? (JIA-2016-0023) RNAi-mediated transgenic rice resistance to Rice stripe virus
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DOI: 10.1016/S2095-3119(16)61369-2
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(JIA-2016-0023) RNAi-mediated transgenic rice resistance to Rice stripe virus
LI Li1*, GUO Cheng2*, WANG Biao1, ZHOU Tong3, LEI Yang1, DAI Yu-hua1, HE Wen1, LIANG Chun1, 2, 4, WANG Xi-feng1
1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
2 Department of Biology, Miami University, Oxford, OH 45056, USA
3 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
4 Department of Computer Science and Software Engineering, Miami University, Oxford, OH 45056, USA
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摘要     【目的】系统分析基于RNA干涉(RNA interference, RNAi)原理获得的转水稻条纹病毒(Rice stripe virus, RSV)核衣壳蛋白(nucleocapsid protein, NCP)基因植株的分子特征,阐明NCP基因的发夹结构在转基因水稻产生抗病性方面的理论基础。【方法】构建NCP基因的RNAi载体,在农杆菌EHA105介导下遗传转化水稻品种爱知旭(Oryza sativa cv. Aichiasahi)的成熟胚愈伤组织,获得再生植株(T0代)。从T2代开始逐代接种RSV进行抗性筛选和分子鉴定,获得T5代高抗RSV、且能稳定遗传发夹结构的株系。采用长链PCR、基因组步移和Southern blotting分析发夹结构在水稻基因组的整合位点和拷贝数,利用Northern blotting定性分析发夹结构是否产生RSV来源的sRNA(RSV-siRNA)及sRNA的长度;最后利用Illumina Small RNA library kit及高通量技术进一步测定RSV侵染或未侵染的转基因和野生型植株产生sRNA序列,利用Tag2siRNA和GSNAP软件分析siRNA的特征及与RSV和水稻基因组匹配对比,明确RSV-siRNA的数量、种类及特征,解析RSV-siRNA与抗病性的相互关系。【结果】构建了NCP基因的RNAi载体,在EHA105介导下遗传转化爱知旭的成熟胚愈伤组织,经潮霉素筛选,获得了27个再生植株(T0代),其中12株PCR扩增到NCP基因。通过对转基因再生植株及后代的抗病筛选和分子鉴定,获得对RSV侵染不表现明显症状、且能稳定遗传发夹结构的3个T0株系的T5代材料。对T5代植株的分子分析表明,NCP基因发夹结构成功整合到了水稻的的基因组中;T-DNA区的插入取代了水稻1号染色体的32,158,773到32,158,787 nt的15个碱基;T-DNA区距上游基因(LOC_Os01g55840)2,630 nt,距离下游基因(LOC_Os01g55850)6,653 nt;转基因植株主要产生了21、22和24 nt的小RNA(sRNA),而野生型没有检测到sRNA。高通量测序及注释结果则表明,无论是RSV侵染或不侵染的转基因植株(T4-B1-V 和T4-B1-VF)均产生了10倍于野生型的siRNA,说明插入的NCP的发夹结构诱导产生了大量的siRNAs。RSV侵染也会诱导转基因和野生型植株的siRNAs剧增,约增加了月50%。siRNAs的起源分析发现,野生型植株产生的siRNA序列主要是水稻内生的(95.37%),没有检测到RSV-siRNA;而在T4-B1-V 和T4-B1-VF上,RSV-siRNA分别增加到61.69%和53.45%,同时来源于水稻基因组的siRNA 序列却分别降至3.75% 和5.59%。对转基因植株上RSV-siRNAs的特征分析发现,RSV-siRNAs长度从20-25 nt,主要为21 nt 、 22 nt和24 nt,如在T4-B1-V上21 nt RSV-siRNAs占34.90%, 22 nt占32.45%, 24 nt占17.74%,这一结果与northern blotting分析结果一致;siRNAs的 5’端多为A或U,如T4-B1-V产生的siRNAs 5’端是A的占32.57%,是U的占28.71% ;RSV NCP基因上的存在产生siRNAs的热点区域,在T4-B1-V上47%的siRNAs起源于 NCP基因的594 到832 nt(占NCP全长基因的24.66%)的序列。【结论】RSV的 NCP基因的发夹结构诱导转基因植株产生大量的RSV-siRNAs,其是抗病性产生的根本原因。【创新性】从基因组学水平上系统分析了抗RSV转基因水稻植株的siRNA特征,阐明了NCP基因的发夹结构在转基因水稻产生抗病性方面的作用。
Abstract     Rice stripe virus (RSV) often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs (siRNAs) with transgenic virus resistance of plants using RNA interference (RNAi) is known for decades, no systematical research have been done on the profiing of siRNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein (NCP) gene. In this paper, three independent RSV-resistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of siRNAs were confimed by Southern blotting and Northern blotting analyses, and the majority of siRNAs were in lengths of 21, 22, and 24 nucleotides (nt), which have validated a connection between the presence of the RSV NCP homologous siRNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines (T4-B1), the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confimed the large population of NCP-derived siRNAs in transgenic plants, and the RSV-infected library (T4-B1-V) possessed more siRNAs than its mock inoculated libraries (T4-B1-VF), these results indicating the inverted repeat construct and RSV could introduce abundance of siRNAs in transgenic rice. Moreover, a varied expression level of specifi siRNAs was found among different segments of the NCP gene template, about 47% of NCP-derived siRNAs reads aligned with the fragment from 594 to 832 nt (239 nt in length) in NCP gene (969 nt in length) in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the fist study to address the siRNA profie in RSV-resistant transgenic plant using next generation sequencing (NGS) technique, we confimed that the massive abundance of siRNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.
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LI Li
GUO Cheng
WANG Biao
ZHOU Tong
LEI Yang
DAI Yu-hua
HE Wen
LIANG Chun
WANG Xi-feng
Key wordsRice stripe virus (RSV)    genic rice     deep sequencing     siRNA     resistance     
Received: 2016-01-08; Published: 2016-05-24
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Financial support was provided by the National Key Basic Research of China (2012CB114004), the Special Fund for Agro-Scientifi Research in the Public Interest, China (201303021) and the National R&D Project of Transgenic Crops of China (2012ZX08009001).

Corresponding Authors: WANG Xi-feng, Tel: +86-10-62815928, E-mail: xfwang@ippcaas.cn; LIANG Chun, E-mail:liangc@miamioh.edu   
About author: LI Li, E-mail: lli@ippcaas.cn; GUO Cheng, E-mail: guoc2@miamioh.edu
Cite this article:   
LI Li,GUO Cheng,WANG Biao et al. (JIA-2016-0023) RNAi-mediated transgenic rice resistance to Rice stripe virus[J]. Journal of Integrative Agriculture, Doi:10.1016/S2095-3119(16)61369-2
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http://www.chinaagrisci.com/Jwk_zgnykxen/EN/10.1016/S2095-3119(16)61369-2      or     http://www.chinaagrisci.com/Jwk_zgnykxen/EN/Y0/V/I/0
 
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