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首页> 外文期刊>Cotton Reseach Journal >Engineering Leaf Curl Virus Resistant Cotton through RNA Interference Approach
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Engineering Leaf Curl Virus Resistant Cotton through RNA Interference Approach

机译：RNA干扰法工程化抗卷叶病毒棉花

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Cotton Leaf Curl disease is one of the serious limitations to cultivation of cotton in North India. Most predominant tetraploid hybrids and cultivars are susceptible to Cotton Leaf Curl Virus (CLCuV) disease. It takes several years to develop a variety by conventional breeding. Often by the time a resistant variety is derived, new strain with altered specificity might originate to overcome the resistance. During 2009-10, we characterized six recombinant strains of CLCuV, one of which G6DC, knocked down resistance of many hitherto resistantpopudar varieties and hybrids of cotton. Pathogen derived resistance based on cross protection and antisense approach have long been employedfor management of viral diseases in plants. Discovery of RNA interferencehas opened up entirely new arena for its therapeutic potential in controlling virus diseases. The phenomenon is evolutionary conserved in plants, animals and fungi and is known to protect cells from invading viruses and trarisposons. With aim to developvirus resistant transgenic cotton, RNAi-mediated approach was employed to target CLCuV. Primers with engineered restriction sites were designed to amplify five conserved target sequences, ranging from 109-367 bp, in DNA-A and p-DNA components of the virus DNA strands for conserved regions of each of these ORF sequences: MP, CP, AC2, betaC4 and betaV4,flanked with Kprd and Xbal sites to facilitate cloning in sense and antisense orientations respectively, were PCR amplified and cloned in pGemT. Two strands for each sequence was cloned individually in sense and antisense orientations in plasmid pBSK-int, 3.125 kb (HQ343203), developed by cloning 125 bp cotton chitin gene intron in plasmid pBSK (3.0 kb, Stratagene, USA). The resulting five inverted repeatconstructs viz., pBSK-int-MP-SA, 3.343 kb (HQ681274), pBSK-int-CP-SA, 3.495 kb (HQ681275), pBSK-int-AC2-SA, 3.425 kb (HQ681276), pBSK-int-betaC4-SA, 3.549 kb (HQ681278) and pBSK-int-betaV4-SA, 3.479 kb (HQ681277) were sub-cloned in two binary vectors pBin-AR(l 1 Kb) andpGreen (4.6 Kb) and introduced in Agrobacterium tumefaciens strain EIIA 105 by tri-parental mating. The resulting constructs were transformed in two popular G. hirsutum cultivars, F846 and HS6 which are otherwise highly susceptible to CLCuV. Putative transformants with pBSK-int-AC2-SA was documented by PCR for presence of viral sequence, both in sense and antisense orientations. Evidence of its integration in cotton and generation of siRNA are being ascertained by Southern hybridizationand Northern blotting, respectively. Transformation and screening of remaining constructs are in progress.
机译：棉叶卷曲病是印度北部棉花种植的严重限制之一。大多数主要的四倍体杂种和品种对棉花卷叶病毒（CLCuV）疾病敏感。通过常规育种开发品种需要花费数年的时间。通常在获得抗性品种时，可能会产生具有改变的特异性的新菌株来克服抗性。在2009-10年期间，我们鉴定了六株CLCuV重组菌株，其中一株为G6DC，其击落了许多迄今具有抗性的popudar品种和棉花杂交种的抗性。基于交叉保护和反义方法的病原体衍生抗性长期以来一直用于植物病毒疾病的管理。 RNA干扰的发现为控制病毒疾病的治疗潜力开辟了一个全新的领域。该现象在植物，动物和真菌中是进化保守的，并且已知能保护细胞免受入侵的病毒和trarisposon的侵害。为了开发抗病毒的转基因棉花，采用RNAi介导的方法靶向CLCuV。设计具有限制性酶切位点的引物，可扩增病毒DNA链的DNA-A和p-DNA组分中五个保守的靶序列，范围为109-367 bp，用于这些ORF序列（MP，CP，AC2）的每个保守区PCR扩增并克隆到pGemT中，分别与Kprd和XbaI位点侧接以促进有义和反义方向克隆的betaC4和betaV4。每个序列的两条链以有义和反义方向分别克隆到质粒pBSK-int 3.125 kb（HQ343203）中，该质粒通过将125 bp棉花几丁质基因内含子克隆到质粒pBSK（3.0 kb，Stratagene，美国）中而开发。所得的五个反向重复构建体即pBSK-int-MP-SA，3.343 kb（HQ681274），pBSK-int-CP-SA，3.495 kb（HQ681275），pBSK-int-AC2-SA，3.425 kb（HQ681276），将pBSK-int-betaC4-SA，3.549 kb（HQ681278）和pBSK-int-betaV4-SA，3.479 kb（HQ681277）亚克隆到两个二元载体pBin-AR（11 Kb）和pGreen（4.6 Kb）中并引入通过三亲交配，在根癌农杆菌菌株EIIA 105中获得抗性。将得到的构建体转化到两个流行的陆地棉栽培种F846和HS6中，否则它们对CLCuV高度敏感。通过PCR记录了具有pBSK-int-AC2-SA的推定转化体在正义和反义方向上都存在病毒序列。分别通过Southern杂交和Northern印迹确定了其在棉花中整合和siRNA产生的证据。其余构建体的转化和筛选正在进行中。

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