多靶向沉默里氏木霉碳代谢阻遏物对纤维素酶活性和表达的调控研究

【目的】构建多靶向siRNA表达载体对里氏木霉碳阻遏抑制因子CRE1、CRE2、CRE3和CRE4进行同时多靶向siRNA干扰,以研究其对里氏木霉纤维素酶基因表达的调控作用。【方法】根据此前研究筛选出沉默cre1、cre2、cre3和cre4基因的4个最佳siRNA序列,设计并构建了A多靶向表达载体,另根据cre1、cre2、cre3和cre4基因中所含有的5个共有序列设计并构建了B多靶向表达载体,将两者转化至里氏木霉QM9414。经筛选后分别在48 h和120 h对各转化子进行纤维素酶酶活力测试(CMC活力测试和滤纸酶酶活力测试)及利用qPCR检测相关基因的表达。【结果】通过RT-qPCR测定结果表明,两种表达载体均可同时抑制里氏木霉的分解代谢物阻遏基因cre1、cre2、cre3和cre4的表达,纤维素酶活力比出发菌株明显升高,多靶向抑制菌株的CMC酶活和滤纸酶活比出发菌株平均提高了1.95倍和2.66倍。纤维素酶基因cbh1和egl1的表达水平比出发菌株也有明显提升,平均提高了3.83倍和3.95倍。纤维素酶相关基因xyr1的表达水平与出发菌株相比也明显上升,平均提高了2.78倍。【结论】多靶向沉默里氏木霉的碳代谢阻遏蛋白有利于解除葡萄糖效应,提高非还原糖的利用,从而提高纤维素酶的产量,使纤维素酶的表达得到更大的提升,为里氏木霉表达纤维素酶在分解代谢物阻遏基因调控方面提供了实验依据和新的技术思路。     

沉默里氏木霉组蛋白赖氨酸甲基转移酶基因对纤维素酶表达的影响

【背景】里氏木霉(Trichoderma reesei)是一种比其他真菌小很多的多细胞真核微生物,在工业上受到广泛应用,而里氏木霉QM9414是目前研究最多基因产纤维素酶丰富的突变菌株。【目的】构建里氏木霉中组蛋白赖氨酸甲基化酶(Histone Lysine Methyltransferase)基因hkmt的siRNA沉默载体和过表达载体来降低或者增强hkmt在里氏木霉QM9414中的表达量,以分析其对里氏木霉纤维素代谢的调控作用。【方法】根据里氏木霉hkmt序列设计siRNA沉默片段并用反转录的方法获得过表达hkmt片段。将沉默片段和过表达片段克隆至里氏木霉组成型表达载体中,构建沉默hkmt的载体和过表达hkmt的载体,并将其转化里氏木霉QM9414。通过荧光显微镜观察重组菌的菌丝生长情况,此外对各重组菌进行纤维素酶的滤纸酶活性(Filter Paper Enzyme Activity,FPA)和羧甲基纤维素钠酶活性(Carboxymethyl Cellulose Enzyme Activity,CMCA)的测试;利用荧光定量PCR的方法检测hkmt、纤维素酶基因cbh1、egl1及木聚糖酶激活因子xyr1的表达量变化。【结果】通过使用荧光显微镜观察,发现沉默、过表达hkmt重组菌的菌丝形态均与出发菌株无明显差异。荧光定量PCR测定结果表明,沉默载体和过表达载体可以分别沉默和促进hkmt的表达。沉默hkmt重组菌株中FPA和CMC酶活力相比出发菌平均升高2.5倍。此外,纤维素酶相关基因和激活因子在沉默hkmt重组菌中的表达量均有所增加,但是在过表达hkmt重组菌株中以上相应指标均呈现相反的趋势。【结论】组蛋白赖氨酸甲基转移酶基因表达产物负调控里氏木霉产纤维素酶基因的表达,这为提高里氏木霉产纤维素酶水平提供了参考,并为里氏木霉产纤维素酶的表观遗传调控研究提供了新的证据。     

敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

[背景]里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一,表观遗传调控是不涉及DNA序列变化的可遗传变化,组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase,HDAC)负责脱乙酰化,敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。[目的]通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase,hdac)建立了里氏木霉hdac缺失突变株(T.reesei△hdac),以研究对纤维素酶基因表达的调控作用。[方法]利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern blotting验证正确后,对突变体T.reesei△hdac连续7 d检测滤纸酶活(filter paper activity,AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity,CMCA),利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。[结果]突变体T.reesei△hdac两种酶活力均显著高于出发菌株,分别高出8.00、30.00 IU/mL。突变体T.reesei△hdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T.reesei QM9414的6.50、6.01和4.51倍。[结论]里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控,这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。     

里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定

为了后续研究里氏木霉(Trichoderma reesei)纤维素酶基因的表达与调控,利用overlap PCR及分子克隆技术构建了含有Col E1原核复制起始位点、氨苄青霉素抗性、里氏木霉的丙酮酸脱羧酶启动子、丙酮酸脱羧酶终止子、潮霉素B抗性的筛选标记并能表达增强型绿色荧光蛋白(Zs Green)的表达载体p LXT-Zs Green。将该载体转化里氏木霉QM9414原生质细胞,使用潮霉素B筛选平板得到阳性转化子,随后使用荧光显微镜在488 nm激发光下观察菌丝,并随机挑取4个转化菌株进行Western-blot验证。结果显示,里氏木霉菌丝体可发出明亮的绿色荧光,而且Western-blot验证了该载体能够在里氏木霉中有效地表达增强型绿色荧光蛋白。上述研究表明,载体p LXT-Zs Green在里氏木霉中能够稳定高效地表达外源基因,为研究里氏木霉的基因表达调控奠定了实验基础。     

同源过表达mhr2基因可提高嗜热毁丝霉纤维素酶活性

为寻找新型的与纤维素酶相关转录调控因子,以嗜热毁丝霉(Myceliophthora thermophila ATCC42464)为研究材料,通过克隆嗜热毁丝霉mhr2基因序列,构建重组过表达载体,转化并筛选到转化子Mt O24中mhr2基因表达量比野生型菌株高204倍。蛋白浓度及酶活测定的结果显示,诱导培养72 h,转化子胞外蛋白浓度和滤纸酶活分别是野生菌的1.58和1.30倍;非诱导培养144 h,转化子胞外蛋白浓度和滤纸酶活分别是野生菌的1.87和1.49倍。实时荧光定量PCR的结果表明,转化子中主要纤维素酶基因egl1、egl3和cbh1、cbh2的表达量均有显著提高。研究初步证实了mhr2基因具有调控纤维素酶基因表达的功能。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

强启动子glaA介导cbhB基因在黑曲霉中的表达

黑曲霉纤维素酶三类不同酶系中,纤维二糖水解酶基因表达处于很低水平,导致纤维素酶总体活力水平不高。为构建黑曲霉纤维素酶高产菌株,采用基因工程方法,全基因合成拼接黑曲霉高表达葡萄糖淀粉酶基因glaA的强启动子片段与纤维二糖水解酶基因cbhB编码区片段,然后将杂合基因克隆到二元载体pCAMBIA1301上,重组质粒通过农杆菌介导转化黑曲霉分生孢子,携带杂合基因的T-DNA片段插入到黑曲霉转化子的染色体上,共筛选到48个具有潮霉素抗性的转化子。纤维素酶活力水平测定结果显示,转化子A3-9的CMC酶活力最高,为野生型黑曲霉菌株的1.31倍;转化子B1-7与A3-6的滤纸酶活力最高,为野生型黑曲霉菌株2.51倍。另外,初步分析了杂合基因在黑曲霉中的表达所需的诱导条件。     

