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1、 毕业论文农杆菌介导的水稻转化体系的优化摘要水稻是世界上最重要的粮食作物之一，为约40%的人口提供了主要食粮。虫害是造成水稻减产减收的主要原因之一。水稻虫害种类繁多，我国每年因虫害造成的产量损失占到水稻总产量的 5% 以上，给水稻的生产带来了极大危害。目前，我国控制水稻虫害最主要的方法是使用化学杀虫剂，但化学防治带来一系列问题，如环境污染，生产成本提高和食用安全性降低等。因此，通过培育自身能够抵抗虫害的水稻品种来防治水稻虫害无疑是最经济安全的有效策略。从已有的研究来看，仅依靠常规育种难以解决抗虫种质资源稀缺的问题，因此，通过基因工程手段导入外源抗虫基因，进而培育抗虫转基因水稻越来越受到人们的重
2、视。近年来转Bt基因抗虫水稻已经在生产上得到了广泛的应用，随着抗虫转基因水稻的大规模种植，Bt基因抗虫谱相对狭窄与害虫易产生耐受性等问题相继引起了人们的关注。因此，从自然界中分离克隆出更多、更有效的基因成为解决这些问题的方法之一。籼稻是亚洲栽培稻的重要亚种，全世界水稻栽培品种中有80%以上属于籼稻，其组织培养研究具有重要意义。而籼稻的组培特性普遍较差，再生率很低，导致一些生产上应用价值较大的优良品种转化仍然难以成功或转化频率低，因而研究优质籼稻的愈伤组织形成和植株再生能力对于遗传转化率提高十分必要。农杆菌介导法因具有转化拷贝数低等独特的优势，而广泛应用于转基因抗虫水稻研究中。然而，水稻不同品种
3、间遗传差异性比较大，且转化反应对水稻基因型依赖性强，从而增加了农杆菌介导的水稻遗传转化的复杂性和随机性。因此，通过对农杆菌介导的水稻转化体系的优化提高转化率十分必要。本研究主要进行了以下方面的工作：1. 在新型Bt基因Cry30Fa1和Cry54Aa1序列的两端分别增加酶切位点序列，送基因合成公司合成，然后将目的基因片段与中间克隆载体pUPROK分别进行双酶切，连接，获得含目的基因的中间载体。测序鉴定得到序列正确的阳性载体，用切酶切下目的基因片段并插pCDMAR-Hyg载体，最终获得高效表达双元载体pCDMAR-Cry30Fa1-Hyg与pCDMAR-Cry54Aa1-Hyg，并通过酶切和测序
4、的方法鉴定出上述载体的正确性。2. 通过对愈伤组织继代次数与继代培养基中加入抗坏血酸（Vc）浓度的优化，调整愈伤组织生长状态，提高胚性愈伤组织数量。结果表明R125与R818的愈伤组织继代2-3次，分化率最高；继代培养基中加入Vc浓度为40mg/L，能减少愈伤组织褐化率且对其分化能力无影响。3. 通过对洗菌后与分化前愈伤组织的干燥处理，改良农杆菌介导的水稻遗传转化体系。结果表明洗菌后进行干燥与分化前愈伤组织在滤纸上干燥2天，能够加快分化速度，提高分化率。4. 农杆菌介导法将外源基因Cry30Fa1与Cry54Aa1转入三系恢复系R125与R818中，通过PCR检测与潮霉素溶液检测转基因植株。结
5、果表明Cry30Fa1与Cry54Aa1转R125未获得转基因阳性植株；Cry54Aa1转R818也未获得转基因阳性植株；Cry30Fa1转R818共获得233株转基因再生植株，经过潮霉素溶液检测和分子检测，其中46株为含目的基因的阳性植株。关键词：籼稻；Cry30Fa1基因；Cry54Aa1基因；抗虫；农杆菌介导法AbstractRice is one of the most important grain crops in the world, for about 40% of the worlds population taking it as their staple food. Ho
6、wever，insect damage is one of the main reasons to decrease the rice yield. There are a large variety of rice pests in China. The rice production loss is more than 5% of total yield because of insect damage. At present, the main method to control rice pest is to utilize chemical pesticides. However,
7、it caused a lot of problems, such as environmental pollution, higher production cost, food safety, etc. Breeding new rice varieties that produce insecticidal proteins by themselves is undoubtedly the most economical and security strategy. On the basis of previous reports, it is difficult to solve th
8、e problem by traditional breeding because of rice germplasm resources are scarce. Given this, it is more important to use genetic engineering to breed resistant rice.In recent years, pest resistant rice with Bt genes in production have already been on a wide range of applications. With large-scale c
9、ultivation of insect-resistant transgenic rice, Bt gene zoophobous spectrum is relatively narrow and tolerance to pests and other problems have been attracted the attention of people. Therefore, separating and cloning new pest resistant genes from the natural world became more and more effective met
10、hods to solve these problems.Indica rice is a major subspecie of Asian cultivated rice, and more than 80% of the rice cultivars in the world belongs to indica rice, so it is important significance to study tissue culture. Tissue culture of indica rice is generally poor and regeneration rate is very
11、low, causing some production value on a large varieties of transformation are still difficult to successfully transform or frequency low. Therefore, the study of high quality indica rice callus formation and plant regeneration ability of genetic transformation rate is necessary.Agrobacterium tumefac
12、iens-mediated transformation has technique advantages including low copies, and is widely used in the study of insect-resistant transgenic rice. However, the relatively large genetic differences between different varieties of rice, and the transformation strong dependence on rice genotypes, increase
13、 complexity and randomness of the Agrobacterium tumefaciens-mediated transformation in rice. Therefore, the optimization of Agrobacterium tumefaciens-mediated rice transformation system to improve transformation rates is necessary.This research has the following main areas of work:1. Seqences of new
14、 Bt genes Cry30Fa1 and Cry54Aa1 that had been added cleavage site sequences at both ends, were sent to gene company to synthesis. Then all of target gene fragments and middle cloning vector pUPROK were cut separately by two enzymes and connected to form middle carriers of the target gene fragments.
15、Positive vectors were got by sequences identification, and were digested by endonuclease to separate target gene fragments. Then target gene fragments were inserted into pCDMAR-Hyg vector to form eventual expression binary vectors pCDMAR-Cry30Fa1-Hyg and pCDMAR-Cry54Aa1-Hyg. All of positive plasmids
16、 were identified by digestion and sequencing.2. By optimizing callus subculture time and different concentration of Ascorbic acid (Vc) in subculture media, callus growth state was adjusted and embryo callus were increased. Results showed that when R125 and R818 callus were subcultured 2 to 3 times, the differentiation rate was the highest; Subculture media with 40 mg/L Vc could re
