【作者】 张淑娟；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2011， 博士

【摘要】 由于化石能源的不可再生性和最终枯竭的不可避免性,能源危机已经成为世界性的问题。可再生的清洁的生物质能源已成为人们关注的热点,其中燃料乙醇的研究与生产最引人注目。目前,燃料乙醇主要是用玉米、甘蔗、薯类等农作物发酵生产,成本过高,广泛应用受限。寻找新的生产原料和技术、降低燃料乙醇的生产成本,已成为国内外热点研究课题。玉米作为一种饲料、粮食和能源作物,具有很高的综合利用价值。其作为C4植物具有高光合速率和生长速率、能高效利用水资源、适应区广、生育期较短等特点,是既产粮食又产生大量生物质的生产者。利用玉米淀粉生产燃料乙醇因“与人争粮”而面临尴尬,而利用玉米生物质生产乙醇可充分利用玉米的生长特点,具有广阔的前景。玉米生物质的主要成分是木质纤维素,而利用木质纤维素生产乙醇由于生产成本一直居高不下而阻碍了其规模化生产。利用纤维素酶的催化反应代替高温高压的化学反应将纤维素水解为葡萄糖,再通过发酵可生产乙醇等有机化工原料和燃料等在实践中存在较大困难。主要原因之一是纤维素酶的生产及木质纤维素预处理过程成本非常高。若能降低纤维素水解复合酶的应用成本则使纤维素乙醇的生产成本大幅度降低。通过植物基因工程的方法来生产纤维素酶、半纤维素酶以及通过改变木质素含量或成份来减少预处理过程都是可解决这些问题的途径。本论文工作的主线是利用转基因技术培育能自身表达高活性纤维素酶的转基因玉米植株和培育低木质素的有利于纤维素酶降解茎叶的玉米材料,以获得适宜纤维素乙醇生产的玉米新种质,为降低生物质生产乙醇的成本而探索。(一)在玉米中过表达微生物基因编码的纤维素酶依据已发表资料,从丝状真菌瑞氏木霉中克隆了编码外切葡聚糖酶基因的CBHI和编码内切葡聚糖酶基因的EGⅢ。为了控制转基因表达,克隆出丝状真菌构巢曲霉(Aspergillus nidulans)的乙醇诱导型启动子αlc系统。测序结果表明,后者碱基序列与已报道的序列有所差异,但是转录起始位点、TATA box和与转录因子alcR结合位点中的高度保守序列位置及序列均完全相同。用获得的启动子构建带有GUS融合基因的植物表达载体,利用基因枪法轰击玉米胚芽鞘进行瞬时表达分析,染色结果确定了该启动子具有乙醇诱导表达的功能。将纤维素酶基因和乙醇诱导启动子构建的融合基因插入到植物表达载体中,通过农杆菌介导的玉米芽尖转化法转入玉米,获得了转基因的玉米株系。对转基因玉米植株的后代进行PCR和Southern杂交分析,肯定了外源基因在玉米基因组中稳定整合并遗传给后代,转基因拷贝数为1-2个。分别检测3叶期和10叶期的转基因玉米,了解乙醇诱导启动子在玉米中能否启动诱导表达,以及不同乙醇浓度、诱导时间、持续诱导等因子对乙醇诱导基因表达强度的影响。在玉米植株中,alc系统可以严谨的控制目的基因的表达。在非诱导状态下,alc系统在转基因玉米中处于关闭状态,通过根部浇灌酒精后可启动目的基因的表达。该系统非常灵敏,用0.1%(v/v)酒精诱导就能检测到外源基因的表达。采用MUC的方法测定转基因植株CBHI酶活性,得出酶活性变化与转基因表达强度的变化是一致的。随着浇灌的酒精浓度升高,转基因植株CBHI酶活性增加,在2%(v/v)时达到最高峰,24h时酶活性达到2400 pmol MU min-1 mg TSP-1,之后酶活性不再升高。因此,选用2%的酒精浓度作为最佳浓度进行后续试验。该浓度酒精对玉米植株无可见的毒害作用。不同酒精诱导时间的试验得出,随着诱导时间的延长,1nRNA及酶活性均有升高,最高点在96小时,酶活性达到4000 pmol MU min-1 mg TSP-1。若在第一次诱导72小时后再施用酒精进行诱导,酶活性能进一步提高,达到5000 pmol MU min-1 mg TSP-1,这种水平可一直持续。2%的酒精诱导24小时之后,转基因玉米的不同器官都检测到酶活性,介于1278 pmol MU min-1 mg TSP-1到2026 pmol MU min-1 mg TSP-1,以叶中的酶活性最高。用2%(v/v)的酒精以根部浇灌的方式诱导转EGⅢ基因植株,随着酒精诱导时间的延长,EGⅢ基因表达强度和酶活性逐步升高。无论是在酒精诱导前后,转基因植株和未转基因对照植株的生长发育和形态特征未出现显著差异。转基因玉米植株产生的纤维素酶在不加任何保护剂的情况下分别在不同pH值和不同温度下测定酶活性,并与最高的酶活性相比较,计算其相对活性(%)。外切葡聚糖酶CBHⅠ在pH5.0、温度45-50℃时较为稳定,而内切葡聚糖酶在pH4.5-5.0、温度为50℃时较为稳定。与大肠杆菌中表达的酶相比,具有更高的热稳定性及更宽广的pH值适宜范围。将转基因植株叶片提取液分别在50℃和60℃下保温,两种纤维素酶的活性都随着保温时间延长而下降,变化趋势较为一致。在50℃条件下放置2h,酶活性能够保持在80%左右,而在60℃条件下放置1h时,酶活性就降低到40%以下。因此,转基因玉米表达的纤维素酶可以耐受较长时间的50-60℃高温,具有一定的热稳定性。将转基因玉米叶片在不同温度下保存,然后测定其酶活性。两种酶活性变化趋势类似,在常温(28℃)条件下保存时,1天后酶活性降低到最初的85%左右,3天时降低到63%左右,5天的时为41%左右。若将叶片保存在-20℃,5天后酶活性为最初活性的85%。因此,转基因玉米植株可以在低温储存条件下保持较长时间纤维素酶活性。将两种纤维素酶液混合,用于降解纤维素,用DNS法测定还原糖的生成量。结果表明,两种纤维素酶在降解纤维素方面具有协同作用,两种酶一同降解纤维素的效果要比单酶降解效果好,能够产生更多的还原性糖。并且随着加入的酶蛋白增多,还原性多糖的生成量增加。(二)玉米木质素合成酶基因的抑制表达对木质素含量的影响木质素合成是由多个酶促反应组成的复杂过程,肉桂醇脱氢酶(CAD夕和肉桂酰辅酶A还原酶(CCR)基因编码木质素合成途径中催化最后两步的关键酶。以玉米为材料,从cDNA中克隆出CAD和CCR基因,分别构建它们的RNAi结构。利用载体pFGC5941构建出由组成型启动子CaMV35S启动RNAi结构和以除草剂抗性基因bar为筛选标记的植物表达载体,通过农杆菌介导的玉米芽尖转化方法获得玉米转化植株。经过PCR检测和Southern杂交鉴定,确定了RNAi结构已整合到玉米的基因组中且稳定遗传。采用RT-PCR方法检测靶基因的表达,得出未转基因对照植株和转基因植株均出现扩增产物,转基因植株的扩增产物亮度明显弱于未转基因对照的。采用Real-time RT-PCR检测靶基因的表达强度,得出不同转基因株系的表达丰度有一定差异。在抑制CAD表达的转RNAi结构株系中,CAD基因表达丰度为对照植株的40%～89%。在抑制CCR表达的转RNAi结构株系中,CCR基因表达丰度为对照植株的36%-94%。即RNAi结构的导入,转基因植株的内源CAD和CCR基因的转录本减少。转基因植株的酶活性测定表明,在抑制CAD基因的株系中酶活性是对照植株的65%-93%,在抑制CCR基因的株系中酶活性是对照的59%-90%。在这些转基因株系中CAD和CCR基因的表达变化与酶活性的变化趋势一致,表明是RNAi结构的表达导致这两个基因的表达下降而引起植株CAD或CCR酶活性降低。对转基因植株性状观测发现,转CAD基因株系与野生型对照相比,株高和叶片大小大致相同,抗倒性和抗病性变化不明显,开花和结籽正常。在转CCR基因的株系中,有些株系的植株与野生型大致相同,也有部分株系的植株与野生型相比株高偏低,但开花结籽正常,抗倒性和抗病性无明显变化。说明转基因植物中木质素足以维持植物正常的生理活动。对生长3个月的灌浆期转基因植株进行Klason木质素分析,与对照植株相比,转基因株系的Klason木质素都有不同程度的下降,占细胞壁干重19-20%。RNAi抑制CCR基因表达对木质素含量的影响要强于CAD的抑制作用。在转CAD基因株系中,木质素减少的变化范围为7.5-14.7%；在转CCR基因株系中,木质素减少的变化范围为11.7-18.9%。可见应用RNAi技术能有效抑制靶基因表达,进而降低转基因植株的木质素含量,但降低幅度仍待提高。在转基因植株中,尽管木质素含量下降,但综纤维素含量与对照相比并没有明显差别,说明木质素合成被抑制没有影响综纤维素的合成和积累。在本实验中,无论是干扰CAD还是干扰CCR的表达,在转基因植株中都能检测到PAL转录本丰度明显提高,与之对应的是可溶性总酚含量明显提高。植物体内大多数酚类物质的合成经历苯丙烷类合成途径,PAL是该条途径的关键酶,PAL表达强度的提高和可溶性酚类物质的增加表明细胞碳代谢可能发生重大调整。有可能木质素合成的下游步骤被限速,刺激了植株产生相应的反应,促进中间物向酚类物质合成途径分配,以保证有足量得可溶性糖类物质流入细胞次生代谢。由于酚类物质是植保素的最重要成员,其含量增多意味着植物抗病能力的提高。阿魏酸在细胞壁的积累可能是由于CAD及CCR活性降低的结果,很可能是木质素含量下降而植株抗病性和抗倒性无明显变化的原因。从转基因玉米植株表型来看,木质素适当降低可以维持玉米植株的正常生理活动和生长发育。综上所述,αlc系统在转基因玉米中能够严谨的控制外源基因的表达,这是在单子叶植物中运用化学可诱导系统成功控制外源基因表达的一个成功实例,为玉米基因功能研究提供了一个有用的工具。本工作得到了能在转基因植株中表达异源纤维素酶和降低了木质素含量的玉米骨干自交系工程株系,为进一步利用玉米生物质进行燃料乙醇的生产提高了重要资料和植物材料。更多还原

