水稻抗黑条矮缩病转基因植株的构建与鉴定

发布时间：2018-12-14 01:44

【摘要】：水稻黑条矮缩病是我国主要的水稻病害之一,感病水稻会表现出植株矮化、分蘖增多、结实率低、生长发育畸形等症状,严重危害水稻产量。水稻黑条矮缩病由水稻黑条矮缩病毒(Rice black streaked dwarf virus, RBSDV)引起,而RBSDV以灰飞虱(Laodelphax striatellus)作为媒介,能以不经卵方式持久传播,传播速度快且范围广。人们对水稻黑条矮缩病的防治需从RBSDV和灰飞虱两方面入手。本研究同时构建了RBSDV S3基因和灰飞虱海藻糖酶基因(Memb)的RNAi转基因水稻,并进行了阳性转基因植株的筛选及阳性转基因植株对RBSDV的抗性鉴定,以期获得抗RBSDV的水稻新品种,主要研究结果如下：1.构建了Memb RNAi克隆,通过菌落PCR、酶切鉴定和测序鉴定,确定Memb RNAi载体构建成功,并将该载体转化至对RBSDV敏感的水稻品种武陵粳中。2.获得稳定遗传的Memb RNAi转基因水稻和RBSDV S3 RNAi转基因水稻。先用潮霉素筛选到潮霉素抗性植株,再用PCR扩增确定目的片段的插入情况,最后利用荧光定量PCR (qPCR)分析阳性转基因植株的目的片段表达水平。目前共获得T2代Memb RNAi转基因水稻表达良好的9个株系和T3代S3阳性RNAi转基因水稻表达良好的6个株系。3.对表达水平较高的转基因植株通过人工室内接种RBSDV进行抗性鉴定,发现T2代Memb RNAi转基因水稻和T3代RBSDV S3 RNAi转基因水稻对RBSDV都具有明显抗性,且T3代S3 RNAi转基因水稻的抗性更加明显。通过农艺性状分析,Memb RNAi和RBSDV S3 RNAi转基因水稻在大田中的农艺性状与对照相比并没有明显改变。本文研究结果为水稻抗黑条矮缩病分子育种提供了重要基因资源、参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.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.111.4

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