| 王巧慧，谷建华，郭 欢，吕士凯，吉万全,张 宏.白粉病菌侵染条件下小麦酵母双杂交文库的构建及可用性检测[J].麦类作物学报,2021,(2):127 |
| 白粉病菌侵染条件下小麦酵母双杂交文库的构建及可用性检测 |
| Construction and Utilization of Wheat Yeast Two-Hybrid Library under the Infection of Blumeria graminis f.sp.tritici |
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| DOI：10.7606/j.issn.1009-1041.2021.02.01 |
| 中文关键词: 白粉菌 小麦 cDNA文库 酵母双杂交 HSP40-70 |
| 英文关键词:Powdery mildew Wheat cDNA Library Yeast two-hybrid HSP40-70 |
| 基金项目:国家自然科学基金项目（31971941） |
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| 摘要点击次数: 817 |
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| 中文摘要: |
| 为解析白粉病菌侵染小麦以及小麦防御的分子机制，以抗白粉病种质N9134为材料，剪取白粉病菌（Blumeria graminis f.sp.tritici,Bgt）接种48 h后的叶片，提取总RNA，利用SMART技术进行反转录合成双链cDNA（ds-cDNA），并进行均一化处理和SfiI酶切处理，处理后的ds-cDNA分别连接pGADT7-SfiI三框载体，将连接产物转化至感受态细胞HST08中构建文库。通过滴度测定和PCR鉴定其库容量和基因重组率，最后收集细菌并提取质粒。经鉴定，该文库库容量大于1.0×106 cfu·mL-1，插入片段主要分布在 400～2 000 bp之间，重组率为100%，冗余度为3%。利用酵母双杂交系统筛选cDNA文库，获得2个与热休克蛋白HSP40-70互作的蛋白质，分别为DnaJ类蛋白（DnaJ-like）和E3泛素蛋白连接酶AIP2（AIP）。综上表明，文库构建质量较好，为后续筛选抗逆关键基因奠定了基础。 |
| 英文摘要: |
| In order to analyze the molecular mechanism of Blumeria graminis f.sp.tritici(Bgt) infecting wheat and wheat defense,the leaves of N9134 with high resistance to powdery mildew were collected at 48 hours post inoculation(hpi) of Bgt. After total RNA extraction,the SMART technology was used to reverse transcription and synthesize double-stranded cDNA(ds-cDNA),and homogenization treatment and SfiI digestion treatment were performed. The processed ds-cDNA was respectively constructed into the pGADT7-SfiI three-frame vector,and then the ligation product was transformed into competent cell HST08 to construct the library. Furthermore,its capacity and gene recombination rate were identified by titer determination and PCR,and finally the bacteria were collected to extract plasmid. It was identified that the library capacity was greater than 1.0×106 cfu·mL-1,and the length of insert fragments mainly ranged from 400 to 2 000 bp,with a recombination rate of 100% and a redundancy of 3%. Screening the cDNA library though yeast two-hybrid system,two potential proteins interacting with the heat shock protein HSP40-70 were obtained. Sequence alignment showed that they are DnaJ-like protein and E3 ubiquitin protein ligase AIP2(AIP),respectively. In summary,the quality of library construction guaranteed subsequent screening key genes for resistance to powdery mildew. |
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