| Although studies on the Nuclear Genetic Engineering play positive roles in improvement of the yield, quality, disease-resistance and insect-resistant of cotton , the Nuclear Genetic Engineering exist various defects such as Gene Inactivation, Gene Silencing, Position Effect, Pollen Dispersal and difficulties of multiple gene transformation. Chloroplast Genetic Engineering as an emerging technology can overcome the shortcomings of the Nuclear Genetic Engineering. To solve the problem, we initiated the research of the Chloroplast transformation of cotton. We constructed the vectors for chloroplast transformation and try to find standard program of cotton chloroplast transformation.Based on the published cotton chloroplast genome sequence (gene bank NC007944), two pairs of primers were synthesized to amplify two adjacent cotton plastome sequence as homologous fragments LHRR and RHRR. Then the vector pBSKRR was constructed by enzyme digesting and ligating the homologous fragments and plasmid pBluesript SK. Through adding the strong promoter (Prrn, PpsbA ) and 3'UTR structures (Trps16, TpsbA ) to the end of the reporter gene gfp and the selective marker nptII which amplified by PCR methods, we constructed two intermediate vectors that contains Prrn—gfp—Trps16 and PpsbA—nptII—TpsbA expression cassettes. Finally, the vector pBRGN was constructed by enzyme digesting and ligating the two expression cassettes and the vector pBSKRR. We successfully transformed this vector in the tobacco chloroplast, then we detected the expression of gfp and nptII gene by amplification and found that the transformed seedlings conferred green fluorescent activity. But the result was not very ideal in cotton.Although there are many difficulties on the cotton chloroplast transformation, the experimental results confirmed the feasibility of our experimental considerations. And this research builds a solid base for the future cotton chloroplast transformation.
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