Study On The Agronomy Characters And Resistance To Sclerotinia Sclerotinorium Of Transgenic Rape Expressing Harpin_Xooc

Harpins elicit hypersensitive cell death (HCD) in non-host plants of the bacteria and induce resistance to pathogens and insects, promote growth in many plants, which are encoded by hrp genes of gam-negative bacteria. Harpins have some characteristics: rich in Gly; lack of Cys; stable to heat; sensitive to proteinase. Treatment with harpin to plants can induce resistance to fungi, bacterium, nematode and enhance plant growth. Furthermore, harpin expression in transgenic tobacco, rice and pear enhance resistance to pathogen. This research was aimed at transgenic rape with hrf2 gene encoding harpin_xooc, whose resistance and output may be meliorated.Hrf2 was derived from Xanthomonas oryzae pv. oryzicola strain RS 105.This gene is 414bp in length, its production has 138 amino acid with molecular weight 15.3 Kda. harpin_xooc encoded by hrf2 has similar characteristics and functions with harpins, which are able to elicit disease and insect in plants, and promote plant growth.In this study, hrf2 gene of Xanthomonas oryzae pv. oryzicola strain RS105 was amplified and proper restriction enzyme sites were added at each end by PCR. It was cloned into pBI121 to make recombinant pBI121::hrf2, which was proved to be constructed successfully by restriction enzyme analysis. Then, we excised the hrf2 gene and its promoter and terminator from pBI121::hrf2 by digestion with restriction enzymes Hindâ…¢and EcoRâ… , and inserted them into the transformation vector pCAMBIA1301. The resulting recombinant vector, pCAMBIA1301::hrf2, was transferred to E. coli DH5a. After verifying correct orientation of genes, this transgenic construct was transferred into Agribacterium tumefaciens LBA4404 by the freeze-thaw method. The positive clones were determined by PCR and restriction enzyme digestion.The cotyledonal petiole segments from rapeseed variety Yangyou 4 were infected and transformed by A. tumefaciens strain LBA4404/pCAMBIA1301::hrf2, and transgenic rape plants were obtained by screening with hygromycin. Genomic integration and expression of the transferred genes were determined by PCR, Southern blot hybridization, reverse transcriptase PCR and Gus analysis. Soluble proteins extracted from plants transformed with hrJ2 could elicit HR on tobacco leaves.Under the same culture conditions, there was no apparent difference between the transgenic and control plants in heights, size of leaves. The transgenic plants growed normally, without macroscopical HR. The flowering time of transgenic plants is later than that of non-transgenic plant. The seed setting rates are various in transformed plants. The numbers of seed per pod of all transgenic plant are lower than that of control, while the 1000-kermel weights of some transgenic plants are higher. T1 generation transgenic rape seeds were obtained by screening with hygromycin 50 mg/L. Disease bioassays showed that, the rape plants transformed with hrf2 enhanced resistance to Sclerotinia Sclerotinorium.