论文标题:农杆菌介导海藻糖合酶基因遗传转化甘蔗研究
Trehalose Synthase Gene Genetic Transformation of Sugarcane(Saccharum officinarum L.) Mediated by Agrobacterium tumefaciens
论文作者 王自章
论文导师 李杨瑞;陈如凯,论文学位 博士,论文专业 作物栽培学与耕作学
论文单位 福建农林大学,点击次数 144,论文页数 96页File Size5524k
2001-04-01论文网 http://www.lw23.com/lunwen_14443757/ 海藻糖合酶基因,农秆菌介导遗传转化,甘蔗,干旱诱导性启动子
Trehalose synthase gene; Agrobacterium turn efaciens meidated genetic transformation; Sugarcane; Tobacco; Drought responsive promoter; Trehalose-6-phosphate synthase gene
甘蔗是我国重要的工业原料作物，但85％左右的种植面积是旱地，而各蔗区每年或轻或重、或早或迟都要受到季节性干旱的影响，干旱是限制我国甘蔗生产的首要环境因素。甘蔗是高度杂合、遗传背景复杂的无性繁殖作物，常规的抗旱选育方法盲目性大，周期长，效果也有限。基因工程方法可以将外源的抗旱基因在人为调控操纵设计下导入优良品种，通过基因表达提高甘蔗的抗旱性，这就为甘蔗抗旱育种开辟了一条新的途径。 本研究将来自担子菌灰树花的海藻糖合酶基因TSase插入到中间载体pBBB双拷贝35s启动子下面构建植物表达载体pBBBT，然后导入超毒力农杆菌EHA105菌株。通过农杆菌介导法转化甘蔗的胚性愈伤组织，在PPT的选择压力下，筛选抗性植株，通过PCR和斑点Southern杂交鉴定海藻糖合酶基因的转化整合。同时，为了检验所使用基因在异位表达的功能还进行了烟草转化试验；为了比较不同类型启动子和不同海藻糖合成途径在甘蔗体内表达的效率，还克隆了拟南芥干旱诱导型启动子Prd29A及大肠杆菌海藻糖磷酸合酶基因OtsA，植物表达载体正在构建之中。 研究工作获得如下几个方面的结果： 1、构建了灰树花海藻糖合酶基因植物表达载体pBBBT。该载体的目的基因启动子为双拷贝的CaMV35s启动子，抗性选择标记为bar基因。 2、在国内首次获得农杆菌介导转化的甘蔗转基因植株。经过连续的PPT选择抗性筛选，获得了56株抗性植株，对其中8株进行PCR及斑点Southern杂交检测，有3株呈阳性反应，证明外源基因已整合到甘蔗基因组中。 3、建立一个可行有效而又较简捷的甘蔗农杆菌介导转化系统。以甘蔗胚性愈伤组织为受体细胞，并通过短时的亚培养及干燥处理提高其感受能力；采用超毒菌株EHA105介导；同时组合了各种便于Vir基因活化和T-DNA转移的转化条件，如附加AS及果糖和葡萄糖、酸性环境感染、较低温度共培养等；使用抗PPT的bar基因作选择标记基因，在0.75mg/L浓度下连续筛选。 4、观察不同培养龄期甘蔗愈伤组织的生长状态，并进一步观察胚状体的发生及分化，为甘蔗转化提供了可靠的受体细胞。 5、以农杆菌EHA105菌株介导将海藻糖合酶基因转化烟草叶盘，经分子检测证明，已获得了转基因烟草植株。 6、克隆了拟南芥干旱诱导型启动子Prd29A及大肠杆菌海藻糖磷酸合酶基因OtsA。 7、观察到部分甘蔗转化子形态上的改变，如植株生长慢，株型矮小纤细，叶片曲折长出或尖细直立，发根困难，根变粗短或且变纤细曲折，并常带有绿、褐等颜色。
Sugarcane (Saccharum officinarum L.),which is the important industrial material crop in China, is affected by drought stress at some time and some degree every year. Drought is the most serious environmental factor limiting production of sugarcane because more than 85% of sugarcane growing area is upland and seasonal drought happens frequently. Moreover sugarcane is a nonsexual propagation crop of highly polyploidy and complex genetic background, use of traditional breeding techniques to improve drought tolerance will be a formidable task. In contrast, genetic engineering enables the specific up- or down-regulation of naturally occurring process or genetic modifications involving the ectopic expression of genes from non-plant sources, which will be an alternative pathway accessible. A plant expression vector, pBBBT, was constructed by inserting trehalose synthase gene from a basidiomycete, Grjfoiafrondosea, into pBBB, with a 2 copy CaM V35S as promoter and bar as selectable marker gene. Then, It was introduced into Agrobacterium tumefaciens strain EHA 105 via triparental mating. The protocol of transformation has been optimized. Sugarcane抯 embryonic calli was transformed by Agrobacterium tumefaciens-mediated method. The transformed caili and shoots were screened under the selective pressure of PPT. The transformed plants were detected by PCR and dot Southern bolting. Meanwhile, Introduction of tobacco(Nicoiiana tabacum L.) transformation and Escherichia coil expression of the genes to identif~r the ectopic expression of the gene and its function. In order to compare the expression efficiency of different types of promoters and different pathways of trehalose synthesis in transgenic sugarcane, a drought responsive promoter, prd29A, form Arabidopsis thaliana and a trehalose-6-phosphate synthase gene, OtsA, from Escherichia coil were cloned. The results were as follows: I A plant expression vector, pBBBT, was constructed by inserted the trehalose synthase gene from Gnfola frondosa into pBBB, which contained a double CaMV3SS promoter and a PPT resistance gene(bar). 2 2 Transgenic sugarcane plants were recovered from co-culture of embryonic calli with Agrobacterium turn efaciens, 56 transformants were obtained after long PPT resistance selection, 8 of them have been detected by PCR and dot Southern blotting, and the DNA of 3 plants showed positive. 3 An efficient protocol for sugarcane transformation mediated by Agrobacterium turnefaciens has been established. Some factors may be important of the success:( I )Embryonic calli were induced as competent receptor cells, including subculture on fresh medium for a few days and dried treatment before infection, (2)Supervirulent strain EHA 105 was used;(3)Several transformation conditions were combined to improve the activation of vir genes and T-DNA transfer, such as added AS, fructose and glucose,and acid infection condition and low co-culture temperature;(4)bar gene, expression to resist PPT, was used as selectable marker gene. 4 The developmental process of calluses and embryoids have been investigated. 5 Trehalose synthase gene transformation of tobacco, mediated by Agrobacterium tumefaciens, was carried out. 6 A drought responsive promoter, Prd29A, from Arabidopsis thaliana and a trehaiose-6-phosphate synthase gene, OtsA, from Escherichia co/i were cloned. 7 Some obvious morphological changes including d
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