共轭亚油酸异构酶基因克隆与表达研究进展

从微生物共轭亚油酸异构酶基因的克隆,重组共轭亚油酸异构酶基因在原核和真核细胞中的表达等方面对共轭亚油酸异构酶基因克隆与表达研究的最新进展进行了综述。     

Chalcone suppresses lignin biosynthesis in illuminated soybean cells

Lignin and its related metabolites play critical roles in plant growth and development. Thus, lignin biosynthesis has attracted interest as a novel target site of plant growth inhibitors. Chalcone has been shown to not only inhibit lignin biosynthesis in plants, but also to suppress the growth of many annual plant species. In order to know the direct effect of chalcone on plant metabolism, the effects of chalcone on the activities of key enzymes in lignin biosynthesis and on the related metabolites were clarified with a time‐course study by using light‐induced suspension cultures of soybean cells. The fresh weight and packed cell volume of the soybean cells were inhibited after 8 h of chalcone treatment. The activities of phenylalanine ammonia lyase (EC 4.3.1.24) and 4‐coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) were largely inhibited 4 h after the treatment with 0.15 mmol L^?1 chalcone. Unlike these two enzymes, the activity of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) was not inhibited until 16 h after the chalcone treatment. The content of the 4CL substrates and lignin in the soybean cells became relatively lower than the control under the light condition within 4 h and 8 h after the chalcone treatment, respectively. These results suggest that the growth suppression of soybean cells is positively associated with the inhibition of lignin biosynthesis by exogenous chalcone.     

植物乳杆菌亚油酸异构酶基因在大肠杆菌中的表达和功能鉴定

应用基因重组表达技术将一个推定的植物乳杆菌亚油酸异构酶基因(pli18)克隆到pET-30a载体的T7启动子的下游,构建pET30-pli原核表达载体。经IPTG诱导和低温培养20h后,在其宿主菌E.coliBL21(DE3)中成功表达了可溶性的PLI18重组蛋白。通过Ni-NTA亲和层析纯化和酶反应产物的气相色谱检测表明,PLI18重组蛋白具有亚油酸异构酶活性,从而证明pli18基因为1个新的亚油酸异构酶基因。为亚油酸异构酶的进一步研究及其在农产品加工中的应用打下了基础。     

Pin1 inhibitor juglone induces apoptosis in human cervical cancer SiHa cells

AIM: To explore the effect of peptidyl-prolyl cis/trans isomerase (Pin1) inhibitor juglone on apoptosis of human cervical cancer SiHa cells. METHODS: Cultured SiHa cells were incubated with juglone at concentrations of 10, 20, 50, 80 and 100 μmol/L for 24 h. The SiHa cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay. The cell apoptosis was analyzed by flow cytometry with Hoechst 33258 staining. The protein levels of cleaved caspase-3,8,9 and PTEN was determined by Western blotting. RESULTS: In different doses of juglone groups, the SiHa cell growth was greatly inhibited (P<0.05) in a dose-dependent manner as compared with control group. The IC₅₀ of juglone was 20.4 μmol/L. After treatment with juglone at concentration of 20 μmol/L for 12 h, the apoptosis of SiHa cells was induced, and the typical morphological changes of cell apoptosis such as karyopyknotic pyknic hyperfluorescence bolus, nuclear fragmentation and apoptotic body were observed by Hoechst 33258 staining. The early apoptotic rate was increased significantly as compared with the control. The protein levels of cleaved caspase-3, 8, 9 and PTEN were also increased significantly as compared with control group. CONCLUSION: Juglone significantly inhibits the cell activity and induces the apoptosis of SiHa cells in vitro by inhibiting the caspase pathway and increasing the expression of anti-oncogene.     

