hrpZ_（Psgl2）基因植物表达载体构建及转化水稻的研究
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摘要
水稻(Oryza sativa L.)是世界上最重要的粮食作物之一,为了提高水稻抗逆、抗病、抗虫能力,转基因技术已经广泛开展。水稻抗病育种的基因来源较为广泛,其中hrp基因来源于微生物,该基因大多存在于革兰氏阴性植物病原细菌中,是决定病原菌对寄主植物致病性或诱导非寄主植物过敏性反应(hypersensitive response, HR)的基因。hrp基因编码的harpin蛋白富含甘氨酸,热稳定,对蛋白酶敏感,可诱导植物产生过敏性坏死反应。本研究所使用的hrpZPsg12基因来源于大豆细菌性斑点病菌(Pseudomonas syringae pv. glycinea,Psg)12号菌株(Psgl2),该基因编码的蛋白不但能引起非寄主植物的HR,还能诱导黄瓜产生抗猝倒病和白粉病的能力,而且还能促进植株生长。因此,通过基因工程技术将该基因转入水稻,以期为培育广谱抗病转基因水稻奠定基础。
本研究主要结果如下：
(1)用Sad, XbaI双酶切质粒pCAMBIA1300和pGM-hrpZPsg12,将hrpZPsg12基因同载体pCAMBIA1300进行连接,构建以bar为选择标记基因的植物表达载体pCAMBIA1300-bar-hrpZpsg12。
(2)用XhoI单酶切p390R及pCAMBIAl 300-bar-hrpZpsg12,以pmi基因替换掉pCAMBIA1300载体中的bar基因,成功构建以甘露糖为选择剂植物表达载体pCAMBIA1300-pmi-hrpZPsg12。
采用热激法将重组子转入大肠杆菌DH5α感受态细胞,提取阳性质粒,用冻融法将重组质粒转入农杆菌EHA105中,为后续水稻遗传转化提供条件。
(3)水稻遗传转化体系的建立。灭菌的水稻种子转至含2,4-D(2 mg/L)的N6D培养基上,32-C持续光照10 d。愈伤组织在OD600值约0.1的农杆菌侵染30 min,共培养3 d及选择培养14 d后转入含NAA(0.1 mg/L)+KT(2 mg/L)+PPT(5 mg/L)(10 g/L甘露糖)MS分化培养基(无机成分：MgSO4-7H2O 185 mg/L+ZnSO4·7H2O 1.72 mg/L+MnSO4·H2O 3.38 mg/L+CuSO4·5H2O 0.005 mg/L),光照培养16 h,暗培养8 h,10 d左右可长出绿色抗性细胞,40 d左右可长出抗性小苗。分化前干燥16 h及分化前期暗培养1周能提高愈伤组织的分化率及降低褐化率。将抗性小苗转入不含植物激素的1/2MS生根培养基,7 d左右可生根。
(4)通过PCR检测初步证明,bar选择标记的3株“黑粳八号”转化水稻植株为阳性。
Rice is one of the most important food crops in the world, in order to promote the resisting adversity property, the ability to resist disease and insect resistance of rice, transgenic technology have widely used. Sources of breeding for disease resistance were widely distributed. Hrp gene comes from microorganism and it is generally found in Gram-negative bacteria, which can cause HR in resistant host plants or non-host plants. Harpins encoded by hrp have similar to the other harpins:aboundant glycine, stable to heat, and sensitive to proteinase. In this study, hrpZPsg12 derived from strain Psg12 of Pseudomonas syringae pv. glycinea. The obtained systemic resistance to damping off and powdery mildew of cucumber were induced by the harpin coded by hrpZPsg12. Hypersensitive response of nonhost was induced by the harpin. Plants acquire systemic resistance and growth potential could be elicited by the harpin. It will be an effective way to cultivate resistance rice by transplanting the gene into rice.
The main results are as follows:
1. The pGM-hrpZPsg12 and vector pCAMBIA1300 digested with SacI/XbaI, then the hrpZPsg12 gene and pCAMBIA1300 ligated with T4DNA ligase. The expression vector pCAMBIA1300-bar-hrpZPsg12 was successfully constructed.
2. Based on pCAMBIA1300-bar-hrpZpsg12, the plant expresion vector was constructed by substituting pmi for bar gene. The security selection marker expression vector was successfully constructed and named pCAMBIA1300-pmi-hrpZPsg12. The two recombined vector was transferred into DH5α. After verifying correct orientation of genes, this transgenic construction was introduced into Agrobacterium tumefaciens strain EHA105 by using frozen-melt method.
3. The tissue culture system was optimized. Sterile seeds inoculate on N6D medium with 2 mg/L 2,4-D, cultured under continuous light at 32℃for 10 d.Bacteria cell concentration was adjusted to 0.6 at OD600, immerse callus in this Agrobacterium for 30 min, co-cultivate the callus at 25℃in the dark for 3 d.,and selective culture for 14 d.The explants were transferred to MS culture-medium containing 0.1 mg/L NAA,2 mg/L KT and 5 mg/L PPT(or 10 g/L mannose)(major inorganic constituents:185 mg/L MgSO4·7H2O,1.72 mg/L ZnSO4·7H2O,3.38 mg/L MnSO4·H2O and 0.005 mg/L CuSO4·5H2O),culture under at 28℃for light 16 h and dark 8 h.The resistant callus come into being after 10 d,and resistant plantlets were screened about 40 d. Drying treatment of calli for 16 h and culture dark for one week before differentiation,which can increase plant regeneration efficiency and reduce browing. Then, transfer resistant plantlets to root media for inducing roots about 7 d.
