Studies On The Cloning, Expression And Evolution Relationships Of Homologous BcMF2 And BcMF4 Genes In Cruciferae

Cruciferae crops are one of the most important vegetable crops and also are a kind of crops of the most successful in utilizing of heterosis in China. In order to completely elucidate the mechanism of plant male sterility, we must know the genes related to plant pollen development. In recent years, an increasingly amount of research has been devoted to the molecular biology of the pollen development of flowering plant. The cloning and characterization of pollen development related genes is important to analysis the mechanism of male sterility in Cruciferae and construct male sterility line. At the same time, because of the emergence of different morphological characters and many transitional types in Cruciferae crops, there are still many ambiguities and debates on phylogentic evolutionary relationship of Cruciferae. With the development of molecular biology and bioinformatics, it is possible to identify and classify species and evaluate the evolutionary relationship in molecular level. In this study, the full length cDNA and DNA of pollen development related BcMF2 and BcMF4 gene was cloned and characterized from Chinese cabbage pak-choi (Brassica campestris L. ssp. chinensis Makino). Homologous genes with the BcMF2 and BcMF4 were cloned from 11 species in 6 genera of Cruciferae by PCR amplification, with two pairs of primers designed according to the sequence of BcMF2 and BcMF4 and Phylogenetic trees of BcMF2 and BcMF4 gene were constructed based on the alignment of their homologous genes, respectively. The results were as follows:(1) The full length DNA and cDNA of BcMF2 gene encoding pollen-specific polygalacturonase were cloned from B. campestris ssp. chinensis cv. Aijiaohuang. The largesteset open reading frame is 1266 bp and encodes a protein of 421 amino acids with a predicted molecular mass of 43.9 kDa, interrupted by three introns of 142 bp, 75 bp and 83 bp in length. Sequence analysis revealed that it has three potential N-glycosylation sites and one polygalacturonase active position (RVTCGPGHGLSVGS). Four domains which are highly conserved in the all plant and fungal PGs is present in BcMF2. Phylogenetic analysis showed that BcMF2 falls into the category of clade-C, which includes PG related to pollen. These results test that BcMF2 acts as pollen-specific polygalacturonase.(2) We firstly studied on prokaryotic expression of BcMF2. A cDNA sequence was inserted into pET-32a (+) and a recombinant plasmid expressing the full length BcMF2 fusion protein was constructed. By using IPTG inducing, overexpression of BcMF2 fusion protein in E.coli BL21 was achieved. The result showed that there is a specific band at about 70 kDa in size, which is identical withthe expected molecular weight of the recombinant protein. And by optimizing the inducing parameters, we find when the recombinant plasmid was induced at 30℃ for 6 h and the corresponding IPTG was added to the media to the final concentration of 0.4 ~ 1 mmol·L-1, Expression amount of the recombinant protein was the largest.(3) BcMF2 Homologous genes were isolated from 11 species in 6 genera of Cruciferae by PCR amplification. The Clustal X program was used to align the homologous sequences from different 13 species of Cruciferae and to produce NJ, ME and MP phylogentic trees based on distances estimated by the two-parameter method with 1000 bootstrap samples using MEGA 2.1. The result shows three kinds of phylogentic trees have the strictly accordant topologic structure. B. rapa L. and B. campestris L. ssp. pekinensis cv. Xiaoqingkou form a specific clade (BCL≥91%) and B. campestris L. ssp. pekinensis cv. Xiaoqingkou and B. campestris L. ssp. pekinensis cv. Hugangya-14 belong to different clade, which is consistent with the results from morphology and cytology.(4) By using GenBank, EMBL and DDBJ database, we downloaded 86 PG sequences from bacteria, fungi and plants. Then the homology analysis was performed and the phylogenetic tree was constructed. The amino acids sequence alignment confirmed that there are four strictly conserved sequence segments, which...