烟粉虱内共生菌groEL基因的原核表达条件研究

为了获得较多的G roEL蛋白,深入研究其性质,对烟粉虱内共生菌groEL基因表达的条件进行了研究。结果表明:诱导groEL基因原核表达的最佳IPTG浓度为100μmol/L;在35℃诱导培养,能够获得较高的蛋白表达量;最佳诱导培养时间为4~5h;最佳诱导培养的初始pH值为8.0;在振荡转速为120 r/m in、诱导培养时间为4~5h时,增大接种量,有利于原核基因的表达;添加少量的NH₄⁺有利于提高groEL基因原核表     

毛白杨两个Phi类GST基因的克隆及生化特性

谷胱甘肽转移酶(glutathione S-transferase,GST)在植物的抗逆反应、细胞信号转导和抗病等方面发挥着重要作用.从毛白杨(Populus tomentosa)中克隆到2个Phi类GS7因(PtoGSTF1和PtoGSTF2),它们分别编码215和218个氨基酸残基的蛋白质.DNA序列分析显示这2个基因均含有2个内含子.组织表达模式分析表明,这2个基因在毛白杨的根、茎、叶、韧皮部和顶芽组织中均表达,是组成型表达基因.在大肠杆菌中表达这2个基因并纯化重组蛋白.酶活性分析显示PtoGSTF1和PtoGSTF2蛋白对底物CDNB、NBD-CI、NBC和Cum-OOH都具有酶学活性,但活性差异较大.动力学分析显示,PtoGSTF2对NBD-CI的亲和力是PtoGSTF1的2.5倍,但PtoGSTF1的催化效率是PtoGSTF2的56.8倍.热力学稳定性分析显示,PtoGSTF1比PtoGSTF2具有更高的热力学稳定性.因此,酶学性质的差异预示着这2个Phi类GS7基因可能存在功能上的分化.     

木聚糖酶生产菌Saccharothrix variisporea YJ的筛选、发酵条件及酶学性质研究

以Azo-xylan为底物,利用双层平板法从堆肥中筛选到可降解木聚糖的菌株,16S rRNA测序分析显示该菌株与糖丝菌属（Saccharothrix variisporea）的同源性最高（99.33%）,命名为S. variisporea YJ。研究发现以酵母提取物或(NH₄)₂SO₄作为氮源、甘蔗叶作为碳源、初始pH值 7.0、发酵温度40 ℃、发酵时间5 d时,发酵液中木聚糖酶的酶活性最高。酶学性质研究表明该木聚糖酶的最适反应温度及pH值分别为55 ℃和8.0,在55 ℃以下及pH值 4.0~10.0的范围内保持较高稳定性。Na⁺能有效提高木聚糖酶活性,Mg²⁺和Mn²⁺没有明显影响,Cu²⁺则严重抑制木聚糖酶活性。此外,发酵液还可以直接对天然底物玉米芯进行降解。     

文心兰乙烯不敏感基因EIN2的克隆及表达分析

该研究采用PCR、RACE方法,对文心兰‘南茜’的乙烯不敏感蛋白基因（ethylene insensitive 2,EIN2）进行克隆及生物信息学分析,并采用qRT PCR技术,对该基因在文心兰不同组织器官和不同花期中的表达模式进行分析。结果表明：（1）成功克隆得到文心兰_EIN2基因序列,命名为OnEIN2（MH497388）;该基因cDNA序列全长为4 177 bp,其中开放阅读框3 879 bp,编码1 292个氨基酸,3′非编码区长208 bp,5′非编码区长90 bp。（2）生物信息学分析显示OnEIN2是一个不稳定的疏水蛋白,含有跨膜结构,分子式为C₆₄₀₆H₉₉₈₈N₁₆₇₀O₁₈₉₇S₄₇;蛋白质分子量142.22 kD,理论等电点5.80。多序列比对和系统进化分析表明,文心兰EIN2与铁皮石斛EIN2的相似度最高（81.98%）,二者亲缘关系也最为接近。（3）实时荧光定量PCR分析发现,OnEIN2基因在根、茎、叶花中均有表达,在花中表达量最高,茎中表达量最低;而在不同花期中,盛开期表达量最高,其次是衰老期。研究表明,文心兰OnEIN2基因在开花和衰老过程中可能有重要的作用。     

