Studies On The Transcription Factors Related To Seed Oil Content And Screening Mutants Of Alleles In Brassica Napus

Oilseed rape (Brassica napus), as the third biggest oil crop in the world and the most important one in China, supplies human with good edible oil and important industrial material. Therefore, it will be strategic necessary to study the regulatory mechanism of seed oil accumulation in oilseed rape. Seed oil content is a typical quantitative trait, belongs to genetic model of major gene and polygene, and can be increased by converging allele with high oil content. Previous papers showed seed oil content can be regulated through modifying key enzymes in oilseed rape lipid biosynthesis. However, more recent studies have indicated that seed oil accumulation appears to be controlled by a coordinated regulatory mechanism, which is not only pertinent to major steps of lipid metabolism pathways, but also requires coordination of key components in carbohydrate metabolism, in particular the regulation of sucrose and hexose flux. Thus, identification of the key genes involved in seed oil content at the level of carbohydrate metabolism represents another strategy for increasing seed oil bioassembly. In contrast to oilseed rape, considerable advances have been made in understanding the genetic and biochemical basis of seed oil accumulation in the closely related Arabidopsis species. Screens of Arabidopsis mutants have identified a number of transcription factors (For example, AtWRINKLED1 and AtGLABRA2), which haven been shown to play important roles in controlling oil accumulation in seed.Based on the genetic characteristics of seed oil content, this paper firstly studied the effect of the two transcription factors (BnWRINKLED1, BnWRI1 and GLABRA2, GL2) related to seed development on seed oil accumulation, using multiple functional methods such as overexpression, antisense and RNAi. Subsequently, the mutants of B. napus two GL2 alleles were screened in an EMS mutation population by a new reverse genetics method (TILLING technology), in order to increase seed oil content by converging identified allele with high oil content. Our results laid foundations for studying the mechanism of seed oil content control and breeding new cultivars. The main results are as follows:1. Functional analysis of B. napus BnWRI1To study the relationship between B. napus BnWRI1 and seed oil content in oilseed rape, the paper employed RT-PCR to isolate a 384 bp fragment from the coding region of BnWRI1, which was then inserted into the RNAi vector of pGΩ4ARi in both forward and reverse directions to form a plant expression vector pG?4ARi-WRI1. The vector was introduced into oilseed rape cv. Zhongshuang 9 via a pollen-tube pathway method. Three transgenic plants were confirmed by PCR and Southern blot analysis that the foreign fragment integrated into the plant genomes. BnWRI1 transcript levels and seed oil contents were detected by Semi-quantitative RT-PCR and improved Soxhlet extraction, respectively. The results showed that BnWRI1 expression is inhibited and seed oil content drastically decreases in each transgenic plant, compared with non-transgenic plants. Our results suggested BnWRI1 could positvely regulate seed oil content in oilseed rape. 2. Isolation of four Brassica GL2 orthologues and analysis of their expression patternsTo clarify the impact of GL2 on seed oil content in oilseed rape, we isolated four homologuos GL2 genes from B. napus (A C genome), B. rapa (A genome), and B. oleracea (C genome), respectively, using a overlapping-PCR strategy. The four genes have identical structures which contain nine exons and eight introns. The proteins deduced from the GL2 exon sequences all have 750 amino acids and belong to HD-ZIPⅣsubfamily. Sequence alignments revealed that the A genome sequences of BnaA.GL2.a from B. napus and BraA.GL2.a from B. rapa are more similar than the others, and likewise the C genome sequences of BnaC.GL2.b from B. napus and BolC.GL2.a from B. oleracea are more similar. BnaA.GL2.a and BraA.GL2.a from the A genome are highly expressed in roots, whilst BnaC.GL2.b and BolC.GL2.a from the C genome are preferentially expressed in seeds.3. Characterization of B. napus BnaC.GL2.b function and development of functional markersTo further study the effect of B. napus BnaC.GL2.b on seed oil content, we constructed the BnaC.GL2.b overexpression and antisense vectors to transform them into Arabidopsis. The results showed that suppression with B. napus BnaC.GL2.b can negatively regulate trichome development and promote oil accumulation in Arabidopsis seed. Therefore, we presume that BnaC.GL2.b might play an important role in seed oil accumulation of oilseed rape. As a result of comparing the four GL2 genes, three A/ C genome-specific primer sets were developed and a C genome-specific EcoRV cleavage site was identified, which can be used as functional markers to distinguish these orthologues within Brassica species.4. Construction and evaluation of a B. napus EMS mutation populationA mutation population provides an important resource for carrying out function genomics research. In this study, we constructed a B. napus EMS mutant population via treating B. napus cv. Zhongshuang 9 (a“double low and high oil”cultivar) with a dosage of 0.5% EMS solution. The mutation population contained 9814 M2 plants (3188 lines). All M2 plants were scrutinized for visible varant phenotypes during the growing season, such as plant architecture, floral, leaf, stem, pod and other morphology. Oil content of M2 seeds was also measured. The mutation population in this paper contained lots of outstanding mutants which can be used for not only breeding a cultivar, but also studing gene function by TILLING technology.5. Establishment of TILLING platform in B. napus and screening GL2 alleles using the techniqueTo identify the mutants of B. napus two GL2 alleles in oilseed rape, we successively established a TILLING platform in B. napus. To date, BnaA.GL2.b, one of the two B. napus GL2 genes, was screened in 1632 M2 plants in the mutant population with TILLING and 18 mutations were obtained. Succesive work such as mutations sequencing and M3 genetics analysis is on progress. We believe that our results laid foundations for further studies of seed oil content control in oilseed rape.