抗纹枯病转基因水稻植株的创制
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摘要
纹枯病菌(Rhizoctonia solani)是水稻纹枯病的病原菌,由于在自然状态下水稻缺乏有效的抗源,该病害有越来越严重的趋向,部分地区的危害程度甚至超过了稻瘟病。随着分子生物学技术的发展,利用转基因技术培育抗纹枯病种质资源的方法已被广泛采用。
为了挖掘和使用不同类型的基因资源,本课题组之前从稻瘟病菌中克隆了一个几丁质酶(CHI)基因。实验表明该基因编码产物具有较强的活性。此外,纹枯病菌与菌核病菌同为草酸产生菌,课题组之前证实了转枯草芽孢杆菌的草酸脱羧酶基因(OXD)的模式植物拟南芥增强了对核盘菌的抗性。同时,有报道表明,将几丁质酶基因和葡聚糖酶基因同时转入植物中,可以进一步提高植物对病原菌的抗病性。据此,本研究分别从稻瘟菌ku-70,枯草芽孢杆菌和水稻品种日本晴中克隆了稻瘟菌几丁质酶基因(CHI),草酸脱羧酶基因(OXD),水稻葡聚糖酶(GLU)基因用于转基因水稻的研究。
用几丁质酶基因、葡聚糖酶基因和草酸脱羧酶基因,分别构建了三个植物表达载体pCAMBIA1301-ubi-chi、pCAMBIA1301-ubi-glu和
pCAMBIA1301-ubi-oxd。利用农杆菌介导转化法,将稻瘟菌几丁质酶基因和枯草芽孢杆菌草酸脱羧酶基因转化到水稻品种明恢86和kitaaki中。经提取转基因水稻基因组DNA的PCR验证和GUS染色鉴定,结果表明,两个基因成功被导入水稻植株中。提取转基因水稻RNA经RT-PCR扩增,进一步证实目的基因在水稻植株中成功转录。通过对部分T1代的转几丁质酶基因和草酸脱羧酶基因株系的抗纹枯病鉴定的结果表明,相对于Lemont和Kitaake两个对照品种,转基因株系的纹枯病抗性均得到增强,且转草酸脱羧酶基因的转基因水稻株系较转几丁质酶基因更抗纹枯病。此外,我们也将几丁质酶和葡聚糖酶基因共转化水稻愈伤组织,以期获得共转转基因植株,该方面相关工作仍在进行中。
转基因水稻植株的获得,对进一步开展相应基因的功能研究具有一定的理论和实际意义。
Rice sheath blight is one of the most important diseases of rice caused by Rhizoctonia solani. The disease has become more and more severe, even becoming the most inportant disease in certain areas, for short of resistant cultivars. However, today, transgenic technique has been widely used to cultivate resistant varieties,. In order to reduce the severity of the disease, different kinds of genes were used to improve rice resistance to R. solani in this paper. Firstly, we chosen gene MGG-08054.5 which from the Magnaporthe grisea, encodes chitinase. It has been demonstrated that MGG-08054.5 encodes a chitinase with strong activity. In addition,
we also focused our study on the OXD gene which encodes oxalate oxd decarboxylation enzyme. Previous research has demonstrated that OXD improved Arabidopsis thaliana’s resistance to Sclerotinia sclerotiorum, which produces of oxalic acid as well as R. solani. Further more, it has been reported that co-transformation of the chitinase gene andβ-1,3-glucanase gene into a plant can improve its resistance to diseases. Therefore, aβ-1,3-glucanase gene (GLU) from Oryza sativa japonica was cloned.
Three plant expression vectors, pCAMBIA1301-ubi-chi, pCAMBIA1301-ubi-glu and pCAMBIA1301-ubi-oxd were constructed and transformed into Minghui 86 and kitaake cultivar mediated by Agrobacterium tumefaciens. The results of PCR assay and GUS dying showed that chitinase and oxd genes have been transformed into rice plants successfully. The RT-PCR assay further confirmed that target genes have been transformed into rice plants. Moreover, the resistance of transgenic plants to R. solani were increased. In addition, pCAMBIA1301-ubi-glu and pCAMBIA1301-ubi-chi co-transformation work also has been done and some transgenic rice plants are coming out soon..
