赤桉单萜合成酶基因的分子克隆与生物信息学分析

桉叶油是一种从桉树叶分泌出来的挥发性芳香精油,主要含有1,8-桉叶油素和萜类化合物等物质,具有重要的药用活性.为深入了解桉树萜类化合物的生物合成途径和分子调控机理,该研究通过扩增从赤桉嫩叶中获得了1条单萜合成酶基因组序列的保守区段后,利用 RACE 技术得到此基因的 cDNA 序列,将其命名为 EC-MS 基因,并进一步获得此基因的 DNA 序列.通过生物信息学分析发现,EC-MS 基因的 DNA 序列长为3763 bp,含有7个外显子和6个内含子,cDNA 长度为1734 bp,编码577个氨基酸,并且具有 MS 蛋白特有的保守序列和相关特征,与蓝桉和白千层等植物的 MS 蛋白具有较高的相似性.EC-MS 的等电点为5．615,存在1个跨膜区,Arg21为信号肽剪切位点,同时,EC-MS 的立体结构以α-螺旋为主,几个底物结合点经过空间折叠后形成了“口袋型”底物结合区.     

Effects of nitric oxide synthase-3 overexpression on post-translational modifications and cell survival in HepG2 cells

Hepatocarcinoma is the fifth most common neoplasm and the third cause of cancer-related death. The development of genetic- and/or molecular-based therapies is urgently required. The administration of high doses of nitric oxide (NO) promotes cell death in hepatocytes. NO contributes to cell signaling by inducing oxidative/nitrosative-dependent post-translational modifications. The aim of the present study was to investigate protein modifications and its relation with alteration of cell proliferation and death in hepatoma cells. Increased intracellular NO production was achieved by stable nitric oxide synthase-3 (NOS-3) overexpression in HepG2 cells. We assessed the pattern of nitration, nitrosylation and carbonylation of proteins by proteomic analysis. The results showed that NOS-3 cell overexpression increased oxidative stress, which affected proteins mainly involved in cell protein folding. Carbonylation also altered metabolism, as well as immune and antioxidant responses. The interaction of nitrosative and oxidative stress generated tyrosine nitration, which affected the tumor marker Serpin B3, ATP synthesis and cytoskeleton. All these effects were associated with a decrease in chaperone activity, a reduction in cell proliferation and an increased cell death. Our study showed that alteration of nitration, nitrosylation and carbonylation pattern of proteins by NO-dependent oxidative/nitrosative stress was related to a reduction of cell survival in a hepatoma cell line.     

不同贮藏条件下豆薯种子的脂质过氧化研究

豆薯(Pachyrhizuus erosus(L.)Urban)种子分别在4℃、15℃相对湿度(RH)45%及室温开放条件下贮藏,或新鲜种子进行超干处理将其含水量降到6.43%后密封贮存,以探讨种子劣变的生理机制.结果表明,豆薯种子在室温开放贮藏12个月的发芽率降至50%,24个月后发芽力完全丧失.4℃下贮藏豆薯种子最有效,贮藏后种子的电导率和紫外吸收值较低,过氧化氢酶、过氧化物酶和超氧化物歧化酶活性仍保持较高水平,种子萌发时释放出的挥发性醛也较低.适当的超干处理效果与15℃45%RH相似,但不及4℃的.     

辽宁碱蓬甜菜碱醛脱氢酶基因(BADH)启动子分离及序列分析

根据已知的辽宁碱蓬BADHcDNA5′端序列设计两个基因特异的反向引物(BR1,BR2),通过衔接头PCR获得了BADH基因起始密码子上游265bp的序列。根据所获得的序列设计两个基因特异的反向引物(BR3,BR4),用BR2、BR3、BR4分别与4个简并引物配对,通过TAILPCR扩增,获得了约2kb的序列。经Sequencer软件拼接上述两段序列,获得了BADH基因起始密码子上游2055bp的序列。用TSSPTCM软件分析此序列,预测出转录起始点(T)位于起始密码子上游62bp处,由此获得了1993bp的SlBADH启动子序列。用PLACE软件分析此序列,发现该序列具有启动子的基本元件TATAbox、CAATbox,包含多个胁迫诱导元件,如盐诱导元件GAAAAA,抗冻、缺水、脱落酸、抗寒元件CANNTG,伤害诱导元件ANATTNCNN,热激元件ATAAATGT等,是一个强的胁迫诱导启动子。     

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ‐tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral’s effect on microtubules was both dose‐ and time‐dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ‐tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP‐Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral’s effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules.     

Activation of farnesoid X receptor attenuates hepatic injury in a murine model of alcoholic liver disease

Alcoholic liver disease (ALD) is a common cause of advanced liver disease, and considered as a major risk factor of morbidity and mortality worldwide. Hepatic cholestasis is a pathophysiological feature observed in all stages of ALD. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, and plays an essential role in the regulation of bile acid, lipid and glucose homeostasis. However, the role of FXR in the pathogenesis and progression of ALD remains largely unknown. Mice were fed Lieber-DeCarli ethanol diet or an isocaloric control diet. We used a specific agonist of FXR WAY-362450 to study the effect of pharmacological activation of FXR in alcoholic liver disease. In this study, we demonstrated that FXR activity was impaired by chronic ethanol ingestion in a murine model of ALD. Activation of FXR by specific agonist WAY-362450 protected mice from the development of ALD. We also found that WAY-362450 treatment rescued FXR activity, suppressed ethanol-induced Cyp2e1 up-regulation and attenuated oxidative stress in liver. Our results highlight a key role of FXR in the modulation of ALD development, and propose specific FXR agonists for the treatment of ALD patients.     

Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.     

转BADH基因紫花苜蓿山苜2号品种的抗盐性鉴定及系统选育

利用转基因技术创造苜蓿新种质已成为牧草新技术育种的重要组成部分。为了有效地从苜蓿转基因植株及其后代中选育出优良品种,深入研究转基因苜蓿的植物学性状及其产量十分重要。以通过农杆菌介导技术获得的T0代转BADH基因苜蓿为试材,利用分子生物学方法对其自交株系的世代群体连续进行抗盐性鉴定筛选和系统选育,首次获得了具有抗盐碱能力的转基因苜蓿稳定株系。同时,通过品种比较实验、区域实验和生产实验,表明在不同盐碱地条件下,转BADH基因的苜蓿植株产草量明显高于对照（未转基因的中苜1号）,生产实验的干草增产率介于13.11%–24.98%之间。上述结果表明,外源目的基因主要特性的遗传稳定,进而从实践上验证了转BADH基因工程操作的实用性。     

Effect of salinity and abscisic acid on accumulation of glycinebetaine and betaine aldehyde dehydrogenase mRNA in Sorghum leaves (Sorghum bicolor)

The effects of salt stress and abscisic acid (ABA) on the expression of betaine aldehyde dehydrogenase (BADH) were determined in sorghum (Sorghum bicolor L.) plants. BADH mRNA expression was induced by salinity, and the timing coincided with the observed glycinebetaine (betaine) accumulation. The leaf water potential in the leaves of the sorghum plants was significantly affected by salinity. In response to salinity, betaine, ABA, Na and Cl accumulations increased 6-, 16-, 90-, and 3-fold, respectively. In the leaf disks from unsalinized plants incubated on NaCl, or ABA solution, the BADH mRNA level was lower than in the ABA-treated disks. Exogenous application of the ABA biosynthetic inhibitor fluridone to the NaCl-treated disks reduced the ABA accumulation and BADH mRNA levels compared with NaCl-treated leaves. The results indicate that the salt-induced accumulation of betaine and BADH mRNA coincides with the presence of ABA.