Study On The Monoterpene Synthase AtTPS03 Gene Over-expression In Xiangyou 15(Brassica Napus)

(3-Ocimene is one of monoterpene, an important plant communication signaling molecule. In nature there are two isomers:cis-β-Ocimene and trans-β-Ocimene. AtTPS03 was identified as one of the large AtTPS gene family and encodes a monoterpene synthase from Arabidopsis thaliana. AtTPS03 can efficiently convert geranyl diphosphate (GPP) intoβ-ocimene. So, AtTPS03 is the key enzyme for synthesisingofβ-ocimene. The existing research results indicated that (E)-β-ocimene emitted from herbivore-damaged plants can change metabolism and induce defense response in the adjacent plants around the damaged plants to resist pathogen infection or to repel herbivores.The main content of this study:full-length cDNA of AtTPS03 was cloned from JA-treated Arabidopsis, construction of over expression vector pHB-O,the use of Agrobacterium-mediated genetic transformation,expression vector pHB-O is transferred into recptor Brassica Xiangyou 15 (It is characterized by a low erucic acid,low sulfur glycoside,that is also the nature of winter oilseed rape. Mainly in the Yangtze River), those resistant seeding were detected by the way of PCR for transgenic plants to determine. We use molecular approach to detecting for defensed genes such as BnPR1 and BnPDF1.2 expression. The theory is preliminary inquiried, which is related to defense gene regulation patterns and access to the following conclusions:1. Construction of AtTPS03 gene over-expression vector (pHB-O). We designed and synthesized one pair primers for cloning AtTPS03'cDNA sequences in Arabidopsis, which has been published in the GenBank website for producingβ-ocimene. Full-length cDNA for AtTPS03 was amplified from total Arabidopsis RNA by RT-PCR method, the Arabidopsis had been treated under the concentration of specific JA,then sequenced and compared online. The cDNA is the homology fragment in GenBank, which was inserted into over expression vector pHB. We constructed the over expression vector pHB for plant AtTPS03.2. We select the petiole for acting as explants, Agrobacterium mediated transformation of receptor Brassica napus Xiangyou 15, from 7932 explants, obtained 132 resistant seedlings and 24 positive plants by PCR test. At last, one genetic stability transgenic plant with AtTPS03 gene over-expression by 5-times PCR detection. The transformation rate is 0.08%.3. Semi-quantitative RT-PCR analysis showed that defense gene BnPDF1.2 and BnPR1 were expressed in the transgenic seedings, but those were not expressed in the Xiangyou 15 plant, which had not been transformed.