Functional Confirmation of a Polygalacturonase Gene BcMF9 in Chinese Cabbage (Brassica Campestris L. ssp. Chinensis Makino)

A polygalacturonase (PG) gene BcMF9 was obtained in floral bud of Chinese cabbage pak-choi (B. campestris ssp. chinensis (L.) Makino, syn. B. rapa ssp. chinensis (L.) Makino) between A line and B line by cDNA-AFLP technology and rapid amplification of cDNA ends (RACE) technology. The multi gene family of PG expressed in the different development stages and different tissues of plants, such as fruit softening,pollen maturation and so on, which related to fruit ripen, cell separation and others. The result of Northern hybridization and RT-PCR confirmed that gene BcMF9 was inhibited obviously in the male sterile line, and the gene only expressed in floral bud but not in leaf and stem. These results shows that BcMF9 may expressed in pollen development. In order to find out the function of gene BcMF9 in detail, we analyse the sequence of BcMF9 and construct the plant expressing plasmid vector and the explants were inoculated with Agrobacterium tumefaciens strain LBA4404 containing pBI35S-BcMF9A or pBIA9-BcMF9A carrying antisense gene BcMF9. Then, the expressing characteristics of gene BcMF9 were analyzed by Northern and Southern hybridization in transgenic plantlets, non-transgenic plantlets of the genic male sterile AB line. At the same time, in order to understand the biological function of gene BcMF9, morphology and the development course of microsporogenesis were observed in transgenic plantlets and non-transgenic plantlets. The primary results obtained from the research provide some clues for the study on the biological function of gene BcMF9A and molecular mechanism of the male sterility. The main results are as follow:(1) The largest open reading frame of BcMF9 is 1188 bp and encodes a protein of 395 amino acids with the DNA sequence of BcMF9 is 1481bp, full-length cDNA is 1286 bp.it interrupted by two introns of 32 bp, 129 bp. The similarity of BcMF9 and PGA3, a PG gene related to plant pollen development in Arabidopsis thaliana is 88%. Phylogenetic analysis showed that BcMF9 falls into the category of clade-C, which includes PG related to pollen. These results test that BcMF9 may acts as a pollen-specific polygalacturonase.(2) After the constitutive type plant expressing plasmid vector (pBI35S-BcMF9A) and the tapetum-spccific plant expressing plasmid vector (pBIA9-BcMF9A) were constructed, we introduced them into Agrobacterium tumefaciens strain LBA4404.(3) More than 100 plantlets Kan~R seedlings are obtained based on the efficient genetic transformation system which have been obtained in our lab. PCR, Southern blotting, Northern blottingand GUS expression staining method of histochemistry and pollen germinateion have done. The frequency of positive to PCR is 90.56%. The result of GUS expression staining method of histochemistry show that there are GUS expression in the pBI35S-BcMF9A transgenic plantlets leaves and in the pB!A9-BcMF9A transgenic plantlets anther, but none in the non-transgenic plantlets. The Southern blotting show the antisense BcMF9 gene was integrated into flowering Chinese cabbage genome.(4) The result of Northern hybridization indicated that antisense gene BcMF9 was inhibited obviously in the transgenic plantlets, and it confirmed that antisense gene BcMF9 has been expressed in flowering Chinese cabbage, and the antisense gene BcMF9 fragment of the transgenic plant has been transcripted actually to inhibit the normal expression of gene BcMF9.lt hints that the antisense gene BcMF9 were transcripted and expressed correctly. Only 30% ~ 40% pollen germination in the transgenic plantlets,but more than 90% in the non-transgenic plantlets. It shows that pollen germination of transgenic plantlets were disturbed. These results predicted that BcMF9 gene was related to the pollen development, whether it expressed effected the normal germination of pollen.