Cloning And Evolution Analysis Of VERNALIZATION INSENSITIVE3Gene In Brassica Napus And Its Relative Parents

The transition from vegetative to reproductive growth is the major developmentswitch in plant life cycle. The timing of flower initiation is critical for a successfulreproduction, and vernalization is indispensable for flowering. Brassica napus (AnAnCnCn,2n=38), Brassica olerecea (CoCo,2n=18), Brassica rapa (ArAr,2n=20) are three importantcrops worldwide. They are winter biennial, and need a prolonged period of cold time, aprocess named vernalization to induce flowering. The reproductive development of a plantis highly important in crop production for generative and vegetative crops alike. Cultivatethe early flowering varieties are the main object in oilseed crop breeding in China inrecent years. Vernalization is necessary for flowering. In Arabidopsis, vernalizationpromotes flowering by silencing the flowering repressor gene FLOWERING LOCUS C(FLC), and the VIN3is the direct inhibiting factor of FLC. Lots of researches revealedthat the gene VIN3and its orthologues play an important role in the integration offlowering signals within Arabidopsis and other species. So understanding of the genomicstructure and evolutionary relationship in the level of VIN3gene of these three species isof significance for genetic improvement and better knowing the flowering regulation inBrassica.A total of60kinds of B. napus, B. rapa, and B. olerecea were tested in this report, theVIN3gene was cloned in the level of DNA and cDNA.(1) The full length DNA and cDNA of BnVIN3gene were cloned from B. napus. Thelargest open reading frame is2550bp and encodes a protein of559amino acids with apredicted molecular mass of59.9KDa, interrupted by four introns. We have identified fourBnVIN3paralogues from the AnCngenomes by blasted them online, they are respectivelylocated in the chromosome A2and A3in B. rapa and C2and C3in B. oleracea. So wenamed them Bn.A2VIN3, Bn.A3VIN3, Bn.C2VIN3, Bn.C3VIN3.(2) We used these four copies do the fluorescent quantitation research to detect thecontribution in flowering time.(3) BnVIN3homologous genes were isolated from B. rapa and B. oleracea by PCRamplicication. When we blasted them among B. napus, B. rapa and B. oleracea, we found the key sequence, this sequence which we named SqA can distinguish the AA and CCgenome. The copy which in the second intron inverted32bp to the other three copiesshowed96%-99%identities to Argenome, however the other copy which lack of32bpshowed96%-99%identities to Cogenome. Furthermore, when we blasted the VIN3genein B. oleracea, we found that about20bp fragment inverted in the first intron of B.oleracea were not existed in B. napus and B. rapa as for VIN3gene, we named it SqB, andwe still found that the SqB is not existed in the wild type of B. oleracea. It means thatVIN3paralogues in B. oleracea inverted some fragments in the evolution, and the invertedfragments’ function is still a mystery. The Clustalx and MEGA5program was used toalign the homologous sequences from different species to produce NJ phylogentic trees.