Isolation And Functional Analysis Of Genes Encoding Key Enzymes In Gibberellin Biosynthesis Pathway In Rapeseed (Brassica Napus)

Heterosis is a common phenomenon and has been used to increase yield and toimprove quality in crops. In order to apply heterosis more efficiently, scientists have for along time poured a lot of efforts to investigate the genetic and molecular mechanismunderlying heterosis. Rapeseed is one of the most important crops in China, to whichheterosis has been successfully applied. Exploring the mechanism underlying heterosis inBrassica napus could enrich the genetic knowledge about crop heterosis. The analysis ofdifferential gene expression and the study on signal transduction would shed light on cropheterosis at molecular level.With the development of modern technology, oilseed rape is not only consumed as food,but also used as raw materials for industry that are difficult to obtain by conventionalbreeding program. More and more genetically modified cultivars of oilseed rape has beengenerated since the first successfully transformed rapeseed in 1985. Transgenictechnology is a powerful tool for scientists to characterize gene function and to improverapeseed as well, thus complementing conventional breeding.The gibberellin (GA) hormones act throughout the life cycle of plants, influencing seedgermination, stem elongation, flower induction, anther development, and seed andpericarp development. It has been shown that some agronomic traits are associate withGA contents in F₁ hybrid, exhibiting faster metabolism rate and more growth vigor thanparental lines. Some of the genes encoding key enzymes in GA metabolic pathway in F₁may be expressed more than in parents and GAs may play an important role in heterosis.Taking into account the above previous studies, we isolated and defined the function ofthree genes encoding key enzymes in GA biosynthesis in order in the future to investigatemolecularly the role of GAs in heterosis. Moreover, Agrobacterium-mediatedtransformation system for rapeseed were optimized. The main results were as follows:1. BnGA3ox1, BnGA3ox2, BnGA20ox1, the genes encoding key enzymes in GAsmetabolism in Brassica napus were isolated. Sequencing analysis indicated that:BnGA3ox1 contains 1648 nucleotides with an intron of 561bp and an open reading frame(ORF) of 358 amino acids; BnGA3ox2 contains 1077 nucleotides, encoding an ORF of358 amino acids; BnGA20ox1 contains 1134 bp, encoding a 377 aa ORF. At the level ofamino acids, BnGA3ox1, BnGA3ox2, and BnGA20ox1 are homologous to theirArabidopsis counterparts by 81.6%, 87.6% and 81.7%, respectively.2. Plant overexpression vectors pBnGA3ox1, pBnGA3ox2, pBnGA20ox1 and RNAinterference expression vectors pBnGA3ox2RNAi, pBnGA20ox1RNA/containing CaMV35S promoter were constructed.3. BnGA3ox1, BnGA3ox2, and BnGA20ox1 transformed tobacco showed the typical GA effects such as higher growth rate and bigger biomass and early flowering. GA assayrevealed that the transgenic plants contained more Gas than the control plants andNorthern blotting demonstrated these genes were overexpressed in the transgenic plants.Taking together, our results indicate that these three genes isolated from Brassica napusare founctional.4. Agrobacterium-mediated transformation system for rapeseed (Brassica napus) usingcotyledonary petiole as explants was optimized, bringing 16,17,2 rapeseed plants(ZHONG SHUANG 9) transformed with pBnGA3ox1, pBnGA20ox1 andpBnGA20ox1RNAi The shoot differentiation and regeneration of cotyledon petioleswere observed, demonstrating ZHONG SHUANG 9 is an appropriate transformationhost.5. The high content Kanamycin (Kan) suppressed the callus formation, bud polarizationand root development of the explants of rapeseed severely. The optimum Kanconcentration was 15mg/L. The resistant buds that were cut from selective medium werenot transferred to Kan-containing medium again.