【Abstract】 As the fossil-fuel is non-renewable and will be inevitably exhausted, energy crisis has become a global issue. There is now emphasis on developing technologies that enable a sustainable, cost-effective biofuels to reduce the world’s dependence on non-renewable resources. Biofuels such as bioethanol are becoming a viable alternative to fossil fuels. But the bioethanol production costs were too high nowadays with the sugarcane、potato and other crops and can not be widely applied. Searching for new raw materials and technologies to reduce the cost of the bioethanol production has drawn much interest both at home and abroad. Maize as a feed、food and energy crop, has high utilization value. Besides its high biomass production and large areas planted annually, it is the C4 plants which has high photosynthetic and growth rates with shorter growth period, and could efficiently use water resources. Ethanol derived from corn grain is the most common renewable fuel today. However, concerns related to land delineation and distribution of maize grown for energy versus food was raised. Utilizing the maize biomass for bioethanol production could take full use of maize growth characteristics with broad prospects. The main component of maize is lignocellulose, whose cost for bioethanol production has been high and hinders the large-scale application.Hydrolysis of cellulose to glucose using cellulase enzymes instead of chemical reaction of high temperature and pressure, and finally through fermentation that converting sugars into ethanol or other chemical materials has difficulties in practice. One major reason is the high cost of the current cellulase production systems and lignocellulose pretreatment technology. If we can lower the cellulase application cost, the costs of cellulosic ethanol production could then be significantly reduced. Heterologous expression of cellulases and other plant-cell-wall-degrading enzymes in plants or reducing lignin content with lessen pretreatments through plant genetic engineering may be the solvents of these problems.Based on the problems proposed above, transgenic approaches were used to create new transgenic maize materials in the present study. Biological activity of the heterologous cellulases was produced in transgenic maize plants, and low-lignin maize plants which could help cellulase degradation of plant materials were also obtained. The new maize materials may be suitable for cellulosic ethanol production, which could help reduce the overall processing cost. The main results of this research were summarized as follows:(一) Heterologous expression of microbial cellulases in transgenic maizeBase on the published data, we cloned the Trichoderrna reesei exo-1, 4-β-glucanase gene (CBHI) and endo-1,4-β-glucanase gene (EGⅢ). And the alc gene-expression system derived from the filamentous fungus Aspergillus nidulans was used to flexibly control the expression of the target genes. The sequencing results showed that there are some differences in the base sequences, but the transcription start sites、TATA box and the highly