RNAi创制水稻抗黑条矮缩病材料的初步研究

发布时间：2018-02-13 11:23

本文关键词： 水稻水稻黑条矮缩病毒灰飞虱 RNAi 转基因株系　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：水稻黑条矮缩病是由水稻黑条矮缩病毒(Rice black streaked dwarf virus, RBSDV)引起的水稻病毒病,严重危害水稻安全生产。本研究利用RNAi (RNA interference)技术,构建针对RBSDV病毒本身及其传播媒介灰飞虱的RNAi载体,转化RBSDV水稻高感品种武陵粳1号,以期获得抗水稻黑条矮缩病和灰飞虱的水稻新种质,并探讨抗水稻黑条矮缩病机制。结果如下：1.以RBSDV的S4、S8和S9序列为候选基因片断,利用两步法(pENTR-pANDA)快速高效构建了5个RNAi载体,通过农杆菌介导的转化法转化武陵粳1号并获得转基因T1代阳性植株,其中含有S9干扰片段的转基因植株]0株,S8转基因13株,目前S4仍在转化过程中。同时,对含有S8干扰片段的转基因T2代植株进行温室RBSDV接种,初步实验结果表明,沉默S8的转基因株系对RBSDV呈现一定抗性。2.利用基因序列拼接手段将RBSDV的S9片段分别与S4、S8进行拼接,组合成较大的干扰片段S4＆S9(一个拼接组合)和S8S9(两个拼接组合),共构建3个RNAi拼接载体,分别转化武陵粳1号,获得了沉默S4S9和S8S9的转基因T1代阳性植株分别有8株系和66株系。3.构建了分别针对灰飞虱关键基因激活性蛋白激酶C受体(Receptor for activated protein kinase C, RACK)基因、60S核糖体蛋白L5 (60S ribosomal protein L5, RPL5)基因、甘油醛-3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase, GAPDH3)基因、60S核糖体蛋白L7a (60S ribosomal protein L7a, RPL7a)基因、60S核糖体蛋白L8(60Sribosomal protein L8, RPL8)基因、线粒体NADH脱氢酶亚基4和5基因(NADH dehydrogenase subunit 4 and NADH dehydrogenase subunit 5, ND4 and ND5)和线粒体NADH脱氢酶亚基2基因(NADH dehydrogenase subunit 2, ND2),共计7个基因的14个RNAi载体,转化武陵粳1号,获得转基因T1代阳性植株。4.利用灰飞虱传毒,建立了室内离体叶片RBSDV接种和活体植株RBSDV接种体系。
[Abstract]:Rice black stripe dwarf disease is caused by rice black stripe dwarf virus Rice black streaked dwarf virus (RBSDV), which seriously endangers the safe production of rice. In this study, a RNAi vector targeting RBSDV virus and its transmission vector was constructed by using the RNAi RNA interference technique. In order to obtain new rice germplasm resistant to rice black stripe dwarf disease and ash planthopper, and to explore the mechanism of resistance to black stripe dwarf disease of rice, the results are as follows: 1. Using the S4S8 and S9 sequences of RBSDV as candidate gene fragments, we transformed Wulingjing 1, a highly susceptible rice variety, into a new rice germplasm resistant to rice black stripe dwarf disease and ash planthopper. Five RNAi vectors were constructed rapidly and efficiently by two-step pENTR-pANDA.The transformation of Wulingjing 1 by Agrobacterium tumefaciens was carried out to obtain the transgenic T1 generation positive plants, including the transgenic plants containing S9 interference fragments. At present, S4 is still in the process of transformation. At the same time, transgenic plants containing S8 interference fragments were inoculated with RBSDV in greenhouse. The transgenic lines silencing S8 showed resistance to RBSDV. The S9 fragment of RBSDV was spliced with S4 S8 by gene sequence splicing. S4 & S9 (a splicing combination) and S8S9 (two splicing combinations) were combined to construct three RNAi splicing vectors, which were transformed into Wulingjing 1, respectively. Eight lines and 66 lines of transgenic T1 generation positive plants with silencing S4S9 and S8S9 were obtained respectively. The gene of 60S ribosomal protein L560S ribosomal protein L5 (RPL5) was constructed for the key gene activated protein kinase C receptor (Receptor for activated protein kinase C, RPL5) of planthopper, respectively. Glyceral dehyde-3-phosphate dehydrogenase (GAPDH3) gene of 60S ribosomal protein L7a, RPL7a) gene of 60S ribosomal protein L8 (RPL8). Mitochondrial NADH dehydrogenase subunits 4 and 5, nadh dehydrogenase subunit 4 and NADH dehydrogenase subunit 5, ND4 and ND5) and mitochondrial NADH dehydrogenase subunit 2, nadh dehydrogenase subunit 2, ND2, were transformed into Wulingjing 1 by 14 RNAi vectors of 7 genes. Transgenic T1 generation positive plants. 4. The indoor RBSDV inoculation system in vitro and RBSDV inoculation system in living plantlets were established by using planthopper transfered virus.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S435.111.4

【相似文献】