Chen Yun, Zhu Wenping, Shi Shudan, Wu Lina, Du Shuanglin, Jin Liangshen, Yang Kuan, Zhao Wenjia, Yang Jiaxin, Guo Longbiao, Wang Zhongwei, Zhang Yi
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Research Center for Perennial Rice Engineering and Technology in Yunnan, School of Agriculture, Yunnan University, Kunming, China.
College of Agronomy and Biotechnology, Southwest University, Chongqing, China.
Front Plant Sci. 2021 Sep 6;12:728193. doi: 10.3389/fpls.2021.728193. eCollection 2021.
Gametophytic male sterility (GMS) plays an important role in the study of pollen development and seed propagation of recessive nuclear male sterile lines insensitive to the environmental conditions in hybrid rice breeding. Since the inherent phenotypic and genetic characteristics of GMS, it is very difficult to find and identify the GMS mutants. However, due to the abundance of gene transcription data, a large number of pollen-specific genes have been found, and most of them may be associated with GMS. To promote the study of these genes in pollen development and heterosis utilization, in this study, an easy and efficient method of creating and identifying GMS was established using RNAi and as a reporter. First, the / gene involved in anthocyanin synthesis was modified, and we have validated that the modified is workable as the same as the pre-modified gene. Then, the ascorbic acid oxidase gene was downregulated using RNAi, driven by its own promoter that resulted in abnormal pollen tube growth. Finally, the RNAi elements were linked with and transformed into an mutant, and the distortion of purple color segregation was found in T and F generations. This indicates that the GMS was prepared successfully. Compared to current methods, there are several advantages to this method. First, time is saved in material preparation, as one generation less needs to be compared than in the conventional method, and mutation screening can be avoided. In addition, for identification, the cost is lower; PCR, electrophoresis, and other processes are not needed; and no expensive chemicals or instruments are required. Finally, the results are more accurate, with much lower background effects, and no damage to the plant. The result is an easy, efficient, low-cost, and accurate method of preparing and identifying GMS genes.
配子体雄性不育（GMS）在杂交水稻育种中对花粉发育和隐性核雄性不育系种子繁殖的研究起着重要作用，这些不育系对环境条件不敏感。由于GMS固有的表型和遗传特性，很难找到和鉴定GMS突变体。然而，由于基因转录数据丰富，已发现大量花粉特异性基因，其中大多数可能与GMS相关。为了促进这些基因在花粉发育和杂种优势利用方面的研究，本研究利用RNA干扰技术并以[具体基因名称未给出]作为报告基因，建立了一种简便高效的创建和鉴定GMS的方法。首先，对参与花青素合成的[具体基因名称未给出]基因进行了修饰，并且我们已经验证修饰后的[具体基因名称未给出]与修饰前的基因一样可行。然后，利用RNA干扰技术在其自身启动子的驱动下下调抗坏血酸氧化酶基因[具体基因名称未给出]，导致花粉管生长异常。最后，将RNA干扰元件与[具体基因名称未给出]连接并转化到[具体突变体名称未给出]突变体中，在T1和F1代中发现紫色分离出现畸变。这表明成功制备了[具体基因名称未给出] GMS。与现有方法相比，该方法具有几个优点。首先，在材料准备上节省了时间，因为比传统方法少比较一代，并且可以避免突变筛选。此外，在鉴定方面成本较低；不需要PCR、电泳等过程；也不需要昂贵的化学试剂或仪器。最后，结果更准确，背景效应低得多，并且对植物无损害。结果是一种简便、高效、低成本且准确的制备和鉴定GMS基因的方法。