conserved sequences in the location of the alcR transcription factor binding sites are identical. The plant expression vector which contained the alc promoter driving the beta-glucuronidase (GUS) fusion protein was constructed and then transformed into the maize coleoptile using the particle bombardment. The staining results confirmed that the obtained promoter had the ability to drive the gus expression after ethanol induction. The cellulase genes fused to the alc promoter were constructed in the plant expression vector respectively and transgenic maize plants was produced by Agrobacterium-mediated transformation of injured shoot tips. Stable integration of the transgene in the maize genome was confirmed by PCR and Southern blot analysis. The results showed that the foreign gene was integrated into maize genome and transmitted to the progenies stably, and the copy numbers of integrated foreign gene was one or two.We characterized the gene expression pattern in transgenic maize plants at two different development stages (three-leaf stage and ten-leaf stage) under the control of the ethanol-inducible promoter. Induction experiments with different ethanol concentration、induction time and sustained induction were performed in detail. Under the conditions tested, the alc regulon was functional and could be tightly regulated in maize. In transgenic plants, the level of non-induced expression mediated by the alc regulon is negligible. On the application of the ethanol inducer through root drench, alc-mediated expression was rapidly induced. The system is very sensitive to ethanol, induction being observed on application of 0.1%(v/v) ethanol. The enzyme activity of CBHI was examined by the MUC method, and the changes of enzyme activity and transgene expression is consistent. The expression level was proportional to the concentrations of ethanol applied. The highest enzyme activity at 2400 pmol MU min-1 mg TSP-1 was observed with the 2%(v/v) ethanol concentration, and no longer increased after then. Therefore,2%(v/v) was chosen as the optimum concentration for the further experiments. This ethanol concentration had no visible toxic effects on maize plants. The results of different induction time showed that the mRNA level and the enzyme activity increased as time prolonged, and the highest enzyme activity was observed at 4000 pmol MU min-1 mg TSP-1 after 96h ethanol application. The second ethanol application was perfomred after 72h of the first ethanol application and made the expression level continue rising to a higher level, nearly 5000 pmol MU min-1 mg TSP-1, and this level could be sustained. After 2%(v/v) ethanol application, several parts of mature plants were assayed separately to determine the enzyme activity after 24h induction. All tissue samples showed MUCase activity, from 1278 pmol MU min-1 mg TSP-1 to 2026 pmol MU min-1 mg TSP-1, and the leaves had the highest level of activity.The EGIII transgenic plants were examined with 2%(v/v) ethanol application according to the ethanol induction conditions of CBHI, and the transgene expression and the enzyme activity was also increased as the ethanol concentration elevated and induction time prolonged. And in this study, we did not observe any obvious deleterious effects on the growth and development of transgenic maize plants both prior to and after the ethanol application compared with control maize plants. Cellulase produced from transgenic maize plants were extracted and measured at different pH and different temperatures respectively, and the relative activity was