Creating Transgenic Maize Materials Suitable For Cellulosic Ethanol Production By Transgenic Approaches

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.