【摘要】：水稻黑條矮縮病是我國(guó)主要的水稻病害之一,感病水稻會(huì)表現(xiàn)出植株矮化、分蘗增多、結(jié)實(shí)率低、生長(zhǎng)發(fā)育畸形等癥狀,嚴(yán)重危害水稻產(chǎn)量。水稻黑條矮縮病由水稻黑條矮縮病毒(Rice black streaked dwarf virus, RBSDV)引起,而RBSDV以灰飛虱(Laodelphax striatellus)作為媒介,能以不經(jīng)卵方式持久傳播,傳播速度快且范圍廣。人們對(duì)水稻黑條矮縮病的防治需從RBSDV和灰飛虱兩方面入手。本研究同時(shí)構(gòu)建了RBSDV S3基因和灰飛虱海藻糖酶基因(Memb)的RNAi轉(zhuǎn)基因水稻,并進(jìn)行了陽性轉(zhuǎn)基因植株的篩選及陽性轉(zhuǎn)基因植株對(duì)RBSDV的抗性鑒定,以期獲得抗RBSDV的水稻新品種,主要研究結(jié)果如下：1.構(gòu)建了Memb RNAi克隆,通過菌落PCR、酶切鑒定和測(cè)序鑒定,確定Memb RNAi載體構(gòu)建成功,并將該載體轉(zhuǎn)化至對(duì)RBSDV敏感的水稻品種武陵粳中。2.獲得穩(wěn)定遺傳的Memb RNAi轉(zhuǎn)基因水稻和RBSDV S3 RNAi轉(zhuǎn)基因水稻。先用潮霉素篩選到潮霉素抗性植株,再用PCR擴(kuò)增確定目的片段的插入情況,最后利用熒光定量PCR (qPCR)分析陽性轉(zhuǎn)基因植株的目的片段表達(dá)水平。目前共獲得T2代Memb RNAi轉(zhuǎn)基因水稻表達(dá)良好的9個(gè)株系和T3代S3陽性RNAi轉(zhuǎn)基因水稻表達(dá)良好的6個(gè)株系。3.對(duì)表達(dá)水平較高的轉(zhuǎn)基因植株通過人工室內(nèi)接種RBSDV進(jìn)行抗性鑒定,發(fā)現(xiàn)T2代Memb RNAi轉(zhuǎn)基因水稻和T3代RBSDV S3 RNAi轉(zhuǎn)基因水稻對(duì)RBSDV都具有明顯抗性,且T3代S3 RNAi轉(zhuǎn)基因水稻的抗性更加明顯。通過農(nóng)藝性狀分析,Memb RNAi和RBSDV S3 RNAi轉(zhuǎn)基因水稻在大田中的農(nóng)藝性狀與對(duì)照相比并沒有明顯改變。本文研究結(jié)果為水稻抗黑條矮縮病分子育種提供了重要基因資源、參考信息以及新的策略。
[Abstract]:Rice black stripe dwarf disease is one of the main rice diseases in China. Susceptible rice will show the symptoms of plant dwarfing, increasing tiller, low seed setting rate, abnormal growth and development, which seriously endangers rice yield. Rice black stripe dwarf disease is caused by rice black stripe dwarf virus (Rice black streaked dwarf virus, RBSDV), while RBSDV, with (Laodelphax striatellus) as the vector, can be transmitted continuously without eggs, and the transmission speed is fast and the range is wide. The prevention and control of rice black stripe dwarf disease should be carried out from two aspects: RBSDV and ash planthopper. In this study, RNAi transgenic rice of RBSDV S3 gene and trehalase gene (Memb) of planthopper were constructed, and the screening of positive transgenic plants and the identification of RBSDV resistance of positive transgenic plants were carried out in order to obtain new varieties resistant to RBSDV. The main results are as follows: 1. The Memb RNAi clone was constructed and identified by PCR, digestion and sequencing. The Memb RNAi vector was successfully constructed and transformed into Wuling japonica, a rice cultivar sensitive to RBSDV. Stable Memb RNAi transgenic rice and RBSDV S3 RNAi transgenic rice were obtained. Hygromycin was used to screen hygromycin resistant plants, then PCR amplification was used to determine the insertion of the target fragment. Finally, the target fragment expression level of positive transgenic plants was analyzed by fluorescence quantitative PCR (qPCR). At present, 9 lines of T2 generation Memb RNAi transgenic rice and 6 T3 generation S3 positive RNAi transgenic rice lines were obtained. The transgenic plants with high expression level were identified by inoculation with RBSDV in vitro. It was found that both T2 generation Memb RNAi transgenic rice and T3 generation RBSDV S3 RNAi transgenic rice had obvious resistance to RBSDV. The resistance of T 3 generation S 3 RNAi transgenic rice was more obvious than that of T 3 generation S 3 RNAi transgenic rice. The agronomic characters of, Memb RNAi and RBSDV S3 RNAi transgenic rice in the field were not significantly changed compared with the control. The results of this study provide important genetic resources, reference information and new strategies for molecular breeding of rice resistant to black stripe dwarf disease.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.111.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 孫楓;徐秋芳;程兆榜;范永堅(jiān);周益軍;;中國(guó)水稻黑條矮縮病研究進(jìn)展[J];江蘇農(nóng)業(yè)學(xué)報(bào);2013年01期

2 牟宗順;邵長(zhǎng)新;;水稻黑條矮縮病的發(fā)生特點(diǎn)與防控技術(shù)[J];中國(guó)種業(yè);2012年10期

3 張倩;魯鼎浩;蒲建;吳敏;韓召軍;;灰飛虱海藻糖酶基因的克隆及RNA干擾效應(yīng)[J];昆蟲學(xué)報(bào);2012年08期

4 施秀珍;吳青君;王少麗;徐寶云;謝文;張友軍;;RNA干擾在昆蟲中的作用機(jī)理及應(yīng)用進(jìn)展[J];生物安全學(xué)報(bào);2012年03期

5 鄧金奇;戴良英;;水稻黑條矮縮病毒的研究進(jìn)展[J];中國(guó)農(nóng)學(xué)通報(bào);2012年18期

6 顧永林;;水稻矮縮病的研究[J];農(nóng)業(yè)災(zāi)害研究;2012年01期

7 陳軍;劉遜忠;謝光禮;;水稻黑條矮縮病發(fā)生危害與綜合防控技術(shù)[J];農(nóng)業(yè)科技通訊;2011年08期

8 周彤;王英;吳麗娟;范永堅(jiān);周益軍;;水稻品種抗黑條矮縮病人工接種鑒定方法[J];植物保護(hù)學(xué)報(bào);2011年04期

9 郭榮;周國(guó)輝;張曙光;;水稻南方黑條矮縮病發(fā)生規(guī)律及防控對(duì)策初探[J];中國(guó)植保導(dǎo)刊;2010年08期

10 陳潔;吳麗娟;周彤;魏邦慶;周益軍;;江蘇省主栽水稻品種對(duì)條紋葉枯病與灰飛虱的抗性評(píng)價(jià)[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);2010年04期

相關(guān)博士學(xué)位論文 前1條

1 周彤;水稻黑條矮縮病抗性鑒定技術(shù)和遺傳研究[D];南京農(nóng)業(yè)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 李祥宇;RBSDV和SRBSDV安徽分離物全基因組特征及SRBSDV mp基因的功能分析[D];安徽農(nóng)業(yè)大學(xué);2013年

2 倪躍群;水稻矮縮病毒和水稻黑條矮縮病毒單克隆抗體的制備及其應(yīng)用[D];浙江大學(xué);2013年

，

本文編號(hào)：2377677