本文關(guān)鍵詞：水稻蛋白二硫鍵異構(gòu)酶基因沉默載體構(gòu)建及其轉(zhuǎn)基因后代的高溫結(jié)實(shí)特性分析，由筆耕文化傳播整理發(fā)布。

作物學(xué)報(bào) ACTA AGRONOMICA SINICA 2013, 39(5): 816 826

ISSN 0496-3490; CODEN TSHPA9

E-mail: xbzw@

DOI: 10.3724/SP.J.1006.2013.00816

水稻蛋白二硫鍵異構(gòu)酶基因沉默載體構(gòu)建及其轉(zhuǎn)基因后代的高溫結(jié)實(shí)特性分析

劉光快 曹珍珍 韋克蘇 潘 剛 蘇 達(dá) 張春嬌 程方民*

浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院, 浙江杭州310058

摘 要: 采用RT-PCR法亞克隆了PDI基因保守區(qū)內(nèi)450 bp的靶標(biāo)序列作為干擾區(qū)段, 構(gòu)建了含有內(nèi)含子hpRNA (ihpRNA)的雙元表達(dá)載體pTCK303-RiOsPDI, 經(jīng)農(nóng)桿菌介導(dǎo)轉(zhuǎn)化日本晴, 獲得轉(zhuǎn)基因植株; 通過(guò)在T0代對(duì)其潮霉素(Hyg)抗性基因的PCR鑒定, 確定攜帶有干擾片段的T-DNA區(qū)已整合到水稻基因組中, 且在轉(zhuǎn)基因T1代符合3∶1的分離模式。半定量PCR和熒光定量PCR的檢測(cè)結(jié)果表明, PDI基因沉默轉(zhuǎn)基因陽(yáng)性植株不同器官中的PDI表達(dá)量均顯著降低, 尤其是其籽粒中表達(dá)量較微, 幾乎能引起靶基因80%左右沉默。對(duì)轉(zhuǎn)基因T2代植株的高溫結(jié)實(shí)特性和籽粒理化品質(zhì)的檢測(cè)結(jié)果, PDI基因沉默會(huì)引起高溫脅迫處理下結(jié)實(shí)率的大幅度降低, 耐熱性顯著下降, 但其在常溫處理下的結(jié)實(shí)率與對(duì)照之間無(wú)顯著差異。此外, PDI基因沉默后, 稻米的透明度下降、堊白度增加, 但對(duì)籽粒粗蛋白總量和直鏈淀粉含量的影響不甚明顯。

關(guān)鍵詞: 水稻; 蛋白二硫鍵異構(gòu)酶; 基因沉默; RNAi載體構(gòu)建; 高溫脅迫; 結(jié)實(shí)特性

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

LIU Guang-Kuai, CAO Zhen-Zhen, WEI Ke-Su, PAN Gang, SU Da, ZHANG Chun-Jiao, and CHENG Fang-Min*

College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China

Abstract: To clarify the effect of PDI 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 T0 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.

Keywords: Rice (Oryza sativa L.); Protein disulfide isomerase (PDI); Gene silence; RNA interference; High temperature stress; Seed setting characteristics

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(31071366, 31271655, 30871488)資助。

*

通訊作者(Corresponding author): 程方民, E-mail: chengfm@

第一作者聯(lián)系方式: E-mail: liukuai2006@

Received(收稿日期): 2012-11-22; Accepted(接受日期): 2013-01-15; Published online(網(wǎng)絡(luò)出版日期): 2013-02-19. URL:

  本文關(guān)鍵詞：水稻蛋白二硫鍵異構(gòu)酶基因沉默載體構(gòu)建及其轉(zhuǎn)基因后代的高溫結(jié)實(shí)特性分析，，由筆耕文化傳播整理發(fā)布。