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马铃薯Y病毒(PVY)是危害烟叶生产的主要病害之一。已有研究表明,烟草对PVY的抗性由隐性抗性基因e IF4E-6控制。本研究利用CRISPR-Cas9技术定向敲除烟草e IF4E-6基因以改良优质烤烟品种K326的PVY抗性。我们构建了2个特异靶向e IF4E-6基因的CRISPR-Cas9载体g1和g2。构建的载体转化烟草品种K326,PCR检测潮霉素B抗性标签鉴定阳性T0代转基因烟草。PCR扩增阳性转基因烟株靶位点周围DNA序列,对PCR产物进行直接测序或克隆测序,筛选自靶位点处发生突变的烟株,得到2个载体各约50株阳性转化T0代烟株。序列分析结果表明,有2株g1载体转化的烟株在靶位点处出现杂合型单碱基缺失,分别有3和4株g1和g2载体转化的烟株在靶位点处出现杂合型1~2个碱基的替换。这些目的基因杂合型突变的T0代烟株为后续自交获得目的基因纯合敲除后代材料并最终获得PVY抗性改良的烟草提供了研究基础。
Potato virus Y (PVY) is one of the major diseases that endanger tobacco production. Studies have shown that the resistance of tobacco to PVY is controlled by the recessive resistance gene e IF4E-6. In this study, CRISPR-Cas9 was used to knock down the tobacco e IF4E-6 gene to improve the PVY resistance of high-quality tobacco K326. We constructed two CRISPR-Cas9 vectors g1 and g2 that specifically target the e IF4E-6 gene. The constructed vector was transformed into tobacco variety K326 and PCR was used to detect hygromycin B resistant tag to identify positive T0 transgenic tobacco. PCR amplification of DNA fragments around the target site of the positive transgenic tobacco plants, direct sequencing or cloning of PCR products were screened from the target site mutated tobacco plants obtained two vectors of about 50 positive transformed T0 generation tobacco plants . The results of sequence analysis showed that there were 2 heterozygous single base deletions at the target sites in two g1 vector-transformed tobacco plants. Three and four strains of tobacco plants transformed with g1 and g2 vectors, respectively, appeared heterozygous at the target site Type 1 to 2 base replacement. The T0 generation tobacco plants with heterozygous mutations of the target gene provided the research basis for the subsequent selfing of obtaining the target gene homozygous knockout progeny material and finally obtaining the improved tobacco with PVY resistance.