地衣芽孢杆菌植酸酶基因的克隆及其在大肠杆菌中的表达

根据已发表的Bacillus licheniformis植酸酶基因DNA序列(13eneBank AF469936),设计并合成了一对引物,应用PCR技术,以地衣芽孢杆菌AB91062的总DNA为模板,扩增出了植酸酶基因phyL的编码区,并将其克隆到pMD18-T载体上,进行了序列测定,测序结果经Genetool序列分析表明该基因编码区长1146bp,编码381个氨基酸,与已发表的Bacillus lichenjformis植酸酶基因序列在核苷酸水平上与所发表的序列有92％的同源性;在氨基酸水平上与已发表的衣芽孢杆菌植酸酶相似性为93％．并将该基因编码区克隆到大肠杆菌表达质粒pHBM625上,并在大肠杆菌中实现了高效表达,表达产物具有正常的生物学活性．     

Cloning of two plastid division ftsZ genes from Nicotiana tabacum and their expression in E.coli

Two cDNAs of plastid division gene NtFtsZ1-1 and NtFtsZ1-2 are isolated from Nicotiana tabacum by RT-PCR and rapid amplification cDNA ends (RACE) method. Analysis of the deduced amino acid sequences encoded by NtFtsZ1-1 and NtFtsZ1-2 indicate that these two proteins possess the typical conservative motifs and GTP binding sites existing in all FtsZ proteins. The existence of putative plastid transit peptide in their N-terminal suggests that there are at least two transit-peptide containing FtsZ proteins in higher plants. Phylogenetic analysis based on amino acid sequences of FtsZ proteins also supports this interference. These two NtFtsZ genes demonstrate a similar expression pattern during the plant development, detected by Northern blot. Expression of NtFtsZ1-1 and NtFtsZ1-2 in E.coli interrupts the normal division process of host cells. These results suggest the diverse functions of FtsZ proteins in higher plants.     

人血管生成素基因的克隆及表达

利用RI-PCR方法从培养的人黑色素瘤细胞系A375中扩增得到了人血管生成素cDNA片段,测序正确后克隆入表达载体pET-28a( )中并转化于E.coli BL21宿主菌中.经IPTG诱导,表达了N端融合6个组氨酸标签(6His-tag)的血管生成素融合蛋白.利用6His-tag与过渡态金属离子Ni2 高亲和力结合的性质,经镍柱纯化,获得了高纯度的血管生成素融合蛋白,为进一步研究其生物活性及应用奠定了基础.     

杉木ClWRKY44基因克隆及其表达特性分析

【目的】以杉木幼叶为试验材料,通过克隆杉木ClWRKY44基因,分析其表达特性,为揭示杉木抗逆生理的分子机制提供理论依据。【方法】采用RT-PCR技术克隆杉木ClWRKY44 基因,对其进行生物信息学分析和亚细胞定位研究,并利用实时荧光定量PCR技术分析其表达情况。【结果】杉木ClWRKY44 基因的开放阅读框(ORF)为1 776 bp,编码591个氨基酸,具有WRKY结构域。系统进化树分析结果显示ClWRKY44 基因与拟南芥(Arabidopsis thaliana)ATWRKY44 的亲缘关系较密切,其在杉木的不同器官(叶、根、茎) 中均有表达,嫩叶中的表达量最高,茎中次之,根中最低。低温胁迫处理6 h,ClWRKY44 基因相对表达量最高,约是对照组的79.8倍。干旱胁迫处理24 h,ClWRKY44 基因在高浓度干旱胁迫处理下的相对表达量最高,约是对照组的46.6倍。【结论】杉木ClWRKY44基因编码的蛋白质序列与拟南芥ATWRKY44的同源性很高,在调控植物逆境胁迫应答过程中可以发挥功能,为杉木幼苗生长发育的适应性提供参考。     

米根霉L-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

文章以米根霉基因组DNA为模板,根据已公布的米根酶L-乳酸脱氢酶基因(ldnL)序列设计引物,PCR扩增得到含有ldnL的DNA片段;PCR产物T/A克隆后再双酶切,将ldnL片段连接到大肠杆菌表达载体pET17b,得到重组表达质粒pET17b-ldnL;pET17b-ldnL转化大肠杆菌BL21(DE3),摇瓶培养诱导表达后的菌体经SDS-PAGE电泳分析,结果表明克隆的ldnL基因在大肠杆菌中实现表达。     