calculated and compared with the highest activity which was defined as 100%. The exo-glucanase CBHI was relatively stable at pH 5.0 and 45-50℃, while endo-glucanase EGIII was relatively stable at pH4.5-5.0 and 50℃. The enzymes had higher thermo-stability and wider pH optimum ranges compared to the E.coli expressed enzymes. The enzymes extracted from the transgenic maize plants were incubated at 50℃and 60℃for different time intervals and the enzyme activities were examined. Both of the two enzymes’activities decreased with time prolonged. The enzyme activity could maintain at 80% with 50℃for 2h, and below 40% with 60℃for 1h. Thus, the cellulase enzyme of transgenic plants could tolerant natural environment of 25-37℃and higher temperature at 50-60℃with shorter time to maintain certain stability. In addition, the leaves of maize plants were preserved under different conditions, and were then used to determine the enzyme activity. The enzymatic activity of CBHI and EGIII were similar, after 1 day,3day and 5 day storage at 28℃, was respectively 85%,63% and 41% of the initial activity. However, the enzyme activity could still remain 85% of the initial activity after storage at -20℃for 5 days. Therefore, cellulase enzymes of the transgenic maize plants could maintain longer period of enzymatic activity under the freezing circumstances.We mixed the enzyme extracts from transgenic maize plants that expressed CBHI or EGIII for cellulose degradation, and the reducing sugars content was determined by the DNS method. The results showed that the reaction produced much higher reducing sugars than that of with either extract alone, which meant the two enzymes in combination could effectively hydrolyze crystalline cellulose in a synergistic fashion. Moreover, higher reducing sugars were observed with more TSP amounts added.(二) RNAi-mediated suppression of lignin biosynthetic pathway enzymes in maize impacts lignin contentLignin biosynthesis is a complex process composed of many enzymatic reactions. Cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) genes encode two key enzymes of the last two steps of the lignin biosynthetic pathway. We cloned the two genes from the maize cDNA and constructed their plant expression RNAi vector respectively. The pFGC5941 vector with the constitutive promoter CaMV35S and the selective marker gene (bar) was constructed and then transformed into maize by the Agrobacterium-mediated transformation of injured shoot tips. The PCR and Southern blot analysis confirmed that the RNAi fragments were integrated into the maize genome and transmitted to the progenies stably. The RT-PCR results showed the amplified bands in both of the transgenic and WT plants, but the brightness of the PCR products from transgenic plants was significantly weaker than that of the WT plants. The Real-time RT-PCR analysis showed that the transgene transcript abundance of the target genes differed between different transgenic lines. RNAi-CAD down-regulated maize lines showed differential CAD expression levels from 40% to 89% of the WT plants, while RNAi-CCR down-regualted maize lines showed differential CCR expression levels from 36% to 94% of the WT plants. This indicated that the decreased CAD and CCR endogenous transcripts were as a result of the introduction of the RNAi fragments. The CAD enzymatic activity of transgenic plants was 65%-93% of the WT control plants, while the CCR enzymatic activity of transgenic plants was 59%-90% of the WT plants. The enzymatic activity was consistent with the changes in their transgene expression