拟南芥抗致病疫霉突变体的筛选及T-DNA侧翼序列分析
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摘要
拟南芥(Arabidopsis thaliana)属十字花科芸薹属植物，在显花植物中，基因组最小(大小为125Mb)，体细胞中仅5对染色体，并且基因组序列已测序完成。
本研究将致病疫霉不同菌株接种到拟南芥Col-0生态型激活标签突变体上后，发现其抗感反应不同，并筛选出了感致病疫霉的突变体类型。以筛选出的拟南芥感病突变体基因组DNA为模板，利用热不对称交错PCR方法(Thermal asymmetric interlaced PCR，TAIL-PCR)，进行了抗性相关基因的研究。通过TAIL-PCR技术，获得了拟南芥T-DNA插入侧翼序列，利用NCBI提供的拟南芥全基因组序列进行了序列同源性比对，主要结果如下：
测定拟南芥258个激活标签突变体接种5个致病疫霉菌株后发现，AtTM31、AtTM34、AtTM40、AtTM113、AtTM246对致病疫霉的菌株17-B-1表现为重度感病，AtTM114对菌株17-B-1表现为中度感病，AtTM32、AtTM144、AtTM243、AtTM244对菌株17-B-1表现为轻度感病；AtTM32、AtTM34、AtTM40、AtTM113对菌株W040604表现为重度感病，AtTM31、AtTM244对菌株W040604表现为中度感病，AtTM114、AtTM144、AtTM243、AtTM246对菌株W040604表现为轻度感病；AtTM31对菌株W041002表现为重度感病，AtTM32、AtTM34、AtTM40、AtTM113、AtTM243、AtTM246对菌株W041002表现为中度感病，AtTM114、AtTM144、AtTM244对菌株W041002表现为轻度感病；AtTM32、AtTM40对菌株W041904表现为重度感病，AtTM34、AtTM144、AtTM243、AtTM246对菌株W041904表现为中度感病，AtTM31、AtTM 113、AtTM114、AtTM243对菌株W041904表现为轻度感病；AtTM31、AtTM32、AtTM40、AtTM246对菌株W041406表现为重度感病，AtTM34、AtTM114、AtTM243、AtTM244对菌株W041406表现为中度感病，AtTM113、AtTM144对菌株W041406表现为轻度感病。
TAIL-PCR可以有效地扩增T-DNA插入侧翼拟南芥基因组序列，简并引物AD1、AD2、AD4和特异引物(Lex2、Lex4和Lex5)、(Lex3、Lex4和Lex5)组成的引物组合是最佳引物组合。对10个TAIL-PCR特异产物测序分析，发现2个T-DNA插入在基因上，2个则插入在基因间隔区中，5个是载体序列。
T-DNA在AtTM34突变体基因组上插入第3条染色体133bp左右，处在第1个外显子上，该基因属于氨基酸透性酶家族蛋白，与SP／Q9WTR6胱氨酸／谷氨酸转座子有低的相似性(Amino acid transport system xc-){Mus musculus}；包含Pfam profile PF00324：Amino acid permease。T-DNA在AtTM246突变体基因组上插入第5条染色体2672bp左右，处在第5个外显子上，该基因属于PPR模体蛋白(pentatricopeptide repeat containing protein)。
通过Southern杂交试验验证，突变体AtTM34中含一个拷贝的T-DNA插入，突变体AtTM246中含两个拷贝的T-DNA插入。
Arabidopsis thaliana is a plant of Crucifereae. The Arabidopsis Genome is 125Mb,having five chromosomes, which had been sequenced in 2000.
Several Phytophthora infestans-susceptible mutants had been obtained based onthe different reactions of Arabidopsis activated tagging mutants after being inoculatedby the fungus, and flanking sequence of Arabidopsis T-DNA insertion had also beenobtained by TAIL-PCR technology and genomic DNA from disease-susceptible mutantsas template. Some disease resistant-related genes had been found using NCBI sequencehomology analysis.
