【作者】 罗园园；

【导师】 赵艳；

【作者基本信息】 浙江工商大学 ， 生物化工， 2011， 硕士

【摘要】 全世界盐碱地面积约占世界总耕地面积的20%。由于干旱,不合理的灌溉以及化肥的过度使用,工业污染加剧,土壤盐碱化正不断扩大,可供耕种的土地面积正日趋减少。水稻是世界1/3人口的主食,但一直处于供不应求的状况,因此,提高水稻产量,改良稻米品质关系到人类的生存和发展。在盐碱地区,利用转基因手段来获得高耐盐的转基因水稻,推广具有广泛适应性的水稻耐盐优异品种,是开辟盐碱荒地和水稻增产的一个新增长点,目前运用最广泛的转基因水稻方法之一是农杆菌介导转化法。研究建立农杆菌介导耐盐基因高效水稻转化系统具有重要理论和实际指导意义。根据外源基因是耐盐基因的特点,建立农杆菌介导无选择标记耐盐基因水稻转化系统对转基因水稻商品化应用具有重大意义,避免了抗性标记基因可能存在对食品和生态环境的潜在危害。制约农杆菌介导转化水稻效率的主要因素是周期长和转化受体的状态。本文从这两方面入手,建立高效农杆菌介导转化水稻系统。根据外源基因是耐盐基因,探讨NaCl作为筛选剂可行性,但传统确定NaCl筛选浓度需要绘制NaCl浓度梯度对愈伤组织增重率之间的生长曲线。此过程比较繁琐且容易造成污染和误差。探讨利用NaCl对水稻种子发芽率的抑制效果作为确定水稻耐盐基因转化中愈伤组织筛选所需NaCl使用浓度的可行性,同时比较NaCl与潮霉素的筛选效果。最后,将两者有机结合,对不同类型的耐盐基因进行农杆菌介导无标记高效转化水稻,证明该系统是完全可行和有效的。主要实验结果如下：(1)快速诱导水稻愈伤组织的建立在32℃暗培养7天诱导水稻愈伤组织的快速诱导系统,日本晴、秀水11、,春江06的出愈率均在60%以上,分别是92.87%、82.63%、64.83%；在28℃光培养7天诱导水稻愈伤组织的快速诱导系统,日本晴、秀水11、春江06的出愈率均在70%以上分别是95.5%、93.07%、74.87%。愈伤表面突起,呈淡黄色,具有活力,完全符合转化的要求,从传统的愈伤组织诱导需要15天缩短到7天。(2)高效农杆菌介导转化水稻的建立采取热激处理,中华11、秀水11、春江06转化效率提高率分别在58.50%、21.96%、27.85%；液体共培养感染,三种水稻转化率提高有60.57%、23.54%、25.17%,建立高效农杆菌介导转化水稻。(3)简便获得NaCl有效筛选浓度法的建立根据常规的NaCl浓度梯度对愈伤组织增重率之间的生长曲线,确定愈伤组织增重抑制率为80%左右的NaCl浓度为合适的筛选浓度,此相应NaCl浓度对四个品种水稻种子的发芽抑制率均达50%以上。因此,可选取对水稻种子的发芽抑制率达50%以上的NaCl浓度作为耐盐基因转化中筛选时NaCl有效浓度的重要参考。(4)证明转化耐盐基因中NaCl筛选的有效性和可行性根据选取的外源基因为耐盐基因,以NaCl作为筛选剂,达到了筛选的目的,对中华11来说,NaCl筛选下转化率为11.98%,潮霉素筛选下转化率为14.43%；对秀水11来说,NaCl筛选下转化率为32.06%,潮霉素筛选下转化率为42.64%；对春江06来说,NaCl筛选下转化率为32.03%,潮霉素筛选下转化率为41.39%,说明NaCl筛选效果与潮霉素筛选相近。(5)安全高效农杆菌介导转化水稻的建立采用NaCl筛选与高效农杆菌介导转化水稻相结合,发现秀水11转化效率提高率29.9%,建立安全高效农杆菌介导转化水稻。(6)证明安全高效农杆菌介导耐盐基因转化日本晴的可行性和有效性选取不同耐盐机制的耐盐基因,将其作为外源基因采用建立的农杆菌介导无选择标记耐盐基因高效转化水稻系统对日本晴进行转化,实验表明：BADH、CMO、PKCC的转化效率均在40%以上,分别是44.03%、46.29%、44.68%,证明农杆菌介导无标记耐盐基因高效转化日本晴是可行和有效的。综上所述,安全,快速,高效的农杆菌介导耐盐基因转化水稻系统已经建立。即32℃光培养7天后,进行43℃水浴30min,冰浴1min,移至事先浸润液体共培基的三层滤纸上暗培养2天,转至含盐筛选培养基,进行筛选,植株再生的水稻转化系统。更多还原