丝状真菌里氏木霉中shRNA沉默红色荧光基因

报道了在里氏木霉中建立的一种以红色荧光蛋白(DsRed)为报告基因的RNA干扰方法。首先,将构建的表达DsRed质粒p ANRed1转化里氏木霉QM9414,得到抗潮霉素B抗性并能稳定表达DsRed的菌株DsRed-T.reesei。其次,以丙酮酸脱氢酶启动子Ppdc和纤维二糖水解酶I终止子cbh I为原件,克隆到载体p PHL上构建质粒p PHL-Ppdc-Tcbh1。根据DsRed基因序列设计特定的siRNA干扰序列和另一条无同源序列的siRNA作为阴性对照,克隆到载体p PHL-Ppdc-Tcbh1得到重组质粒。将其转化到DsRed-T.reesei中,用含有100μg/m L潮霉素B和250μg/m L腐草霉素的PDA平板筛选转化子。结果表明,约79%的转化子出现红色荧光沉默现象,其中一些转化子DsRed的表达几乎完全被抑制。荧光定量PCR和Western印迹分析显示DsRed基因的表达受到不同程度的下调。以上结果提示,在里氏木霉中可用此方法研究基因表达调控。     

里氏木霉中小分子RNA对纤维素酶表达调控的作用

目的:通过过表达小分子RNA和抑制小分子RNA功能,研究小分子RNA对里氏木霉QM9414纤维素酶表达的影响。方法:利用含里氏木霉强启动子Ppdc的质粒p-Ppdc'-Tcbh1,分别构建过表达载体和Tough Decoy(Tu D)序列,对里氏木霉QM9414的3个小分子RNA(mil R5、mil R7和mil R10)进行过表达和抑制,过表达和抑制后的小分子RNA表达量通过荧光定量PCR检测,最终测定转化菌株的滤纸酶活,研究过表达和抑制小分子RNA后对纤维素酶表达的影响。结果:过表达载体能提高小分子RNA的表达量,过表达重组菌株OE-mil R7的滤纸酶活明显提高;Tu D序列表达载体可抑制小分子RNA的功能,抑制小分子RNA的重组菌株Tu D7酶活略有下降。结论:mil R7可能参与纤维素酶表达调控,为对里氏木霉的产纤维素酶能力进行改造提供了新的靶点。     

siRNA介导的ERA1表达下调对拟南芥抗旱性的影响

ERA1是控制植物气孔开闭的一个重要基因,根据其保守域构建RNA干扰(RNAi)载体并转化拟南芥,考察转基因植株的生长、气孔导度、离体叶片失水率以及ERA1和相关基因表达,探讨siRNA介导的ERA1表达下调对拟南芥抗旱性的影响。结果表明:转基因拟南芥株系中ERA1的表达受到明显抑制,其离体叶片失水率低于野生型,但并未出现ERA1缺失突变体的负面生长表型;转基因株系对ABA处理比野生型更敏感,其ABA处理株的根长显著变短,气孔孔径更小;转基因株ABI1、ABI2、ATHB6的表达量降低,而RAB18、RD29B、ADH1的表达量升高,siRNA介导的ERA1表达下调可能会激活RAB18、RD29B等逆境响应元件。研究发现,采用RNAi技术可以有效下调ERA1表达,在没有过多负面生长表型的前提下提高拟南芥的抗旱性,且ERA1表达下调可能通过ABA途径正面影响拟南芥的抗旱性。     

Contributions of XyIR, CcpA andcre to diauxic growth ofBacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulatexyl operon expression on diauxic growth and expression of axylA-lacZ fusion.xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation ofxylR yields a two-fold increase in expression ofxylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. WhenxylR andcre are inactivated together a residual two-fold repression ofxylA is found. Inactivation ofxylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion ofccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivatedcre site inxylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Therapeutic potential of BAK gene silencing in aluminum induced neural cell degeneration

Previous studies have demonstrated robust BAK gene silencing via RNA interference (RNAi). To investigate whether BAK RNAi may serve as a co-therapeutic agent in neural cell death, we herein established a cell degeneration model using a human neuroblastoma cell line (SH-SY5Y) treated by aluminum (Al). Combining cell viability assays and expression analyses by QRT (quantitative real-time)-PCR and immunocytochemistry, we selected and validated the optimal small interfering RNA (siRNA) from three candidate siRNAs for the BAK gene. Our data identified siRNA1 as the most effective siRNA; the optimal concentration of the transfection agent was 10 nM and the optimal incubation period was 24 h. The transfection and knockdown efficiency was 93% and 58%, respectively, which closely correlated with the BAK protein expression. SH-SY5Y cells with BAK knockdown showed a clear resistance against cell death and Al-induced apoptosis. These results indicate that genetic inactivation of BAK could be an effective strategy in delaying the onset of apoptosis in Al-treated cells, and exemplify the therapeutic potential of RNAi-based methods for the treatment of neural cell degeneration.     