HPLC-ESI-MS~n法分析西藏秦艽花和川西秦艽花中的7种成分

目的:采用HPLC和电喷雾质谱联用技术分析西藏秦艽花与川西秦艽花中的7种化学成分.方法:液相色谱条件色谱柱:Agilent Zorbax ODS(5μm,4.6 mm×250 mm);流动相:乙腈(A)-0.4%醋酸水溶液(B)进行梯度洗脱;体积流速为0.4 m L/min;检测波长254 nm;电喷雾质谱采用负离子模式采集.结果:采用HPLC-ESI-MS~n法分析西藏秦艽花和川西秦艽花中的7种成分,分别为獐牙菜苦苷、异牡荆黄素、落干酸、龙胆苦苷、异荭草苷、獐牙菜苷及6'-O-β-D-葡萄糖基龙胆苦苷.结论:HPLC-ESI-MS~n联用分析法可以鉴定分析西藏秦艽花和川西秦艽花中的7种成分,为该属植物成分的质量控制提供了科学依据.     

拟南芥HIS1-3基因克隆及表达载体构建

文章提取拟南芥植株总RNA,通过RT-PCR扩增出HIS1-3基因的cDNA片段,连接到pEASY-T1Simple载体上,转化到Trans1-T1感受态细胞内,筛选阳性单克隆并抽提质粒。利用限制性内切酶Eco91Ⅰ和NcoⅠ对质粒pEASY-T1-HIS1-3和植物表达载体pCAMBIA2301进行完全双酶切,通过电转化法,将重组表达载体pCAMBIA2301-HIS1-3导入根癌农杆菌C58中以期获得携带HIS1-3基因的根瘤农杆菌株,为研究HIS1-3基因的抗逆分子机理以及遗传改良作物抗逆性奠定了基础。     

<Emphasis Type="Italic">ftsZ</Emphasis> gene and plastid division

As the important cellular organelles in plants, plas-tids comprise one of the primary features that distinguish plant cells from those of other eukaryotes. Seen from the origin, plastids derive from endosymbiotic photosynthetic bacteria. Subsequently, plastids have evolved to become essential components for plant cell function. Besides the important role of chloroplasts in photosynthesis, some water-soluble proteins that involved in biosynthesis of starch, fatty acids, amino acids, nucleic aci…     

β葡聚糖酶杂合基因bglHAM的克隆及在P.pastoris中的表达

采用PCR的方法,以Bacillus macerans总DNA为模板,构建出β-1,3-1,4葡聚糖酶杂合基因bglHAM,与巴斯德毕赤酵母表达载体pPIC9K重组,得到重组质粒pPIC9K-HAM,电脉冲转化法转化酵母菌GS115,KM71,在MM,MD平板上筛选表型,YPD-G418平板上筛选多拷贝重组子,重组菌株经甲醇诱导后,刚果红平板染色法检测到β-葡聚糖酶活性,SDS-PAGE证明表达产物的分子量约为24kD,摇瓶诱导培养筛选到的重组菌株,胞外每mL发酵液最高酶活达240U。     

胡萝卜DcPS基因的克隆、进化及根发育中表达分析

补骨素合成酶(PS)是胡萝卜营养物质补骨素生物合成的关键酶.从'黑田五寸'胡萝卜中克隆得到DcPS基因,对其进行了进化和在胡萝卜根发育过程中表达水平分析.结果表明,DcPS基因长1518 bp,编码氨基酸505 aa,属于细胞色素P450超级基因家族.其氨基酸序列含有10个明显的特征,其中含有1个跨膜区,为亲水性蛋白....     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-l in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2 . On the basis of the research on ftsZl family, we analyzed the function of NtFtsZ2-l gene in Nicotiana tabacum . Microscopic analysis of the sense and antisense NtFtsZ2-l transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-l gene is involved in plant chloroplast division.     

褐飞虱羧酸酯酶基因cDNA片段的克隆及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱羧酸酯酶基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为396 bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自铜绿蝇、家蝇、沟鼠、黑腹果蝇、线虫和埃及伊蚊的羧酸酯酶的片段存在高度同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,羧酸酯酶基因表达水平明显升高.以上结果表明,羧酸酯酶基因的表达受抗性水稻的诱导,该基因在有毒化学物质解毒及增强褐飞虱对抗性水稻的耐受性方面可能起着重要作用.     

Cloning and expression pattern of a dehydrin-likeBDN1 gene from drought-tolerantBoea crassifolia Hemsl.

