【作者】 权瑞党；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 发育生物学， 2003， 博士

【摘要】 基因枪轰击法和农杆菌介导法是植物遗传转化的两大主流方法。农杆菌介导法与基因枪轰击法、电穿孔法、PEG法等其它DNA直接转移方法相比具有很大的优越性,例如可以转移大片段DNA,整合的外源基因拷贝数较少,并且基因重排频率较低。玉米作为一种单子叶植物不是农杆菌的天然宿主,农杆菌介导的玉米转化难度较大,其发展落后于农杆菌介导的双子叶植物的转化,目前玉米遗传转化的常用方法在大多数实验室仍为基因枪轰击法。尽管农杆菌介导的玉米模式种质A188自交系和Hi-Ⅱ杂交种（来源于A188×B73）的转化获得突破,建立了高效的转化体系,但是具有生产价值的玉米骨干自交系大部分难以通过农杆菌介导法转化。而且随着玉米分子生物学的发展,玉米基因组学研究也需要一种高效的玉米遗传转化方法。因此,建立农杆菌介导的玉米骨干自交系高效遗传转化体系不仅具有理论意义,而且具有重要的实用价值。 在本研究中,以农杆菌LBA4404侵染玉米骨干自交系齐319、掖515、掖502、DH4866等胚性愈伤组织,经过潮霉素筛选后由抗性愈伤组织再生植株,自交结实。研究发现,侵染时农杆菌悬浮液浓度、共培养时间、真空渗入处理、愈伤组织部分酶解和超声波处理等能影响转化率。 农杆菌悬浮液浓度OD₆₀₀0.5-0.7,侵染5 min,共培养3-4天,掖515和齐319胚性愈伤组织的转化率较高（约6%）。而增加或减少农杆菌悬浮液浓度,延长或缩短共培养时间,转化率均降低。原因可能是增加农杆菌悬浮液浓度使吸附在愈伤组织上的农杆菌数目增加,延长共培养时间使农杆菌增殖增加,但同时伴随着玉米愈伤组织死亡率的增加;而减少农杆菌悬浮液浓度,吸附在愈伤组织上的农杆菌数目减少,玉米细胞转化率下降;缩短共培养时间可能造成农杆菌不能有效完成T-DNA的转移和整合过程。 农杆菌侵染玉米胚性愈伤组织时,在50kPa下真空渗入5min,掖515和更多还原

【Abstract】 Microprojectile bombardment and Agrobacterium-mediated transformation are two main methods for plant transformation. As compared with other direct DNA transfer methods such as microprojectile bombardment, electroporation and polyethylene glycol-mediated transformation methods, Agrobacterium-mediated transformation facilitates the delivery of larger segments of foreign DNA, and results in the integration of low numbers of gene copies into the plant chromosome and relatively few gene rearrangements. Maize, a monocotyledonous plant, is not a natural host of Agrobacterium. Therefore, Agrobacterium-mediated maize transformation is relatively difficult, and its development lags behind Agrobacterium-mediated dicotyledons. At present, in most laboratories the method routinely used for maize transformation is microprojectile bombardment. Breakthroughs in Agrobacterium-mediated transformation of model maize germplasms including A188 inbred line and Hi-II (with A188xB73 background) hybrid have been made, and efficient transformation systems have been established thereof. However, most elite maize inbred lines with agronomic value are recalcitrant to transformation mediated by Agrobacterium. Moreover, with the advances in maize molecular biology, the research of maize genome will require an efficient transformation system. Thus, the establishment of an efficient transformation system of maize elite inbred lines mediated by Agrobacterium is not only of theoretical significance, but also of great value for agriculture.In this study, embryogenic calli of elite maize inbred lines Qi319, Ye515, Ye502 and DH4866 were infected by Agrobacterium tumefaciens LBA4404, and fertile plants were recovered from the resistant calli after selection by hygromycin. The results . demonstrated that the transformation efficiency was infected by the concentration ofAgrobacterium suspension, the duration of co-cultivation, vacuum infiltration, partial enzymolysis of calli and sonication.With the concentration of Agrobacterium at OD600 0.5-0.7, the duration of infection of 5 min. and the duration of co-cultivation between 3-4 days, the transformation efficiency of embryogenic calli of Ye515 and Qi319 was highest (about 6%). However, the transformation efficiency decreased when the concentration of Agrobacterium suspension was increased or decreased, and the duration of co-cultivation was prolonged or shortened. The reasons are probably that the number of Agrobacterium attached to calli increased with the increase of the concentration of Agrobacterium, and the proliferation increased with the elongation of the duration of co-cultivation, meanwhile, the mortality rate of infected calli increased. Whereas the reduction in the concentration of Agrobacterium suspension resulted in the decrease in the number of Agrobacterium attached to calli, the transformation efficiency of maize cells decreased; and the process of T-DNA transfer and integration might not have been accomplished when the duration of co-cultivation was shortened.Vacuum infiltration at 50 kPa for 5 min during infection of maize embryogenic calli by Agrobacterium increased the transformation efficiency of Ye515 and Ye502 from 6.3% and 4.5% to 8.7% and 7.8%, respectively. This might result from the fact that assisted with vacuum infiltration during infection Agrobacterium could enter into the inside of calli, and was beneficial to more calli cells to contact with Agrobacterium, and to increase the chances of transformation of maize cells.Partial enzymolysis of embryogenic calli with 0.2% Macerozyme R-10 for 10 min before infection improved transformation efficiency of Ye515 and Ye502 to 8.3% and 8.9%, respectively. This might result from the fact that the partial enzymolysis of maize calli could degrade cell wall, increase intercellular space, and stimulate cell division, all of which were in favor of the attachment and infection of plant cells by Agrobacterium.After