绿色木霉内切几丁质酶基因的克隆及其毛壳菌转化

利用PCR方法人Ttichoderma viride的基因组DNA中克隆了一个42kDa的内切几丁质酶基因,扩增的长度为1672bp其中包含了启动子和mRNA的编码区。将该基因与来自构巢曲霉的色氨酸启动子相连后,通过原生质体转化将该基因导入球壳毛壳菌CG10。内切几丁质酶活性的测定结果表明,约1/3转化子的内切丁质酶活性得到了明显提高。本实验为利用基因工程方法提高毛壳菌的生防能力打下了较好的基础。     

嗜酸氧化亚铁硫杆菌启动子探针载体的构建

目的：为了研究嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）启动子结构与功能。方法：以pSV-β-galactosidase质粒为骨架,通过定点突变的方法引入一个新的BstBⅠ单酶切位点,构建能在大肠杆菌（Escherichia coli）中正常复制的启动子探针载体。利用PCR的方法将A.ferrooxidans菌cycA2基因上游5＇段上游DNA片段克隆到探针载体β-半乳糖苷酶基因上游以替代其原有启动子（gpt启动子）,并将重组的质粒转化E.coliDH5α菌株。通过检测宿主细胞的β-半乳糖苷酶活性,来鉴定启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。结果：pSV-β-galactosidase质粒被正确突变,成功构建了启动子探针载体pSVB。来源于A. ferrooxidans菌的启动子片段可驱动β-半乳糖苷酶基因在E.coli细胞中表达,转化子酶活性约为gpt 启动子驱动下活性的70 %。结论：启动子探针载体（pSVB）可用于A. ferrooxidans菌或者其它原核生物启动子的分离及进一步的分析研究。酶活性分析结果表明,来源于A. ferrooxidans菌cycA2基因上游5＇段上游DNA片段具有显著启动子活性。     

电脉冲穿孔法将几丁质酶基因导入巨大芽胞杆菌

为了探索适宜于巨大芽胞杆菌电脉冲转化条件,将带有沙雷氏菌几丁质酶编码基因的穿梭质粒转化到植病生防菌—巨大芽胞杆菌B1301中。以芽胞杆菌B1301对数期细胞为感受态细胞,采用不同的电击转化条件进行转化,通过转化率和几丁质酶活性表达认为巨大芽胞杆菌的最佳电击转化条件为电压1000 V/mm,电容25μF,电阻400Ω,转化率为9.6×104/μg质粒,几丁质酶活性表达的菌株几率为41.67%。     

球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

一种新型几丁质酶基因LcChi2在转基因水稻中的功能分析

LcChi2是从羊草中克隆获得的一种新型几丁质酶基因,生物信息学分析表明该基因表达一个Ⅱ类几丁质酶,属于19家族。在双子叶模式植物烟草中过表达该基因表现为抗真菌病害的生物学功能提高,然而在单子叶植物中是否具有抗病功能至今未知。以吉林省主栽水稻品种吉粳88为供试材料,构建了含LcChi2基因和除草剂筛选标记Bar基因的双价植物表达载体,利用农杆菌介导的遗传转化方法成功获得LcChi2和Bar基因过表达的转基因水稻。T_1代转基因水稻的PCR、RT-PCR和几丁质酶活性检测结果表明LcChi2基因已成功整合到水稻基因组中,并且表达产物表现出较高的外源几丁质酶活性;稻瘟病活体接种实验结果证明该基因显著提高了水稻的抗病性;抗除草剂鉴定结果表明获得的转基因水稻新材料同时具有除草剂抗性。研究结果证明LcChi2基因可有效提高单子叶植物的抗病性,该基因在利用现代生物技术开展抗病作物遗传改良方面具有重要的应用价值。     

几丁质酶表达质粒pKChiA和pMChiA的构建及表达

从质粒pLCHIA中切出含粘质沙雷氏菌（Serratia marcescens）几丁质酶基因（chiA）的1.8kb HinfI片段,将其插入到表达载体pKK223-3强启动子Ptac下游的SmaI位点内,构建成几丁质酶表达质粒pKChiA。再用内切酶BamHI从pKChiA质粒中切出2.1kb Ptac-ChiA片段,并将其重组到质粒pMC71A的单一BamHI位点内,构建成另一种几丁质酶表达质粒pMChiA。这两种质粒可在大肠杆菌HB101和JM105中高效表达,几丁质酶表达水平比质粒pLCHIA高1－3倍。     

杆状病毒几丁质酶基因结构与功能的研究进展*

杆状病毒几丁质酶基因是杆状病毒的非必需基因 ,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶 ,该酶N端具信号肽 ,中部是酶的活性区 ,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性 ,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的 ,同时它还是原组织蛋白酶 (pro V Cath)的分子伴侣 ,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

以报告基因分析霍乱弧菌噬菌体VP1的预测启动子

VP1为感染并裂解霍乱弧菌的噬菌体,全基因组为环状双链DNA。通过测定该基因组序列,预测出15个可能的启动子区,利用报告基因质粒转化及全噬菌体共感染的策略分析了这些推测启动子在霍乱弧菌中的活性,将预测的启动子区分别克隆到启动子探测lacZ融合质粒载体pRS1274,在转化于大肠埃希氏菌受体菌株JM109中时,所有克隆子均呈现兰斑。同时将质粒电击到缺失了lacZ基因的霍乱弧菌菌株7743△Z,然后用噬菌体VP1感染转化菌株。在转化成功的13个含预测启动子片段的重组质粒中,通过检测β_半乳糖苷酶活性表达随感染后时间的变化,提示P17为早期启动子,P2、P3、P9等为中期启动子,P18为晚期启动子。     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

