RNA干扰抑制STAT6基因表达的初步研究

目的:构建针对信号转导子和转录激活子6(Singnal transducers and activators of transcription 6,STAT6)基因的干扰质粒,并观察其对STAT6蛋白表达的抑制效果。方法:将构建的短发夹干扰质粒(Short hairpin RNA,shRNA)表达载体,与合成的pIRES2-EGFP-STAT6基因表达质粒共同转染COS7细胞,荧光显微镜下观察干扰效果,并利用Western blot观察蛋白表达的变化。结果:将pIRES2-EGFP-STAT6重组载体转染COS7细胞后,结果显示STAT6蛋白可以在细胞中高水平表达;与阴性对照组相比,STAT6 shRNA-1干扰组和STAT6 shRNA-2干扰组STAT6蛋白表达水平分别是23.1%和9.6%。结论:成功构建并筛选到具有显著干扰效率的STAT6干扰质粒,为哮喘的基因治疗研究奠定了基础。

Oxygen-glucose deprivation of neurons transfected with toll-like receptor 3-siRNA Determination of an optimal transfection sequence

Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.

小鼠Dppa2基因shRNA载体的构建及干扰效果的检测

目的:构建有效的针对小鼠Dppa2基因的shRNA(short hairpin RNA)干扰载体。方法:设计合成2对针对小鼠Dppa2基因的shRNA序列以及1对与哺乳动物基因组无同源性的shRNA序列作为对照,构建pSUPER.Retro.puro干扰载体并进行PCR,酶切和测序验证。进一步将各干扰载体分别转染小鼠胚胎干细胞(embryonic stem cells,ESCs),RT-PCR检测干扰效率。结果:PCR,酶切和测序验证均表明各shRNA载体构建成功。将空载体及各重组载体分别转染小鼠ESCs发现,干扰组Dppa2基因表达水平相对于空载体对照组和阴性shRNA载体对照组明显下调。结论:成功构建了有效的针对小鼠Dppa2基因的shRNA干扰载体,为进一步研究Dppa2基因在维持小鼠ESCs不分化过程中的作用提供了基础。

Molecular and functional characterization of a candidate sex pheromone receptor OR1 in Spodoptera fitura

Olfaction is primarily mediated by highly specified olfactory receptors （ORs）. Here, we cloned and identified an olfactory receptor, named SlituOR1 （Genbank no. JN835269）, from Spodoptera litura and found evidence that it is a candidate pheromone receptor. It exhibited male-biased expression in the antennae, where it was localized at the base of sensilla trichoidea, the antennal sensilla mainly responsive to pheromones in moths. Conserved orthologues of this receptor, found among known pheromone receptors within the Lepidoptera, and SlituOR1 were placed among a clade of candidate pheromone re- ceptors in a phylogeny tree of insect OR gene sequences. SlituOR1 showed differential expression in S. litura populations attracted to traps baited with different ratios of the two sex pheromone components （9Z,11E）-tetradecadienyl acetate （Z9E11-14：OAc） and （9Z,12E）-tetradecadienyl acetate （Z9E12-14：OAc）. Knocking down of SlituOR1 by RNA interference reduced the electroantennogram （EAG） response to Z9E11-14：OAc, and this result is consistent with the field trapping experiment. We infer that variation in transcrip- tion levels of olfactory receptors may modulate sex pheromone perception in male moths and could provide some of the flexibility required to maintain the functionality of com- munication with females when a population is adapting to a new niche and reproductive isolation becomes an advantage.

Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry （Fragaria vesca L.） genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry over- expressing FvYUC6, which showed typical high-auxin pheno- types. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesta. Additionally, specific repression of FvYUC6 expression by RNA interference signifi- cantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm （Spodoptera litura） and RNA interference to evaluate its role in commonly used insecticides

Insect cytochrome P450 monooxygenases （CYPs or P450s） play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference （RNAi） and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, β-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal develop- ment indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up- regulated by chlorpyrifos,β-cypermethrin and methomyl with both lethal concentration at 15% （LC15） （50, 100 and 150 μg/mL, respectively） and 50%（LC50） dosages （100,200 and 300μg/mL, respectively）. Addition of piperonyl butoxide （PBO） significantly increased the toxicity ofchlorpyrifos,β-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi- mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin, respectively, at the LCso dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and β-cypermethrin detoxification in S. litura.

Two hydroxypyruvate reductases encoded by OsHPR1 and OsHPR2 are involved in photorespiratory metabolism in rice

Mutations in the photorespiration pathway dis- play a lethal phenotype in atmospheric air, which can be fully recovered by elevated C02. An exception is that mutants of peroxisomal hydroxypyruvate reductase （HPR1） do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice （Oryza sativo L. cv. Zhonghua 11）. Results from reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase （SGAT） activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hprl x hpr2 was generated by crossing individual mutant of hprl and hpr2. The phenotypes of all transgenic lines were determined in ambient air and C02-elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPRI and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPRI and OsHPR2 are involved in photorespiratory metabolism in rice.