4. Transformation was confirmed by PCR, totally 3 regenerated plants were obtained.
本研究主要结果如下：
(1)用Sad, XbaI双酶切质粒pCAMBIA1300和pGM-hrpZPsg12,将hrpZPsg12基因同载体pCAMBIA1300进行连接,构建以bar为选择标记基因的植物表达载体pCAMBIA1300-bar-hrpZpsg12。
(2)用XhoI单酶切p390R及pCAMBIAl 300-bar-hrpZpsg12,以pmi基因替换掉pCAMBIA1300载体中的bar基因,成功构建以甘露糖为选择剂植物表达载体pCAMBIA1300-pmi-hrpZPsg12。
采用热激法将重组子转入大肠杆菌DH5α感受态细胞,提取阳性质粒,用冻融法将重组质粒转入农杆菌EHA105中,为后续水稻遗传转化提供条件。
(3)水稻遗传转化体系的建立。灭菌的水稻种子转至含2,4-D(2 mg/L)的N6D培养基上,32-C持续光照10 d。愈伤组织在OD600值约0.1的农杆菌侵染30 min,共培养3 d及选择培养14 d后转入含NAA(0.1 mg/L)+KT(2 mg/L)+PPT(5 mg/L)(10 g/L甘露糖)MS分化培养基(无机成分：MgSO4-7H2O 185 mg/L+ZnSO4·7H2O 1.72 mg/L+MnSO4·H2O 3.38 mg/L+CuSO4·5H2O 0.005 mg/L),光照培养16 h,暗培养8 h,10 d左右可长出绿色抗性细胞,40 d左右可长出抗性小苗。分化前干燥16 h及分化前期暗培养1周能提高愈伤组织的分化率及降低褐化率。将抗性小苗转入不含植物激素的1/2MS生根培养基,7 d左右可生根。
(4)通过PCR检测初步证明,bar选择标记的3株“黑粳八号”转化水稻植株为阳性。
Rice is one of the most important food crops in the world, in order to promote the resisting adversity property, the ability to resist disease and insect resistance of rice, transgenic technology have widely used. Sources of breeding for disease resistance were widely distributed. Hrp gene comes from microorganism and it is generally found in Gram-negative bacteria, which can cause HR in resistant host plants or non-host plants. Harpins encoded by hrp have similar to the other harpins:aboundant glycine, stable to heat, and sensitive to proteinase. In this study, hrpZPsg12 derived from strain Psg12 of Pseudomonas syringae pv. glycinea. The obtained systemic resistance to damping off and powdery mildew of cucumber were induced by the harpin coded by hrpZPsg12. Hypersensitive response of nonhost was induced by the harpin. Plants acquire systemic resistance and growth potential could be elicited by the harpin. It will be an effective way to cultivate resistance rice by transplanting the gene into rice.
The main results are as follows:
1. The pGM-hrpZPsg12 and vector pCAMBIA1300 digested with SacI/XbaI, then the hrpZPsg12 gene and pCAMBIA1300 ligated with T4DNA ligase. The expression vector pCAMBIA1300-bar-hrpZPsg12 was successfully constructed.
2. Based on pCAMBIA1300-bar-hrpZpsg12, the plant expresion vector was constructed by substituting pmi for bar gene. The security selection marker expression vector was successfully constructed and named pCAMBIA1300-pmi-hrpZPsg12. The two recombined vector was transferred into DH5α. After verifying correct orientation of genes, this transgenic construction was introduced into Agrobacterium tumefaciens strain EHA105 by using frozen-melt method.
3. The tissue culture system was optimized. Sterile seeds inoculate on N6D medium with 2 mg/L 2,4-D, cultured under continuous light at 32℃for 10 d.Bacteria cell concentration was adjusted to 0.6 at OD600, immerse callus in this Agrobacterium for 30 min, co-cultivate the callus at 25℃in the dark for 3 d.,and selective culture for 14 d.The explants were transferred to MS culture-medium containing 0.1 mg/L NAA,2 mg/L KT and 5 mg/L PPT(or 10 g/L mannose)(major inorganic constituents:185 mg/L MgSO4·7H2O,1.72 mg/L ZnSO4·7H2O,3.38 mg/L MnSO4·H2O and 0.005 mg/L CuSO4·5H2O),culture under at 28℃for light 16 h and dark 8 h.The resistant callus come into being after 10 d,and resistant plantlets were screened about 40 d. Drying treatment of calli for 16 h and culture dark for one week before differentiation,which can increase plant regeneration efficiency and reduce browing. Then, transfer resistant plantlets to root media for inducing roots about 7 d.
4. Transformation was confirmed by PCR, totally 3 regenerated plants were obtained.
引文
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