National Research Centre on Plant Biotechnology, Pusa campus, New Delhi, India.
Shobhit University, Meerut, Uttar Pradesh, India.
PLoS One. 2018 Jun 25;13(6):e0198293. doi: 10.1371/journal.pone.0198293. eCollection 2018.
To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to Hsp90 was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of TaHsp90 (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of TaHsp90 was validated in E. coli BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene TaHsp90 isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.
为了生成热应激响应基因/EST 的遗传资源，我们在一种耐热耐旱的印度面包小麦品种 C306 中构建了抑制性消减杂交（SSH）文库。在灌浆期，93 天大的植株在 37°C 和 42°C 的高温下分别经历不同时间的热应激（30 分钟、1 小时、2 小时、4 小时和 6 小时）。从 37°C 和 42°C 热应激的叶片样本中分离 RNA，制备了两个消减 cDNA 文库。通过反向Northern dot blot 杂交对获得的 EST 进行了重新确认。通过基因本体论分析，从 1728 个 EST 中获得了 175 个连续群和 403 个单倍体。通过 qRT-PCR 对一些选定基因（10 个）的热应激下的差异表达进行了验证。在 C306 中，观察到一个与 Hsp90 同源的转录本在热应激下上调（7.6 倍）。从 C306 中分离并鉴定了 TaHsp90（登录号 MF383197）的 CDS。TaHsp90 在大肠杆菌 BL21 中的异源表达通过蛋白凝胶印迹和 MALDI-TOF 分析得到了验证。进行了基于计算的分析，以了解 TaHsp90 的分子功能。开发的热应激响应 SSH 文库导致鉴定出许多对热响应的基因/EST，可用于揭示小麦的耐热机制。本研究中分离和鉴定的基因 TaHsp90 可用于开发耐热转基因作物。
