球孢白僵菌抗逆相关功能基因克隆与基因工程菌株构建研究
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摘要
虫生真菌作为重要的天敌生物资源,具有广阔的应用前景,尤其是其可贵的流行潜力始终吸引着人们。球孢白僵菌Beauveria bassiana(以下简称白僵菌)是害虫微生物防治中一种经典的丝孢类虫生真菌,可侵染15个目149个科的700多种昆虫和若干种蛛螨类。在自然界中它具有分布广、感染力强和扩散流行的特点;同时也具有不污染环境,易于培养,以及防治效果好等优点。因此白僵菌也是研究最多、应用最广和最具有开发潜力的虫生真菌。然而,如同其他真菌杀虫剂一样,球孢白僵菌因为击倒害虫时间长、防效不稳定以及自身抗逆性较差等缺点,目前在农药市场上的占有率依然很低。为延长白僵菌孢子储藏期,增强其环境稳定性,有必要利用基因工程手段对其进行改造,以获得适合市场需要的产品,促进其商品化生产和应用。
海藻糖是广泛存在于各种生物体内的抗逆境剂,具有增强生物体对高温、脱水、干旱、冷冻、高渗透性、重金属及有毒物质等逆境的抵抗能力。胞内海藻糖积累与丝状真菌孢子的储藏期延长密切相关。我们推测,通过基因工程技术改变球孢白僵菌体内海藻糖代谢途径,可以提高孢子内海藻糖含量,增加其抗逆能力。
同样,具有保护细胞功能的热休克蛋白70是热休克蛋白家族中最重要的一类家族成员。它的研究已成为目前生命科学领域研究的热点之一。HSP70蛋白作为一种非特异性保护蛋白,能够防止蛋白质变性和帮助已变性的蛋白重新折叠,恢复正确的构象,具有“分子伴侣”、细胞保护、抗凋亡等独特复杂的生物学功能。因此,对于细胞的生存起着至关重要的作用。通过克隆球孢白僵菌hsp70基因,并深入探讨其表达规律和作用机理对虫生真菌抗逆机制研究具有重要的意义。
我们通过SMART RACE RT-PCR技术从球孢白僵菌Bb202菌株中克隆了海藻糖合成途径中的两个重要基因(海藻糖6-磷酸合成酶基因tps1和海藻糖-6-磷酸磷酸酯酶基因tps2)的cDNA序列(GenBank登录号分别为:FJ769373和FJ769375)。并通过DNA步移技术获得了它们各自的上游启动子序列(GenBank登录号:FJ769374,FJ769376)。序列分析表明:球孢白僵菌tps1结构基因含有一个长度为55bp的内含子,位于结构基因的313碱基至367碱基处。其cDNA全序列为1906bp。开放式阅读框由1563个核苷酸组成,编码520个氨基酸,成熟蛋白理论分子量为58.3kDa,理论等电点为5.63。5'非翻译区(5'-UTR)与3'-非翻译区(3'-UTR)分别为112bp和263bp。在终止密码子下游3'-UTR有polyA加尾信号AATATA和有26 bp的polyA结构。球孢白僵菌tps1基因上游序列的转录起始位点位于第2785位G碱基处。该上游序列含有TATA-box、GC-box,同时也存在其它多个潜在转录因子结合位点。如:Oct-1结合位点,CRE-BP结合位点,CdxA结合位点以及可与Ⅳ型锌指蛋白(转录因子)相结合的GATA元件等。球孢白僵菌tps2结构基因含有两个长度分别为140bp和62bp的内含子。它们分别位于结构基因的127碱基至266碱基处和2209碱基至2270碱基处。它们均符合真核生物内含子剪接位点5'-GT…AG-3'规则。tps2的cDNA全序列为3219bp。开放式阅读框由2619个核苷酸组成,编码872个氨基酸,成熟蛋白理论分子量为97.8kDa,理论等电点为6.32。5'非翻译区(5'-UTR)与3'-非翻译区(3'-UTR)分别为310bp和290bp。在终止密码子下游含有28bp的polyA结构。球孢白僵菌tps2基因上游序列的转录起始位点位于第4105位C碱基处。该上游序列含有TATA-box、CAAT-box和GC-box,并且也存在其它多个潜在转录因子结合位点。如:cap结合位点,Adf-1结合位点,Oct-1结合位点,C/EBP结合位点,CdxA结合位点等。其中可与Ⅳ型锌指蛋白(转录因子)相结合的GATA元件含量相当高。
使用同样方法,我们从球孢白僵菌中克隆出了完整的热休克蛋白HSP70基因编码区序列及上游序列。该基因cDNA全长2405bp,5'端非翻译区171bp,3'端非翻译区263bp,开放阅读框(ORF)1971bp,编码656个氨基酸。成熟蛋白理论分子量为71.3kDa,理论等电点为4.92。得到的上游序列长度3559bp,其中有305bp序列与cDNA序列重叠。分析表明,该上游序列的转录起始位点位于第3255位G碱基处,没有明显的TATA-box和CAAT-box,但含有CCAAT-binding factor、GC-box等重要的转录因子结合位点,以及热激应答元件HSE和GATA元件等启动子顺式调控元件。
通过Realtime-PCR技术,我们对球孢白僵菌tps1和hsp70基因在高温、低温、紫外照射等不同胁迫条件下的转录情况进行了检测。从mRNA转录水平探讨了不同胁迫条件对球孢白僵菌这两个基因表达的影响。结果表明,球孢白僵菌菌丝与孢子中的tps1基因在三种胁迫下转录表达差异较小。而三种胁迫均能诱导球孢白僵菌热休克蛋白基因hsp70的快速高效转录表达。说明球孢白僵菌中tps1与hsp70基因,对于外界逆境影响,具有不同的应答机制。
将克隆得到的球孢白僵菌tps1基因连接到分泌性表达载体pPIC9K上后,通过电转化,转入毕赤酵母GS115。经甲醇诱导获得了60kDa左右的表达蛋白,与预测的结果(58.3kDa)及相关报道结果接近。同时,酶活测定结果表明,表达蛋白具有海藻糖-6-磷酸合成酶生物活性,诱导的粗酶液酶活最高达到1.384U/ml。
将克隆得到的球孢白僵菌tps1基因连接到含有构巢曲霉3-磷酸甘油醛脱氢酶基因启动子的载体pBARGPE1后,通过芽孢转化,重新转入球孢白僵菌中,对该基因进行组成型表达。抗逆性评价表明tps1基因的组成型表达,在低温下可一定程度增强重组菌株的贮藏性能,相对于出发菌株,重组菌株的活孢率同比提高了约35%。在一定热胁迫范围内,重组菌株耐热性能也得到了提高,相对于出发菌株,重组菌株的活孢率同比提高了约30%。但重组菌株抗紫外能力并没有增强。生物学特性观测表明,重组工程菌株相对于出发菌落生长较慢,且产孢量降低。这些都说明利用组成型表达载体改造虫生真菌抗逆基因,在一定程度上增强了重组菌株孢子的耐贮性和耐热性,但也存在一定的负作用。
As microbial control agents,entomopathogenic fungi have wide application prospects. Beauveria bassiana,as one of the most common and most important entomopathogenic fungi,has been widely used for fungal insecticide development either in China or outside China.It has wide range host range including many mites and more than 700 insect species belonging to 149 families of 15 orders.It invades insects mainly through cuticle,an incomparable tribute for sucking insects as compared to other insect pathogens.Just as other fungal pesticides,however,the acceptance of B.bassiana products for pest control has been limited,because it kills insects slowly and is usually sensitive to adverse environment and loses their effectiveness rapidly.These drawbacks hinder the utilization of B.bassiana against insects.Currently,genetic engineering techniques are needed to prolong its shelf life and enhance its environmental stability for promotion of application of B.bassiana products.
Trehalose exists in a large range of organisms,such as bacteria,fungi,animal and plant.It plays an important role in protection of stress,such as heat,dehydration,drought, freezing,high osmotic pressure,heavy metal salts and toxic substances.There is a strong correlation between accumulation of intracellular trehalose and shelf life of conidia in filamentous fungi.It is speculated that increasing trehalose content in conidia is feasible by genetic engineering techniques to change trehalose metabolism pathway.
Heat shock proteins(HSPs) are highly conserved proteins which are synthesized in all organisms responding to environmental stresses.HSP70 is the most important member of them.As a universal cytoprotection protein,HSP70 is ubiquitous molecular chaperone. Moreover,it plays important roles in cellular protection and anti-apoptotic effect.It could help to refold the misfolding proteins and repair the damaged polypeptides.So it is significance to clone hsp70 gene from B.bassiana and study its experssion and action mechanism.
The full-length cDNA of two trehalose synthesis relevant genes tps1 and tps2 (GenBank accession No:FJ769373 and FJ769375) were cloned from B.bassiana b202 using SMART RACE RT-PCR.The sequence of their respective upstream promotors (GenBank accession No:FJ769374 and FJ769376) were amplified by genome walking. Sequence analysis showed that there was an intron with size of 55bp in the structural gene of tps1,which was located at the 313th～367th base.The full-length cDNA of tps1 gene was 1906bp,with 1563bp,112bp and 263bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region(3'-UTR),respectively.The open reading frame(ORF) encoded 520 amino acids.The mature protein had a molecular mass of 58.3 kDa with a calculated pI of 5.63.The 3'-UTR contained polyadenylation signal AATATA and 26bp PolyA.The transcription initiation site of B.bassiana tps1 gene was located at G base of position 2785 of upstream sequence.The upstream sequence contained TATA-box, GC-box and several other regulatory elements,such as Oct-1-binding factor, CRE-BP-binding factor,CdxA-binding factor and GATA.There were two introns with size of 140bp and 62bp in the structural gene of tps2,located at the 127th～266th base and 2209th～2270th base,respectively.Their sequences complied with the rule of "5'-GT" and "AG-3'".The full-length cDNA of tps2 gene was 3219bp,with 2619bp,310bp and 290bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region (3'-UTR),respectively.The open reading frame(ORF) encoded 872 amino acids.The mature protein had a molecular mass of 97.8 kDa with a calculated pI of 6.32.The 3'-UTR contained 28bp PolyA.The transcription initiation site of B.bassiana tps2 gene was located at C base of position 4105 of upstream sequence.The upstream sequence contained TATA-box,CAAT-box,GC-box and other regulatory elements,such as cap-binding factor, Adf-1-binding factor,Oct-1-binding factor and GATA.In additional,the content of GATA in the upstream sequence was very high.
The same method was used to clone the full-length cDNA of hsp70 gene and its upstream sequence from B.bassiana.Analysis of the complete cDNA cloned,with a whole sequence of 2405bp,showed that it encompassed an open reading frame(ORF) with 1971bp encoding 656 amino acids.The mature protein had a molecular mass of 71.3 kDa with a calculated pI of 4.92.The analyses indicated that the upstream sequence contained several regulatory elements,such as CCAAT-binding factor,GC-box,HSE and GATA,but TATA-box and CAAT-box were not be found.