Agroinfiltration as a Tool for Transient Expression of <Emphasis Type="Italic">cre</Emphasis> Recombinase <Emphasis Type="Italic">in vivo</Emphasis>

Agroinfiltration was used to express transiently cre recombinase from bacteriophage P1 in planta. Activation of gfp expression after cre-mediated excision of a bar intervening sequence served as a marker to monitor site-specific recombination events in lox-target N. benthamiana plants. Gfp expressing regenerants from A. tumefaciens infiltrated leaves were obtained with an efficiency of about 34%. In 20% of the regenerants bar gene excision was due to the expression of stably integrated cre gene, whereas in 14% of plants site-specific recombination was a consequence of transient cre expression. Phenotypic and molecular data indicated that the recombined state has been transferred to the T₁ generation. These results demonstrate the suitability of agroinfiltration for the expression of cre recombinase in vivo.     

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.     

香菇HMG-box转录因子lelcrp1基因的功能

【目的】研究香菇(Lentinula edodes) HMG-box转录因子LELCRP1 (Lentinula edodes lignocellulase genes regulation protein 1)在木质纤维素降解相关酶基因表达中的功能与作用。【方法】通过double-joint及同源重组方法构建lelcrp1基因RNAi载体,采用根癌农杆菌介导转化的方法转入香菇异核菌株W1菌丝中,筛选得到RNAi转化子,通过Southern杂交检测插入片段在菌株W1基因组中的拷贝数量。采用荧光定量PCR检测RNAi转化子木质纤维素降解酶基因表达水平变化,并在含有3.5μg/mL潮霉素的MYG平板上测定RNAi转化子的菌丝生长速度。【结果】获得了4个lelcrp1基因表达水平与出发菌株W1相比显著下调6–7倍的RNAi转化子。Southern杂交结果显示,lelcrp1基因RNAi片段已成功整合至香菇菌株W1基因组内,并以单拷贝形式存在。对其中2个RNAi转化子的26个木质纤维素降解酶基因表达水平进行分析,发现其中9个纤维素酶基因、1个半纤维素酶基因、2个辅助酶AA9基因和1个锰过氧化物酶基因的表达水平均表现出明显的下调。平板生长试验表明,RNAi转化子菌丝生长速度均显著慢于出发菌株W1。【结论】通过RNAi技术成功抑制了香菇异核菌株中lelcrp1基因表达水平,并导致部分纤维素及木质素酶基因表达水平相应下调,首次发现HMG-box结构域的转录因子能调控木质纤维素降解相关酶基因表达。     

粘虫颗粒体病毒增效蛋白在苏云金芽胞杆菌中的表达及增效活性

【目的】在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中表达截短后的转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus-Ps, PuGV-Ps)增效蛋白,为构建增效Bt工程菌提供理论基础。【方法】通过对截短后增效蛋白的密码子进行优化,构建增效蛋白及其融合蛋白表达载体,分析不同启动子指导下增效蛋白表达量的变化,明确增效蛋白对Bt的增效活性。【结果】本研究构建了表达载体pHTP_cry1AcCoEn81、 pHTRHCoEn81和pHTNCCoEn81, SDS-PAGE结果显示pHTP_cry1AcCoEn81和pHTNCCoEn81分别可以产生81 kDa和134 kDa的重组蛋白。启动子Pcry1Ac和P_cry8E指导下的增效蛋白表达量和重组增效蛋白产量均无显著性差异。生物测定结果表明,重组增效蛋白可以显著增加Bt对小菜蛾的杀虫活性。【结论】研究结果表明,密码子优化的PuGV-Ps增效蛋白可以在Bt中表达并具有显著增效活性,为高效苏云金芽胞杆菌工程菌的构建及...