温度与CO2浓度升高对集胞藻PCC 6803修复UV损伤的关键基因转录量的影响

通过Taqman探针绝对定量法研究了集胞藻PCC 6803在5种不同的环境条件下:(1)25℃+400μmol/mol CO₂,(2)29℃+400μmol/mol CO₂,(3)25℃+800μmol/mol CO₂,(4)29℃+800μmol/mol CO₂,(5)25℃+1200μmol/mol CO₂,其phrA/psbA1/psbA2/psbA3等UV修复基因和16S rRNA基因的转录本拷贝数的变化情况。结果表明:温度与CO₂浓度的升高可以导致集胞藻PCC 6803的psbA2/psbA3基因和16S rRNA转录本拷贝数的大幅减少,说明温室效应将有可能导致蓝藻的UV损伤修复能力与核糖体合成能力的下降;温度升高和CO₂浓度升高对psbA2/psbA3基因和16S rRNA转录本拷贝数的联合作用表现为互相抵消,说明温度升高与CO₂浓度升高的联合作用的机制较复杂,值得深入研究。     

小麦TaWRKY47基因的克隆与表达分析

为了探究小麦WRKY基因的功能,该研究采用RT PCR方法,在小麦叶片组织中克隆WRKY基因,并对其进行生物信息学和不同逆境胁迫下的表达分析。结果表明：(1)成功克隆得到1个小麦WRKY基因,命名为TaWRKY47。(2)TaWRKY47基因开放阅读框长度为900 bp,编码299个氨基酸,含有一个WRKY保守结构域和一个C₂HC锌指结构域,属于WRKY基因家族的第Ⅲ类成员。(3)亚细胞定位分析结果显示,TaWRKY47蛋白定位于细胞核。(4)荧光定量PCR结果表明,TaWRKY47基因在小麦根、茎、叶、雄蕊和雌蕊中均有表达,其中在雌蕊中表达量最高,且受低温、干旱、盐、ABA和H₂O₂等胁迫表达增强,推测TaWRKY47基因参与了小麦的逆境胁迫过程。该研究结果为进一步研究TaWRKY47基因功能与抗逆机制奠定了理论基础。     

华丽龙胆GsF3′5′H和GsFNS基因的克隆及表达分析

该研究以华丽龙胆(Gentiana sino ornata)5个不同开放阶段(H₁~H₅)的蓝色花冠为试材,利用RT PCR技术克隆GsF3′5′H和GsFNS全长序列,并进行生物信息学分析,比较GsF3′5′H和GsFNS在不同组织和不同开放阶段的基因表达模式。结果显示：(1)所克隆的GsF3′5′H和GsFNS基因分别包含1 560 bp和1 590 bp开放阅读框(OFR),并分别编码520和529个氨基酸。(2)结构分析显示,GsF3′5′H和GsFNS均具有典型的F3′5′H和FNSⅡ蛋白保守结构域。(3)系统进化树分析表明,GsF3′5′H和GsFNS亲缘关系最近的物种是三花龙胆(Gentiana triflora)。(4)qRT PCR结果显示,GsF3′5′H和GsFNS基因在根、茎、叶和花冠中均表达,其中GsF3′5′H基因在花冠H₃阶段表达量最高。GsFNS在根中表达量最高,其次在花冠H₄阶段两基因的表达均较高。研究推测,GsF3′5′H基因表达产生的飞燕草素苷和GsFNS表达产生的黄酮共着色作用可能使华丽龙胆的花冠呈更稳定艳丽的蓝色,为蓝色花分子育种提供重要的基因资源。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

岩溶环境因子对细菌胞外碳酸酐酶表达及活性的影响*

以从西南岩溶生态系统分离出的一株细菌(编号GLRT102Ca)为例,研究了温度、pH、金属离子和阴离子等主要岩溶环境因子对其胞外碳酸酐酶(CA)表达及活性的影响。结果显示,在实验温度(10℃~50℃)和pH(5·5~9·0)范围内,实验菌株均能表达出不同程度的胞外CA活性,其中20℃~30℃以及中性偏碱性条件下的胞外酶活性较高。钙、镁、锌、钴等4种金属离子以及SO₄^2-、H₂PO₄^-、NO相似文献   

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。