【作者】 张艳；

【导师】 杨传平；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 我国土壤重金属污染问题不断恶化，亟待解决。深入研究植物的耐重金属机理，克隆相关基因并利用基因工程技术提高植物对重金属的抗性，是净化污染土壤的一条有效途径。金属硫蛋白（metallothionein，MT）是一类广泛存在于生物中的低分子量，富含半胱氨酸可结合重金属的蛋白质，在土壤重金属污染的治理中越来越引起人们的重视。应用生物信息学技术从柽柳和星星草cDNA文库中分别克隆了柽柳和星星草的MT基因，对该基因编码的MT进行了蛋白质家族、疏水性、信号肽、等电点及分子量的预测，并选取了几种植物的MT基因氨基酸序列进行了多序列比对分析及分子进化树绘制，结果表明克隆的柽柳和星星草MT基因符合植物MT基因所具有的典型特征。将从柽柳中克隆到的MT基因构建到载体pROKⅡ上，通过农杆菌介导的叶盘法对烟草进行遗传转化，共获得75株卡那霉素抗性苗，转化率达到78％。选取10株长势良好的转基因烟草进行分析，PCR检测结果均为阳性；对其中6个转基因株系进行PCR-Southern杂交和Northern杂交，结果均为阳性，证明外源基因已整合到烟草基因组中并得到表达。将在柽柳和星星草中克隆到的MT基因分别连接到载体pROKⅡ上，构建了柽柳和星星草MT基因双价植物表达载体，对烟草进行遗传转化，共获得83株卡那霉素抗性苗，转化率达到75％。选取10株长势良好的转基因烟草进行分析，PCR检测结果均为阳性；对其中7个转基因株系分别用柽柳和星星草MT基因标记的探针进行PCR-Southern杂交，结果均为阳性；再从中选取5个转基因株系进行Northern杂交，结果均为阳性，证明外源基因已整合到烟草基因组中并得到表达。对非转基因对照烟草、转柽柳MT基因烟草和转柽柳、星星草MT基因烟草T₀代组培苗进行了重金属Cd和Cu胁迫试验，测定并分析了各转基因烟草及非转基因对照烟草胁迫后的生根率、增重和株高变化。结果显示，T₀代转基因烟草在不同浓度CdCl₂和CuSO₄胁迫条件下，生根率、增重和株高均高于非转基因对照烟草。在CdCl₂胁迫下，非转基因对照烟草能在100μmol·L^-1 CdCl₂浓度下生长，转柽柳MT基因烟草能在200μmol·L^-1 CdCl₂浓度下生长，而转柽柳、星星草MT基因烟草能在300μmol·L^-1 CdCl₂浓度下生长。在CuSO₄胁迫下，在75μmol·L^-1 CuSO₄浓度下，三者均能正常生长；在150μmol·L^-1 CuSO₄浓度下，对照烟草不能生长，转柽柳MT基因烟草生长缓慢，植株矮小，转柽柳、星星草MT基因烟草大部分能正常生长；在225μmol·L^-1 CuSO₄浓度下，转柽柳、星星草MT基因烟草不能生长，但生长量有所增加。试验表明，MT基因的导入提高了T₀代转基因烟草对Cd和Cu的耐受力，其中转基因烟草对Cd的抗性要高于对Cu的抗性，并且转双价MT基因比转单价MT基因更能提高转基因烟草对重金属的耐受力。将T₀代自交得到的转基因烟草种子播种于含100 mg·L^-1 Kan的MS培养基上培养20 d后，对8个转基因株系的绿白苗之比进行统计，结果表明转柽柳MT基因烟草T₁-5、T₁-8和转柽柳、星星草MT基因烟草T₁^′-2、T₁^′-3的绿色苗与白化苗分离规律基本符合（2²-1）∶1；转柽柳MT基因烟草T₁-6和转柽柳、星星草MT基因烟草T₁^′-9株系的绿色苗与白化苗分离规律基本符合（2⁴-1）∶1。对8个转基因株系卡那霉素抗性植株进行PCR检测，结果均为阳性，初步证明了MT基因仍整合在烟草子一代基因组中。对8个转基因烟草株系子一代（T₁）进行了Cd胁迫试验，测定并分析了CdCl₂胁迫下转基因烟草生长量的变化。结果显示，转基因烟草T₁代组培苗在根长、根数、苗增重、叶片数方面均高于非转基因对照烟草。转柽柳MT基因烟草能够在CdCl₂浓度为100μmol·L^-1培养基上生长良好，平均增重是非转基因对照的3.07倍，平均根长是非转基因对照的1.96倍；转柽柳、星星草MT基因烟草能够在CdCl₂浓度为200μmol·L^-1培养基上生长，平均增重是非转基因对照的3.85倍，平均根长是非转基因对照的2.74倍；而非转基因对照烟草在CdCl₂浓度为100μmol·L^-1时就表现为生长缓慢，根系受到明显抑制。试验表明转双价MT基因烟草比转单价MT基因烟草具有更强的Cd抗性。证明了外源MT基因可以在烟草子一代中遗传，该基因的表达提高了烟草对Cd胁迫的耐受能力。更多还原

【Abstract】 Heavy metal pollution in soil has been a serious problem and a matter of great urgency to resolve in our country. Studying the heavy metal tolerance mechanism of plant, cloning the related genes and improving plant ability of heavy metal tolerance by genetic engineering technique have become a valid path to decontaminate the polluted soil. Metallothionein （MT） is a kind of low molecular, cysteine-rich and heavy metal-binding protein. MT already caused the people to value highly in controlling the soil heavy metal pollution.Two MT genes were cloned from Tamarix androssowii cDNA library and PuccineUia tenuiflora cDNA library using bioinformatics technology. Results of sequence analysis revealed two MT genes were the member of plant MT family.The MT gene from T. androssowii was cloned into the plant express vector pROKⅡ. The recombinant plasmid was transformed into tobacco by Agrobacterium mediated genetic transformation. 75 kanamycin-resistant buds were obtained in study, the transform efficiency was 78 percent. We selected 10 excellent lines to PCR test. All the lines were positive in PCR. In PCR-Southern blotting and Northern blotting hybridization, we checked 6 lines selected from 10 transgenic lines randomly. The results demonstrated that foreign target gene had been integrated into tobacco genome already and could be expressed in tobacco.The MT genes from T. androssowii and P. tenuiflora were cloned into the plant express vector pROKⅡ. The recombinant plasmid was transformed into tobacco by Agrobacterium mediated genetic transformation. 