A 500-bp cDNA fragment was amplified via RT-PCR from drought-induced total RNA of the drought-tolerant B. crassifolia Hemsl. using primers based on the sequence of published dehydrin conserved region. By using 5′RACE, full-length coding region (1 148 bp) of BDN1 gene was produced. It is a new member of the dehydrin gene family. Southern analysis indicated that BDN1 is present in the B. crassifolia genome as a single-copy gene. Northern analysis revealed that its expression is inducible by drought and cold stresses as well as ABA application.     

祁连龙胆愈伤组织和再生植株的生长及其两种药效成分分析

通过不同激素组合的培养基筛选出祁连龙胆（Gentiana Przewalskii Maxim.）的快速生长细胞系并获得再生植株. 采用反相高效液相色谱法测定愈伤组织及其再生植株中獐牙菜苦甙（Swertiamarin）、龙胆苦甙(Gentiopicroside)的含量. 结果表明：(1）祁连龙胆愈伤组织在不同培养基上诱导率和生长速度不同,在MS+2?mg/L 2,4 D +1?mg/L 6 BA +6?mg/L GA的培养基上诱导率最高,达78.0％. 在MS+3?mg/L 2,4 D +0.5?mg/L KT的培养基上生长最快,增殖倍数达到7.18; (2）祁连龙胆愈伤组织及其再生植株有效成分及含量与原植株相比均有差别,其中,愈伤组织含有0.265%的龙胆苦甙,低于原植株(4.225%),再生植株含有0.279%的獐牙菜苦甙和1.579%的龙胆苦甙,前者高于原植株（0.260％）,但后者略有下降. 按生长速度推算,愈伤组织及其再生植株积累的药效成分含量明显高于原植株,值得进一步开发利用.     

大熊猫肠道菌抗生素抗性菌转座酶的基因克隆和表达

对大熊猫肠道大肠杆菌抗生素抗性质粒pAm08CD7339全序列的生物信息学分析结果表明,该质粒中存在一个编码转座酶的DNA片段,两条DNA链均可编码转座酶,其编码区长度分别为717 bp和600 bp,编码238个和199个氨基酸残基（分别命名为Transposase238和Transposase199）.以pAm08CD7339质粒DNA为模板,利用PCR方法对2个转座酶基因进行扩增,构建相应表达质粒,转化不同大肠杆菌菌株进行表达,结果表明：Transposase199可以在E.coli BL2     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-1 in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2. On the basis of the research on ftsZ1 family, we analyzed the function of NtFtsZ2-1 gene in Nicotiana tabacum. Microscopic analysis of the sense and antisense NtFtsZ2-1 transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-1 gene is involved in plant chloroplast division.     

褐飞虱细胞色素P450基因cDNA片段的克隆、测序及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱细胞色素P450基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为237bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自烟草天蛾、棉铃虫、埃及伊蚊、家蝇、黑腹果蝇和线虫的CYP6家族的P450的氨基酸序列存在同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,P450基因的表达水平明显升高.以上结果表明,P450基因的表达受抗性水稻的诱导,该基因在褐飞虱对抗性水稻的耐受性和解毒方面可能起着重要作用.     

龙胆草中6-去甲氧基-7-甲基茵陈色原酮分离和波谱表征

为研究龙胆草地上部分的化学成分,利用硅胶柱色谱对其乙醇提取物进行了分离纯化,根据理化性质和波谱方法鉴定化合物的结构,分离得到6-去甲氧基-7-甲基茵陈色原酮(Ⅵ)和其他5种已知化合物:β-香树脂醇(Ⅰ)、β-谷甾醇(Ⅱ)、β-香树脂醇乙酸酯(Ⅲ)、乌苏醇(Ⅳ)、齐墩果酸(Ⅴ).其中化合物Ⅵ属首次从龙胆科植物中分离得到的具有2-苯氧基色原酮骨架的天然酚类成分,对其1D和2D NMR谱的特征及EI质谱主要碎片的可能裂解方式进行了讨论,并利用单晶X射线衍射方法对其晶体结构进行了分析.结果表明化合物Ⅵ属正交晶系,Pbca空间群,晶胞参数a=1.291 83(19)nm,b=0.721 19(15)nm,c=2.923 7(5)nm,α=90°,β=90°,γ=90°,V=2.723 9(8)nm3,Z=8,Dc=1.464 g/m3,μ=0.114 mm-1,F(000)=1 248.B环平面与A/C环平面的两面角为99.6°,分子间存在π-π堆积作用.