The expression of tps1 and hsp70 gene from B.bassiana under several stress conditions was detected using Realtime-PCR.The effects of different stress conditions on expression of tps1 and hsp70 gene were explored based on mRNA level.The results showed that the change of tps1 gene transcriptional expression in mycelium and conidia of B.bassiana was not significant under three stress conditions,but the expression of hsp70 gene from B.bassiana increased rapidly under the same conditi6ns,suggesting that tpsl and hsp70 gene from B.bassiana have different response mechanisms for external stresses. Tpsl gene into secreting expression vector pPIC9K was cloned and then transformed into Pichia pastoris GS115.After continuous methanol induction,expression protein with molecular weight of ca.60kDa was obtained,which was closed to predicted value and those in related reports.Enzymatic activity assay revealed that the expression protein had bioactivity of trehalose-6-phosphate synthase.The maximum enzymatic activity of crude enzyme liquid was 1.384U/mL. The tpsl gene of B.bassiana was inserted into the cloning site of pBARGPE1 vector, which contained promoter of the glyceraldehyde-3-phosphate gene from Aspergillus nidulans.After the recombinant plasmid was transformed into B.bassiana,tpsl gene was constitutively expressed in the recombinant strain.In comparison with wild-type strains, under lower temperture the shelf life of the recombinant strains was enhanced by nearly 35%.The heat tolerance of recombinant strains was enhanced by nearly 30%within a certain high temperature stress.The UV tolerace of recombinant,however,was not be enhanced.In addition,the growth rate and sporulation of the recombinant strains were lower than those of wild-type ones.It is suggested that constitutive expression of adversity gene of entomogenous fungi enhanced stress resistance of recombinant strains,however, with some negative effects.
海藻糖是广泛存在于各种生物体内的抗逆境剂,具有增强生物体对高温、脱水、干旱、冷冻、高渗透性、重金属及有毒物质等逆境的抵抗能力。胞内海藻糖积累与丝状真菌孢子的储藏期延长密切相关。我们推测,通过基因工程技术改变球孢白僵菌体内海藻糖代谢途径,可以提高孢子内海藻糖含量,增加其抗逆能力。
同样,具有保护细胞功能的热休克蛋白70是热休克蛋白家族中最重要的一类家族成员。它的研究已成为目前生命科学领域研究的热点之一。HSP70蛋白作为一种非特异性保护蛋白,能够防止蛋白质变性和帮助已变性的蛋白重新折叠,恢复正确的构象,具有“分子伴侣”、细胞保护、抗凋亡等独特复杂的生物学功能。因此,对于细胞的生存起着至关重要的作用。通过克隆球孢白僵菌hsp70基因,并深入探讨其表达规律和作用机理对虫生真菌抗逆机制研究具有重要的意义。
我们通过SMART RACE RT-PCR技术从球孢白僵菌Bb202菌株中克隆了海藻糖合成途径中的两个重要基因(海藻糖6-磷酸合成酶基因tps1和海藻糖-6-磷酸磷酸酯酶基因tps2)的cDNA序列(GenBank登录号分别为:FJ769373和FJ769375)。并通过DNA步移技术获得了它们各自的上游启动子序列(GenBank登录号:FJ769374,FJ769376)。序列分析表明:球孢白僵菌tps1结构基因含有一个长度为55bp的内含子,位于结构基因的313碱基至367碱基处。其cDNA全序列为1906bp。开放式阅读框由1563个核苷酸组成,编码520个氨基酸,成熟蛋白理论分子量为58.3kDa,理论等电点为5.63。5'非翻译区(5'-UTR)与3'-非翻译区(3'-UTR)分别为112bp和263bp。在终止密码子下游3'-UTR有polyA加尾信号AATATA和有26 bp的polyA结构。球孢白僵菌tps1基因上游序列的转录起始位点位于第2785位G碱基处。该上游序列含有TATA-box、GC-box,同时也存在其它多个潜在转录因子结合位点。如:Oct-1结合位点,CRE-BP结合位点,CdxA结合位点以及可与Ⅳ型锌指蛋白(转录因子)相结合的GATA元件等。球孢白僵菌tps2结构基因含有两个长度分别为140bp和62bp的内含子。它们分别位于结构基因的127碱基至266碱基处和2209碱基至2270碱基处。它们均符合真核生物内含子剪接位点5'-GT…AG-3'规则。tps2的cDNA全序列为3219bp。开放式阅读框由2619个核苷酸组成,编码872个氨基酸,成熟蛋白理论分子量为97.8kDa,理论等电点为6.32。5'非翻译区(5'-UTR)与3'-非翻译区(3'-UTR)分别为310bp和290bp。在终止密码子下游含有28bp的polyA结构。球孢白僵菌tps2基因上游序列的转录起始位点位于第4105位C碱基处。该上游序列含有TATA-box、CAAT-box和GC-box,并且也存在其它多个潜在转录因子结合位点。如:cap结合位点,Adf-1结合位点,Oct-1结合位点,C/EBP结合位点,CdxA结合位点等。其中可与Ⅳ型锌指蛋白(转录因子)相结合的GATA元件含量相当高。
使用同样方法,我们从球孢白僵菌中克隆出了完整的热休克蛋白HSP70基因编码区序列及上游序列。该基因cDNA全长2405bp,5'端非翻译区171bp,3'端非翻译区263bp,开放阅读框(ORF)1971bp,编码656个氨基酸。成熟蛋白理论分子量为71.3kDa,理论等电点为4.92。得到的上游序列长度3559bp,其中有305bp序列与cDNA序列重叠。分析表明,该上游序列的转录起始位点位于第3255位G碱基处,没有明显的TATA-box和CAAT-box,但含有CCAAT-binding factor、GC-box等重要的转录因子结合位点,以及热激应答元件HSE和GATA元件等启动子顺式调控元件。
通过Realtime-PCR技术,我们对球孢白僵菌tps1和hsp70基因在高温、低温、紫外照射等不同胁迫条件下的转录情况进行了检测。从mRNA转录水平探讨了不同胁迫条件对球孢白僵菌这两个基因表达的影响。结果表明,球孢白僵菌菌丝与孢子中的tps1基因在三种胁迫下转录表达差异较小。而三种胁迫均能诱导球孢白僵菌热休克蛋白基因hsp70的快速高效转录表达。说明球孢白僵菌中tps1与hsp70基因,对于外界逆境影响,具有不同的应答机制。
将克隆得到的球孢白僵菌tps1基因连接到分泌性表达载体pPIC9K上后,通过电转化,转入毕赤酵母GS115。经甲醇诱导获得了60kDa左右的表达蛋白,与预测的结果(58.3kDa)及相关报道结果接近。同时,酶活测定结果表明,表达蛋白具有海藻糖-6-磷酸合成酶生物活性,诱导的粗酶液酶活最高达到1.384U/ml。
将克隆得到的球孢白僵菌tps1基因连接到含有构巢曲霉3-磷酸甘油醛脱氢酶基因启动子的载体pBARGPE1后,通过芽孢转化,重新转入球孢白僵菌中,对该基因进行组成型表达。抗逆性评价表明tps1基因的组成型表达,在低温下可一定程度增强重组菌株的贮藏性能,相对于出发菌株,重组菌株的活孢率同比提高了约35%。在一定热胁迫范围内,重组菌株耐热性能也得到了提高,相对于出发菌株,重组菌株的活孢率同比提高了约30%。但重组菌株抗紫外能力并没有增强。生物学特性观测表明,重组工程菌株相对于出发菌落生长较慢,且产孢量降低。这些都说明利用组成型表达载体改造虫生真菌抗逆基因,在一定程度上增强了重组菌株孢子的耐贮性和耐热性,但也存在一定的负作用。
As microbial control agents,entomopathogenic fungi have wide application prospects. Beauveria bassiana,as one of the most common and most important entomopathogenic fungi,has been widely used for fungal insecticide development either in China or outside China.It has wide range host range including many mites and more than 700 insect species belonging to 149 families of 15 orders.It invades insects mainly through cuticle,an incomparable tribute for sucking insects as compared to other insect pathogens.Just as other fungal pesticides,however,the acceptance of B.bassiana products for pest control has been limited,because it kills insects slowly and is usually sensitive to adverse environment and loses their effectiveness rapidly.These drawbacks hinder the utilization of B.bassiana against insects.Currently,genetic engineering techniques are needed to prolong its shelf life and enhance its environmental stability for promotion of application of B.bassiana products.
Trehalose exists in a large range of organisms,such as bacteria,fungi,animal and plant.It plays an important role in protection of stress,such as heat,dehydration,drought, freezing,high osmotic pressure,heavy metal salts and toxic substances.There is a strong correlation between accumulation of intracellular trehalose and shelf life of conidia in filamentous fungi.It is speculated that increasing trehalose content in conidia is feasible by genetic engineering techniques to change trehalose metabolism pathway.
Heat shock proteins(HSPs) are highly conserved proteins which are synthesized in all organisms responding to environmental stresses.HSP70 is the most important member of them.As a universal cytoprotection protein,HSP70 is ubiquitous molecular chaperone. Moreover,it plays important roles in cellular protection and anti-apoptotic effect.It could help to refold the misfolding proteins and repair the damaged polypeptides.So it is significance to clone hsp70 gene from B.bassiana and study its experssion and action mechanism.
The full-length cDNA of two trehalose synthesis relevant genes tps1 and tps2 (GenBank accession No:FJ769373 and FJ769375) were cloned from B.bassiana b202 using SMART RACE RT-PCR.The sequence of their respective upstream promotors (GenBank accession No:FJ769374 and FJ769376) were amplified by genome walking. Sequence analysis showed that there was an intron with size of 55bp in the structural gene of tps1,which was located at the 313th～367th base.The full-length cDNA of tps1 gene was 1906bp,with 1563bp,112bp and 263bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region(3'-UTR),respectively.The open reading frame(ORF) encoded 520 amino acids.The mature protein had a molecular mass of 58.3 kDa with a calculated pI of 5.63.The 3'-UTR contained polyadenylation signal AATATA and 26bp PolyA.The transcription initiation site of B.bassiana tps1 gene was located at G base of position 2785 of upstream sequence.The upstream sequence contained TATA-box, GC-box and several other regulatory elements,such as Oct-1-binding factor, CRE-BP-binding factor,CdxA-binding factor and GATA.There were two introns with size of 140bp and 62bp in the structural gene of tps2,located at the 127th～266th base and 2209th～2270th base,respectively.Their sequences complied with the rule of "5'-GT" and "AG-3'".The full-length cDNA of tps2 gene was 3219bp,with 2619bp,310bp and 290bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region (3'-UTR),respectively.The open reading frame(ORF) encoded 872 amino acids.The mature protein had a molecular mass of 97.8 kDa with a calculated pI of 6.32.The 3'-UTR contained 28bp PolyA.The transcription initiation site of B.bassiana tps2 gene was located at C base of position 4105 of upstream sequence.The upstream sequence contained TATA-box,CAAT-box,GC-box and other regulatory elements,such as cap-binding factor, Adf-1-binding factor,Oct-1-binding factor and GATA.In additional,the content of GATA in the upstream sequence was very high.
