Research On The Methods Of The Genetic Transformation Of Blakeslea Trispora

Blakeslea trispora is the main filamentous fungus to industrially produce naturalβ-carotene at present. At the meantime, Haematococcus pluvialis is a monoplast alga which fastly and largely accumulated astaxanthin in adversity intimidation conditions, so it is an ideal alga to produce astaxanthin at present. It's known thatβ-carotene is catalyzed into astaxanthin by hydroxylase and ketolase. In this research, the genes of hydroxylase and ketolase were transformed into Blakeslea trispora by gene engineering. When we construct the available genetic transformation methods of Blakeslea trispora, we also hope that they could extend the metabolic pathway fromβ-carotene to astaxanthin at the same time. Although efforts had been made to construct the methods of the genetic transformation of Blakeslea trispora, it has not been yet achieved. The three methods of genetic transformation, including transformation mediated by protoplast, Agrobacterium tumefaciens and particle bombardment, were firstly applied into Blakeslea trispora.(1) Theβ-carotene hydroxylase and ketolase genes were cloned from Haematococcus pluvialis, and the bidirectional promoter gene was cloned from Blakeslea trispora. Fusion primers were designed to obtain the complete expression sequences of WPZ gene by fusion PCR, and the fusion gene was linked to expression vector called pCAMBIA1303, resulting to the vector pCAMBIA1303-WPZ.(2) The regeneration of Blakeslea trispora protoplast and the genetic transformation system of protoplast-mediated transformation was established, and the results revealed the ratio of mixed enzyme including Lywallzyme, Lysozyme, Cellulase and Snailase was 0.5:0.25:0.25:1, pH ranges from 6 to 7, the mycelium which had grown nearly 16 h was digested for 4 h. In that condition the number of the protolasts could reach the peak, but we had not got the positive transformants.(3) We researched the genetic transformation system of Blakeslea trispora by Agrobacetrium tumefaciens-mediated transformation. The concentration of the Agrobacterium tumefaciens and AS were OD600=0.5 and 200μg/mL respectively; the temperature and the time of co-culture were 28℃and 48 h respectively.(4) We also researched the genetic transformation system of Blakeslea trispora by particle bombardment-mediated transformation. And the transformants which expressed green fluorescence were obtained. Based on the results, a rupture disc pressure of 1100 psi , a vacuum pressure of -12 psi and a stopping-screen-to-target distance of 6.5 cm for one time were held constant in all subsequent experiments aimed at optimizing parameters.Among the three genetic transformation methods, the Agrobacterium tumefaciens and the particle bombardment-medicated transformation are more effective and stable genetic transformation systems . The positive transformants could express GFP in the dark under GFP excitation .