黄花蒿法呢基焦磷酸合酶、紫穗槐二烯合酶双功能酶基因的构建、原核表达与功能鉴定

试验将青蒿素生物合成途径中催化两步连续反应的酶(法呢基焦磷酸合酶和紫穗槐二烯合酶)的基因进行融合,经大肠杆菌表达后鉴定其融合蛋白的功能。结果表明:融合蛋白具有了双功能酶活性。双功能酶基因的构建,为进一步将双功能酶基因转入黄花蒿,提高青蒿素含量奠定了基础。     

甘蔗B家族蔗糖磷酸合成酶基因SotSPSB的克隆及原核表达

【目的】克隆甘蔗B家族s0心PsB基因并进行原核表达,为进一步研究甘蔗SPS酶学特性及SPS活性调控机制奠定基础。【方法】在进化分析的基础上,通过同源克隆获得甘蔗SofSPSB基因部分序列,再结合RACE技术获得全长cDNA序列。扩增SofSPSB基因ORF并连到原核表达载体pETBlue－2上导入大肠杆菌BL21（DE3）中表达。【结果】通过比对B家族中进化关系很近的玉米（Zeamays）ZmSPS1和水稻（Oryzasativa）OsSPS1基因序列,并在保守区设计一对引物扩增获得甘蔗B家族SPS基因（SolSPSB）2330bp序列。结合5’－RACE和3’－RACE技术获得3481 bp SofSPSB基因全长cDNA序列,该序列包含一个3225bp的开放阅读框（0I江）;起始密码子（ATG）位于转录起始位点后56bp处,终止密码子（TGA）后有一段201bp的非编码序列,并带有真核生物典型的polyA尾巴;编码1074个氨基酸,SofSPSB与Zm—SPS1、OsSPS1的核苷酸序列同源性分别为94．7％和81．3％,氨基酸序列同源性分别为96．0％和83．9％;其理论分子量Mw=118．96kDa,等电点pI=6．30。经原核表达后纯化获得带6xHis标签的融合蛋白。【结论】克隆获得甘蔗B家族Sol-SPSB基因全长cDNA序列,成功构建了SoPSPSB基因原核表达载体,使其在大肠杆菌BL21（DE3）中表达。     

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.     

选择与适应:兰科植物查尔酮合酶基因家族成员的分子进化和功能趋异

回顾了近十五年来国内外对各科植物查尔酮合酶基因CHS分子进化的研究成果,并结合作者近年来对兰科植物CHS基因分子进化的研究结果,对兰科植物CHS基因分子进化和功能趋异的机制展开综述,揭示了植物中普遍存在的CHS基因分子进化以及伴随的功能趋异的现象和机制,初步探讨了CHS基因分子进化与生物进化和自然选择的关系。     

A target-site mutation is present in a glyphosate-resistant Lolium rigidum population

Annual ryegrass (Lolium rigidum) is the only weed species to have evolved resistance to the broad‐spectrum herbicide glyphosate in Australia. A population that had failed to be controlled by glyphosate was collected from a vineyard in the Adelaide Hills region of South Australia. Dose–response experiments on this population (SLR 77) showed that it was glyphosate resistant, with an LD₅₀ that was 1.9–3.4 times higher than that of a susceptible population (VLR 1). The movement of radiolabelled glyphosate within SLR 77 plants showed that this population did not have the differential glyphosate translocation mechanism of resistance common to several other Australian glyphosate‐resistant populations. Subsequent analysis of shikimic acid accumulation within the plant after glyphosate treatment showed that this population accumulated significantly less shikimic acid than a susceptible population, but more than a glyphosate‐resistant population with the translocation mechanism, indicating the possible involvement of another mechanism of resistance. Sequencing of a portion of the SLR 77 5‐enolpyruvylshikimate‐3‐phosphate synthase gene was carried out and a mutation causing an amino acid change at position 106 from proline to threonine was identified. This mutation is likely to be responsible for glyphosate resistance in this population, as mutations in this position have been found to be responsible for glyphosate resistance in goosegrass (Eleusine indica) from Malaysia. This paper represents the first report of target‐site glyphosate resistance in L. rigidum and provides evidence that this species has at least two mechanisms of glyphosate resistance present in Australia.     