The same method was used to clone the full-length cDNA of hsp70 gene and its upstream sequence from B.bassiana.Analysis of the complete cDNA cloned,with a whole sequence of 2405bp,showed that it encompassed an open reading frame(ORF) with 1971bp encoding 656 amino acids.The mature protein had a molecular mass of 71.3 kDa with a calculated pI of 4.92.The analyses indicated that the upstream sequence contained several regulatory elements,such as CCAAT-binding factor,GC-box,HSE and GATA,but TATA-box and CAAT-box were not be found.
The expression of tps1 and hsp70 gene from B.bassiana under several stress conditions was detected using Realtime-PCR.The effects of different stress conditions on expression of tps1 and hsp70 gene were explored based on mRNA level.The results showed that the change of tps1 gene transcriptional expression in mycelium and conidia of B.bassiana was not significant under three stress conditions,but the expression of hsp70 gene from B.bassiana increased rapidly under the same conditi6ns,suggesting that tpsl and hsp70 gene from B.bassiana have different response mechanisms for external stresses. Tpsl gene into secreting expression vector pPIC9K was cloned and then transformed into Pichia pastoris GS115.After continuous methanol induction,expression protein with molecular weight of ca.60kDa was obtained,which was closed to predicted value and those in related reports.Enzymatic activity assay revealed that the expression protein had bioactivity of trehalose-6-phosphate synthase.The maximum enzymatic activity of crude enzyme liquid was 1.384U/mL. The tpsl gene of B.bassiana was inserted into the cloning site of pBARGPE1 vector, which contained promoter of the glyceraldehyde-3-phosphate gene from Aspergillus nidulans.After the recombinant plasmid was transformed into B.bassiana,tpsl gene was constitutively expressed in the recombinant strain.In comparison with wild-type strains, under lower temperture the shelf life of the recombinant strains was enhanced by nearly 35%.The heat tolerance of recombinant strains was enhanced by nearly 30%within a certain high temperature stress.The UV tolerace of recombinant,however,was not be enhanced.In addition,the growth rate and sporulation of the recombinant strains were lower than those of wild-type ones.It is suggested that constitutive expression of adversity gene of entomogenous fungi enhanced stress resistance of recombinant strains,however, with some negative effects.
引文
[1]王翠玲,覃荣,席永士.生物农药的研究与开发.西藏科技,2003,7:16-18
[2]陈为京,陈建爱,王未名.发展生物农药产业的建议.科学与管理,2002,22(1):39-41
[3]蒲蛰龙,李增智.昆虫真菌学.合肥:安徽科学技术出版社.1996
[4]胡加付.白僵菌工业化生产与防治天牛技术的研究:[硕士毕业论文].合肥:安徽农业大学,2001
[5]袁哲明,刘树生,孟小林.小菜蛾病原微生物研究进展.中国生物防治,1999,15(2):85-89
[6]李永红.农药的发展与人类的健康.生物学通报,2001,36(5):12-14
[7]李增智,樊美珍.真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.北京:科学出版社.2000,115-121
[8]St Leger RJ,Screen SE.Prospects for strain improvement of fungal pathogens of insects and weeds.In:Fungi as biocontrol agent,Butt T M,Jackson C,Magan N(eds),CABI international,2001,219-237
[9]Bogo MR,Rota CA,Pinto H,et al.A chitinase encoding gene(chitl gene) from the entomopathogen Metarhizium anisopliae:isolation and characterization of genomic and full length cDNA.Current Microbiology,1998,37(4):221-225
[10]张爱文,邓春生,农向群.虫生真菌育种方法的进展.微生物学杂志,1991,11(2):89-92
[11]Viaud M,Couteaudier Y,Levis C,et al.Genome organization in Beauveria bassiana:electrophoretic karyotype,gene mapping,and tolorneric fingerprint.Fungal Genetics and Biology,1996,20(3):175-183
[12]Viaud M,Couteaudier Y,Riba G.Molecular analysis of hypervirulent somatic hybrids of the entomopathogenic fungi Beauveria bassiana and Beauveria sulfurescens.Applied and Environmental Microbiology,1998,64(1):88-93
[13]St Leger RJ,Frank DC,Roberts DW,et al.Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae.European Journal of Biochemistry,1992,204(3):991-1001
[14]Smithson SL,Paterson IC,Bailey AM,et al.Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.Gene,1995,166(1):161-165
[15]方卫国,张永军,杨星勇等.球孢白僵菌降解昆虫表皮蛋白酶基因CDEP-1克隆与序列分析.遗传学报,2002,29(2):278-282
[16]黄勃,张中,彭凡等.粉棒束孢几丁质酶基因CDNA全序列克隆及结构特征分析.菌物学报,2006,25(4):633-638
[17]St Leger RJ,Joshi L,Bidochka MJ,et al.Construction of an improved mycoinsecticide overexpressing a toxic protease.Proceedings of the National Academy of Sciences of the United States of America,1996,93(13):6349-6354
[18]St Leger RJ,Staples RC,Roberts DW.Cloning and regulatory analysis of starvation-stress gene,ssgA,encoding a hydrophobin-like protein from the entomopathogenic fungus,Metarhizium anisopliae.Gene,1992,120(1):119-124
[19]Xia YX,Gao MY,Clarkson JM,et al.Molecular cloning,characterisation,and expression of a neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.Journal of Invertebrate Pathology,2002,80(2):127-137
[20]胡宗利,王中康,彭国雄等.金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析.微生物学报,2005,45(6):890-894
[21]胡宗利,陈国平,王中康.绿僵菌NTL基因上游调控序列的克隆及分析.重庆大学学报(自然科学版),2005,29(4):73-76
[22]Cai ZJ,Peng GX,Cao YQ,et al.Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae:clone,expression and properties of the recombinant.Journal of Bioscience and Bioengineering,2009,107(5):499-505
[23]Barrett J.Anhydrobiotic nematodes.Agriculturalzoology Reviews,1989,4:161-176
[24]Arg(u|¨)elles JC.Physiological roles of trehalose in bacteria and yeasts:a comparative analysis.Archives of Microbiology,2000,174(4):217-224
[25]Richards AB,Krakowka S,Schmid H,et al.Trehalose:a review of properties,history of use and human tolerance,and results of multiple safety studies.Food and Chemical Toxicology,2002,40(7):871-898
[26]Wingler A.The function of trehalose biosynthesis in plants.Phytochemistry,2002,60(5):437-440
[27]De Virgilio C,Hottiger T,Dominguzz J,et al.The role of trehalose synthesis for the acquisition of thermotolerance in yeast.European Journal of Biochemistry,1994,219(1-2):187-190
[28]Hottiger T,Schmutz P,Wiemken A.Heat induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.Journal of Bacteriology,1987,169(12):5518-5522
[29]Laere AV,Francois A,Overloop K,et al.Relation between germinination,trehalose and the status of water in Phycomyces blakeleeamus spores as measured by protein-NMR.Journal of General Microbiology,1987,169(2):239-245
[30]Zachariassen KE.Physiology of cold tolerance in insects.Physiological Review,1985,65: 799-803
[31]Tottiger T,Boller T,Wiemken A.Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts.FEBS Letters,1987,220(1):113-115
[32]Jacobson K,Papahadjopoulos D.Phase transition and phase separations in phospholipids membranes induced by changes in temperature,PH and concentration of bivalent cation.Biochemistry,1975,14(1):152-161
[33]Mozhaev W,Martinek K.Structure-stability relationships in proteins:aguide to approaches to stabilizing enzymes.Advanced Drug Delivery Reviews,1990,4(3):387-419
[34]李浩明,高兰.海藻糖在生物制品干燥与保存中的应用研究.食品与发酵工业,1994,20(4):49-52
[35]Valenzuela-Soto EM,Marquez-Escalante J,Iturriaga G,et aI.Trehalose-6-phosphate synthase from Selaginella lepidophylla:purification and properties.Biochemical and Biophysical Research Communications,2004,313(2):314-319
[36]Kwon HB,Yeo ET,Hahn SE.Cloning and characterization of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatese from Zygocaccharomyces rouxii.FEMS Yeast Research,2003,3:433-440
[37]Ritossa F.A new puffing pattern induced by temperature shock and DNP in drosophila.Cellular and Molecular Life Sciences,1962,18(12):571-573
[38]Tissieres A,Mitchell HK,Tracy UM.Protein synthesis in salivary glands of Drosophilla melanogaster:relation to chromosome puffs.Journal of Molecular Biology,1974,85(3):389-398
[39]Becker J,Craig EA.Heat-shock proteins as molecular chaperones.European Journal of Biochemistry,1994,219(1-2):11-23
[40]Kiang JG,Tsokos GC.Heat shock protein 70kDa:molecular biology,biochemistry and physiology.Pharmacology and Therapeutics,1998,80(2):183-201
[41]Ellis RJ.The general concept of molecular chaperones.Philosophical Transactions Biological Sciences,1993,339:257-261
[42]Georgopoulos C,Welch WJ.Role of the major heat shock proteins as molecular chaperones.Annual Review of Cell Biology,1993,9:601-634
[43]Welch WJ.Heat shock proteins functioning as molecular chaperones:their roles in normal and stressed cells.Philosophical Transactions Biological Sciences,1993,339:327-333
[44]Yang XD,Feige U.Heat shock proteins in autoimmune disease.From causative antigen to specific therapy? Cellular and Molecular Life Sciences,1992,48(7):650-656
[45]Carper SW,Duffy JJ,Gerner EW.Heat shock proteins in thermotolerance and other cellular processes.Cancer Research,1987,47:5249-5255
[46]Lindquist S,Craig EA.The heat-shock proteins.Annual Review of Genetics,1988,22:631-677
[47]Morimoto RI,Cell in stress:transcriptional activation of heat shock genes.Science,1993,259:1409-1410
[48]Beckmann RP,Mizzen LE,Welch WJ.Interaction of Hsp70 with newly synthesized proteins:implications for protein folding and assembly.Science,1990,248:850-854
