Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.     

白桦BpNAC012基因调控白桦木质部发育表达谱分析

NAC转录因子成员被认为是调控植物次生壁合成的开关。前期研究结果显示BpNAC012基因的表达能够调控白桦次生细胞壁的合成。为研究BpNAC012调控的下游靶基因,本研究分别以该基因的过表达,抑制表达株系茎为材料构建转录组,以野生型为对照分析差异表达基因。结果显示:与对照相比,过表达株系OE中上调的基因有627条,下调的基因有229条,抑制表达株系中上调的基因有299条,下调表达的基因有207条。过表达BpNAC012相对于抑制表达能够调控更多的基因表达变化。而抑制表达BpNAC012更多的是影响蛋白修饰和转运类基因的表达变化。在差异表达基因中,涉及受体信号通路,营养代谢,氨基酸合成,及苯丙烷生物合成相关代谢通路基因比较富集。BpNAC012能够调控纤维素、木质素合成及木质部发育相关基因的表达变化,同时能够调控多种转录因子的表达变化。该研究为深入分析BpNAC012在白桦次生细胞壁合成的分子调控机制奠定基础。     

Tuber aestivum and Tuber uncinatum: two morphotypes or two species?

Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, beta-tubulin and EF 1-alpha genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.     

抑制素α基因表达下调对鹅卵泡颗粒细胞凋亡和增殖的影响

抑制素α基因是控制家禽排卵率的一个重要候选基因。为研究抑制素α基因对体外培养的鹅卵泡颗粒细胞增殖凋亡的遗传作用,构建psiRNA-INHα1和psiRNA-INHα2两个抑制素α基因干扰载体,以降低抑制素α基因表达。体外培养鹅F1和闭锁卵泡的颗粒细胞,经抑制素α基因干扰48h后,荧光显微镜检测转染效率,同时用流式细胞术检测抑制素α基因表达水平,以及细胞生长周期、细胞凋亡指数和增殖指数的变化,用放免法测定细胞培养上清中雌激素和孕酮水平。结果表明,抑制素α基因干扰后卵泡颗粒细胞抑制素表达水平比对照组降低30%～40%（P〈0.05）;细胞凋亡指数和增值指数显著高于对照组（P〈0.05）;细胞分泌雌激素水平显著下降（P〈0.05）。由此推断,抑制素α基因对颗粒细胞具有显著的抗凋亡效应。     

Genetic variability of Tuber uncinatum and its relatedness to other black truffles

Genetic variability is one of the major survival strategies developed by symbiotic fungi. We focused on the ectomycorrhizal fungus Tuber uncinatum Chatin that produces edible ascomata. In order to understand the degree of its variability and its relatedness to another morphologically-similar truffle, T. aestivum Vittad., ascomata of T. uncinatum were collected from a single natural truffle-ground located in the north of Italy and compared with samples from other Italian sites, as well as with T. aestivum ascomata from other European regions. We used multi-locus approaches, such as microsatellite-primed PCR (polymerase chain reaction), and single locus markers, such as mitochondrial and nuclear ribosomal DNA on 30 samples. The results demonstrate that the level of genetic polymorphism among isolates of T. uncinatum was higher than in other Tuber species, like T. melanosporum. Neighbour-joining analyses were carried out on a binary data matrix on 12 ascomata of T. uncinatum and T. aestivum, and on 15 internal transcribed spacer (ITS) sequences of these species and 5 from other Tuber species. Taken together, they clustered T. uncinatum and T. aestivum in two separate groups. The mitochondrial rDNA primers, NMS1 and NMS2, were not able to differentiate morphologically related and unrelated truffles. Moreover, a pair of primers, intentionally designed to differentiate isolates of T. aestivum and T. uncinatum from other Tuber species, successfully amplified DNA from all the samples of T. aestivum and T. uncinatum considered in our analysis. In conclusion, different molecular approaches separate T. aestivum and T. uncinatum according to their spore reticulum and their taste and smell.     

Downregulation of protease activated receptor expression and cytokine production in P815 cells by RNA interference

Background  

Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. Recently, it was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. In order to further investigate the issues, RNA interference (RNAi) technique was employed and small interfering RNAs (siRNA) of PARs were transfected in P815 cells.     

Insulin receptors regulate the fecundity of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Two InR (insulin receptor) genes have been identified in the Nilaparvata lugens. In this study, we used RNA interference (RNAi) to investigate the role of InR genes in the fecundity of N. lugens. The expression of NLInR1 and NLInR2 genes was simultaneously silenced with mixture of dsInR1 and dsInR2 (dsInRs) injection. Our results showed that larvae RNAi against both NLInR1 and NLInR2 reduced the number of eggs laid by N. lugens and some eggs as well as ovaries were abnormal. In addition, the relative expression of Vg (vitellogenin) and VgR (vitellogenin receptor) was significantly reduced on the 4th and 6th days after insects treated with larvae RNAi reached the adult stage. We also determined the relative expression levels of insulin/insulin-like signaling (IIS) related genes in RNAi-treated larvae and found that the expression levels of Chico (homologous receptor substrate), Akt (protein kinase B), PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog) genes decreased whereas FOXO (forkhead box O) and GSK3 (glycogen synthase kinase-3) levels increased on the 4th and 6th days after insects reached the adult stage. These results indicate that silencing of NLInR1 and NLInR2 reduces the fecundity of N. lugens through the IIS pathway.     

RNA interference in the Colorado potato beetle,Leptinotarsa decemlineata: Identification of key contributors

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using ³²P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.     

RNAi抑制萝卜过氧化物酶基因的效果受光照影响

农杆菌介导的RNAi技术已广泛应用于研究植物基因的功能.本实验应用小块萝卜肉质根体外培养,探讨光照对干扰萝卜过氧化物酶基因Rsprx1表达的影响.结果表明,干扰萝卜过氧化物酶基因Rsprx1表达后,抑制组中过氧化物酶活性显著低于对照组,光照减弱RNAi的抑制作用;抑制作用始于浸染后4 h, 过氧化物酶活性减低时,花青素含量增加,但光照增加花青素含量;HPLC结果显示,与对照组比,抑制组中花青素苷种类和含量有较大差异;花青素合成相关基因（RsCHS、RsDFR和RsLDOX）的mRNA水平在处理后明显上调.此外,过氧化氢酶活性和H2O2含量相应升高.由此表明,光照可影响农杆菌介导的RNAi效果,干扰萝卜过氧化物酶基因Rsprx1表达可以通过影响花青素合成相关基因的表达和过氧化氢含量,从而影响花青素代谢.