Genetic Transformation Of Mthsp70in Rice And LPA1,LPA2in Arabidopsis

Rice is one of the most important staple food crops in the worldwide as well as a model plant for monocotyledon research. Previous study indicated that Mitochondrial hsp70may play an important role in programmed cell death. For further research on the fuction of Mitochondrial hsp70, the aim of the study was to extablish an transgenic system for Mitochondrial hsp70in rice. Comparing with nuclear transformation, plastid transformation has many advantages, such as more bio-safety, higher expression efficiency, and expressing prokaryotic genes without modification. We initiated the study on tobacco chloroplast transformation of lpal and lpa2(low PSII accumulation), which built a solid base for the following research. The main results are given as follows:Two pairs of primers were synthesized to amplify Mitochondrial hsp70in rice. hsp70(+) and hsp70(-) were inserted into pCAMBIA1304. After Agrobacterium-mediated transformation and screening of plants resistant to antibiotic, we have obtained resistant callus.Transfered resistant callus to medium for differentiation and then regenerated into plant. Transferred the other resistant callus into liquid MS medium and cultured under condition of28℃,120rpm/min,transgenic cell suspention line was obtained. PCR analyses showed that hsp70(+) and hsp70(-) were transferred into rice successfully. Semi-quantitative RT-PCR analyses indicated that on transcriptional level, hsp70(+) and hsp70(-) transgenic rice had an overexpression and suppressexpression respectively.Amplified aadA gene encoding the Aminoglycoside Adenylytransferase from plasmid pCN1. Prrn and Trbcl from tobacco chloroplast genome were fused as promoter and terminator. Amplified lpa1and lpa2from Arabidopsis. Promoter PpsbA was amplified from tobacco chloroplast genome together with terminator of psbA gene3’sequence. Through analyzing the sequence of tobacco chloroplast genome, amplified Ty region and inserted into pUC19, digested Ty region into two fragments as left and right homologous recombination sites. Constrcted chloroplast expression vector by cloning aadA and lpal/lpa2espression cassette between two homologous recombination sites. To establish tobacco tissue culture system,we induced callus from sterile leaf and regenerated it into plant. Based on the chloroplast expression vector and the regenerationg system, we tried to carried out tobacco chloroplast transformation mediated by biolistic bombardment, the parameters was listed as follows:500μg and1μg per bombardment of gold particles and DNA,1100psi acceleration pressure,6cm bombardment distance.We discussed the parameters of bombardment in order to optimize tramsformation system which include raising the amount of plasmid DNA, changing the bombardment distance,increasing the numbers of bombardment.