| Currently,important factors in China's rapeseed industry is unable to achieve large-scale mechanized harvest. Mainly, because there is no suitable height of Brassica napus varieties. Lodging can be reduced while dwarfi ng increasing harvest index, dwarfism is a very important molecular properties of crop.Gibberellins(GAs) are one of the main hormones controlling plant height. As one of the six hormones,GAs participated in the regulation of plant growth and development of the whole process. Content of seed dormancy and germination, cell division and elongation, plants bolting and flowering, fruits and seeds mature are closely related to bioactiveGAs.GA2 ox gene is able to produce dioxygenase hydroxylated 2C position o fGAs leading to inactivation of activeGAs.GA 2-oxidase can reduce the content of bioactiveGAs, leading to plant shorter. AppliedGA 2-oxidase to rapeseed will provide broad ideas to cultivation semi-dwarf varieties. However, more research is focused onGA2 ox in Arabidopsis, rice and Phaseolus coccineus etc., less research in Brassica napus. T herefore to carry out this study, and obtained the following relevant results:(1) According to the Brassica EST sequences, SevenGA2 ox fragment were cloned. Using NCBI's tools Blastp, we foundGA2 ox gene of Brassica napus share very high identity with ArabidopsisGA2 ox. So, they were named BnGA2ox1-2, BnGA2ox1-5, BnGA2ox2, BnGA2ox4, BnGA2ox6-1, BnGA2ox6-2 and BnGA2ox8.(2) Gene clustering and phylogenetic results sh ow that: BnGA2ox1-2, BnGA2ox1-5, BnGA2ox2, BnGA2ox4, BnGA2ox6-1, BnGA2ox6-2 were classified as C19-GA2 ox gene. BnGA2ox8 was classified as C20-GA2 ox gene.(3) Analysis of Brassica napusGA2 ox expression patterns in differ ent tissues and organs show: BnGA2ox1, BnGA2ox2, BnGA2ox4, BnGA2ox6 and BnGA2ox8 were expressed in various parts of the organization. High expression concentrated in the cotyledon and silique. BnGA2ox1, BnGA2ox2, BnGA2ox4, BnGA2ox6 and BnGA2ox8 reached the highest expression respectively in different silique mature period, indicating thatGA2 ox gene expression in the silique presents spatiotemporal specificity.(4) Bioinformatics projections indicate: BnGA2ox6 encodes a 329 amino acid protein; there are two conserved domains DIOX_N and 2OG-Fell_Oxy; BnGA2ox6 and BrGA2ox6, AtGA2ox6 and CsGA2ox6 show high homology, and they are in the same evolutionary branches.(5) We successfully constructed expression vector p EGAD-BnGA2ox6, transformed Arabidopsis by floral dip method. T he BnGA2ox6 overexpression plants exhibited a series of distinct phenotypes. Specific performance shorter hypocotyls, dwarf plant, flowering delay, chlorophyll content increased, shorter silique. At the same time, embryonic development show close to normal. The germination rate declined 6~6.66%. BnGA2ox6 transgenic plants were able to reply to the phenotype of wild-type Col-4 treatment with exogenousGA3.(6) Overexpression BnGA2ox6 affects the expression of endogenousGA metabolism and signaling-related genes.GA biosynthetic g ene AtGA20ox1, AtGA20ox2, AtGA3ox1 and AtGA3ox2 transcription levels were significantly elevated in BnGA2ox6 overexpressing lines; besides AtGA2ox3 gene, 4 of 5 C19-GA2 ox genes AtGA2ox1, AtGA2ox2, AtGA2ox4 and AtGA2ox6 expression levels are reduced, but the C20-GA2 ox AtGA2ox7 and AtGA2ox8 genes transcription levels are raised; moreover,GA receptor gene GID1 a, GID1 b, GID1 c, and DELLA proteins gene RGA,GAI, RGL1, RGL2 and RGL3, transcription levels are elevated in transgenic plants.Summary: SevenGA2 ox genes in Brassica napus L. were cloned. BnGA2ox6 gene plays an important role in plant height, chlorophyll synthesis, flower ing, fruit pods and seed development. We hypothesized that BnGA2ox6 gene affects the growth and development of plants by reducing the content of activeGAs. |
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