Cloning An Oleosin Gene From Brassica Napus And Construction Of Plant Expression Vectors Harboring Oleosin-GUS Fusion

The construction of a suitable oleosin-fusion expression vector is not only useful in promoting plant bioreactor development, but also useful in researching in sterility, self-incompatibility, anti-disease and insect, pollen nutrition, high crop yield, the safety of gene engineering. It is valuable both in theoretic and practice. In this paper, an oleosin gene was cloned from brassica napus, three expression vectors harboring oleosin-GUS fusion were constructed and transformed leaf discs of Petunia .The main experiment and result are as follows:1. Cloning and sequence analysis of OTS gene fragment from Brassica napusThe oleosin gene fragment was amplified by means of PCR using genomic DNA of Brassica napus as template and a pair of specific oligonucleotides at 5 ' - and 3 ' -end as primer, and cloned into E.coli vector. The vector was named pUC-OTS. Results showed that the DNA sequence consisted of 889 bp and encoded 196 amino acid residues deduced from DNA sequence, which had an intron. Except for the extra 29 bp, comparison with previously published sequence showed 79.20% homologies in nucleotide sequence and 91.21% in amino acid sequence respectively.2. Obtaining of OTS-GUS fusion gene and predicting the character of fusion proteinOleosin gene from Brassica napus and GUS gene were connected by thrombin with six amino acids, forming a fusion protein. The fusion protein gene has a coding region of 2415 base pairs encoding 804 amino acids and a terminator code. The software of protein analysis (Antheprot.5.0) was used, and results showed that the fusion protein keeps physical and chemical characters as good as the original two proteins, and maintained the essential secondary structures of three proteins. By cloning the fusion gene to vector pUC121, the intermediate vector pUCOTSGUS was constructed.3. Construction of plant expression vectorThe plant expression vectors pBI35SOTS121, pBILeaOTS121 and pBINapin!21 drove respectively by CaMV35S, Lea and Napin promoters, were constructed. Each of constructs was introduced into the Agrobacterium tumefaciens strain LBA4404.4. Detection of transient GUS expressionAgrobacterium tumefaciens harboring each construct was used to transform leaf discs of Petunia. After three days, GUS activity was measured according to Wang Guanlin. Detection of transient GUS expression showed that the OTS-GUS drove respectively by CaMV35S, Lea and Napin promoters in this experiment could express.