Study Of RNAi Based Transgenic Boehmeria Nivea L. Modified By Introduction Of Pratylenchus Coffeae Pat-10 Gene

Boehmeria nivea (L.) Gaudich. alias piemarker, belongs to the family Urticaceae, Boehmeria, is perennial fiber herbaceous plant. China is a major ramie production country in the world, and Hunan is the most important province to grow it. The ramie is the important economic crop with wide application. Ramie root rot is the major disease causing production reduction, and there is no effective way to control it. The Pratylenchus cqffeae (Zimmermann) is main protozoon which belongs to Tylenchida Thorne, Tylenchina Chitwood, Tylenchoidea, Pratylenchidae, Pratylenchinae, Pratylenchus.In this study, the author collected root and soil around the ramie root in Yuanjiang city, Hunan province, for separation of Pratylenchus coffeae. Cloned pat-10 gene, this gene is essential for muscle contraction and completion of embryonie morphogenesis and elongation. By RNAi, inverted-repeat dsRNA plant expression vector with pat-10 was constructed and transformed into Boehmeria nivea (L.).The results show that:1. Separation and identification of the Pratylenchus coffeaeTwo methods were used to separate the nematodes in root and soil around the ramie root from Yuanjiang city in Hunan province. Their forms were observed by light-microscope, and the parameter of their characteristics were measured. In addition, the method of molecular biology was used to characterize those nematodes. Nematodes rDNA-ITS was propagated by PCR, and the sequences of the products were compared with the other sequences in GenBank. The homo logy is 99%. Combining with the results of morphological analysis, author can conclude that those nematodes are Pratylenchus coffeae. The cultivation Pratylenchus coffeae was performed by inoculation to healthy ramie. The substantial amount of Pratylenchus coffeae was acquired three month after inoculation.2. Clone Pat-10 gene of Pratylenchus coffeaeAfter achieved Pratylenchus coffeae, author used pat-10 sequence in Genbank to design primer, after RNA purification and RT-PCR, pat-10 gene was obtained. Using PCR selection and sequencing the pat-10 gene was definite. Comparation of pat-10 gene with other sequences from GenBank show that the homology is 97%.3. Construct the dsRNA plant expression vector with pat-10 geneUsing RNAi, inverted-repeat dsRNA plant expression vector with pat-10 gene was constructed. Based on the sequence which obtained by clone, primers were designed containing restriction enzyme (AscⅠ, SwaⅠ, AvrⅡand XbaⅠ, SmaⅠ), and the size was 400 bp. The fragment was inserted into the expression vector pGFC5941. The recombinant inverted-repeat dsRNA vector plasmid was consistent with expected results by PCR selection and restriction enzyme digestion.4. The genetic transformation of Boehmeria nivea (L.)Using agrobacterium-mediated transformation, The dsRNA plant expression vector was transformed into Boehmeria nivea (L.). After tissue culture and PCR analysis,28 transgenic ramies with the positive rate of 87.5% were achieved.