PtDRG01基因原核表达分析及遗传转化的初步研究
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摘要
毛白杨(Populus tomentosa Carr.)是我国特有的乡土杨树,具有速生、优质和抗逆性强等特性,在我国北方,尤其是在黄河中下游林业生产和生态环境建设中占有重要地位。但随着栽培面积的不断扩大,一些病害的发生严重影响着它的推广。叶锈病是危害最为严重的病害之一,以马格栅锈菌(Melampsora magnusiana Wanger)致病性为最强。它通常侵害幼苗及成年树的叶片和芽,从而形成黄色小斑点,致使植物光合效率降低,造成受害叶片提早落叶,严重时则会形成大型枯斑,病叶和病芽枯死,严重影响了杨树的生长和发育。而目前有关毛白杨抗病方面的研究还很少,尤其在抗病遗传资源筛选、抗病品种选育和抗病基因定位与克隆等方面还未见报道,因此,在对毛白杨抗病遗传资源研究的基础上,分离抗叶锈病基因并对其产物的功能进行研究显得尤为必要,最终获得抗叶锈病的毛白杨转基因植株,将对毛白杨抗病性状的遗传改良具有重要的理论意义和生产价值。本研究克隆了NBS型抗病相关基因PtDRG01,并对其进行了原核表达分析和毛白杨RNAi基因转化研究,得到了如下主要结论:
1.在已分离的NBS型抗病候选基因PtDRG01的基础上,构建了其原核表达载体pGEX-KG-Pt01,并导入大肠杆菌XA90,经IPTG诱导表达后,SDS-PAGE电泳分析显示,PtDRG01表达的蛋白分子量约为79kD。同时对原核表达体系以及融合蛋白的表达特性进行了优化与分析。结果表明,最佳诱导表达条件为1mmol/L IPTG在37℃下诱导4h,所表达的融合蛋白为胞内分泌的可溶性蛋白。利用谷胱苷肽-琼脂糖亲和层析柱纯化获得了电泳纯级的目的蛋白,然后采用改良钼蓝比色法对PtDRG01蛋白进行生化功能分析。结果显示该蛋白仅能微弱地催化ATP的水解,其生化特性还有待采用同位素标记法等更灵敏的方法进一步研究。
2.本研究成功建立了三倍体毛白杨无性系[(P. tomentosa×P. bolleana)×P. tomentosa]高效的叶片再生体系,构建了PtDRG01基因的RNAi表达载体,并采用基因枪法进行三倍体毛白杨高抗无性系的遗传转化研究,获得了一批卡那霉素抗性植株,PCR检测证实外源基因已整合到三倍体毛白杨基因组内,进一步的分子检测与抗病试验正在进行之中。
本研究为毛白杨抗病基因的功能鉴定及抗病性状的分子育种提供了重要的科学理论依据,具有重要的应用价值。
The Chinese white poplar (Populus tomentosa Carr.), indigenous to China, is a fast growing poplar species, with an excellent wood quality and outstanding resistance to abiotic stress. It has played a key role in forest production and ecological environment construction along the Yellow River. However, it is susceptible to a couple of diseases, particularly the leaf rust caused by Melampsora magnusiana Wagner. It is normally invade into the seedlings and leaves and shoots located in the senior tree, resulted in yellow dot of pathogen and in turn lowered the efficiency of photosynthesis, this consequently severely affected the growth and development of the poplar. But, it is little known about the research of disease resistance in P. tomentosa, especially in the study of disease resistance, including selection of germplasm and breeding, gene mapping and map based cloning of disease resistant gene had not been reported. Therefore, it is indispensable to isolate the gene controlling of leaf rust and analyze the function of gene’s product, based on the germplasm of disease resistance, and eventually the transgenic plants with disease resistant genes would be produced. These will have the significance in the theory and practical value for the improvement of disease resistance in P. tomentosa. In this study, the prokaryotic expression of a candidate disease resistant gene PtDRG01 and genetic transformation of RNAi constructed PtDRG01 had been conducted. The major results and conclusions are described as follows.
1. The prokaryotic expression vector pGEX-KG with the full-length open reading frame of isolated disease resistance candidate gene PtDRG01 isolated form P. tomentosa ( Accession number:EF157840)) was successfully constructed and transferred into an expression host E. coli strain XA90. The fusion protein of PtDRG01 induced by IPTG treatment was separated by SDS-PAGE electrophoresis, and the result indicated that the size of the fusion protein was about 79 kD, which was consistent with the predicted value. The prokaryotic expression system was also optimized. The result suggested that 1 mmol/L IPTG treatment for 4 h at 37 oC was most effective, and the product was predominately soluble and not extra-cellular secreting. Further, the fusion protein was purified with the affinity chromatography column of Glutathione Sepharose 4B. Catalyses ability of the hydrolysis of ATP with weakly effect by the purified protein with the modified colorimetry of molybdenum blue method was determined.