[49]Gething MJ,Sambrook J.Protein folding in the cell.Nature,1992,355:33-45
[50]Welch WJ.Mammalian stress response:cell physiologt,structure/function of stress proteins,and implications for medicine and disease.Physiological Reviews,1992,72(4):1063-1081
[51]Hartl FU,Martin J.Molecular chaperones in cellular protein folding.Current Opinion in Structural Biology,1995,5(1):92-102
[52]Moseley PL.Heat shock proteins and heat adaptation of the whole organism.Journal of Applied Physiology,1997,83(5):1413-1417
[53]Hemmingsen SM,Woolford C,Vies VD,et al.Homologous plant and bacterial proteins chaperone oligomeric protein assembly.Nature,1988,333(6171):330-334
[54]Parsell DA,Morimoto RI.The biology of heat shock proteins and molecular chaperones.New York:Cold Spring Harbor Laboratory Press,1994:457-494
[55]Dahlgaard J,Loeschcke V,Michalak P,et al.Induced thermotolerance and associated expression of the heat-shock protein Hsp70 in adult Drosophila melanogaster.Functional Ecology,1998,12:786-793
[56]Guttman SD,Glover CV,Allis CD,et al.Heat shock,deciliation and release from anoxia induce the synthesis of the same polypeptides in starved T.pyriformis.Cell,1980,22:299-307
[57]Hahn GM,Li GC.Thermotolerance and heat shock proteins in mammalian cells.Radiation Research,1982,92:452-457
[58]Barbe MF,Tytell M,Gower DJ,et al.Hyperthermia protects against light damage in the rat retina.Science,1988,241(4874):1817-1820
[59]Sciandra JJ,Subjeck JR.The effect of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells.Journal of Biological Chemistry,1983,258(20):12091-12093
[60]Weitzel G,Pilatus U,Rensing L.Similar dose response of heat shock protein synthesis and intracellular pH change in yeast.Experimental Cell Research,1985,159(1):252-256
[61]Marber MS,Mestril R,Chi SH,et al.Overexpression of the rat inducible 70-kDa heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.The Journal of Clinical Investigation,1995,95(4):1446-1456
[62]Das DK,Engelman RM,Kimura Y.Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia.Cardiovascular Research,1993,27(4):578-584
[63]Peter ME,Heufelder AE,Hengartner MO.Adances in apoptosis research.Proceedings of the National Academy of Sciences of the United States of America,1997,94(24):12736-12737
[64]Kroemer G,Reed JC.Mitochondrial control of cell death.Nature Medicine,2000,6:513-519
[65]Zamzami N,Kroemer G.The mitochondrion in apoptosis:How Pandora's box opens.Nature Reviews Molecular Cell Biology,2001.2:67-71
[66]Saleh A,Srinivasula SM,Balkir L,et al.Negative regulation of the Apaf-1 apoptosome by Hsp70.Nature Cell Biology,2000,2:476-483
[67]Beere HM,Wolf BB,Cain K,et al.Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.Nature Cell Biology,2000,2:469-475
[68]Sreedhar AS,Csermely P.Heat shock proteins in the regulation of apoptosis:new strategies in tumor therapy:a comprehensive review.Pharmacology and Therapeutics,2004,101(3):227-257
[69]Garrido C,Gurbuxani S,Ravagnan L,et al.Heat shock proteins:endogenous modulators of apoptotic cell death.Biochemical and Biophysical Research Communications,2001,286(3):433-442
[70]Creagh EM,Carmody J,Cotter TG,et al.Heat shock protein 70 inhibits caspase-dependent and independent apoptosis in Jurkat T cells.Experimental Cell Research,2000,257(1):58-66
[71]Ravagnan L,Gurbuxani S,Susin SA,et al.Heat-shock protein 70 antagonizes apoptosis-inducing factor.Nature Cell Biology,2001,3:839-843
[72]Case ME,Schweizer M,Kushner SR,et al.Efficient transformation of Nelurospora crassa by utilizing hybrid plasmid DNA.Proceedings of the National Academy of Sciences of the United States of America,1979,76(10):5259-5263
[73]Jackson CW,Heale JB.Parasexual crosses by hyphal anastomosis and protoplast fusion in the entomopathogen Verticillium lecanii.Journal of General Microbiology,1987,133(12):3537-3547
[74]Inglis PW,Tigano MS,Valadares-inglis MC.Transformation of the entomopathogenic fungi,Paecilomyces fumosoroseus and Paecilomyces lilacinus(deuteromycotina:hyphomycetes) to benomyl resistence.Genetics and Molecular Biology,1999,22(1):119-123
[75]Bernier L,Cooper RM,Charnley AK,et al.Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance.FEMS Microbiology Letters,1989,60(3):261-266
[76]李增智.虫生真菌研究展望.安徽农学院学报,1987,3:61-72
[77]Fincham JR.Transformation in fungi.Microbiology and Molecular Biology Reviews,1989,53(1):148-170
[78]Pfeifer TA,Khachatourians GG.Beauveria bassiana protoplast regeneration and transformation using electroporation.Applied Microbiology and Biotechnology,1992,38(3):376-381
[79]Fungaro MHP,Rech E,Muhlen GS,et al.Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia.FEMS Microbiology Letters,1995,125(2):293-297
[80]Vianey OM,Carlos CP,Alfredo HE.Three decades of fungal transformation key concepts and applications.Methods in Molecular Biology,2004,267:297-313
[81]Ying SH,Feng MG.Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.Applied Microbiology and Biotechnology,2006,72(1):206-210
[82]St Leger RJ,Shimizu S,Joshi L,et al.Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants.FEMS Microbiology Letters,1995,131(3):289-294
[83]Bogo MR,Vainstein MH,Aragao FJ,et al.High frequency gene conversion among benomyl resistance transformants in the entomopathogenic fungus Metarhizium anisopliae.FEMS Microbiology Letters,1996,142(1):123-127
[84]Jiang Q,Ying SH,Feng MG.Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation.Journal of Microbiological Methods,2007,69(3):512-517
[85]Bundock P,Dulk-Ras AD,Beijersbergen A,et al.Trans-kingdom T-DNA transfer from Agrobacterium tumefaeiens to Saccharomyces cerevisiae.The EMBO Journal,1995,14(13):3206-3214
[86]De Groot MJ,Bundock P,Hooykass PJ,et al.Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnology,1998,16:839-842
[87]Cantone FA,John D.Genetic transformation and mutagenesis of the entomopathogenic fungus Paecilomyces fumosoroseus.Journal of lnvertebrate Pathology,1999,74(3):281-288
[88]Fang WG,Zhang YJ,Yang XY.Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker.Journal of Invertebrate Pathology,2004,85(1):18-24
[89]Duarte RTD,Staats CC,Fungaro MHP,et al.Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var.acridum.Letters in Applied Microbiology,2007,44(3):248-254
[90]Punt PJ,Oliver RP,Dingemanse MA,et al.Transformation of Aspergillus based on the hygromycin B resistance marker form Escherichia coli.Gene,1987,56(1):117-124
[91]Sandhu SS,Kinghorn JR,Rajak RC,et al.Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.Indian Journal of Experimental Biology,2001,39(7):650-653
[92]李维,张义正,以潮霉素B磷酸转移酶基因为遗传标记的启动子探针型载体的构建.四川大学学报(自然科学版),1999,36(4):736-740
[93]Thompson CJ,Movva NR,Tizard R,et al.Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.EMBO Journal,1987,6(9):2519-2523
[94]Cantone FA,Vandenberg JD.Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts..Journal of Invertebrate Pathology,1999,74(2):193-197
[95]Goettel MS,St Leger RJ,Bhairi S,et al.Pathogenicity and growth of Metarhizium anisopliae stale transformed to Benomyl resistance.Current Genetic,1990,17(2):129-132.
[96]Furlaneto MC,Paiao FG,Pinto FG,et aL Transformantion of the entomopathogenic fungus Metarhizium flavoviride to high resistance to benomyl.Canadian Journal of Microbiology,1999,45(10):875-878
[97]Frey PM.Biocontrol agents in the age of molecular biology.Trends in Biotechnology,2001,19(11):432-433
[98]Gressel J.Potential failsafe mechanisms against the spread and introgression of transgenic hypervirulent biocontrol fungi.Trends in Biotechnology,2001,19(4):149-154
[99]Charnley AK,Cobb B,Clarckson JM.Towards the improvement of fungal insecticides BCPC symposium proceedings.British Crop Protection Council,1997,68:115-126
[100]Gornova IB,Feofilova EP,Tereshina VM,et al.VM,Effect of carbohydrate content of Aspergillus japonicus spores on their survival in storage and subsequent germination.Mikrobiologiya,1992,61:549-554
[101]Harman GE,Jin X,Stasz TE,et al.Production of conidial biomass of Trichoderma harzianum for biological control.Biological Control,1991,1(1):23-28
[102]董志扬,方宣钧,林敏等.海藻糖的生物合成及抗逆机理.核农学研究进展,1996:115-120
[103]Goddijn WJM,Dun KV.Trehalose metabolism in plants.Trends in Plant Science,1999,4(8):315-319
[104]Pilon-Smits EAH,Terry N,Sears T.Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress.Journal of Plant Physiology,1998, 152(4-5):525-532
[105]Holmstrom KO,Mantyla E,Welin B,et al.Drought tolerance in tobacco.Nature,1996,397(6567):683-684
[106]王自章,张树珍,杨本鹏等.甘蔗根癌农杆菌介导转化海藻糖合酶基因获得抗渗透胁迫能力增强植株.中国农业科学,2003,36(2):140-146
[107]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses.Proceedings of the National Academy of Sciences of the United States of America,2002,99(25):15898-15903
[108]Avonce N,Leyman B,Thevelein J,et al.Trehalose metabolism and glucose sensing in plants.Transactions,2005,33:276-279
[109]Vogel G,Fiehn O,Boller T,et al.Trehalose metabolism in Arabidopsis:occurrence of treholase and molecular cloning and characterization of trehalose-6-phosphate synthase homologues.Jorunal of Experimental Botany,2001,52(362):1817-1826
