Characterization Analysis Of Structural Variation Of Four Flowering Genes Induced By Allopolyploidization In Resynthesized Brassica Napus

The instability of genome or phenotype in resynthesized polyploidy species is an important factor to restrict high-efficient polyploidy breeding. Sequence elimination and novel sequence generation often occurs in polyploidization. The sequence variations induced by polyploidization in previous reports were mainly involved with repetitive sequences or random sequences but not with genes. Our previous research found that the four varied DNA fragments had higher homologies with the flowering genes(LHP1,CRY1, TOE2, GAI) containing simple repeat sequence in the resynthesized alloployploid Brassica napus derived from the cross between diploid Brassica oleracea and Brassica rapa, and subsequent chromosome doubling.We will use the different plants of F1 to F4 generations of the resynthesized Brassica napus as experimental materials, to clone the full-length genes and cDNAs to explore their rules of variation characteristics and spatiotemporal expression. This study plays a great role in clarifying the effect of varied genes induced by polyploidization on polyploidy generation. It also is beneficial to provide the theory to guide polyploid breeding. The main results are as follows:1. To screen for genetic variation lines and clone, by sequence alignment, Primers were designed for gene segments. Four genes have 15 pairs of primers could amplify clear 31 polymorphic banding patterns in the F1-F4 population, of which 13 for the new band type, LHP1 have seven, CRY1 have one, TOE2 there are four, GAI have one. Statistics found that variation in four genes distributed in 57 resynthesized Brassica napus lines, indicating that most strains are likely to mutate at some sites. LHP1, CRY1, TOE2 and GAI are produced plant variation were 29, 3, 49 and 12. This experiment confirmed that the gene is cut into small fragments of suitable length, the primers were designed and PCR amplification in a different material, it is possible to become the development of a new gene-based functional molecular marker.2. According to the BRAD database has been published sequence of rapeseed four flowering genes. Using primer premier5 software design primers by RT-PCR technology from the blade of Brassica napus were cloned into the full-length of four flowering gene CDS. And four full-length gene CDS is connected to the pGEM-T cloning vector and sequenced. Find out the existence of different fragments of lines.3. The full-length of CRY1 gene was 2522 bp, a total of 20 SNP and two InDel is detected, there are three SNP mutations occur in exon, SNP mutation did not change the amino acid sequence. The full-length of GAI gene was 423 bp, a total of 4 SNP and one InDel are detected, Mutation occurred in exon and changes the amino acid sequence and number. The full-length of LHP1 gene was 1842 bp, a total of 8 SNP and two InDel is detected, there are two SNP mutations occur in exon, SNP mutation alters the amino acid sequence. The full-length of TOE2 gene was 2005 bp, a total of 6 SNP and three InDel are detected, there are four SNP mutations and one InDel occur in exon, SNP mutation and InDel changes the amino acid sequence and number. CRY1, GAI, LHP1 and TOE2 gene nucleotide polymorphisms(Pi) were: 0.00793, 0.00946, 0.00434 and 0.00301. In addition, Analysis compared four mutant genes and the wild-type genes GO function. The results showed that: molecular function CRY1 and TOE2 have not changed; and biological processes involved in mutant and wild-type GAI differences, comments can not participate in the regulation of nitrogen utilization and phloem transport; molecular function LHP1 also changed, compared to wild type, mutant LHP1 also involved in gene silencing by RNA, the primary metabolic processes, cellular macromolecule metabolic process and single-organism organelle organization.4. Whole CDS of the four differences flowering gene were connected with pMD19-T vector followed by sequencing determination. The recombinant plasmids containing CRY1 were isolated, double digested with BamHI and XbaI, ligated into PBI121 S vector which double digested with BamHI and XbaI. Transformed into DH5ɑ and obtained PBI121S-CRY1 and anti-PBI121S-CRY1 over-expression vector. The recombinant plasmids containing TOE2、LHP1、GAI were isolated, double digested with BamHI and KpnI, ligated into pBI121 S vector which double digested with BamHI and KpnI. Transformed into DH5ɑ and obtained PBI121S-TOE2, anti-PBI121S-TOE2, PBI121S-LHP1, anti-PBI121S-LHP1, PBI121S-GAI, anti-PBI121S-GAI over-expression vector.5. The four plants sense and antisense flowering gene expression vectors were transformed into Agrobacterium EHA105 using freeze-thaw method. The PCR amplification was further carried out in transformed bacteria, expression vectors were confirmed to transfer into the Agrobacterium. Agrobacterium containing the vector for gene functions verification. In this study, four flowering gene cloning and gene variant sequences were analyzed, construct the plant expression vector to prepare for genetic verification experiment.