萎病菌,qRT-PCR检测表明,接种后2 d和4 d的转pTRV2-CfRFN2沉默组植株的CfRFN2基因表达量比接种后同时期的野生型植株显著降低(分别下降34.75%和98.27%),病情指数增加为野生型的1.32倍,初步证明黑籽南瓜CfRFN2基因具有抗枯萎病的功能,推测该基因可能在黑籽南瓜抗枯萎病防御过程中发挥着重要作用。该研究中NBS类基因CfRFN2的克隆和VIGS验证为黑籽南瓜更多优异基因的克隆和功能验证奠定了前期基础,也为发掘黑籽南瓜优异抗病基因和开展瓜类分子育种提供新信息。     

Genetic structure and essential oil composition in wild populations of Salvia multicaulis Vahl.

The phytochemical study on ten populations of Salvia multicaulis Vahl. revealed that their essential oil qualitative profiles contained a significant amount of monoterpene hydrocarbons, which were the most abundant compounds. Besides, α-Pinene was the major constituent in all studied populations' essential oils. Significant correlations were observed between edaphic parameters and some major essential oil compounds. According to clustering analyses of the chemical data, the S. multicaulis populations were divided into three chemotypes: β-caryophyllene, camphene and camphor, and limonene. The population genetics study showed significant molecular differences among the populations. The Mantel test indicated a significant positive correlation between the geographical distances and genetic diversity, exhibiting a low amount of gene flow and a considerable genetic differentiation value. We also detected four genotypes based on the Nei's genetic distance and structure analysis. The identified chemical and genetic similarities/differences among these populations were correlated with edaphic parameters and geographic distances, suggesting that environmental factors are the primary drivers of the chemical polymorphism of essential oils in S. multicaulis populations.     

Prenatal diagnosis of Pallister-Killian syndrome and literature review

Pallister-Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.     

Marinifilum flexuosum sp. nov., a new Bacteroidetes isolated from coastal Mediterranean Sea water and emended description of the genus Marinifilum Na et al., 2009

A facultatively anaerobe, moderately halophilic, Gram-negative, filamentous, non motile and unpigmented bacterium, designated M30^T, was isolated from coastal Mediterranean Sea water in Valencia, Spain. Phylogenetic analysis based on 16S rRNA sequences placed this strain in the phylum “Bacteroidetes” with Marinifilum fragile JC2469^T as its closest relative with 97% sequence similarity. Average nucleotide identity (ANI) values between both strains were far below the 95% threshold value for species delineation (about 89% using BLAST and about 90% using MUMmer). A comprehensive polyphasic study, including morphological, biochemical, physiological, chemotaxonomic and phylogenetic data, confirmed the independent species status of strain M30^T within the genus Marinifilum, for which the name Marinifilum flexuosum sp. nov. is proposed. The type strain of Marinifilum flexuosum is M30^T (=CECT 7448^T = DSM 21950^T).     

B-Transferase with a Pro234Ser Substitution Acquires AB-Transferase Activity

A/B-Transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen, which is responsible for the synthesis of glycoprotein- and glycolipid-conjugates termed A/B-antigens. One polymorphism that causes the Pro234Ser substitution in B-transferase was recently found in a genotyping study, and might be cis-AB. In the present study, we analyzed the phenotypes arising from the enzymatic specificity of B-transferase with the Pro234Ser mutation. To evaluate the effect of the P234S mutation on enzymatic specificity, we generated an expression plasmid for B-transferase with Pro234Ser as well as A-transferase with Leu266Met, which is frequently found in cis-ABs. Transfection of B-transferase/P234S or A-transferase/L266M cDNA into HeLa cells, an O-blood group cell line, resulted in an AB-phenotype by absorption-elution testing and immunostaining, whereas A- and B-transferase-expressing HeLa cells exhibited only their own activity. Molecular simulation indicated that the P234S mutation causes a conformational change in the substrate pocket making it suitable for N-acetylgalactosamine.     

Start Codon Targeted (SCoT) markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species

Start Codon Targeted markers were used to establish phylogenetic relationship among seven species from Citrus L. genus. Twelve SCoT primers were used for their ability to reveal polymorphism of the targeted codon of initiation. A total of 132 amplicons were generated and 93.9% of them were polymorphic. The polymorphism information content of 0.884 and the resolving power of 75.22 illustrate the efficiency of the tested SCoT primers in highlighting polymorphism. The average Nei's (1973) gene diversity (0.376), the Schannon's index (0.548) and the Gst parameter (0.346) describe an important polymorphism at the interspecies level in Citrus genus. Analysis of molecular variance suggested significant genetic differences within species. In fact, 84% of variance occurs within the species, whereas 16% of the variation was recorded among the species of Citrus. The limited gene flow (Nm = 0.941) was recognized as a major factor to explain the partition of the observed diversity. The principal coordinates analyses, Neighbor Joining and the Bayesian clustering approach based on the SCoT markers also confirm the discrimination of the species of Citrus. Our results confirm the relevance and suggest the effectiveness of the SCoT markers for assessing genetic diversity, characterization and identification of the species of Citrus.     

A study of nonrandom mating in a British population of the two-spot ladybird with a high frequency of the melanic morph

In some studies of the two-spot ladybird (Adalia bipunctata), melanic males have been found in excess over the typical morph in matings. Data suggest that a genetic female mating preference is responsible. The mating advantage of melanic males may be important in maintaining a polymorphism between melanic and typical ladybirds in many populations in the United Kingdom (U.K.). It has been reported that preference frequency varies linearly with melanic frequency throughout most of the U.K. One particular population ofAdalia bipunctata near Aberdare, South Wales, is noted for its high frequency of melanic individuals. It has been suggested that local environmental factors account for the high melanic frequency in this population. It is also possible, however, that a female mating preference may be at least partly responsible for the high frequency of melanics (as has been proposed for the rest of the U.K.). In this study, experiments have been performed to determine the level of female mating preference in the Aberdare population. No evidence was found for any mating advantage to melanic males. There was inconsistent and unexpected evidence that melanic females were overrepresented in matings, but the cause for this was unclear. Female mating preference does not appear, therefore, to be responsible for the high melanic frequency in the population ofAdalia bipunctata near Aberdare. There is not a simple association between mating preference and melanic frequency in U. K. populations of the two-spot ladybird.     

Angiotensin converting enzyme gene polymorphism studies: A case-control study

Mild gestational hyperglycemia (MGH) is a very common complication of pregnancy that is characterized by intolerance to glucose. The association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism to MGH has been previously reported. In this study, we evaluated the association between ACE polymorphism and the risk of MGH in a Saudi population. We conducted a case-control study in a population of 100 MGH patients and 100 control subjects. ACE gene polymorphism was analyzed by the novel approach of tetraprimer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR). The frequency of ACE polymorphism was not associated with either alleles or genotypes in MGH patients. Glucose concentration was found to be significantly associated with the MGH group. Our study suggests that ACE genotypes were not associated with ACE polymorphism in a Saudi population.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.