嗜热毛壳菌纤维素酶（CBHⅡ）cDNA的克隆及在毕赤酵母中的表达

嗜热毛壳菌Chaetomium thermophilum CT2是一种土壤腐生菌,可产生具有重要工业生产价值的纤维素酶类。RACE-PCR获得嗜热毛壳菌纤维二糖水解酶Ⅱ（CBHⅡ）的编码基因(cbh2)。DNA序列分析表明cbh2的开放阅读框由1428个碱基组成,编码476个氨基酸。推断的氨基酸序列包含一个典型真菌纤维素酶的糖结合域（CBD）、催化域（CD）以及二者之间富含脯氨酸和羟基氨基酸的连接桥。根据氨基酸序列推算该酶分子量为53kD,属于糖苷水解酶第六家族,具有该家族催化保守区的典型特征。PCR扩增cbh2的成熟蛋白编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶蛋白的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.2 mg/mL。经硫酸铵沉淀、DEAESepharose Fast flow阴离子层析等步骤纯化了该重组表达蛋白。SDS-PAGE得到重组蛋白分子量为67kD,与从嗜热毛壳菌中纯化的该酶分子量一致。该重组纤维二糖水解酶作用的最适合温度50℃,最适pH4.0,在70℃的半衰期为30min,具有较好的热稳定性。     

球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。     

球毛壳菌循环肽HC-毒素基因的序列分析

为了验证Phrap软件是否适合在EST分析中应用,对球毛壳菌循环肽HC-毒素基因进行了序列分析。根据EST分析的结果,从cDNA文库中挑取循环肽HC-毒素基因的克隆进行了测序并序列分析。结果表明cDNA文库中循环肽HC-毒素基因的克隆插入片断大小为1217bp;用Phrap软件拼接出来的循环肽HC-毒素的表达序列标签拼接序列与实际序列不完全一致,因此Phrap软件不适合在EST分析中应用。     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

Role of antibiosis in the biological control of spot blotch (Cochliobolus sativus) of wheat by Chaetomium globosum

Chaetomium globosum Kunze, has been identified as a potential antagonist of Cochliobolus sativus (S. Ito & Kurib.) Deschler ex Dastur. (Syn = Drechslera sorokiniana). Production of antifungal compounds by Chaetomium globosum (Cg) and their role in suppression of spot blotch of wheat caused by this fungus under in vitro and in vivo has been evaluated. Interaction between Chaetomium globosum isolates and C. sativus showed mycoparasitism by isolates Cg 1 and Cg 6 whereas isolates Cg 2, Cg 3, Cg 4 and Cg 5 showed antibiosis. Syringe filtered culture extracts of Cg 2 completely inhibited mycelial growth of C. sativus in liquid broth. In vitro bioassays were undertaken by amending the medium with crude extracts and agar diffusion method in order to assess the fungistatic activity of crude extracts from culture filtrates of different isolates of Chaetomium globosum. Significant differences in antagonism between isolates were observed. Antifungal metabolite profiling, on TLC (Thin Layer Chromatography) plates identified 13 compounds in isolate Cg 2, 11 compounds in Cg 3 and 7 compounds in Cg 6. Isolate Cg 1 produced only two faint bands and Cg 5 produced two bands of the same Rf value but of higher intensity. The production of antifungal compounds by isolates was positively correlated with antagonism to C. sativus on seedlings in glasshouse studies. The results showed high antifungal metabolite production by isolate Cg 2, which also gave maximum bioefficacy under laboratory and glasshouse conditions.     

The genus Chaetomium in Iran, a phylogenetic study including six new species

Twenty-one species of Chaetomium known from Iran were compared on the basis of morphological and molecular characters. Six new species are recognized, five isolated from cereals and one from nematode cysts. A combined sequence dataset of the ITS region, partial LSU rDNA, and β-tubulin gene sufficiently resolved five species groups of Chaetomium that are largely concordant with combined features of peridium structure, ascospore shape and germ pore position. Among the new species C. undulatulum is a close relative of C. globosum, C. rectangulare is close to C. elatum, C. interruptum and C. grande are close to C. megalocarpum, altogether forming the C. globosum species group. Chaetomium iranianum and C. truncatulum are members of the C. carinthiacum species group, characterized by spirally coiled ascomatal hairs and fusiform ascospores. A chrysosporium-like anamorph is newly described for C. acropullum.     

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.     

球毛壳菌组蛋白H3基因克隆与特性分析

构建了球毛壳菌菌丝的cDNA文库,并获得了1410条ESTs序列,用里氏木霉(Hypocrea jecorina,AAM76068)和粗糙脉胞菌(Neurospora crassa,CAA25761)的组蛋白H3基因(Histone H3)蛋白序列对球毛壳菌(Chaetomium globosum)ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌组蛋白H3cDNA序列。cDNA序列全长739bp,开放阅读框411bp,编码136个氨基酸组成的多肽,蛋白分子量为15.4kD。BlastP同源性分析表明该基因与里氏木霉同源性最高为100%;与地钱(Marchantia polymorpha)同源性最低为95%。三级结构预测表明,该蛋白C端为球状结构域,而N端结构对其发挥调控作用起重要作用。该基因的cDNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY669068,AAT74576)。