Cloning And Functional Analysis Of Dwarf Gene Bnrga-ds In Rapeseed (Brassica Napus L.)

Height is an important agromonic trait of rapeseed. Utilization of semi-dwarf gene is an important way to increase lodging resistance and promote merchanized harvesting. It is indispensable to obtain dwarf rapeseed mutant with suitable height and good agronomic traits for dwarf cultivar breeding. Dwarf repeseed mutants are fewer than those of rice and wheat. Among them, only the dwarf gene of Brassica rapa mutant dwf2 was cloned. Therefore, it is significant to screen more valuable dwarf rapeseed mutants and clone their dwarf genes. Semi-dwarf B. napus mutant ds-1 was a double haploid (DH) line derived from EMS-treated microspore cultures of a breeding line 92-10B. In this study, ds-1 mutant was used for genetic analysis, and its semi-dwarf gene was tagged, cloned and functionally analyzed, which would help to use ds-1 gene in rapeseed breeding. Meantime, we obtained the molecular markers linked to ds-1 gene that can be utilized in molecular marker assistant breeding. In additional, ds-1 gene was transformed into rapeseed. Main results are as follows:1. The inheritance of dwarf phenotype was analyzed using the cross between ds-1 and wild-type No.9369. F1 plants have intermediate plant heights between the two parents. Plant heights in F2 population showed a continuous distribution. The segregation for homozygous dwarf, intermediate and homozygous tall plants in F2 population fits an expected Mendelian inheritance ratio of 1:2:1. These data indicated that the dwarf phenotype of ds-1 was controlled by a single semi-dominant allele, supporting previous results.2. Three SSR markers BnEMS1125, BrGMS83 and BrMS226 linked to DS-1 were obtained from chromosome A6. Among them, marker BnEMS1125 derived from B. napus RGA were con-segregated with DS-1. BnRGA was listed as candidate gene for DS-1.The SCAR marker SCM07 closely linked to another B. napus dwarf gene Bzh was used to detect polymorphism and survey the F2 population in this study. SCM07 also showed close linkage to the DS-1 gene at a genetic distance of 0.7 cM, indicating that the Bzh could be allelic to DS-1. 3. Semi-quantative RT-PCR analysis demonstrated that BnRGA genes (BnRGA and Bnrga-ds) had similar transcript level in No.9369 and ds-1, indicating that the dominant dwarf phenotype of ds-1 was not caused by an increase of Bnrga-ds transcription. Amino acid sequence alignment of Bnrga-ds and BnRGA with other DELLA proteins from different plant species and B. napus cultivars indicated that a Pro-to-Leu change in the N-terminal VHYNP motif belonging to the DELLA domain was specific to Bnrga-ds.4. The DNA fragments of BnRGA and Bnrga-ds containing its native promoter, coding sequence and terminator were respectively introduced into wild-type Arabidopsis (Columbia ecotype). The plants carrying homozygous Bnrga-ds transgene were dwarfed compared with wild-type and BnRGA transgenic plants, indicating that Bnrga-ds was responsible for dwarfism of ds-1 mutant.5. Site-direct mutagenesis was performed on wild-type RGA sequence of Arabidopsis (Columbia ecotype) to obtain Atrga-ds encoding an identical mutation as in Bnrga-ds. The transgenic Arabidopsis plants expressing Atrga-ds also showed dwarf phenotype, whereas AtRGA transgenic plants showed normal phenotype. These results demonstrated that the effect of Pro-to-Leu mutation in the VHYNP motif is conserved in different plant species.6. Yeast two-hybrid assay showed BnRGA and AtRGA can interact with GA receptor AtGID1a, whereas the mutated Bnrga-ds and Atrga-ds cannot interact with it in the presence of GA3. These results indicated the Pro-to-Leu mutation in the VHYNP motif abolished DELLA-GID1 interaction, suggesting that this conserved Pro in the VHYNP motif plays an important role in DELLA-GID1 interaction.7. Subcellar localization results showed BnRGA and Bnrga-ds target the nucleus in plant cell, suggesting that BnRGA and Bnrga-ds function as transcription regulators.8. The Bnrga-ds was also introduced into B. napus cultivar Westar through Agrobacterium-mediated method. Fourteen positive transgenic plants were obtained, and some of them have displayed dwarf phenotype. The phenotypes of T1 plants will be further observed.