【Abstract】 Saline land area accounts for 20% of the total arable land in the world. Because of the drought, unreasonable irrigation and fertilizer overuse, aggravation pollution of industry, the area of soil salinization is unceasingly enlarging, available area for farming land is decreasing. Rice is about one-third of the world population’s stable, the yield of the rice has been in the shortage of supply, therefore, increase the rice yield, improve the rice quality relate to human survival and development. High salt tolerance of transgenic rice and extensive adaptability of rice varieties through transgenic technologies are new points of utilization salinity-alkalinity desert and increasing rice yield. Currently one of the most widely applied rice transformation technologies is Agrobacterium mediated transformation. Establishment of high efficient Agrobacterium mediated salt tolerance gene transformation of rice has important theoretical and practical guiding significance. According to salt tolerant character, establishment of high efficient Agrobacterium mediated marker-free salt tolerance gene transformation of rice is of great significance to apply commercialization, and avoid the potential hazards of the resistance marker genes that may exist on food and the ecological environment.The shortcomings of transformation efficience of Agrobacterium mediated transformation are the long cycle and transformation receptor state. This paper establishes high efficient Agrobacterium mediated transformation rice system starting from the two facts. According to salt tolerant character, NaCl used as a screening agent is explored, but traditional determine NaCl screening concentration needs to draw the growth curve between NaCl concentration gradient and callus weight increase ratio. This process is tedious, easy to pollute, and prone to errors. The feasibility of determine NaCl screening concentration through NaCl inhibition effect to rice seed germination is explored, meanwhile screening effect of NaCl is compared with hygohycine. Finally, with high efficient Agrobacterium mediated marker-free various types of salt resistance gene transformation of rice, this system is proved completely feasible and effective. The experimental results are as follows:(1)Establish a high efficient Agrobacterium mediated transformation rice systemIt is found after 32℃dark culture 7d, the callus induction rate of Nipponbare, Xiushui 11 and Chunjiang 06 are 60%above,92.87%,83.62%,64.83% respectively; after 28℃light culture 7d, the callus induction rate of Nipponbare, Xiushui 11 and Chunjiang 06 are 70%above,95.5%,93.07%,74.87%respectively. Callus surface protuberant, pale yellow, vigor that fully meet the transformation requirements, the time for callus induction shorten from 15d to 7d.(2) Establish high efficient Agrobacterium-mediated transformation of riceZhonghua 11, Xiushui 11 and Chunjiang 06 transformation efficiency improve rate are 58.50%,21.96%,27.85%with heat shock treatment; Zhonghua 11, Xiushui 11 and Chunjiang 06 transformation efficiency improve rate are 60.57%,23.54%,25.17%with liquid co-culture infection, efficient Agrobacterium-mediated transformation rice is established.(3)Establish a Simple selection method of effective screening concentration of NaClAccording to the growth curve between NaCl concentration gradient and callus weight increase ratio, it is showed when the weight gain inhibition rate reaches 80%, NaCl concentration appropriate for screening, the same as NaCl concentration when seed germination inhibition rate by 50%or more. Therefore, NaCl concentrations when seed germination inhibition rate by 50%as an important reference for screening in salt resistance genes transformation.(4) NaCl screening in salt-tolerant gene transformation is effective and feasibleTransformation effect of NaCl as screening agent is compared with hygohycine. For Zhonghua 11, the transformation efficient of NaCl screening is 11.98%, and hygohycine is 14.43%; For Xiushui 11, the transformation efficient of NaCl screening is 32.06%, and hygohycine is 42.64%; For Chunjiang 06, the transformation efficient of NaCl screening is 32.03%, and hygohycine is 41.39%, which prove NaCl screening is feasible and effective.(5)Establish safe and high effective Agrobacterium mediated transformation of riceAdopted NaCl screening and high efficient agrobacterium-mediated transformation rice combination, it is found Xiushui 11’s transformation efficiency improving rate is 29.9%, safe and high effective Agrobacterium mediated transformation of rice is established.(6) Safe and high effective Agrobacterium mediated salt-tolerant gene transformation of Nipponbare is feasible and effectiveChoosing salt resistance genes of different salt resistance mechanisms as the exogenous gene using Agrobacterium mediated marker-free salt resistance gene transformation of Nipponbare, the results show that the transformation efficiency of BADH, CMO, PKCC are all above 40%, respectively 44.03%,46.29%,44.68%, which proof Agrobacterium-mediated marker-free salt resistant gene transformation is feasible and effective.All the results suggest that:safe, fast and efficient Agrobacterium mediated salt resistance gene transformation rice system has been established:callus after 32℃light training for 7d,43℃water-bath 30min, ice bath beforehand 1min, moved to three-layer filter paper infiltrated liquid co-culture medium on dark for 2d, turn to salt screening medium, and plantlet regeneration.更多还原