花生不同品种叶片类黄酮含量和相关合成酶活性对PEG胁迫的响应

以花生品种花17、农大818、丰花5号和山花11号为试材,聚乙二醇(PEG)室内砂培法模拟干旱胁迫,研究20%PEG胁迫下花生幼苗叶片中类黄酮含量及相关合成酶活性的变化。20% PEG开始处理后,随胁迫时间延长,幼苗叶中类黄酮含量逐步上升,在胁迫9h达到高峰,之后逐步下降至对照水平,农大818、丰花5号和山花11号均在处理后72h降至对照水平,花17在36h降至对照水平。PEG开始处理后苯丙氨酸解氨酶（PAL）、查尔酮合成酶（CHS）、查尔酮异构酶（CHI）酶活性均逐渐提高,6h后CHS、CHI酶活性达到高峰,9h后PAL酶活性达到高峰,之后下降,4个品种趋势一致。分析表明PEG处理后山花11号和丰花5号的合成酶具有较高的活性和积累速率是其类黄酮快速大量合成的原因,而农大818 CHS酶活性和PAL酶活性提高速率较低限制了其类黄酮的积累,3种酶的活性和积累速率均较低导致花17叶中类黄酮合成少,含量提高慢。相关分析表明PEG胁迫下PAL、CHS、CHI共同调控类黄酮的代谢,胁迫引起PAL、CHS、CHI酶活性改变是类黄酮含量变化的原因。     

苦荞愈伤遗传转化体系的优化及用于FtCHS1的过表达分析

【目的】建立和优化苦荞愈伤组织遗传转化体系,为苦荞基因功能验证及分子育种提供研究工具。【方法】以苦荞品种“西荞二号”为材料,对苦荞愈伤遗传转化条件进行优化,包括苦荞外植体类型、诱导愈伤的激素比例、继代培养基的激素比例及农杆菌类型。利用苦荞类黄酮生物合成关键酶基因FtCHS1的过表达验证优化后的遗传转化体系。通过PCR筛选和荧光观察鉴定阳性株系,采用紫外分光光度法和高效液相色谱法（high performance liquid,HPLC）测定花青素及黄酮醇支路代谢物含量,使用实时荧光定量PCR分析类黄酮合成相关基因的表达,比较FtCHS1过表达愈伤组织与对照组的差异。【结果】苦荞诱导愈伤组织的最佳外植体为下胚轴,其最适诱导培养基为MS+0.8 mg·L^-1 6-BA+3.5 mg·L^-1 2,4-D,诱导率达72%;最优继代培养基为MS+3 mg·L^-1 6-BA+1 mg·L^-1 KT,愈伤组织增殖率与增殖系数分别为98%和1.09;转化过程中的最佳农杆菌是GV3101,转化效率达31.3%;FtCHS1过表达愈伤组织中,花青素、芦丁和杨梅素的含量显著高于对照（P<0.05）,山奈酚和槲皮素的含量极显著高于对照组（P<0.01）;外源FtCHS1的过表达对转基因愈伤组织中5个内源同源基因FtCHSs的表达水平没有影响（P>0.05）,而FtCHI、FtF3H、FtFLS1、FtFLS2、FtFLS3和FtDFR1等黄酮合成途径关键酶基因均上调表达（P<0.05）。此外,特异性正调控黄酮醇合成的转录因子基因FtMYB5和FtMYB6上调表达,而花青素合成抑制子基因FtMYB8的表达降低（P<0.05）。【结论】建立了苦荞愈伤组织遗传转化体系,过表达FtCHS1的苦荞愈伤组织通过上调黄酮合成相关基因的表达增加类黄酮物质的积累。     

人参生长发育过程中茎内5种酶活性的初步研究

以五年生人参茎为供试材料,分别于人参展叶期、开花期、结果期、果后参根生长期、枯萎期采集人参茎样品,采用中性缓冲液制备粗酶液,应用分光光度法测定硝酸还原酶(NR)、超氧化物歧化酶(SOD)、谷丙转氨酶(GPT)、葡萄糖磷酸异构酶(GPI)、延胡索酸酶(FUM)的活力.结果表明:人参生长发育过程中,茎内NR和FUM均在展叶...     