[110]Blasquez MA,Santos E,Flores CL,et al.Isolation and molecular characterization of the Arabidopsis TPS1 gene,encoding trehalose-6-phosphate synthase.Plant Journal,1998,13(5):685-689
[111]Sambrook J,Russell DW.Molecular Cloning.New York:Cold Spring Harbor Laboratory Press,2001
[112]周勇,马玲.人卵透明带3蛋白的二级结构和B细胞抗原表位预测.新疆医科大学学报,2008,31(8):955-958
[113]肖军,吴季辉.杀菌肽及膜蛋白螺旋结构预测.生物物理学报,1994,10(2):281-288
[114]周颖君,杨亦桦,武淑文等.棉铃虫细胞色素P450 CYP9A12基因5'-上游区的克隆及序列分析.昆虫学报,2008,51(2):120-125
[115]王德培,孙伟,李明春等.利用长引物嵌套式反向PCR方法克隆雅致枝霉△6-脂肪酸脱氢酶基因上游序列.生物工程学报,2006,22(4):581-586
[116]胡新立,周迅蕾.Nodal基因5'末端侧翼序列启动子的分析.北京大学学报(自然科学版),2000,36(2):178-185
[117]张红缨,刘洋,张今.海藻糖的生物合成和相关酶的特性.微生物学通报,1998,25(4):236-238
[118]Goddijn OJM,Verwoerd TC,Voogd E,et al.Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.Plant Physiology,1997,113(1):181-190
[119]Becker K,Pan Dao,Whitley CB.Real-time quantitative polymerase chain reaction to assess gene transfer.Human Gene Therapy,1999,10(15):2559-2566
[120]Higgins JA,Ezzell J,Hinnebusch BJ.5' nuclease PCR assay to detect Yersinia pesits.Journal of Clinical Microbiology,1998,36(8):2284-2288
[121]Bieche I,Onody P,Laurendeau I,et al.Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical apolications.Clinical Chemistry,1999,45:1148-1156
[122]Wang XK,Li X,Currie RW,et al.Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1 beta mRNA in ischemic brain tolerance.Journal of Neuroscience Research,2000,59(2):238-246
[123]Higuchi R,Fockler C,Dollinger G,et al.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions.Biotechnology,1993,11(9):1026-1030
[124]张丽靖.球孢白僵菌孢子粉生产、制剂和贮存技术的改进及其淀粉酶特性测定:[硕士学位论文].杭州:浙江大学,2003
[125]Bateman R.The "MycoHarvester":cleaning up locust control.International Pest Control,2003,45:76-77
[126]Hong TD,Gunn J,Ellis RH,et al.The effect of storage environment on the longevity of conidia of Beauveria bassiana.Mycological Research,2001,105(5):597-602
[127]崔旭红,谢明,万方浩.高温胁迫下B型烟粉虱热激蛋白基因hsp70表达量的变化.昆虫学报,2007,50(11):1087-1091
[128]Overbergh L,Valckx D,Waer Mark,et al.Quantification ofmurine cytokine mRNAs using real time quantitative reverse transcriptase PCR.Cytokine,1999,11(4):305-312
[129]杜荣骞.生物统计学.北京:高等教育出版社,施普林格出版社.1999
[130]唐启义,冯明光.实验统计分析及其DPS数据处理系统.北京:科学出版社.2002
[131]Gault E,Michel Y,Dehee A,et al.Quantification of human cytomegalovirus DNA by Real-Time PCR.Journal of Clinical Microbiology,2001,39(2):772-775
[132]李尊华,林健文,马金成等.变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响.微生物学报,2008,48(7):887-892
[133]Liu Q,Ying SH,Feng MG,et al.Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus Beauveria bassiana to thermal stress.Antonie van Leeuwenhoek,2009,95:65-75
[134]Fillinger S,Chaveroche MK,Van-Dijck P,et al.Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans.Microbiology,2001,147:1851-1862
[135]Ocon A,Hampp R,Requena N.Trehalose turnover during abiotic stress in arbuscular mycorrhizal fungi.New Phytologist,2007,174(4):879-891
[136]马向东,郭永豪,余华顺等.三株酵母的抗热激性与tpsl基因表达的关系.郑州大学学报(理学版),2005,37(1):30-33
[137]Emmanuelle BM,Michel P,Francoise B,et al.Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae.Journal of Bacteroilogy,1998,180(5):1044-1052
[138]洪洄,王冬梅.应用酵母进行基因表达的研究进展.生物学通报,1999,34(7):12～14
[139]陈偿.甲醇毕赤氏酵母表达体系.热带农业科学,1999,2:3¨3
[140]Cregg JM,Madden KR.Development of the methylotrophic yeast,Pichia Pastoris,as a host system for the production of foreign proteins.Developments in Industrial Microbiology,1988,29(3):33-41
[141]Allais JJ,Louktibi A,Baratti J.Oxidation of methanol by the yeast Pichia pastoris:purification and properties of alcohol oxidase.Agricultural and Biological Chemistry,1983,47(11):2547-2554
[142]Cregg JM,Madden KR,Barringer KJ,et al.Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.Molecular and Cellular Biology,1989,9(3):1316-1323
[143]Cereghino JL,Cregg JM.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
[144]Thor D,Xiong S,Orazem CC,et al.Cloning and characterization of Pichia pastoris MET2gene as a selectable marker.FEMS Yeast Research,2005,5(10):935-942
[145]Laemmli UK.Cleavage of structural protein during the assembly of the head of bacterphage T4.Nature,1970,227:680-686
[146]Silva Z,Alarico S,Da Costa MS.Trehalose biosynthesis in Thermus thermophilus RQ-1:biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.Extremophiles,2005,9(1):29-36
[147]Romanos M.Advances in the use of Pichia pastoris for high level gene expression.Current Opinion in BiotechnoIogy,1995,6(5):527-533
[148]Hong F,Meinander NQ,Jonsson LJ.Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.Biotechnology and Bioengineering,2002,79(4):438-449
[149]Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene,1997,190(1):55-62
[150]Duman JG,Miele RG,Hong L,et al.O-Mannosylation of Pichia pastoris cellular and recombinant proteins.Biotechnology and Applied Biochemistry,1998,28(1):39-45
[151]Fang WG,Leng Bo,Xiao YH,et al.Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virulence.Applied and Environmental Microbiology,2005,71(1):363-370
[152]Fan YH,Fang WG,Xiao YH,et al.Directed evolution for increased chitinase activity.Applied Microbiology and Biotechnology,2007,76(1):135-139
[153]俞佳,冯明光.基于分生孢子热胁迫反应的球孢白僵菌耐热菌株筛选.菌物学报,2006,25(2):278-283
[154]Bell W,Sun W,Hohmann S,et al.Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex.Journal of Biological Chemistry,1998,273(50):33311-33319
[155]De Silva-Udawatta MN,Cannon JF.Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation.Molecular Microbiology,2001,40(6):1345-1356
[156]郭永豪,孙连海,余华顺等.啤酒酵母中海藻糖合成酶和海藻糖-6-磷酸合成酶的功能及结构分析.酿酒科技,2005,6:40-43
[157]黄惠芳,马飞.热激蛋白的分子进化研究.厦门大学学报(自然科学版),2004,43(8):166-170
[158]王笑虹,苗迎秋,宋光.热休克蛋白70生物学功能研究的新进展.大连大学学报,2003,24(2):103-105
[159]Piano A,Franzellitti S,Tinti F,et al.Sequencing and expression pattern of inducible heat shock gene products in the European flat oyster,Ostrea edulis.Gene,2005,361:119-126
[160]Landais L,Pommet JM,Mita K,et al.Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda.Gene,2001,271(2):223-231
[161]Nover L,Scharf KD.Heat stress proteins and transcription factors.Cellular and Molecular Life Sciences,1997,53(1):80-103
[162]李冰祥,蔡惠罗,陈永林.昆虫的热休克反应和热休克蛋白.昆虫学报,1997,40(4):417-427
[163]李晓鲁,彭毅志.热激蛋白70与热激反应.生命的化学,2002,22(2):145-147
[164]Tsan MF,Gao B.Cytokine function of heat shock proteins.American Journal of Physiology-Cell Physiology,2004,286:739-744
[165]Deane EE,Woo NYS.Cloning and characterization of the hsp70 mutigene family from silver sea bream:modulated gene expression between warm and cold temperature acclimation.Biochemical and Biophysical Research Communications,2005,330(3):776-783
[166]Rezaie S,Ban J,Mildner M,et al.Characterization of a cDNA clone,encoding a 70kDa heat shock protein from the derrnatophyte pathogen Trichophyton rubrum.Gene,2000,241(1): 27-33
[167]Demand J,Luders J,Hohfeld J.The carboxy-terminal domain of HSC70 provides binding sites for a distinct set of chaperone cofactors.Molecular and Cellular Biology,1998,18:2023-2028
[168]Vayssier M,Guerhier FL,Fabien JF,et al.Cloning and analysis of a Trichinella briotovi gene encoding a cytoplasmic heat shock protein of 72 kDa.Parasitology,1999,119:81-93
[169]Craven RA,Tyson JR,Stifling CJ.A novel subfamily of HSP70s in the endoplasmic reticulum.Trends in Cell Biology,1997,7:277-282
[170]焦传珍,王在照,李富花等.编码中国明对虾(Fenneropenaeus chinensis)一种组成型热休克蛋白70(HSC70)cDNA克隆、测序及其表达分析.科学通报,2004,49(21):2178-2186
[171]陈亮,张富春,黄萍等.光滑鳖甲热休克蛋白70基因的克隆及表达.昆虫学报,2007,50(9):883-888
[172]吴莹,徐香玲.植物热激转录因子的研究进展.哈尔滨师范大学自然科学学报,2005,21(2):73-78
[173]Morimotol RI.Regulation of the heat shock transcriptional response:cross talk between a family of heat shock factors,molecular chaperones,and negative regulators.Genes and Development,1998,12:3788-3796
[174]张伟,王登高.热休克基因转录的调节:热休克转录因子(HSF)的结构与功能.第三军医大学学报,2000,22(2):198-200
[175]陈晓红,印莉萍.结合于GATA元件的Ⅳ型锌指蛋白.生命的化学,1999,19(4):179-180
[176]张立国,张琚.实时定量PCR技术的介绍.生物技术,2003,13(2):39-40
[177]Sarrazin C,Cartner BC,Sizmann D,et al.Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 to 5.Journal of Clinical Microbiology,2006,44(3):729-737
[178]谢翎,陈红梅,蒲顺昌等.球孢白僵菌热休克蛋白基因Bbhsp 70的cDNA及上游序列克隆与分析.菌物学报,2009,28(2):283-288
[179]马旭,吕刚.HSP-70与细胞保护的研究进展.大连医科大学学报,2007,29(2):194-196
[180]Shyu WC,Kao MC,Chou WY,et al.Heat shock modulates prion protein expression in human NT-2 cells.Neuroreport,2000,11(4):771-774
[181]Saavedra CA,Hmmell CM,Heath CV,et al.Yeast heat shock mRNAs are exported through a distinct pathway defined by Riplp.Genes and Development,1997,11(21):2845-2856
[182]Vainberg IE,Dower K,Rosbash M.Nuclear export of heat shock and non-heat-shock mRNA occurs via similar pathways.Molecular and Cellular Biology,2000,20(11):3996-4005
[183]Benedetti AD,Baglioni C.Translational regulation of the synthesis of a major heat shock protein in HeLa cells.Journal of Biological Chemistry,1986,261(33):15800-15804
[184]Tamai KT,Liu X,Silar P,et al.Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways.Molecular and Cellular Biology,1994,14(12):8155-8165