Based on the 258 Arabidopsis activated tagging mutants being inoculated by thefive Phytophthora infestans different isolates we found that AtTM31, AtTM34, AtTM40,AtTM113, AtTM246 were higher susceptibility to isolate 17-B-1 and AtTM114 weremediate susceptibility to isolate 17-B-1 and AtTM32, AtTM144, AtTM243, AtTM244were lower susceptibility to isolate 17-B-1; AtTM32, AtTM34, AtTM40, AtTM113were higher susceptibility to isolate W040604 and AtTM31, AtTM244 were mediatesusceptibility to isolate W040604 and AtTM114, AtTM144, AtTM243, AtTM246 werelower susceptibility to isolate W040604; AtTM31 were higher susceptibility to isolateW041002 and AtTM 32, AtTM34, AtTM40, AtTM113, AtTM243, AtTM246 weremediate susceptibility to isolate W041002 and AtTM114, AtTM144, AtTM244 werelower susceptibility to isolate W041002; AtTM32, AtTM40 were higher susceptibilityto isolate W041904 and AtTM34, AtTM144, AtTM243, AtTM246 were mediatesusceptibility to isolate W041904 and AtTM31, AtTM113, AtTM114, AtTM243werelower susceptibility to isolate W041904; AtTM31, AtTM32, AtTM40, AtTM246 werehigher susceptibility to isolate W041406 and AtTM34, AtTM114, AtTM243, AtTM244were mediate susceptibility to isolate W041406 and AtTM113, AtTM144 were lowersusceptibility to isolate W041406.
Thermal asymmetric interlaced PCR (TAIL-PCR) was efficiently used to amplifythe T-DNA flanking regions. Arbitrary primers AD1, AD2, AD4 and specific primers(Lex2, Lex4 and Lex5),(Lex3, Lex4 and Lex5) were the most suitable combination.Among 10 specific tertiary TAIL-PCR products sequenced, 2 mutants were found with T-DNA insertion within gene regions and 2 with T-DNA insertion in the region betweentwo genes and 5 with vector sequences.
In AtTM 34, T-DNA was inserted at 133bp in chromosomeⅢand was in the exonof gene AT3G13620.1 which was Amino acid permease family protein. In AtTM 246,T-DNA was inserted at 2672bp in chromosome V and was in the fifth exon of geneAT5G24830.1. The function was pentatricopeptide repeat containing protein.
Southern blotting analysis showed that AtTM 34 have one copy of T-DNAinsertion and AtTM 246 have two copies of T-DNA insertions.
本研究将致病疫霉不同菌株接种到拟南芥Col-0生态型激活标签突变体上后，发现其抗感反应不同，并筛选出了感致病疫霉的突变体类型。以筛选出的拟南芥感病突变体基因组DNA为模板，利用热不对称交错PCR方法(Thermal asymmetric interlaced PCR，TAIL-PCR)，进行了抗性相关基因的研究。通过TAIL-PCR技术，获得了拟南芥T-DNA插入侧翼序列，利用NCBI提供的拟南芥全基因组序列进行了序列同源性比对，主要结果如下：
测定拟南芥258个激活标签突变体接种5个致病疫霉菌株后发现，AtTM31、AtTM34、AtTM40、AtTM113、AtTM246对致病疫霉的菌株17-B-1表现为重度感病，AtTM114对菌株17-B-1表现为中度感病，AtTM32、AtTM144、AtTM243、AtTM244对菌株17-B-1表现为轻度感病；AtTM32、AtTM34、AtTM40、AtTM113对菌株W040604表现为重度感病，AtTM31、AtTM244对菌株W040604表现为中度感病，AtTM114、AtTM144、AtTM243、AtTM246对菌株W040604表现为轻度感病；AtTM31对菌株W041002表现为重度感病，AtTM32、AtTM34、AtTM40、AtTM113、AtTM243、AtTM246对菌株W041002表现为中度感病，AtTM114、AtTM144、AtTM244对菌株W041002表现为轻度感病；AtTM32、AtTM40对菌株W041904表现为重度感病，AtTM34、AtTM144、AtTM243、AtTM246对菌株W041904表现为中度感病，AtTM31、AtTM 113、AtTM114、AtTM243对菌株W041904表现为轻度感病；AtTM31、AtTM32、AtTM40、AtTM246对菌株W041406表现为重度感病，AtTM34、AtTM114、AtTM243、AtTM244对菌株W041406表现为中度感病，AtTM113、AtTM144对菌株W041406表现为轻度感病。
TAIL-PCR可以有效地扩增T-DNA插入侧翼拟南芥基因组序列，简并引物AD1、AD2、AD4和特异引物(Lex2、Lex4和Lex5)、(Lex3、Lex4和Lex5)组成的引物组合是最佳引物组合。对10个TAIL-PCR特异产物测序分析，发现2个T-DNA插入在基因上，2个则插入在基因间隔区中，5个是载体序列。
