| Rice blast, caused by the Magnaporthe grisea, is one of the most serious rice diseases in rice cultivation areas of the world. It is also one of the main factors limiting rice yield and production astability. And development of disease-resistant rice cultivars is an economic, safe and effective measure. However, the rice blast is a typical host-pathogen co-evolutionary system, whose pathogenicity variation and differentiation often cause cultivars with vertical resistance difficult to maintain their diseas resistance. Therefore, development of rice cultivars with durable disease resistance becomes urgent in rice production.The pi21, a recessive QTL, is the firstly cloned rice gene with a broad-spectrum and durable resistance to rice blast. In this study, a275-bp region of the3’-UTR of Pi21, starting17bp downstream from the stop codon, was used for construction of Pi21RNAi vector (pLYL21). The pLYL21vector was transformed via Agrobacterium into rice Nipponbare embryogenic callus and93independent transgenic plants were generated. Semi-quantitative RT-PCR analysis of To and T1transgenic plants showed that the Pi21transcript level was significantly decreased as compared with wild-type rice plants, and the RNAi plants showed resistance to blast fungus. A Pi21-RNAi transgenic line was inoculation with rice blast fungus and the expression of rice disease related genes in different time after inoculation was analyzed in RT-PCR. The expression of OsWRKY2’8, ABC transporter-1and ABC transporter-2genes in control plants Nipponbare were induced and increased12h after inoculation, while the expression of those in the Pi21RNAi plants were reduced. And PR1, PAL and OsWRKY45genes in both of the Pi21RNAi plants and untransformed controls were induced48h after inoculation.In addition, to establish a system for visual examination of interaction of blast fungus and the Pi21-RNAi rice, red fluorescent protein (RFP) was transformed into the blast fungus strain E2007-046A2via PEG-mediated transformation method and stable transformants were generated, providing materials for subsequent experiments.To establish a high-throughput system for genetic analyses of transgenic rice, the red fluorescent protein, mCherry, was introduced into japonica rice via Agrobacterium mediated transformation using mature embryo derived rice callus and the hygromycin phosp ho trans ferase (HPT) selectable marker. Red fluorescence was detected in transformed rice callus, primary transformed plants (T0) and T1progenies, indicating stable expression of mCherry in transgenic rice. Genetic analysis of139independent transformed rice lines indicated that mCherry should be inherited in the transgenic progenies with multiple patterns, and54.0%of the transgenic lines showed single-locus inheritance. Transgene integration in the rice genome was further confirmed using TAIL-PCR. Sequence analysis of T-DNA integration sites showed that nucleotide deletion, insertion and substitution occurred at the T-DNA left border, whereas the nucleotide deletion only happened in the T-DNA right border, which suggested that the left border region should have far more diverse types of nucleotide variation than the right border region. Furthermore, real-time reverse-transcription PCR of mCherry and HPT in T2transgenic plants revealed positional effects, of which the mCherry and HPT transcript levels varied in the transgenic lines depending on T-DNA integration locations in the rice genome. Overall, the system based on mCherry and other related methods described in this study presented a high-efficient streamline for genetic and molecular analysis of trans genic rice. |
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