Influence of transection of the cervical sympathetic trunk on content of NO and expression of NOS in placenta of rats with pregnancy-induced hypertension syndrome

AIM:To observe the influence of transection of the cervical sympathetic track (TCST) on the content of NO and the expression of eNOS mRNA and iNOS mRNA in placenta of the rats with pregnancy-induced hypertension syndrome (PIH).METHODS: Pregnant Wistar rats were randomly divided into 5 groups: control group (C): saline was injected subcutaneously from 14th day to 20th day of gestation;PIH group 1 (H1) and group 2 (H2): L-NAME was respectively injected with 125 mg/kg and 62.5 mg/kg,respectively,then the other procedures were the same as group C;Operation group (O): TCST was operated on 14th day of the gestation,then the other procedures were the same as group H1;sham operation group (S): the cervical sympathetic trunk was only separated and exposed on 14th day of the gestation,then the other procedures were the same as group H1.RESULTS: (1) Except the base value of the BP and protein in urine of the pregnant rats,all the parameters observed in group H1 and H2 were higher than those in group C significantly (P<0.01),and in group H2 were lower than those in group B1 markedly (P<0.01).(2) In comparison with those in group C,the size and body weigh of fetus in group H1,H2 decreased markedly (P<0.01).The above indexes in group H1 were lower than those in group H2 markedly (P<0.01,P<0.05).The changes of the rate of embryo absorption and fetal death,and deformity rate of the fetal rats were contrary to the above indexes.(3) The content of NO and the expression of eNOS mRNA and iNOS mRNA in placenta in group H1 and H2 were lower than those in group C markedly (P<0.01).Those in group H1 were lower than those in group H2 obviously (P<0.01,P<0.05).Those in group O were higher than those in group H1 markedly (P<0.01).CONCLUSION: TCST protects pregnant rats against PIH,and it was related to the mRNA expression of eNOS and iNOS and the content of NO in placenta tissue.     

果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建

应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶cDNA反义表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。     

Pineapple organic acid metabolism and accumulation during fruit development

Developmental changes in pineapple (Ananas Comosus (L.) Merrill) fruit acidity was determined for a ‘Smooth Cayenne’ high acid clone PRI#36-21 and a low acid clone PRI#63-555. The high acid clone gradually increased in fruit acidity from 1.4 meq/100 ml 6 weeks from flowering, and peaked a week before harvest at ca 10 meq/100 ml. In contrast, the low acid clone increased in acidity 6 to 8 weeks after flowering, peaked 15 weeks after flowering at ca. 9 meq per/100 ml and then sharply declined in 2 weeks to 6 meq/100 ml. The increased in total soluble solids (TSS) of the low acid clone began 6 weeks after flowering and for the high acid clone at 12 weeks after flowering. The increase in titratable fruit acidity (TA) paralleled the changes in the citric acid content of both clones. Citric acid content increased from less than 1 mg/g at 6 weeks after flowering to 6 to 7 mg/g, 9 weeks later. The malic acid concentration in both clones varied between 3 and 5 mg/g and showed no marked changes just before harvest. The developmental changes in fruit potassium were significantly correlated with fruit acidity and fruit total soluble solids in both the high and low acid clones. Developmental changes in acid-related enzymatic activities showed an increase in citrate synthase (EC 4.1.3.7) activity that occurred a week before harvest, coincided with the peak in citric acid in the high acid clone. An increase in aconitase (ACO, EC 4.2.1.3) activity was observed just before harvest as the decline in acidity occurred in the low acid clone. The activities of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and malic enzyme (ME, EC 1.1.1.40) did not parallel any changes in fruit acidity. The results indicated that the change in pineapple fruit acidity during development was due to changes in citric acid content. The major difference in acid accumulation occurred in the low acid clone just before harvest when acidity declined by one-third. The activities of citrate synthase and aconitase possibly played a major role in pineapple fruit acidity changes.