摘要
【目的】揭示水稻化感抗(耐)稗草的转录调控机制,为遗传修饰改良水稻化感性状做铺垫,也为杂草的防控提供新途径。【方法】利用SMART技术,通过同源重组方式,在Y2H菌株中构建水稻受稗草胁迫的酵母cDNA文库;利用限制性内切酶BamHⅠ和NdeⅠ构建OsMYB57 C端缺失的pGBK-OsMYB57诱饵载体,按照Yeastmaker^(TM) Yeast TransformationSystem 2操作检测诱饵自激活活性;用酵母双杂交技术筛选OsMYB57互作蛋白,将所得互作蛋白的编码序列构建至pGAD载体,并与诱饵载体共转化Y2HGold菌株验证蛋白互作,再以BiFC技术进一步验证蛋白间的互作。【结果】酵母cDNA文库库容量为1.36×10^(7) CFU/mL,插入片段在250~2000 bp,平均长度大于1000 bp,重组率为100%,库容量大且质量良好;筛选出4个有注释的互作蛋白,分别是16号RZFP34、50号4HTR、51号BM1PD和74号GTP1;回转验证及BiFC结果显示RZFP34、4HTR、BMIPD、GTP1与OsMYB57在植物细胞中相互作用,其中RZFP34是一个E3亚型泛素连接酶,在植物中响应非生物逆境胁迫应答,且水稻OsRZFP34基因启动子区含有与水稻化感调控有关的水杨酸和茉莉酸等激素响应及MYB转录因子结合域相关元件。【结论】RZFP34通过与OsMYB57互作参与水稻化感转录调控而发挥作用。
【Objective】To explore the transcriptional regulation mechanism on rice allelopathy resistant weeds,and provide theoretical basis for breeding rice of resistant weeds.It provided basis for the genetic modification and improvement of rice allelopathy traits,and also provided a new way for the prevention and control of weeds.【Method】Using SMART technology,cDNA library of rice under barnyard grass stress was constructed in Y2H by homologous recombination.The decoy vector pGBK-OsMYB57 with C-terminal deletion of OsMYB57 was constructed using restriction endonuclease BamHⅠand NdeⅠ,and its self-activation activity was detected according to Yeastmaker^(TM) Yeast Transformation System 2.The interaction protein of OsMYB57 was screened by yeast two-hybrid technology,and pGAD vector was constructed with the interaction protein coding sequence,and the interaction protein of Y2HGold strain was co-transformed with bait vector.The interaction between proteins was further verified by BiFC technology.【Result】The size of yeast cDNA library was 1.36×10^(7) CFU/mL,the inserted fragment was 250-2000 bp,the average length was more than 1000 bp,and the recombinant rate was 100%,showed that the library was large and of good quality.Four annotated interacting proteins,RZFP3416,4HTR 50,BM1PD 51 and GTP174,were screened out.RZFP34,4HTR,BMIPD and GTP1 in-teracted with OsMYB57 in plant cells.The RZFP34 was an E3 subtype ubiquitin ligase that responded to abiotic stress in plants,and the promoter region of rice OsRZFP34 contained elements related to hormone response such as salicylic acid,jasmonic acid and MYB transcription factor binding domain related to allelopathy regulation of rice.【Conclusion】RZFP34 interacts with OsMYB57 and participates in allelopathy transcriptional regulation of rice.
作者
张翠
马继琼
杨奕
许明辉
孙一丁
郭怡卿
ZHANG Cui;MA Ji-qiong;YANG Yi;XU Ming-hui;SUN Yi-ding;GUO Yi-qing(College of Plant Protection,Yunnan Agricul tural University,Kunming 650201,China;Institute of Biotechnology and Genetic Resources,Yunnan Academy of Agricultural Sciences,Kunming 650205,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2022年第1期29-37,共9页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31760523)
云南省科技计划项目(2018FB146,2018FB063)。
