LURP1诱导型启动子驱动下AtWRKY70表达载体构建及对水稻的遗传转化
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摘要
LURP1 (Late Upregulated in Response to Hyaloperonospora parasitica)是在拟南芥中发现的一种在真菌感染后期持续上调的抗性基因。LURP1启动子在拟南芥抗真菌病害中起重要调节作用[1]。植物特异转录因子AtWRKY70是一种在SA和JA介导的信号途径中的一个重要成员。AtWRKY70的表达受到SA的诱导,但是受JA的抑制。SA调控下产生的AtWRKY70是NPR1非依赖型的。通过组成型过表达AtWRKY70增加了植物对于真菌的抵抗性[2-5]。但将两者结合起来,是否具有更强的调控作用还不很清楚。本研究旨在完成相关的载体构建工作并转化水稻,获得水稻转基因植株,为进一步开展LURP1启动子及AtWRKY70转录因子在水稻中的功能分析奠定基础。主要结果如下:
1、构建了带有LURP1诱导型启动子的双元载体,并用此双元载体构建了LURP1诱导型启动子与gusA报告基因的表达载体,以及LURP1诱导型启动子驱动下的转录因子AtWRKY70的表达载体,并进行了测序验证。
2、开展了水稻农杆菌介导的遗传转化,从所获得的转基因植株中,PCR分别鉴定出13株pMLURP1::GUS和11株pMLURP1-WRKY70 cDNA转基因植株。
以上研究结果为进一步研究鉴定诱导型启动子LURP1及AtWRKY70在水稻中的抗性的功能,筛选生物活性小分子物质及进一步研究启动子与转录因子的互作详细机制奠定了基础。
LURP1 (late upregulated in response to Hyaloperonospora parasitica) , a sustained rise of resistance gene in the late of a fungal infection in Arabidopsis. LURP1 promoter plays an important regulatory role in Arabidopsis resistance to fungal disease. Research confirmed that plant-specific transcription factor AtWRKY70 is an important member in SA and JA-mediated signal pathway. AtWRKY70 expression induced by SA, but inhibited by JA. AtWRKY70 regulated by SA is non-dependent NPR1. By constitutive overexpression of AtWRKY70 increased plant resistance to pathogens.But it not clear that put LURP1 and AtWRKY70 together whether have a much stronger resistance to the disease in rice. This study aimed to construct the related vectors then transfer them into rice and obtain the transgenic plants. Furthermore, the study laid the foundation for the further researches on LURP1 promoter and AtWRKY70 transcription factor function analysis in rice. The main results of this study are as follows:
1, constructed the LURP1 inducible promoter binary vector. Using this binary vector constructed two expression vectors through Gateway technology.One is LURP1 inducible promoter and gusA fusion protein expression vector, the other is expression vector of the transcription factor AtWRKY70 driven by LURP1 inducible promoter and completed the sequence test of the two genes.
2, launched a research on the Agrobacterium-mediated genetic transformation of rice.Obtained transgenic plants and identified 13 pMLURP1:: GUS and 11 pMLURP1-WRKY70 cDNA transgenic plants by PCR test.
These results laid the foundation for identifying the resistance functions of the inducible promoter LURP1 and AtWRKY70 transcription factor in rice, screening bioactive small molecules and researching promoter and transcription factor interaction.
1、构建了带有LURP1诱导型启动子的双元载体,并用此双元载体构建了LURP1诱导型启动子与gusA报告基因的表达载体,以及LURP1诱导型启动子驱动下的转录因子AtWRKY70的表达载体,并进行了测序验证。
2、开展了水稻农杆菌介导的遗传转化,从所获得的转基因植株中,PCR分别鉴定出13株pMLURP1::GUS和11株pMLURP1-WRKY70 cDNA转基因植株。
以上研究结果为进一步研究鉴定诱导型启动子LURP1及AtWRKY70在水稻中的抗性的功能,筛选生物活性小分子物质及进一步研究启动子与转录因子的互作详细机制奠定了基础。
LURP1 (late upregulated in response to Hyaloperonospora parasitica) , a sustained rise of resistance gene in the late of a fungal infection in Arabidopsis. LURP1 promoter plays an important regulatory role in Arabidopsis resistance to fungal disease. Research confirmed that plant-specific transcription factor AtWRKY70 is an important member in SA and JA-mediated signal pathway. AtWRKY70 expression induced by SA, but inhibited by JA. AtWRKY70 regulated by SA is non-dependent NPR1. By constitutive overexpression of AtWRKY70 increased plant resistance to pathogens.But it not clear that put LURP1 and AtWRKY70 together whether have a much stronger resistance to the disease in rice. This study aimed to construct the related vectors then transfer them into rice and obtain the transgenic plants. Furthermore, the study laid the foundation for the further researches on LURP1 promoter and AtWRKY70 transcription factor function analysis in rice. The main results of this study are as follows:
1, constructed the LURP1 inducible promoter binary vector. Using this binary vector constructed two expression vectors through Gateway technology.One is LURP1 inducible promoter and gusA fusion protein expression vector, the other is expression vector of the transcription factor AtWRKY70 driven by LURP1 inducible promoter and completed the sequence test of the two genes.
2, launched a research on the Agrobacterium-mediated genetic transformation of rice.Obtained transgenic plants and identified 13 pMLURP1:: GUS and 11 pMLURP1-WRKY70 cDNA transgenic plants by PCR test.
These results laid the foundation for identifying the resistance functions of the inducible promoter LURP1 and AtWRKY70 transcription factor in rice, screening bioactive small molecules and researching promoter and transcription factor interaction.
引文
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