里氏木霉RUT—C30 β—甘露聚糖酶的诱导表达及其cDNA的克隆
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摘要
在里氏木霉Rut C-30的基础培养基(Mandels营养液)中加入固定碳源乳糖和槐豆胶，然后将可变碳源(云杉纤维、玉米芯纤维、麦杆纤维、麦杆木聚糖、玉米芯木聚糖、云杉甘露聚糖)进行单因子、双因子、三因子、四因子、五因子的里氏木霉Rut C-30正交培养实验，并以槐豆胶为底物用3，5二硝基水杨酸法测定培养液中β—甘露聚糖酶的活力。从而确定了酶活最高且菌体生长良好的含云杉纤维、麦杆木聚糖和云杉甘露聚糖的诱导培养基为最佳培养基，用该培养基培养的里氏木霉(T.reesei)Rut C-30使其转录的β-甘露聚糖酶(β-1，4-mannan mannohydrolase EC 3.2.1.78)mRNa量能够满足RT-PCR的要求。
分取诱导培养液中的菌体，用异硫氰酸胍法提取总RNA，总RNA再经无RNA酶的DNA酶处理后用于RT—PCR。在PCR扩增目的基因时，通过优选扩增体系，使镁离子浓度为1.25mM时RT—PCR可顺利地获得目的基因，并能定向克隆到载体pGEM—3Z(amp~r)中。
用克隆载体转化宿主大肠杆菌JM109，通过筛选获取阳性克隆子，对阳性克隆子进行酶切与PCR鉴定，并对载体中插入的目的基因进行测序。其测序结果与genbank中的里氏木霉Rut C-30β-甘露聚糖酶成熟肽cDNA序列进行序列同源性比较，结果二者序列完全一致。
This experiment passing to grope for the carbon source constitutes of the culture medium and using T. Reesei Rut C-30 induced the expression of #-mannanase ( # -1,4-Mannan mannohydrolase EC 3.2.1.78).In this experiment I put the constant carbon source(lactose and locust bean gum) in the foundation culture medium (Mandels nourishment liquid) of T. Reesei Rut C-30,then proceeded the variable carbon source(dragon spruce fiber, com rush pith fiber, wheat straw fiber, wheat straw xylan, corn rush pith xylan, dragon spruce mannan) to single factor,double factor,three factor,four factor and five factor orthogonal experiment.1 determined the activity of P -mannanase using locost bean gum as substract by the 3,5-dinitosalicylic acid method,and observed the growing situation of the gernic At the end I selected the directions for the inducement expression of the #?mannanase from Trichoderma reesei Rut-C30 that contained the dragon spruce fiber, wheat straw xylan, dragon spruce mannan.
The total RNA was purified from the germ in the liquid by the guanidine isothiocyantehod method, then the total RNA digested by DNase that had not RNase was used for RT-PCR.I change the magnesium ion dencity in the PCR system in order to optimize the PCR condition.At the end I selected the magnesium ion density as 1.25 mM.The production of RT-PCR was inserted directionally into pGEM?Z(ampr).The pGEM?Z(ampr) was used to transform E coli JM109.I got a positive clone through culling and identificatin.The DNA sequence inserted into pGEM?Z(ampr) was sequenced and blasted with the cDNA sequence of the # -mannanase mature peptide that got from Genbank. The result of the alignment was 100%.
分取诱导培养液中的菌体，用异硫氰酸胍法提取总RNA，总RNA再经无RNA酶的DNA酶处理后用于RT—PCR。在PCR扩增目的基因时，通过优选扩增体系，使镁离子浓度为1.25mM时RT—PCR可顺利地获得目的基因，并能定向克隆到载体pGEM—3Z(amp~r)中。
用克隆载体转化宿主大肠杆菌JM109，通过筛选获取阳性克隆子，对阳性克隆子进行酶切与PCR鉴定，并对载体中插入的目的基因进行测序。其测序结果与genbank中的里氏木霉Rut C-30β-甘露聚糖酶成熟肽cDNA序列进行序列同源性比较，结果二者序列完全一致。
This experiment passing to grope for the carbon source constitutes of the culture medium and using T. Reesei Rut C-30 induced the expression of #-mannanase ( # -1,4-Mannan mannohydrolase EC 3.2.1.78).In this experiment I put the constant carbon source(lactose and locust bean gum) in the foundation culture medium (Mandels nourishment liquid) of T. Reesei Rut C-30,then proceeded the variable carbon source(dragon spruce fiber, com rush pith fiber, wheat straw fiber, wheat straw xylan, corn rush pith xylan, dragon spruce mannan) to single factor,double factor,three factor,four factor and five factor orthogonal experiment.1 determined the activity of P -mannanase using locost bean gum as substract by the 3,5-dinitosalicylic acid method,and observed the growing situation of the gernic At the end I selected the directions for the inducement expression of the #?mannanase from Trichoderma reesei Rut-C30 that contained the dragon spruce fiber, wheat straw xylan, dragon spruce mannan.
The total RNA was purified from the germ in the liquid by the guanidine isothiocyantehod method, then the total RNA digested by DNase that had not RNase was used for RT-PCR.I change the magnesium ion dencity in the PCR system in order to optimize the PCR condition.At the end I selected the magnesium ion density as 1.25 mM.The production of RT-PCR was inserted directionally into pGEM?Z(ampr).The pGEM?Z(ampr) was used to transform E coli JM109.I got a positive clone through culling and identificatin.The DNA sequence inserted into pGEM?Z(ampr) was sequenced and blasted with the cDNA sequence of the # -mannanase mature peptide that got from Genbank. The result of the alignment was 100%.
引文
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