水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

RNAi Vector Construction for Protein Disulfide Isomerase Gene and Seed Setting Characteristics in Offspring of Transgenic Rice under High Temperature Treatment

采用RT-PCR法亚克隆了PDI基因保守区内450bp的靶标序列作为干扰区段,构建了含有内含子hpRNA(ihpRNA)的双元表达载体pTCK303-RiOsPDI,经农杆菌介导转化日本晴,获得转基因植株;通过在T0代对其潮霉素(Hyg)抗性基因的PCR鉴定,确定携带有干扰片段的T-DNA区已整合到水稻基因组中,且在转基因T1代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明,PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低,尤其是其籽粒中表达量较微,几乎能引起靶基因80%左右沉默。对转基因T2代植株的高温结实特性和籽粒理化品质的检测结果,PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低,耐热性显著下降,但其在常温处理下的结实率与对照之间无显著差异。此外,PDI基因沉默后,稻米的透明度下降、垩白度增加,但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。

To clarify the effect ofPDI gene silence on rice yield traits and grain quality, we sub-cloned a 450 bp RNAi fragment from Nipponbare by RT-PCR, and successfully constructed a binary-vector pTCK303-RiOsPDI containing intron hpRNA （ih-pRNA）, then transformed it into the callus of wild type Nipponbare mediated by EHA105. The T-DNA region for PDI RNA inter-ference in regenerating rice plants was integrated with rice genome via single copy in To generation transgenic plants, and showed a 3：1 genetic mode in T1 transgenic population, which could be conformed by PCR amplifying analysis and Southern blotting identification. The PCR and qRT-PCR for PDI gene expression in different organs showed that there were much lower level of PDI expression in grains, leaves, stem and sheath for transgenic plants compared with those for wild type Nipponbare, with al-most 80% of the dropping in transgenic seeds. The influence of high temperature stress on seed setting traits, panicle agronomic traits and grain quality and also its relation to PDI gene expression in developing grains was further examined with T1 generation of transgenic plants imposed to two temperature treatments under the controlled temperature chambers, the results indicated that PDI silence transgenic plants had a remarkable lower seed setting rate, somewhat lower grain weight compared with its wild phe-notype under the high temperature treatment in despite of no obviously varying with its wild phenotype under normal develop- mental condition, suggesting that PDI gene should be probably responsible for rice tolerance to high temperature stress. More-over, there were lower translucence and higher chalky degree for transgenic plants than for Nipponbare, although no significant difference were observed in grain total protein and amylose content between PDI silence transgenic plant and its CK.