[185]Ruis H,Schuller C.Stress signalling in yeast.Bioessays,1995,17(11):959-965
[186]Rensinga L,Monnerjahna C,Meyera U.Differential stress gene expression during the development of Neurospora crassa and other fungi.FEMS Microbiol Letters,1998,168(2):159-166
[187]Extrucha F.Stress-controlled transcription factors,stress-induced genes and stress tolerance in budding yeast.FEMS Microbiol Reviews,2000,24(4):469-486
[188]Hoffmann AA.Acclimation:increasing survival at a cost.Trends in Ecology and Evolution,1995,10(1):1-2
[189]Coleman JS,Heckathorn SA,Hallberg RL.Heat-shock protein and thermotolerance:linking molecular and ecological perspectives.Trends in Ecology and Evolution,1995,10(8):305-306
[190]杨艳,李增波.海藻糖TPS/TPP生物合成途径的进化研究.科技情报开发与经济,2008,18(16):123-125
[191]Elbein AD,Pan YT,Pastuszak I,et al.New insights on trehalose:a multifunctional molecule.Glycobiology,2003,13(4):417-427
[192]Paul MJ,Primavesi LF,Jhurreea D,et al.Trehalose metabolism and signaling.Annual Review of Plant Biology,2008,59:17-41
[193]Wang Y J,Hao Y J,Zhang ZG;et al.Isolation of trehalose-6-hosphate phosphatase gene from tobacco and its functional analysis in yeast cells.Journal of Plant Physiology,2005,162(2):215-223
[194]马辉,刘桂丰,徐晨曦等.二色补血草海藻糖-6-磷酸磷酸酯酶基因的克隆及分析.东北林业大学学报,2008,36(8):1-3
[2]陈为京,陈建爱,王未名.发展生物农药产业的建议.科学与管理,2002,22(1):39-41
[3]蒲蛰龙,李增智.昆虫真菌学.合肥:安徽科学技术出版社.1996
[4]胡加付.白僵菌工业化生产与防治天牛技术的研究:[硕士毕业论文].合肥:安徽农业大学,2001
[5]袁哲明,刘树生,孟小林.小菜蛾病原微生物研究进展.中国生物防治,1999,15(2):85-89
[6]李永红.农药的发展与人类的健康.生物学通报,2001,36(5):12-14
[7]李增智,樊美珍.真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.北京:科学出版社.2000,115-121
[8]St Leger RJ,Screen SE.Prospects for strain improvement of fungal pathogens of insects and weeds.In:Fungi as biocontrol agent,Butt T M,Jackson C,Magan N(eds),CABI international,2001,219-237
[9]Bogo MR,Rota CA,Pinto H,et al.A chitinase encoding gene(chitl gene) from the entomopathogen Metarhizium anisopliae:isolation and characterization of genomic and full length cDNA.Current Microbiology,1998,37(4):221-225
[10]张爱文,邓春生,农向群.虫生真菌育种方法的进展.微生物学杂志,1991,11(2):89-92
[11]Viaud M,Couteaudier Y,Levis C,et al.Genome organization in Beauveria bassiana:electrophoretic karyotype,gene mapping,and tolorneric fingerprint.Fungal Genetics and Biology,1996,20(3):175-183
[12]Viaud M,Couteaudier Y,Riba G.Molecular analysis of hypervirulent somatic hybrids of the entomopathogenic fungi Beauveria bassiana and Beauveria sulfurescens.Applied and Environmental Microbiology,1998,64(1):88-93
[13]St Leger RJ,Frank DC,Roberts DW,et al.Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae.European Journal of Biochemistry,1992,204(3):991-1001
[14]Smithson SL,Paterson IC,Bailey AM,et al.Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.Gene,1995,166(1):161-165
[15]方卫国,张永军,杨星勇等.球孢白僵菌降解昆虫表皮蛋白酶基因CDEP-1克隆与序列分析.遗传学报,2002,29(2):278-282
[16]黄勃,张中,彭凡等.粉棒束孢几丁质酶基因CDNA全序列克隆及结构特征分析.菌物学报,2006,25(4):633-638
[17]St Leger RJ,Joshi L,Bidochka MJ,et al.Construction of an improved mycoinsecticide overexpressing a toxic protease.Proceedings of the National Academy of Sciences of the United States of America,1996,93(13):6349-6354
[18]St Leger RJ,Staples RC,Roberts DW.Cloning and regulatory analysis of starvation-stress gene,ssgA,encoding a hydrophobin-like protein from the entomopathogenic fungus,Metarhizium anisopliae.Gene,1992,120(1):119-124
[19]Xia YX,Gao MY,Clarkson JM,et al.Molecular cloning,characterisation,and expression of a neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.Journal of Invertebrate Pathology,2002,80(2):127-137
[20]胡宗利,王中康,彭国雄等.金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析.微生物学报,2005,45(6):890-894
[21]胡宗利,陈国平,王中康.绿僵菌NTL基因上游调控序列的克隆及分析.重庆大学学报(自然科学版),2005,29(4):73-76
[22]Cai ZJ,Peng GX,Cao YQ,et al.Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae:clone,expression and properties of the recombinant.Journal of Bioscience and Bioengineering,2009,107(5):499-505
[23]Barrett J.Anhydrobiotic nematodes.Agriculturalzoology Reviews,1989,4:161-176
[24]Arg(u|¨)elles JC.Physiological roles of trehalose in bacteria and yeasts:a comparative analysis.Archives of Microbiology,2000,174(4):217-224
[25]Richards AB,Krakowka S,Schmid H,et al.Trehalose:a review of properties,history of use and human tolerance,and results of multiple safety studies.Food and Chemical Toxicology,2002,40(7):871-898
[26]Wingler A.The function of trehalose biosynthesis in plants.Phytochemistry,2002,60(5):437-440
[27]De Virgilio C,Hottiger T,Dominguzz J,et al.The role of trehalose synthesis for the acquisition of thermotolerance in yeast.European Journal of Biochemistry,1994,219(1-2):187-190
[28]Hottiger T,Schmutz P,Wiemken A.Heat induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.Journal of Bacteriology,1987,169(12):5518-5522
[29]Laere AV,Francois A,Overloop K,et al.Relation between germinination,trehalose and the status of water in Phycomyces blakeleeamus spores as measured by protein-NMR.Journal of General Microbiology,1987,169(2):239-245
[30]Zachariassen KE.Physiology of cold tolerance in insects.Physiological Review,1985,65: 799-803
[31]Tottiger T,Boller T,Wiemken A.Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts.FEBS Letters,1987,220(1):113-115
[32]Jacobson K,Papahadjopoulos D.Phase transition and phase separations in phospholipids membranes induced by changes in temperature,PH and concentration of bivalent cation.Biochemistry,1975,14(1):152-161
[33]Mozhaev W,Martinek K.Structure-stability relationships in proteins:aguide to approaches to stabilizing enzymes.Advanced Drug Delivery Reviews,1990,4(3):387-419
[34]李浩明,高兰.海藻糖在生物制品干燥与保存中的应用研究.食品与发酵工业,1994,20(4):49-52
[35]Valenzuela-Soto EM,Marquez-Escalante J,Iturriaga G,et aI.Trehalose-6-phosphate synthase from Selaginella lepidophylla:purification and properties.Biochemical and Biophysical Research Communications,2004,313(2):314-319
[36]Kwon HB,Yeo ET,Hahn SE.Cloning and characterization of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatese from Zygocaccharomyces rouxii.FEMS Yeast Research,2003,3:433-440
[37]Ritossa F.A new puffing pattern induced by temperature shock and DNP in drosophila.Cellular and Molecular Life Sciences,1962,18(12):571-573
[38]Tissieres A,Mitchell HK,Tracy UM.Protein synthesis in salivary glands of Drosophilla melanogaster:relation to chromosome puffs.Journal of Molecular Biology,1974,85(3):389-398
[39]Becker J,Craig EA.Heat-shock proteins as molecular chaperones.European Journal of Biochemistry,1994,219(1-2):11-23
[40]Kiang JG,Tsokos GC.Heat shock protein 70kDa:molecular biology,biochemistry and physiology.Pharmacology and Therapeutics,1998,80(2):183-201
[41]Ellis RJ.The general concept of molecular chaperones.Philosophical Transactions Biological Sciences,1993,339:257-261
[42]Georgopoulos C,Welch WJ.Role of the major heat shock proteins as molecular chaperones.Annual Review of Cell Biology,1993,9:601-634
[43]Welch WJ.Heat shock proteins functioning as molecular chaperones:their roles in normal and stressed cells.Philosophical Transactions Biological Sciences,1993,339:327-333
[44]Yang XD,Feige U.Heat shock proteins in autoimmune disease.From causative antigen to specific therapy? Cellular and Molecular Life Sciences,1992,48(7):650-656
[45]Carper SW,Duffy JJ,Gerner EW.Heat shock proteins in thermotolerance and other cellular processes.Cancer Research,1987,47:5249-5255
[46]Lindquist S,Craig EA.The heat-shock proteins.Annual Review of Genetics,1988,22:631-677
[47]Morimoto RI,Cell in stress:transcriptional activation of heat shock genes.Science,1993,259:1409-1410
[48]Beckmann RP,Mizzen LE,Welch WJ.Interaction of Hsp70 with newly synthesized proteins:implications for protein folding and assembly.Science,1990,248:850-854
[49]Gething MJ,Sambrook J.Protein folding in the cell.Nature,1992,355:33-45
[50]Welch WJ.Mammalian stress response:cell physiologt,structure/function of stress proteins,and implications for medicine and disease.Physiological Reviews,1992,72(4):1063-1081
[51]Hartl FU,Martin J.Molecular chaperones in cellular protein folding.Current Opinion in Structural Biology,1995,5(1):92-102
[52]Moseley PL.Heat shock proteins and heat adaptation of the whole organism.Journal of Applied Physiology,1997,83(5):1413-1417
[53]Hemmingsen SM,Woolford C,Vies VD,et al.Homologous plant and bacterial proteins chaperone oligomeric protein assembly.Nature,1988,333(6171):330-334
[54]Parsell DA,Morimoto RI.The biology of heat shock proteins and molecular chaperones.New York:Cold Spring Harbor Laboratory Press,1994:457-494
[55]Dahlgaard J,Loeschcke V,Michalak P,et al.Induced thermotolerance and associated expression of the heat-shock protein Hsp70 in adult Drosophila melanogaster.Functional Ecology,1998,12:786-793
[56]Guttman SD,Glover CV,Allis CD,et al.Heat shock,deciliation and release from anoxia induce the synthesis of the same polypeptides in starved T.pyriformis.Cell,1980,22:299-307
[57]Hahn GM,Li GC.Thermotolerance and heat shock proteins in mammalian cells.Radiation Research,1982,92:452-457
