Apriasti Retnosari, Widyaningrum Suvia, Hidayati Weny N, Sawitri Widhi D, Darsono Nurmalasari, Hase Toshiharu, Sugiharto Bambang
Postgraduate Program for Biotechnology, University of Jember, Jember, Indonesia.
Center for Development of Advanced Science and Technology (CDAST), University of Jember, Jl. Kalimantan No 37, Kampus Tegalboto, Jember, 68121, Indonesia.
Mol Biol Rep. 2018 Dec;45(6):2749-2758. doi: 10.1007/s11033-018-4326-1. Epub 2018 Aug 31.
Sugarcane mosaic virus (SCMV) is a plant pathogenic virus of the family Potyviridae that causes chlorosis, stunting and significantly reduced sugar productivity in sugarcane. Pathogen-derived resistance is a method used to develop SCMV-resistant sugarcane by overexpression of viral DNA. In this study, the gene encoding the coat protein (CP) of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. Nucleotide sequence analysis of amplified cDNA indicated that the 998-bp-long cDNA, termed ScMVCp cDNA, codes for the CP of SCMV from the PS881 isolate. The ScMVCp cDNA was inserted into the binary vector pRI101-ON with two constructs, a full nucleotide sequence (p927) and a sequence coding for N-terminally truncated protein (p702). The constructs were then introduced into sugarcane using Agrobacterium-mediated transformation. Southern blot analysis showed a single hybridized DNA copy inserted into the genome of transgenic sugarcane lines. The inserted genes were expressed at both the RNA transcript and protein levels in the transgenic sugarcane. The highest expression was found in transgenic lines 10, 11 and 13 from the p927 construct. Artificial infection by the virus showed that p927 generated a higher resistance to virus compared with p702. This resistance was passed on to the second generation of transgenic sugarcane with 100 and 20-40% levels of resistance in the p927 and p702 transgenic lines, respectively. This report shows that the full sequence of the CP gene is required to disrupt viral assembly and packaging, thereby generating resistance to SCMV infection.
甘蔗花叶病毒（SCMV）是一种属于马铃薯Y病毒科的植物致病病毒，可导致甘蔗出现黄化、发育不良并显著降低糖分产量。病原衍生抗性是一种通过病毒DNA过表达来培育抗SCMV甘蔗的方法。在本研究中，通过逆转录PCR从有症状的甘蔗叶片中扩增出编码SCMV外壳蛋白（CP）的基因，并用于培育转基因甘蔗。对扩增的cDNA进行核苷酸序列分析表明，这个998 bp长的cDNA（称为ScMVCp cDNA）编码来自PS881分离株的SCMV的CP。将ScMVCp cDNA插入二元载体pRI101-ON中，构建了两个构建体，一个是全长核苷酸序列（p927），另一个是编码N端截短蛋白的序列（p702）。然后使用农杆菌介导的转化方法将这些构建体导入甘蔗。Southern杂交分析表明，单个杂交DNA拷贝插入到转基因甘蔗品系的基因组中。插入的基因在转基因甘蔗的RNA转录本和蛋白质水平上均有表达。在来自p927构建体的转基因品系10、11和13中发现了最高表达。病毒人工感染表明，与p702相比，p927对病毒产生了更高的抗性。这种抗性传递给了第二代转基因甘蔗，p927和p702转基因品系中的抗性水平分别为100%和20 - 40%。本报告表明，CP基因的完整序列是破坏病毒组装和包装从而产生对SCMV感染抗性所必需的。
