水稻愈伤诱导过程中生长素通路的初步研究
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国家重大专项“抗虫转基因水稻新品种培育”(2016ZX08001001)


Auxin pathway in process of rice callus induction
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    摘要:

    构建水稻LBD基因家族中CRL1基因的CRISPR/CAS9基因敲除载体,并转化中花11水稻愈伤,获得了8个转基因片段缺失家系;对CRL1基因敲除材料种子进行愈伤诱导实验;结果显示,它们的愈伤组织诱导受到了强烈抑制,与已知的水稻OsIAA10基因的RNA干扰材料表型相似。进行OsIAA10的亚细胞定位实验,结果显示,OsIAA10同时定位在细胞核与细胞膜上。通过酵母双杂交筛库实验,发现OsIAA10能与OsARF家族成员中的OsARF5、OsARF1、OsARF721、OsARF23四个转录因子发生互作。以CRL1基因启动子为诱饵构建载体,进行酵母单杂交点对点实验,结果显示,这4个转录因子中只有OsARF5与OsARF21能特异性结合CRL1基因启动子区的AuxRE基序,激活CRL1基因转录。本研究结果初步证明水稻侧根发育通路与愈伤组织诱导过程存在直接相关,水稻与双子叶植物之间存在保守的生长素调控机制,在水稻种子的愈伤组织诱导过程中发挥着重要的作用。

    Abstract:

    Rice as a monocot model plant is one of the few crops that achieve mature tissue culture,but the physiological processes and specific molecular mechanisms in the process of tissue culture are still rarely understood. In this study,the CRISPR/CAS9 gene knockout vector of the CRL1 gene in the rice LBD gene family was constructed and transformed into callus of rice Zhonghua 11. Eight families with missing transgene fragments were obtained. The result of callus induction on the seeds of CRL1 gene knockout material showed that their callus induction was strongly inhibited,similar to the known RNA interference material phenotype of the rice OsIAA10 gene. The results of a subcellular localization of OsIAA10 showed that OsIAA10 was located on the nucleus and the cell membrane. Through the yeast two-hybrid screening library experiment,it was found that OsIAA10 can interact with four transcription factors including OsARF5,OsARF 17,OsARF 21,and OsARF 23 among members of the OsARF family. A yeast one-hybrid point-to-point experiment was performed using the promoterof CRL1 gene as a bait to construct a vector. The results showed that only OsARF5 and OsARF21 can specifically bind to the AuxRE motif in the promoter region of the CRL1 gene and activate the transcription of CRL1 gene. It is indicated that the development pathway of rice lateral roots is directly related to the process of callus induction. There is a conservative mechanism of auxin regulation between rice and dicots,which plays an important role in the process of callus induction in rice seeds.

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段镇淳,张昭阳,林拥军.水稻愈伤诱导过程中生长素通路的初步研究[J].华中农业大学学报,2021,40(3):98-104

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  • 收稿日期:2020-12-08
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  • 在线发布日期: 2021-06-07
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