[58]Barbe MF,Tytell M,Gower DJ,et al.Hyperthermia protects against light damage in the rat retina.Science,1988,241(4874):1817-1820
[59]Sciandra JJ,Subjeck JR.The effect of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells.Journal of Biological Chemistry,1983,258(20):12091-12093
[60]Weitzel G,Pilatus U,Rensing L.Similar dose response of heat shock protein synthesis and intracellular pH change in yeast.Experimental Cell Research,1985,159(1):252-256
[61]Marber MS,Mestril R,Chi SH,et al.Overexpression of the rat inducible 70-kDa heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.The Journal of Clinical Investigation,1995,95(4):1446-1456
[62]Das DK,Engelman RM,Kimura Y.Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia.Cardiovascular Research,1993,27(4):578-584
[63]Peter ME,Heufelder AE,Hengartner MO.Adances in apoptosis research.Proceedings of the National Academy of Sciences of the United States of America,1997,94(24):12736-12737
[64]Kroemer G,Reed JC.Mitochondrial control of cell death.Nature Medicine,2000,6:513-519
[65]Zamzami N,Kroemer G.The mitochondrion in apoptosis:How Pandora's box opens.Nature Reviews Molecular Cell Biology,2001.2:67-71
[66]Saleh A,Srinivasula SM,Balkir L,et al.Negative regulation of the Apaf-1 apoptosome by Hsp70.Nature Cell Biology,2000,2:476-483
[67]Beere HM,Wolf BB,Cain K,et al.Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.Nature Cell Biology,2000,2:469-475
[68]Sreedhar AS,Csermely P.Heat shock proteins in the regulation of apoptosis:new strategies in tumor therapy:a comprehensive review.Pharmacology and Therapeutics,2004,101(3):227-257
[69]Garrido C,Gurbuxani S,Ravagnan L,et al.Heat shock proteins:endogenous modulators of apoptotic cell death.Biochemical and Biophysical Research Communications,2001,286(3):433-442
[70]Creagh EM,Carmody J,Cotter TG,et al.Heat shock protein 70 inhibits caspase-dependent and independent apoptosis in Jurkat T cells.Experimental Cell Research,2000,257(1):58-66
[71]Ravagnan L,Gurbuxani S,Susin SA,et al.Heat-shock protein 70 antagonizes apoptosis-inducing factor.Nature Cell Biology,2001,3:839-843
[72]Case ME,Schweizer M,Kushner SR,et al.Efficient transformation of Nelurospora crassa by utilizing hybrid plasmid DNA.Proceedings of the National Academy of Sciences of the United States of America,1979,76(10):5259-5263
[73]Jackson CW,Heale JB.Parasexual crosses by hyphal anastomosis and protoplast fusion in the entomopathogen Verticillium lecanii.Journal of General Microbiology,1987,133(12):3537-3547
[74]Inglis PW,Tigano MS,Valadares-inglis MC.Transformation of the entomopathogenic fungi,Paecilomyces fumosoroseus and Paecilomyces lilacinus(deuteromycotina:hyphomycetes) to benomyl resistence.Genetics and Molecular Biology,1999,22(1):119-123
[75]Bernier L,Cooper RM,Charnley AK,et al.Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance.FEMS Microbiology Letters,1989,60(3):261-266
[76]李增智.虫生真菌研究展望.安徽农学院学报,1987,3:61-72
[77]Fincham JR.Transformation in fungi.Microbiology and Molecular Biology Reviews,1989,53(1):148-170
[78]Pfeifer TA,Khachatourians GG.Beauveria bassiana protoplast regeneration and transformation using electroporation.Applied Microbiology and Biotechnology,1992,38(3):376-381
[79]Fungaro MHP,Rech E,Muhlen GS,et al.Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia.FEMS Microbiology Letters,1995,125(2):293-297
[80]Vianey OM,Carlos CP,Alfredo HE.Three decades of fungal transformation key concepts and applications.Methods in Molecular Biology,2004,267:297-313
[81]Ying SH,Feng MG.Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.Applied Microbiology and Biotechnology,2006,72(1):206-210
[82]St Leger RJ,Shimizu S,Joshi L,et al.Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants.FEMS Microbiology Letters,1995,131(3):289-294
[83]Bogo MR,Vainstein MH,Aragao FJ,et al.High frequency gene conversion among benomyl resistance transformants in the entomopathogenic fungus Metarhizium anisopliae.FEMS Microbiology Letters,1996,142(1):123-127
[84]Jiang Q,Ying SH,Feng MG.Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation.Journal of Microbiological Methods,2007,69(3):512-517
[85]Bundock P,Dulk-Ras AD,Beijersbergen A,et al.Trans-kingdom T-DNA transfer from Agrobacterium tumefaeiens to Saccharomyces cerevisiae.The EMBO Journal,1995,14(13):3206-3214
[86]De Groot MJ,Bundock P,Hooykass PJ,et al.Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnology,1998,16:839-842
[87]Cantone FA,John D.Genetic transformation and mutagenesis of the entomopathogenic fungus Paecilomyces fumosoroseus.Journal of lnvertebrate Pathology,1999,74(3):281-288
[88]Fang WG,Zhang YJ,Yang XY.Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker.Journal of Invertebrate Pathology,2004,85(1):18-24
[89]Duarte RTD,Staats CC,Fungaro MHP,et al.Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var.acridum.Letters in Applied Microbiology,2007,44(3):248-254
[90]Punt PJ,Oliver RP,Dingemanse MA,et al.Transformation of Aspergillus based on the hygromycin B resistance marker form Escherichia coli.Gene,1987,56(1):117-124
[91]Sandhu SS,Kinghorn JR,Rajak RC,et al.Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.Indian Journal of Experimental Biology,2001,39(7):650-653
[92]李维,张义正,以潮霉素B磷酸转移酶基因为遗传标记的启动子探针型载体的构建.四川大学学报(自然科学版),1999,36(4):736-740
[93]Thompson CJ,Movva NR,Tizard R,et al.Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.EMBO Journal,1987,6(9):2519-2523
[94]Cantone FA,Vandenberg JD.Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts..Journal of Invertebrate Pathology,1999,74(2):193-197
[95]Goettel MS,St Leger RJ,Bhairi S,et al.Pathogenicity and growth of Metarhizium anisopliae stale transformed to Benomyl resistance.Current Genetic,1990,17(2):129-132.
[96]Furlaneto MC,Paiao FG,Pinto FG,et aL Transformantion of the entomopathogenic fungus Metarhizium flavoviride to high resistance to benomyl.Canadian Journal of Microbiology,1999,45(10):875-878
[97]Frey PM.Biocontrol agents in the age of molecular biology.Trends in Biotechnology,2001,19(11):432-433
[98]Gressel J.Potential failsafe mechanisms against the spread and introgression of transgenic hypervirulent biocontrol fungi.Trends in Biotechnology,2001,19(4):149-154
[99]Charnley AK,Cobb B,Clarckson JM.Towards the improvement of fungal insecticides BCPC symposium proceedings.British Crop Protection Council,1997,68:115-126
[100]Gornova IB,Feofilova EP,Tereshina VM,et al.VM,Effect of carbohydrate content of Aspergillus japonicus spores on their survival in storage and subsequent germination.Mikrobiologiya,1992,61:549-554
[101]Harman GE,Jin X,Stasz TE,et al.Production of conidial biomass of Trichoderma harzianum for biological control.Biological Control,1991,1(1):23-28
[102]董志扬,方宣钧,林敏等.海藻糖的生物合成及抗逆机理.核农学研究进展,1996:115-120
[103]Goddijn WJM,Dun KV.Trehalose metabolism in plants.Trends in Plant Science,1999,4(8):315-319
[104]Pilon-Smits EAH,Terry N,Sears T.Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress.Journal of Plant Physiology,1998, 152(4-5):525-532
[105]Holmstrom KO,Mantyla E,Welin B,et al.Drought tolerance in tobacco.Nature,1996,397(6567):683-684
[106]王自章,张树珍,杨本鹏等.甘蔗根癌农杆菌介导转化海藻糖合酶基因获得抗渗透胁迫能力增强植株.中国农业科学,2003,36(2):140-146
[107]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses.Proceedings of the National Academy of Sciences of the United States of America,2002,99(25):15898-15903
[108]Avonce N,Leyman B,Thevelein J,et al.Trehalose metabolism and glucose sensing in plants.Transactions,2005,33:276-279
[109]Vogel G,Fiehn O,Boller T,et al.Trehalose metabolism in Arabidopsis:occurrence of treholase and molecular cloning and characterization of trehalose-6-phosphate synthase homologues.Jorunal of Experimental Botany,2001,52(362):1817-1826
[110]Blasquez MA,Santos E,Flores CL,et al.Isolation and molecular characterization of the Arabidopsis TPS1 gene,encoding trehalose-6-phosphate synthase.Plant Journal,1998,13(5):685-689
[111]Sambrook J,Russell DW.Molecular Cloning.New York:Cold Spring Harbor Laboratory Press,2001
[112]周勇,马玲.人卵透明带3蛋白的二级结构和B细胞抗原表位预测.新疆医科大学学报,2008,31(8):955-958
[113]肖军,吴季辉.杀菌肽及膜蛋白螺旋结构预测.生物物理学报,1994,10(2):281-288
[114]周颖君,杨亦桦,武淑文等.棉铃虫细胞色素P450 CYP9A12基因5'-上游区的克隆及序列分析.昆虫学报,2008,51(2):120-125
[115]王德培,孙伟,李明春等.利用长引物嵌套式反向PCR方法克隆雅致枝霉△6-脂肪酸脱氢酶基因上游序列.生物工程学报,2006,22(4):581-586
[116]胡新立,周迅蕾.Nodal基因5'末端侧翼序列启动子的分析.北京大学学报(自然科学版),2000,36(2):178-185
[117]张红缨,刘洋,张今.海藻糖的生物合成和相关酶的特性.微生物学通报,1998,25(4):236-238
[118]Goddijn OJM,Verwoerd TC,Voogd E,et al.Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.Plant Physiology,1997,113(1):181-190
[119]Becker K,Pan Dao,Whitley CB.Real-time quantitative polymerase chain reaction to assess gene transfer.Human Gene Therapy,1999,10(15):2559-2566
[120]Higgins JA,Ezzell J,Hinnebusch BJ.5' nuclease PCR assay to detect Yersinia pesits.Journal of Clinical Microbiology,1998,36(8):2284-2288
[121]Bieche I,Onody P,Laurendeau I,et al.Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical apolications.Clinical Chemistry,1999,45:1148-1156
