日本百脉根LjbHLH34基因克隆及耐旱功能鉴定

干旱是影响植物生长发育的重要环境因素。本研究分析了日本百脉根抗旱相关基因LjbHLH34的耐旱功能,初步解析其响应干旱胁迫的分子机制,以期为百脉根抗旱分子育种提供理论基础。本研究克隆得到的LjbHLH34基因大小为711 bp、编码236个氨基酸,属bHLH转录因子家族成员。系统进化树分析显示,LjbHLH34蛋白与拟南芥bHLHⅣ亚家族中AtbHLH34和AtbHLH104亲缘关系较近。实时荧光定量分析表明LjbHLH34在日本百脉根的根中表达量最高,叶中次之,茎中最少,暗示其在日本百脉根多个组织中发挥作用;同时LjbHLH34基因也受聚乙二醇(PEG)和脱落酸(ABA)诱导表达。在酵母中检测发现LjbHLH34具有转录激活活性;亚细胞定位试验表明LjbHLH34蛋白定位于细胞核中。将LjbHLH34基因转入拟南芥获得过表达株系。在200 mmol·L^-1甘露醇胁迫下,LjbHLH34转基因拟南芥的根长明显长于野生型。干旱处理后,野生型拟南芥比转基因拟南芥萎蔫程度更加明显,而转基因株系的相对含水量和超氧化物歧化酶(SOD)活性显著高于野生型,丙二醛(MDA)积累...

Cloning and identification of drought tolerance function of the LjbHLH34 gene in Lotus japonicus

Drought is an important environmental factor affecting plant growth and development. In this paper, we studied the drought tolerance function of the gene LjbHLH34, and conducted a preliminary analysis of its molecular mechanism in response to drought stress, in order to provide a theoretical basis for molecular breeding of Lotus japonicus drought resistance. In this study, the size of LjbHLH34 was found to be 711 bp and to encode 236 amino acids, belonging to the bHLH transcription factor family. Phylogenetic tree analysis showed that LjbHLH34 was closely related to AtbHLH34 and AtbHLH104 in the bHLH Ⅳ subfamily of Arabidopsis thaliana. The expression level of LjbHLH34 was the highest in the roots, the second highest in the leaves, and the lowest in the stems, suggesting that LjbHLH34 plays a role in multiple tissues of L. japonicus.LjbHLH34 gene expression was also induced by polyethylene glycol （PEG） and abscisic acid （ABA）. A yeast assay showed that LjbHLH34 had transcriptional activation activity, and a subcellular localization assay showed that LjbHLH34 was located in the nucleus. Under 200 mmol·L^-1 D-Mannitol stress, the root length of LjbHLH34 transgenic A. thaliana was significantly longer than that of the wild type. After drought treatment, the degree of withering of the wild-type was more obvious than transgenic Arabidopsis. At the same time, the relative water content and superoxide dismutase （SOD） activity of transgenic Arabidopsis were significantly higher than those of the wild-type, while malondialdehyde （MDA） accumulation was significantly lower than in the wild-type. qRT-PCR showed that the expression levels of stress-related genes AtCAT1, AtCAT3 and AtRD22 in transgenic lines were significantly increased after drought treatment. These results indicated that LjbHLH34 positively regulates drought resistance of plants.