83 kanamycin-resistant buds were obtained in study, the transform efficiency was 75 percent. We selected 10 excellent lines to PCR test. All the lines were positive in PCR. In PCR-Southern blotting hybridization, we checked 7 lines selected from 10 transgenic lines randomly. The result demonstrated that two MT genes had been integrated into tobacco genome already. In Northern blotting hybridization, we checked 5 lines selected from 7 transgenic lines randomly. The test showed that two MT genes could be expressed in tobacco.Rooting rate, the average gain and plant height were analyzed after the transgenic tobacco lines To generation and control line were subjected to Cd and Cu stress treatment: The results showed: the Cd and Cu tolerance of transgenic tobacco lines were higher than control. Under CdC12 stress the control line could live in the medium containing 100/zmol.L^-1 CdCl2; The transgenic tobacco lines with MT gene from T. androssowii could live in the medium containing 200/zmol·L^-1 CdCl2; But the transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora could live in the medium containing 300/μmol·L^-1 CdCl2. Under CuSO4 stress three kinds of tobacco lines could grow at the concentration of 75/μmol·L^-1; At the concentration of 150μmol·L^-, the control line couldn’t live and the transgenic tobacco lines with MT genes from T. androssowii and P tenuiflora could grow better than the transgenic tobacco lines with MT gene from T. androssowii. At the concentration of 225μmol·L^-1, the transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora couldn’t live but the growth had increased. The above results confirmed that the MT gene can enhance the Cd and Cu tolerance of transgenic tobacco lines. The tolerance of transgenic tobacco lines to Cd was higher to Cu, and the tolerance of transgenic tobacco lines with two MT genes to Cd and Cu was higher than that with one MT gene.We transplanted transgenic tobacco lines To generation in the greenhouse and obtained T1 generation seeds. We growed them on culture medium of MS at the concentration of 100 mg.L^-1 kanamycin. After 20 days, we count numbers of green plants and white plants. The results showed: the ratios of T₁-5, T₁-8, T₁’-2 and T₁’-3 were accord with （2²-1） : 1, the ratios of T₁-6 and T 1’-9 were accord with （2⁴-1） : 1.We selected 8 transgenic tobacco lines T₁ generation to PCR test, all the lines were positive in PCR. The results demonstrated that foreign target genes still had been integrated into tobacco genome.We studied the growth condition of 8 transgenic tobacco lines T₁ generation and control line under different Cd concentration. The results showed: the transgenic tobacco lines were better than control line in root length, root quantity, the average gain and leave quantity under Cd stress. The transgenic tobacco lines with MT gene from T. androssowii lived well at the concentration of 100μmol.L¹ CdCl2, weighed 3.07 times of the control, and were root length 1.96 times of the control. The transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora could live at the concentration of 200,μmol·L^-1 CdCl2, weighed 3.85 times of the control, and were root length 2.74 times of the control. While the control hardly rooted at the concentration of 100μmol·L^-1 CdCl2. All the results proved that the transgenic tobacco lines T₁ generation with MT genes have the genetic stability, and the expression of MT genes could enhance the resistence of the transgenic tobacco to Cd.更多还原