[122]Wang XK,Li X,Currie RW,et al.Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1 beta mRNA in ischemic brain tolerance.Journal of Neuroscience Research,2000,59(2):238-246
[123]Higuchi R,Fockler C,Dollinger G,et al.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions.Biotechnology,1993,11(9):1026-1030
[124]张丽靖.球孢白僵菌孢子粉生产、制剂和贮存技术的改进及其淀粉酶特性测定:[硕士学位论文].杭州:浙江大学,2003
[125]Bateman R.The "MycoHarvester":cleaning up locust control.International Pest Control,2003,45:76-77
[126]Hong TD,Gunn J,Ellis RH,et al.The effect of storage environment on the longevity of conidia of Beauveria bassiana.Mycological Research,2001,105(5):597-602
[127]崔旭红,谢明,万方浩.高温胁迫下B型烟粉虱热激蛋白基因hsp70表达量的变化.昆虫学报,2007,50(11):1087-1091
[128]Overbergh L,Valckx D,Waer Mark,et al.Quantification ofmurine cytokine mRNAs using real time quantitative reverse transcriptase PCR.Cytokine,1999,11(4):305-312
[129]杜荣骞.生物统计学.北京:高等教育出版社,施普林格出版社.1999
[130]唐启义,冯明光.实验统计分析及其DPS数据处理系统.北京:科学出版社.2002
[131]Gault E,Michel Y,Dehee A,et al.Quantification of human cytomegalovirus DNA by Real-Time PCR.Journal of Clinical Microbiology,2001,39(2):772-775
[132]李尊华,林健文,马金成等.变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响.微生物学报,2008,48(7):887-892
[133]Liu Q,Ying SH,Feng MG,et al.Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus Beauveria bassiana to thermal stress.Antonie van Leeuwenhoek,2009,95:65-75
[134]Fillinger S,Chaveroche MK,Van-Dijck P,et al.Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans.Microbiology,2001,147:1851-1862
[135]Ocon A,Hampp R,Requena N.Trehalose turnover during abiotic stress in arbuscular mycorrhizal fungi.New Phytologist,2007,174(4):879-891
[136]马向东,郭永豪,余华顺等.三株酵母的抗热激性与tpsl基因表达的关系.郑州大学学报(理学版),2005,37(1):30-33
[137]Emmanuelle BM,Michel P,Francoise B,et al.Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae.Journal of Bacteroilogy,1998,180(5):1044-1052
[138]洪洄,王冬梅.应用酵母进行基因表达的研究进展.生物学通报,1999,34(7):12～14
[139]陈偿.甲醇毕赤氏酵母表达体系.热带农业科学,1999,2:3¨3
[140]Cregg JM,Madden KR.Development of the methylotrophic yeast,Pichia Pastoris,as a host system for the production of foreign proteins.Developments in Industrial Microbiology,1988,29(3):33-41
[141]Allais JJ,Louktibi A,Baratti J.Oxidation of methanol by the yeast Pichia pastoris:purification and properties of alcohol oxidase.Agricultural and Biological Chemistry,1983,47(11):2547-2554
[142]Cregg JM,Madden KR,Barringer KJ,et al.Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.Molecular and Cellular Biology,1989,9(3):1316-1323
[143]Cereghino JL,Cregg JM.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
[144]Thor D,Xiong S,Orazem CC,et al.Cloning and characterization of Pichia pastoris MET2gene as a selectable marker.FEMS Yeast Research,2005,5(10):935-942
[145]Laemmli UK.Cleavage of structural protein during the assembly of the head of bacterphage T4.Nature,1970,227:680-686
[146]Silva Z,Alarico S,Da Costa MS.Trehalose biosynthesis in Thermus thermophilus RQ-1:biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.Extremophiles,2005,9(1):29-36
[147]Romanos M.Advances in the use of Pichia pastoris for high level gene expression.Current Opinion in BiotechnoIogy,1995,6(5):527-533
[148]Hong F,Meinander NQ,Jonsson LJ.Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.Biotechnology and Bioengineering,2002,79(4):438-449
[149]Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene,1997,190(1):55-62
[150]Duman JG,Miele RG,Hong L,et al.O-Mannosylation of Pichia pastoris cellular and recombinant proteins.Biotechnology and Applied Biochemistry,1998,28(1):39-45
[151]Fang WG,Leng Bo,Xiao YH,et al.Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virulence.Applied and Environmental Microbiology,2005,71(1):363-370
[152]Fan YH,Fang WG,Xiao YH,et al.Directed evolution for increased chitinase activity.Applied Microbiology and Biotechnology,2007,76(1):135-139
[153]俞佳,冯明光.基于分生孢子热胁迫反应的球孢白僵菌耐热菌株筛选.菌物学报,2006,25(2):278-283
[154]Bell W,Sun W,Hohmann S,et al.Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex.Journal of Biological Chemistry,1998,273(50):33311-33319
[155]De Silva-Udawatta MN,Cannon JF.Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation.Molecular Microbiology,2001,40(6):1345-1356
[156]郭永豪,孙连海,余华顺等.啤酒酵母中海藻糖合成酶和海藻糖-6-磷酸合成酶的功能及结构分析.酿酒科技,2005,6:40-43
[157]黄惠芳,马飞.热激蛋白的分子进化研究.厦门大学学报(自然科学版),2004,43(8):166-170
[158]王笑虹,苗迎秋,宋光.热休克蛋白70生物学功能研究的新进展.大连大学学报,2003,24(2):103-105
[159]Piano A,Franzellitti S,Tinti F,et al.Sequencing and expression pattern of inducible heat shock gene products in the European flat oyster,Ostrea edulis.Gene,2005,361:119-126
[160]Landais L,Pommet JM,Mita K,et al.Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda.Gene,2001,271(2):223-231
[161]Nover L,Scharf KD.Heat stress proteins and transcription factors.Cellular and Molecular Life Sciences,1997,53(1):80-103
[162]李冰祥,蔡惠罗,陈永林.昆虫的热休克反应和热休克蛋白.昆虫学报,1997,40(4):417-427
[163]李晓鲁,彭毅志.热激蛋白70与热激反应.生命的化学,2002,22(2):145-147
[164]Tsan MF,Gao B.Cytokine function of heat shock proteins.American Journal of Physiology-Cell Physiology,2004,286:739-744
[165]Deane EE,Woo NYS.Cloning and characterization of the hsp70 mutigene family from silver sea bream:modulated gene expression between warm and cold temperature acclimation.Biochemical and Biophysical Research Communications,2005,330(3):776-783
[166]Rezaie S,Ban J,Mildner M,et al.Characterization of a cDNA clone,encoding a 70kDa heat shock protein from the derrnatophyte pathogen Trichophyton rubrum.Gene,2000,241(1): 27-33
[167]Demand J,Luders J,Hohfeld J.The carboxy-terminal domain of HSC70 provides binding sites for a distinct set of chaperone cofactors.Molecular and Cellular Biology,1998,18:2023-2028
[168]Vayssier M,Guerhier FL,Fabien JF,et al.Cloning and analysis of a Trichinella briotovi gene encoding a cytoplasmic heat shock protein of 72 kDa.Parasitology,1999,119:81-93
[169]Craven RA,Tyson JR,Stifling CJ.A novel subfamily of HSP70s in the endoplasmic reticulum.Trends in Cell Biology,1997,7:277-282
[170]焦传珍,王在照,李富花等.编码中国明对虾(Fenneropenaeus chinensis)一种组成型热休克蛋白70(HSC70)cDNA克隆、测序及其表达分析.科学通报,2004,49(21):2178-2186
[171]陈亮,张富春,黄萍等.光滑鳖甲热休克蛋白70基因的克隆及表达.昆虫学报,2007,50(9):883-888
[172]吴莹,徐香玲.植物热激转录因子的研究进展.哈尔滨师范大学自然科学学报,2005,21(2):73-78
[173]Morimotol RI.Regulation of the heat shock transcriptional response:cross talk between a family of heat shock factors,molecular chaperones,and negative regulators.Genes and Development,1998,12:3788-3796
[174]张伟,王登高.热休克基因转录的调节:热休克转录因子(HSF)的结构与功能.第三军医大学学报,2000,22(2):198-200
[175]陈晓红,印莉萍.结合于GATA元件的Ⅳ型锌指蛋白.生命的化学,1999,19(4):179-180
[176]张立国,张琚.实时定量PCR技术的介绍.生物技术,2003,13(2):39-40
[177]Sarrazin C,Cartner BC,Sizmann D,et al.Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 to 5.Journal of Clinical Microbiology,2006,44(3):729-737
[178]谢翎,陈红梅,蒲顺昌等.球孢白僵菌热休克蛋白基因Bbhsp 70的cDNA及上游序列克隆与分析.菌物学报,2009,28(2):283-288
[179]马旭,吕刚.HSP-70与细胞保护的研究进展.大连医科大学学报,2007,29(2):194-196
[180]Shyu WC,Kao MC,Chou WY,et al.Heat shock modulates prion protein expression in human NT-2 cells.Neuroreport,2000,11(4):771-774
[181]Saavedra CA,Hmmell CM,Heath CV,et al.Yeast heat shock mRNAs are exported through a distinct pathway defined by Riplp.Genes and Development,1997,11(21):2845-2856
[182]Vainberg IE,Dower K,Rosbash M.Nuclear export of heat shock and non-heat-shock mRNA occurs via similar pathways.Molecular and Cellular Biology,2000,20(11):3996-4005
[183]Benedetti AD,Baglioni C.Translational regulation of the synthesis of a major heat shock protein in HeLa cells.Journal of Biological Chemistry,1986,261(33):15800-15804
[184]Tamai KT,Liu X,Silar P,et al.Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways.Molecular and Cellular Biology,1994,14(12):8155-8165
[185]Ruis H,Schuller C.Stress signalling in yeast.Bioessays,1995,17(11):959-965
[186]Rensinga L,Monnerjahna C,Meyera U.Differential stress gene expression during the development of Neurospora crassa and other fungi.FEMS Microbiol Letters,1998,168(2):159-166
[187]Extrucha F.Stress-controlled transcription factors,stress-induced genes and stress tolerance in budding yeast.FEMS Microbiol Reviews,2000,24(4):469-486
[188]Hoffmann AA.Acclimation:increasing survival at a cost.Trends in Ecology and Evolution,1995,10(1):1-2
[189]Coleman JS,Heckathorn SA,Hallberg RL.Heat-shock protein and thermotolerance:linking molecular and ecological perspectives.Trends in Ecology and Evolution,1995,10(8):305-306
[190]杨艳,李增波.海藻糖TPS/TPP生物合成途径的进化研究.科技情报开发与经济,2008,18(16):123-125
[191]Elbein AD,Pan YT,Pastuszak I,et al.New insights on trehalose:a multifunctional molecule.Glycobiology,2003,13(4):417-427
[192]Paul MJ,Primavesi LF,Jhurreea D,et al.Trehalose metabolism and signaling.Annual Review of Plant Biology,2008,59:17-41
[193]Wang Y J,Hao Y J,Zhang ZG;et al.Isolation of trehalose-6-hosphate phosphatase gene from tobacco and its functional analysis in yeast cells.Journal of Plant Physiology,2005,162(2):215-223
[194]马辉,刘桂丰,徐晨曦等.二色补血草海藻糖-6-磷酸磷酸酯酶基因的克隆及分析.东北林业大学学报,2008,36(8):1-3