National Institute for Plant Biotechnology, Pusa Campus, New Delhi, India.
National Institute for Plant Biotechnology, Pusa Campus, New Delhi, India; Division of Genetics, IARI, Pusa Campus, New Delhi, India.
Plant Physiol Biochem. 2020 Nov;156:7-19. doi: 10.1016/j.plaphy.2020.07.031. Epub 2020 Aug 23.
A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T stage. For drought stress tolerance, three independent lines (T stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.
利用抑制差减杂交（SSH）方法，在耐旱型 Pennisetum glaucum 基因型 841B 中生成了一组干旱胁迫响应基因/ESTs 的基因组资源。将 55 天大的植物在不同的时间间隔（10 天、15 天、20 天和 25 天）停水后进行干旱胁迫处理。从叶片组织分离的 RNA 中制备正向消减 cDNA 文库。通过 RT-qPCR 对选定的 9 个重叠群进行了干旱胁迫下差异基因表达的验证。从基因型 841B 中分离并鉴定出一个在干旱胁迫下上调表达的 Setaria italica ASR3 同源物。在拟南芥中验证了 PgASR3 的异源表达，并在多种非生物胁迫条件下得到证实。通过 Southern blot 在 T 期分析了四个独立的过表达基因 PgASR3 的转基因株系。为了研究耐旱性，在幼苗期通过生化和生理测定分析了三个独立的株系（T 期）。与 WT 幼苗相比，在不同非生物胁迫条件下，转基因幼苗的生长速度（茎和根长）提高。还对三个转基因系进行了干旱胁迫耐受性和 RT-qPCR 分析，在成熟阶段。在干旱胁迫条件下，与 WT 植物相比，成熟的转基因系表现出更高的 RWC、叶绿素和脯氨酸含量，但 MDA 含量较低。本研究中分离和验证的 PgASR3 基因可用于开发抗非生物胁迫作物。