T-DNA在AtTM34突变体基因组上插入第3条染色体133bp左右，处在第1个外显子上，该基因属于氨基酸透性酶家族蛋白，与SP／Q9WTR6胱氨酸／谷氨酸转座子有低的相似性(Amino acid transport system xc-){Mus musculus}；包含Pfam profile PF00324：Amino acid permease。T-DNA在AtTM246突变体基因组上插入第5条染色体2672bp左右，处在第5个外显子上，该基因属于PPR模体蛋白(pentatricopeptide repeat containing protein)。
通过Southern杂交试验验证，突变体AtTM34中含一个拷贝的T-DNA插入，突变体AtTM246中含两个拷贝的T-DNA插入。
Arabidopsis thaliana is a plant of Crucifereae. The Arabidopsis Genome is 125Mb,having five chromosomes, which had been sequenced in 2000.
Several Phytophthora infestans-susceptible mutants had been obtained based onthe different reactions of Arabidopsis activated tagging mutants after being inoculatedby the fungus, and flanking sequence of Arabidopsis T-DNA insertion had also beenobtained by TAIL-PCR technology and genomic DNA from disease-susceptible mutantsas template. Some disease resistant-related genes had been found using NCBI sequencehomology analysis.
Based on the 258 Arabidopsis activated tagging mutants being inoculated by thefive Phytophthora infestans different isolates we found that AtTM31, AtTM34, AtTM40,AtTM113, AtTM246 were higher susceptibility to isolate 17-B-1 and AtTM114 weremediate susceptibility to isolate 17-B-1 and AtTM32, AtTM144, AtTM243, AtTM244were lower susceptibility to isolate 17-B-1; AtTM32, AtTM34, AtTM40, AtTM113were higher susceptibility to isolate W040604 and AtTM31, AtTM244 were mediatesusceptibility to isolate W040604 and AtTM114, AtTM144, AtTM243, AtTM246 werelower susceptibility to isolate W040604; AtTM31 were higher susceptibility to isolateW041002 and AtTM 32, AtTM34, AtTM40, AtTM113, AtTM243, AtTM246 weremediate susceptibility to isolate W041002 and AtTM114, AtTM144, AtTM244 werelower susceptibility to isolate W041002; AtTM32, AtTM40 were higher susceptibilityto isolate W041904 and AtTM34, AtTM144, AtTM243, AtTM246 were mediatesusceptibility to isolate W041904 and AtTM31, AtTM113, AtTM114, AtTM243werelower susceptibility to isolate W041904; AtTM31, AtTM32, AtTM40, AtTM246 werehigher susceptibility to isolate W041406 and AtTM34, AtTM114, AtTM243, AtTM244were mediate susceptibility to isolate W041406 and AtTM113, AtTM144 were lowersusceptibility to isolate W041406.
Thermal asymmetric interlaced PCR (TAIL-PCR) was efficiently used to amplifythe T-DNA flanking regions. Arbitrary primers AD1, AD2, AD4 and specific primers(Lex2, Lex4 and Lex5),(Lex3, Lex4 and Lex5) were the most suitable combination.Among 10 specific tertiary TAIL-PCR products sequenced, 2 mutants were found with T-DNA insertion within gene regions and 2 with T-DNA insertion in the region betweentwo genes and 5 with vector sequences.
In AtTM 34, T-DNA was inserted at 133bp in chromosomeⅢand was in the exonof gene AT3G13620.1 which was Amino acid permease family protein. In AtTM 246,T-DNA was inserted at 2672bp in chromosome V and was in the fifth exon of geneAT5G24830.1. The function was pentatricopeptide repeat containing protein.
Southern blotting analysis showed that AtTM 34 have one copy of T-DNAinsertion and AtTM 246 have two copies of T-DNA insertions.
引文
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