The transgenic plants obtained here may be used to rice resistance breeding in the future.
为了挖掘和使用不同类型的基因资源,本课题组之前从稻瘟病菌中克隆了一个几丁质酶(CHI)基因。实验表明该基因编码产物具有较强的活性。此外,纹枯病菌与菌核病菌同为草酸产生菌,课题组之前证实了转枯草芽孢杆菌的草酸脱羧酶基因(OXD)的模式植物拟南芥增强了对核盘菌的抗性。同时,有报道表明,将几丁质酶基因和葡聚糖酶基因同时转入植物中,可以进一步提高植物对病原菌的抗病性。据此,本研究分别从稻瘟菌ku-70,枯草芽孢杆菌和水稻品种日本晴中克隆了稻瘟菌几丁质酶基因(CHI),草酸脱羧酶基因(OXD),水稻葡聚糖酶(GLU)基因用于转基因水稻的研究。
用几丁质酶基因、葡聚糖酶基因和草酸脱羧酶基因,分别构建了三个植物表达载体pCAMBIA1301-ubi-chi、pCAMBIA1301-ubi-glu和
pCAMBIA1301-ubi-oxd。利用农杆菌介导转化法,将稻瘟菌几丁质酶基因和枯草芽孢杆菌草酸脱羧酶基因转化到水稻品种明恢86和kitaaki中。经提取转基因水稻基因组DNA的PCR验证和GUS染色鉴定,结果表明,两个基因成功被导入水稻植株中。提取转基因水稻RNA经RT-PCR扩增,进一步证实目的基因在水稻植株中成功转录。通过对部分T1代的转几丁质酶基因和草酸脱羧酶基因株系的抗纹枯病鉴定的结果表明,相对于Lemont和Kitaake两个对照品种,转基因株系的纹枯病抗性均得到增强,且转草酸脱羧酶基因的转基因水稻株系较转几丁质酶基因更抗纹枯病。此外,我们也将几丁质酶和葡聚糖酶基因共转化水稻愈伤组织,以期获得共转转基因植株,该方面相关工作仍在进行中。
转基因水稻植株的获得,对进一步开展相应基因的功能研究具有一定的理论和实际意义。
Rice sheath blight is one of the most important diseases of rice caused by Rhizoctonia solani. The disease has become more and more severe, even becoming the most inportant disease in certain areas, for short of resistant cultivars. However, today, transgenic technique has been widely used to cultivate resistant varieties,. In order to reduce the severity of the disease, different kinds of genes were used to improve rice resistance to R. solani in this paper. Firstly, we chosen gene MGG-08054.5 which from the Magnaporthe grisea, encodes chitinase. It has been demonstrated that MGG-08054.5 encodes a chitinase with strong activity. In addition,
we also focused our study on the OXD gene which encodes oxalate oxd decarboxylation enzyme. Previous research has demonstrated that OXD improved Arabidopsis thaliana’s resistance to Sclerotinia sclerotiorum, which produces of oxalic acid as well as R. solani. Further more, it has been reported that co-transformation of the chitinase gene andβ-1,3-glucanase gene into a plant can improve its resistance to diseases. Therefore, aβ-1,3-glucanase gene (GLU) from Oryza sativa japonica was cloned.
Three plant expression vectors, pCAMBIA1301-ubi-chi, pCAMBIA1301-ubi-glu and pCAMBIA1301-ubi-oxd were constructed and transformed into Minghui 86 and kitaake cultivar mediated by Agrobacterium tumefaciens. The results of PCR assay and GUS dying showed that chitinase and oxd genes have been transformed into rice plants successfully. The RT-PCR assay further confirmed that target genes have been transformed into rice plants. Moreover, the resistance of transgenic plants to R. solani were increased. In addition, pCAMBIA1301-ubi-glu and pCAMBIA1301-ubi-chi co-transformation work also has been done and some transgenic rice plants are coming out soon..
The transgenic plants obtained here may be used to rice resistance breeding in the future.
引文
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