sonication for 90-120 s at 100 W during infection of embryogenic calli, the transformation efficiency of was up to 9.1% and 9.4% for Ye515 and Ye502, respectively. This might result from the fact that the sonication of maize calli resulted inmicro-wounding both on the surface of and deep within the target tissues, and the formation of crannies within plant tissues was beneficial to the intrusion of Agrobacterium into the inside of calli to transform inner cells.The above results demonstrated that the transformation efficiency of embryogenic calli of elite maize inbred lines mediated by Agrobacterium was improved by the optimization of the concentration of Agrobacterium suspension and the duration of co-cultivation, and by the assistance of vacuum infiltration, partial enzymolysis and sonication.Abiotic stresses such as drought, salt and low temperature are main factors limiting the yield and the quality of crops. So it is necessary to breed new crop varieties that are more tolerant to these abiotic stresses so that new land can be brought under cultivation to meet the progressively demand of the society. The problem with traditional plant breeding for achieving this end is that it is time consuming and laborious; it is difficult to modify single traits; and it relies on existing genetic variability. However, genetic engineering can now be used as a relatively fast and precise means of achieving improved stress tolerance. The most consistently successful approach is the introduction of genes encoding enzymes that catalyse the conversion of a naturally occurring substrate into a product with osmoprotective properties, and the most studied method is the introduction of glycine betaine synthesis pathway into plants so that the transgenic plants could accumulate higher levers of glycine betaine and acquire enhanced stress tolerance.In this study, betA gene from Escherichia coli coding for choline dehydrogenase was transferred to elite maize inbred lines mediated by Agrobacterium tumefaciens LBA4404. PCR and Southern analysis of the genomic DNA from transgenic plants in the first and subsequent generations showed that foreign genes have integrated into maize genome. The analysis of five transgenic lines of elite maize inbred DH4866 indicated that four transgenic lines accumulated higher levels of glycine betaine and improved tolerance to chilling and drought stresses.In the third generation of transgenic DH4866, five lines with good agronomicaltraits were selected to detect the tolerance to chilling and drought stresses. In these five lines, the level of betaine in the leaves of line 1 was 1.5 umol [g FW]"1 (FW: fresh weight), which was slightly higher than that of non-transgenic control (wild type) plants with 1.2 umol [g FW]"1, whereas the levels of betaine in leaves of lines 2, 3, 4 and 5 were 2.5-4.0 umol [g FW]’1. The betaine level in seeds of non-transgenic control was 2.0 umol [g DW]1 (DW: dry weight), and that of line 1 was 2.3 umol [g DW]’\ but betaine levels in seeds of line 2, 3, 4 and 5 were 4.1-5.8 umol [g DW]*1. The levels of choline, the substrate of the synthesis of betaine catalyzed by choline dehydrogenase, were not affected by the expression of foreign genes.At 25 °C, the germination rate of transgenic and non-transgenic seeds was all above 95%, and did not show significant difference. At 15 °C, the germination of seeds of transgenic lines 2, 3, 4 and 5 was ahead of that of non-transgenic control and transgenic line 1 for about two days. At 10 °C, the final germination rate of line 1 was reduced to 35%, and those of lines 2, 3, 4 and 5 were reduced to 60-80%, however, the final germination rate of non-transgenic control was reduced to 25%.At 25 °C, the shoot growth rate of geminated seeds was not significantly different. At 15 °C, the shoot growth rate decreased to about 60% as compared to 25 °C, but showed no significant difference between transgenic and non-transgenic plants. However, in comparison with that of 25 °C, the shoot growth rate at 10 °C of lines 2, 3, 4 and 5 was about 20% and that of non-transgenic plants was only 10%.After seedlings at three-leaf stage were subjected to 10 days of chilling treatment at 10/5 °C (day/night), the chilling injury of lines 2, 3, 4 and 5 was significantly less than that of non-transgenic control. When these chilling treated seedlings were transferred to 25 °C for 10 days, the survival percentage of lines 2, 3, 4 and 5 was more than 30% higher than that of non-transgenic control.After seedlings at three-leaf stage were subjected to 10 days of chilling treatment at 10 °C, cell membrane damage of lines 2, 3, 4 and 5 was 20% lower than that of non-transgenic control. The photosystem II activity and net photosynthesis rate of lines 2, 3, 4 and 5 were more than 20% higher than those of non-transgenic control after更多还原