紫花苜蓿蛋白质二硫键异构酶mPDI的生物信息学分析与同源建模研究

利用紫花苜蓿蛋白质二硫键异构酶（mPDI）的mRNA（来自NCBI,登录号为Z11499．1）及氨基酸序列（来自UniProt KB／Swiss—Prot数据库及NCBI数据库,其登录号分别为1729828、CAA77575．1）,应用生物信息学软件预测了该蛋白质的理化性质、亲疏水性、信号肽、二级结构、卷曲螺旋结构、跨膜区域、糖基化位点、活性位点、亚细胞定位、功能结构域及高级结构。结果表明：紫花苜蓿mPDI蛋白质是一个整体疏水性蛋白,细胞定位为粗面内质网,含有512个氨基酸,理论等电点为4．98,分子量为57087．4Da,原子组成为C2597H3977N651O786S6摩尔消光系数在280nm处为37610,不稳定系数为40．12,脂肪系数为79．96,总平均亲水性为-0．357。该蛋白质由20种氨基酸组成,其中非极性R基团氨基酸占44．3％,极性R基团氨基酸占28．4％,酸性氨基酸占13．6％,碱性氨基酸占13．1％,Ip、Glu、Val、Ala含量最为丰富,Met和Cys含量最少。信号肽位于1～24号氨基酸。利用ProtScale软件的KyteandDoolittle算法对mPDI蛋白进行亲／疏水性预测,结果表明该蛋白质含有7个高疏水性区域,分别分布在4J0～50区域、95～105区域、120～130区域、190～200区域、285～295区域、340～350区域以及365～375区域。利用SignalP网络工具对mPDI蛋白进行信号肽的预测,神经网络法显示第24～25位点是最可能的剪切点,马可夫模型显示了同样的信号肽酶切位点。mPDI蛋白质二级结构中α-螺旋占26．37％（135AA）、无规则卷曲占53．32％（273AA）、延伸链占20．31％（104AA）;包含3个卷曲螺旋结构、3个糖基化位点（分别为143位的S、148位的T、461位的S）、2个硫氧还蛋白结构域（14～144位、357～485位）、2个硫氧还蛋白活性位点（54～72位、399～417位）、1个内质网靶向序列（509～512位）;Ramachandram结构检测表明此模型的三维结构符合立体化学能量规则。     

超临界CO2在酶法合成共轭亚油酸中的应用

[目的]探讨在超临界CO2中利用嗜酸乳杆菌亚油酸异构酶转化亚油酸、合成共轭亚油酸(CLA)的可行性。[方法]以嗜酸乳杆菌亚油酸异构酶为研究对象,在考察底物溶解性、酶粉经超临界CO2处理后活性变化的基础上,研究超临界CO2温度、压力、酶粉的记忆pH和水分活度对共轭亚油酸合成的影响。[结果]结果表明,底物亚油酸在超临界CO2中的溶解度随压力和温度而改变,当压力超过15 MPa时,底物开始具有较好的溶解性;酶粉经超临界CO2处理后的活性随压力和时间产生较大变化;超临界CO2压力、温度、酶粉的记忆pH和水分活度均对CLA产率产生影响,单因素试验所得的较好条件是压力为15 MPa,温度为37℃,pH值为4.0,水分活度为0.4~0.5。[结论]初步研究表明,在超临界CO2中酶法合成CLA是可行的,但催化反应的条件优化、酶在体系中的选择性等都有待进一步探索。     

Selective growth suppression of five annual plant species by chalcone and naringenin correlates with the total amount of 4-coumarate: coenzyme A ligase

The 4-coumarate: coenzyme A ligase (4CL) is one of the key enzymes in the biosynthesis of lignin monomers. It has been demonstrated that the 4CL is a new potential target site for developing effective plant growth inhibitors. Although previous studies demonstrate that chalcone and naringenin differentially suppress the growth of several annual plant species, we show here that the compounds can inhibit the 4CL enzyme activity in the plants. The enzyme was extracted and partially purified from the leaf tissues of two tolerant plants (wheat and soybean) and three susceptible plants (tomato, barnyard grass, and common chickweed). A maximal 29-fold purification of the enzyme, with a yield of 32% (tomato), was achieved by a six-step procedure, including anion-exchange column chromatography. Naringenin strongly inhibited the 4CL specific activity in wheat, soybean and barnyard grass, whereas chalcone showed the highest inhibitory effect in common chickweed. A good correlation was observed between the level of growth suppression by the compounds and the total 4CL amount in the plants. These results suggest that the inhibitor treatment at the same concentration could not inactivate the entire 4CL enzyme produced in the tolerant plants. Taken together, these results highlight the possibility of the 4CL as a new action site of growth suppression.