Construction Of RNAi Vector Of Panonychus Citri Chitinase Gene And Its Transformation In Citrus

Citrus is the first important fruit crop by total output in the world.138countries and regions such as Brazil, China, USA cultivate citrus plants with a total acreage of about6.67million hectares. At present, citrus growing acreage in China reaches1.8million hectares with an annual output of18million tons, ranking the first and the second in the world, respectively. Diseases and pests causes both qualitative and quantitative yield loss, of which, Panonychus citri is the most serious pests of citrus around the world. Control of P. citri mainly depends on chemical applications, which lead to acaricide resistance in P. citri. Currently this situation is deteriorating because of the improper application of acaricides, leading to resistance to many registered acaricides. It is urgent to select a new measures for this mite control. RNA interference (RNAi) can affect normal expression of chitinase gene and plant-mediated RNAi is a potential approach for controlling insect/mite pest. The chitinase exist in microorganisms and animals, which plays an important role in the decrustation, the growth and development process in insects. In this study, the full length of PcCht2related to the growth and reproduction process was cloned by RT-PCR and RACE. The sequence information was used to synthesize double-strand RNAs (dsRNA), establish the plant expression vector, and the recombination vector was transformed into Agrobacterium tumefaciens. At last, transgenic plants were acquired by Agrobacterium-mediated approach.(1) Unigene7021gene amplification and full-length sequence analysis in P. citriWe select13chitinase genes from P. citri transcriptome data base, then blast sequence in the NCBI and analysis. Finding out the gene that is a conservative high chitinase gene Unigene7021as the goal of this experiment. According to known sequence in the database, we design of5′and3′end amplification primer, with RACE kit to amplify the5′and3′end sequences and analyse with DNAMAN, thereby gaining Unigene7021over the span of2518bp sequence, the tot open reading frame contains1995bp, encoding665amino acids, using DNAstar software analysis the protein molecular weight about239.94KDa, isoelectric point is6.55.(2) Construction of RNA interference vectorsThe experiment select binary expression vector pFGC5941as interference vector, and analyze the full-length sequence of Unigene7021. By RT-PCR, we amplified the forward fragment containing XhoI and AscI restriction sites and reverse fragment containing Xbal and BamHI restriction sites. To obtain ug7021XA-pFGC5941vector with positive interference fragments, double digests was conducted between interference expression vector pFGC5941and plasmid containing forward fragments, and the forward fragments was connected to expression vector with T4ligase. Then we connected the recombinant plasmid to plasmid containing reverse interference fragments with T4ligase after double digests to construct interference expression vector ug7021XA-pFGC5941-ug7021XB with positive interference fragments and reverse interference fragments.(3) The expression of RNAi vectors in citrusThe recombinant vector plasmid was transferred into Agrobacterium strains LBA4404by electroporation. Using jincheng hypocotyl as explants, and transformed RNAi vector plasmid into the Agrobacterium plants by Agrobacterium-mediated infection. A certain number of adventitious buds are obtained. Ten strains transgenic plants are obtained by using PCR analysis.