Genetic Transformation Of Rice With Phenylalanineammonia-lyase Gene And Its Resistance To Brown Planthopper

Plants are constantly exposed to diverse biotic stresses, such as herbivores,pathogens and viruses. During this process, plants produce some low molecularweight compounds to peptides and proteins that are active against insects to defenseor offset the effects of stress. Phenylalanine ammonia lyase (PAL) catalyzes the firststep of phenylpropanoid pathway, converting L-phenylalanine to trans-cinnamic acid,flavonoids, lignins, anthocyanins, phytoalexins, and plant hormones are derived fromthis pathway. Brown planthopper (BPH, Nilaparvata lugens St l) is a sap-suckinginsect pest of rice (Oryza sativa L.), causing devastating losses to rice production.Great efforts, including breeding resistant varieties and exploiting insecticides havebeen taken to control BPH, but serious outbreak still happened quite frequently,mainly owing to BPH’s strong ability in adapting to the host plants or insecticides. Soit is important to explore potential rice insecticidal active substance or resistance geneto control BPH. In our previous study of rice microarray, some genes (include pal)related to flavanoid biosynthetic pathway showed a different expression pattern in riceupon BPH infestation, and was higher in resistant varieties than that in susceptibleTN1. In the study of High Performance Liquid Chromatography (HPLC) fingerprintanalysis, we found that schaftoside was the main ingredients of extract from leafsheathes of rice resistant varieties, indicating that flavanoid biosynthetic pathway takepart in the interaction between the rice and the BPH. Based on the previous studies,the study firstly explored the relationship between pal expression and rice resistancein responding to BPH’s infestation stress; secondly, by genetically engineeringtechniques, the full-length cDNA of OsPAL were cloned and constructed into OsPALover-expresion vector, which successfully expressed in susceptible varieties. Theresistance effect of transgenic rice to BPH was also tested. The main results were asfollows,1. Analysis the expression pattern of OsPAL upon BPH infestation. The results ofreal-time PCR showed that OsPAL mRNA expression was induced in rice plants upon BPH infestation. As to susceptible TN1, the relative expression level was low at0hpoint (normalize to1.0) as well as3h point, and then significantly increased to2.4after BPH infestation for6h. Subsequently, the OsPAL mRNA levels decreasedgradually, reaching the lowest level of0.4at the last stage (72h). It should be notedthat there was still a weak increase at48h, during the process of OsPAL mRNA leveldeclined from6h to72h.2. Construction of expression vector p6-OsPAL. Using full-length cDNA of ricevariety Nipponbare as template, the ORF of the OsPAL was amplified. Primers of palwere designed based on the sequence of rice PAL (NCBI accession: AK100346).Sequence alignment revealed that the cloned OsPAL showed100%similarities to thereference rice PAL gene. The PCR product was fused into the vector pJet. Therecombinant plasmid (pJet-OsPAL) was then introduced into Escherichia coli top10and sequenced from both sides. Recombinant plasmid was digested with Kpn I andSpe I, inserted into the sites which had been digested by the same enzymes, behind themaize ubiquitin (Ubi) promoter in a modified vector p6with the GUS gene asreporter.3. Screening of transgenic rice and determination of BPH resistance. The constructedvector was introduced into rice calli of variety Nipponbare (for over-expression) bythe Agrobacterium-mediated transformation method. Transgenic lines were screenedby planting transgenic seeds on1/2MS medium supplemented with hygromycin.Primary transformants were transferred into plastic box, and transgenic plants werescreened routinely for the presence of the hygromycin gene by PCR amplification andGUS staining. As a result,18transgenic seedlings were screened out. Then theirresistance to BPH as well as the expression of OsPAL mRNA level was examined. Ingeneral, the resistance of transgenic rice to BPH clearly enhanced as compared withnon-transformed WT line. When most (70%) WT rice died after BPH infestation for7days, the transgenic rice survived with high survival rates at about50%-90%, exceptfor transgenic rice3line. Real-time PCR showed that OsPAL mRNA level of thetransgenic rice was higher than that of the WT line (WT normalized to1.0), with a relative expression level varied from2.3to5.3.