level, which showed that expression of the RNAi construct decreased the CAD and CCR gene expression that leading to the reduction of enzymatic activity.Phenotype observation showed that RNAi-CAD down-regulated maize plants exhibited roughly the same plant height and leaf size、unobvious lodging and disease resistance and could normally blossom and set seeds. Some of the RNAi-CCR down-regulated maize plants showed the similar phenotype with WT plants, and some were lower than WT plants, but they could normally blossom、set seeds and had unobvious lodging and disease resistance. This suggested that the residual lignin contents of the transgenic plants were sufficient to maintain the normal physiological activities. Transgenic plants cultivated for 3 month at filling stage were analyzed with Klason lignin analysis. Compared with the WT plants, the transgenic plants had different levels of decreased Klason lignin contents accounting for 19-20% of the cell wall dry weight. The impacts of decreased lignin contents by RNAi the CCR transgene expression was stronger than that of the CAD gene. And the reduction of lignin contents ranged from 7.5% to 14.7% in the CAD down-regulated maize plants, while the reduction of lignin contents ranged from 11.7% to 18.9% in the CCR down-regulated maize plants. It can be seen that the RNAi technology could effectively suppress the target gene expression, regulate the lignin biosynthesis and further reduce the lignin content of transgenic plants, but the reduction extent need to be improved. In addition, in the present study, transgenic lines with lower lignin contents had the similar holocellulose content with the WT control lines, which indicating that the suppression of lignin synthesis had no effect on the holocellulose synthesis process.In the experiment, significant enhanced PAL expression was examined in both the CAD and CCR down-regulated maize plants, and the total soluble phenol content was correspondingly increased. Most of the phenolic compounds synthesis in plants was related to the phenylpropanoid pathway, and PAL was the key enzyme in the pathway. The enhanced PAL expression and the increased total phenolic content might suggest the redistribution of the carbon metabolism. The downstream steps of lignin synthesis may be rate-limited, sitmulating the plant to response correspondingly and promote the intermediate compounds to the phenolic compounds distribution, which could ensure a sufficient quantity of soluble sugars flowing into the secondary metabolism. Since phenolic compounds are the most important member of the phytoalexin, whose elevated content means the increased disease resistance. The accumulation of ferulic acid in cell walls may be due to the decreased CAD and CCR activity, which is likely to be the reason of low-lignin content with unobviously lodging and disease resistance. From the view of the phenotype of transgenic maize plants, appropriately reduce the lignin content could maintain normal physiological activities of the plants.In summary, the ale system could strictly controll the transgene expression in the maize plants, which is the successful example of using chemical induction system controlling the foreign gene expression and thus provides a powerful tool for studying the gene function in maize plants. We obtained the transgenic maize materials with heterologous expression of cellulase enzymes and maize inbred lines with lower lignin content, which could be the foundation for the further use of transgenic maize biomass for bioethanol production.更多还原