2. The regeneration system of genetic transformation with leave disc was established in P. tomentosa and the plant binary expression vector of Pbi121-PtDRG01 RNAi was constructed and transferred into the triploid poplar through particle bombardment. Many transgenic poplars were obtained and the integration of RNAi sequences into the host genome was confirmed by the PCR detection. The further molecular detection and experiment of disease resistance is ongoing.
The above results, therefore, provided the important foundation for function detection of PtDRG01 gene and molecular breeding of new germplasms with disease tolerance in P. tomentosa, have the important practical value in the forest production.
1.在已分离的NBS型抗病候选基因PtDRG01的基础上,构建了其原核表达载体pGEX-KG-Pt01,并导入大肠杆菌XA90,经IPTG诱导表达后,SDS-PAGE电泳分析显示,PtDRG01表达的蛋白分子量约为79kD。同时对原核表达体系以及融合蛋白的表达特性进行了优化与分析。结果表明,最佳诱导表达条件为1mmol/L IPTG在37℃下诱导4h,所表达的融合蛋白为胞内分泌的可溶性蛋白。利用谷胱苷肽-琼脂糖亲和层析柱纯化获得了电泳纯级的目的蛋白,然后采用改良钼蓝比色法对PtDRG01蛋白进行生化功能分析。结果显示该蛋白仅能微弱地催化ATP的水解,其生化特性还有待采用同位素标记法等更灵敏的方法进一步研究。
2.本研究成功建立了三倍体毛白杨无性系[(P. tomentosa×P. bolleana)×P. tomentosa]高效的叶片再生体系,构建了PtDRG01基因的RNAi表达载体,并采用基因枪法进行三倍体毛白杨高抗无性系的遗传转化研究,获得了一批卡那霉素抗性植株,PCR检测证实外源基因已整合到三倍体毛白杨基因组内,进一步的分子检测与抗病试验正在进行之中。
本研究为毛白杨抗病基因的功能鉴定及抗病性状的分子育种提供了重要的科学理论依据,具有重要的应用价值。
The Chinese white poplar (Populus tomentosa Carr.), indigenous to China, is a fast growing poplar species, with an excellent wood quality and outstanding resistance to abiotic stress. It has played a key role in forest production and ecological environment construction along the Yellow River. However, it is susceptible to a couple of diseases, particularly the leaf rust caused by Melampsora magnusiana Wagner. It is normally invade into the seedlings and leaves and shoots located in the senior tree, resulted in yellow dot of pathogen and in turn lowered the efficiency of photosynthesis, this consequently severely affected the growth and development of the poplar. But, it is little known about the research of disease resistance in P. tomentosa, especially in the study of disease resistance, including selection of germplasm and breeding, gene mapping and map based cloning of disease resistant gene had not been reported. Therefore, it is indispensable to isolate the gene controlling of leaf rust and analyze the function of gene’s product, based on the germplasm of disease resistance, and eventually the transgenic plants with disease resistant genes would be produced. These will have the significance in the theory and practical value for the improvement of disease resistance in P. tomentosa. In this study, the prokaryotic expression of a candidate disease resistant gene PtDRG01 and genetic transformation of RNAi constructed PtDRG01 had been conducted. The major results and conclusions are described as follows.
1. The prokaryotic expression vector pGEX-KG with the full-length open reading frame of isolated disease resistance candidate gene PtDRG01 isolated form P. tomentosa ( Accession number:EF157840)) was successfully constructed and transferred into an expression host E. coli strain XA90. The fusion protein of PtDRG01 induced by IPTG treatment was separated by SDS-PAGE electrophoresis, and the result indicated that the size of the fusion protein was about 79 kD, which was consistent with the predicted value. The prokaryotic expression system was also optimized. The result suggested that 1 mmol/L IPTG treatment for 4 h at 37 oC was most effective, and the product was predominately soluble and not extra-cellular secreting. Further, the fusion protein was purified with the affinity chromatography column of Glutathione Sepharose 4B. Catalyses ability of the hydrolysis of ATP with weakly effect by the purified protein with the modified colorimetry of molybdenum blue method was determined.
2. The regeneration system of genetic transformation with leave disc was established in P. tomentosa and the plant binary expression vector of Pbi121-PtDRG01 RNAi was constructed and transferred into the triploid poplar through particle bombardment. Many transgenic poplars were obtained and the integration of RNAi sequences into the host genome was confirmed by the PCR detection. The further molecular detection and experiment of disease resistance is ongoing.
The above results, therefore, provided the important foundation for function detection of PtDRG01 gene and molecular breeding of new germplasms with disease tolerance in P. tomentosa, have the important practical value in the forest production.
引文
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