摘要：
Seed coat colour was one of the most important agronomic traits in Brassica napus L.,which was believed to be related with oil content and meal quality.Traditional hypothesis on the relation of seed coat colour with oil content and protein content focused on the reduction of fibre content in the yellow seeds.However in different reports,results on the hypothesis varied.The effects of genetic backgrounds and environments on oil content and protein content were believed to be responsible for the variation.In order to investigate the effects of seed coat colour on oil content and protein content more accurately,the genetic backgrounds and environments need to be kept identical as much as possible for the seeds with different colours.In this study,we isolated a single gene controlled yellow seed coat mutation of B.napus L.,on which yellow seeds and brown seeds were seeded in single silique simultaneously.Based on the yellow seeds and brown seeds isolated from the single plants,effects of seed coat colour on oil content and protein content were precisely measured at identical genetic backgrounds and environments.In this research,oil contents of yellow seeds and brown seeds isolated from single plants were measured by Soxhlet lipid extraction method.Protein content was measured by Hitachi L-8900 amino acids analyzer.Results showed that oil contents of yellow seeds and brown seeds isolated from Plant 1 were (49.58±0.26)%and (49.90±0.28)%,respectively.Oil contents of yellow seeds and brown seeds from Plant 2 were (49.65 ± 0.27)% and (49.36±0.25)%,respectively.That from Plant 3 and Plant 4 were (48.68±0.21)% and (48.82± 0.17)%,(49.41±0.19)% and (49.46±0.13)%,respectively.The difference was not statistically significant (P=0.87).The yellow seeds and the brown seeds derived from the single plant showed the same oil content. However,the protein content was significantly improved in the yellow seeds.The total hydrolyzed amino acids contents in the yellow seed meal and the brown seed meal were (2139.48±17.59)μmol/g and (2063.31±24.53)μmol/g,respectively.The difference was significant (P=0.0194).The most dramatically improved amino acids were tyrosine (9.0% improved)and phenylalanine (8.5% improved)in the yellow seed meal.Aspartate and lysine contents were as well improved 6.5% and 5.2% in the yellow seed meal,respectively.Electron microscopy revealed that the cell wall of inner cell layer of seed coat was decreased in yellow seeds.It was deduced that this may be one of the causes to improve the protein content in yellow seeds.Acidic hydrolysate of seed meal showed that phenylalanine (tyrosine was from phenylalanine)was the most dramatically improved amino acid in the yellow seed meal.Real-time qPCR revealed that the genes encodingβ-D-glucan exohydrolase and phenylalanine ammonia-lyase were related on the yellow seed coat mutation.Transcription of BnaA06g17630D (encodingβ-D-glucan exohydrolase)in the brown seed coat was increased by 1.6 folds as compared with the yellow seed coat.Transcription of BnaA05g28470D (encoding phenylalanine ammonia-lyase)in the yellow embryo was increased by 1.8 folds as compared with the brown embryo.We hypothesized that the phenylalanine used for pigment synthesis in the brown seeds was used for protein synthesis in the yellow mutation so that protein content was improved.The amount of protein increase and fibre decrease in yellow seeds might be fair so that oil content was unaffected.It was interesting that lysine content in the brown and yellow oil seed meal was 7.35% and 7.42%,respectively,much higher than that in soybean (5.4%).Thus,it was valuable to be used as lysine additive for grain feed.In conclusion,the yellow seed coat mutation of B.napus was independent with seed oil content but improved protein content significantly.Phenylalanine was the most dramatically improved amino acid in the yellow seed coat mutation.The lysine content was improved as well.The yellow seed meal was valuable to be as lysine additive for grain feed.Transcription of the genes encoding β-D-glucan exohydrolase (BnaA06g17630D)and phenylalanine ammonia-lyase (BnaA05g28470D)were related to yellow seed coat mutation.%籽粒颜色是油菜重要的农艺性状之一,传统观点认为黄籽油菜由于降低了纤维素含量从而提高了含油量和蛋白质含量,但在不同的研究中,黄籽与含油量和蛋白质含量的关系并不一致,这可能是环境因素对其有重要影响.本研究在获得的一个单基因黄籽突变材料的基础上,从同一个单株上分离出黄籽油菜和褐籽油菜,从而最大可能地降低了环境因素的影响.通过对同一单株来源的黄籽和褐籽油菜含油量和水解氨基酸含量的测定,发现黄籽突变不影响含油量,但显著提高蛋白质含量.电子显微观测显示黄籽种皮内层细胞壁显著变薄,推测该因素导致饼粕中纤维素含量降低从而提高了蛋白质含量.同时水解氨基酸测定结果显示苯丙氨酸在黄籽中提升幅度最大,暗示苯丙氨酸代谢与该黄籽突变基因功能有关,定量PCR结果也显示与细胞壁合成有关的β-葡聚糖水解酶基因和种皮色素合成相关的苯丙氨酸解氨酶基因与该黄籽突变有关,与TT基因无关.推测原用于种皮色素合成的苯丙氨酸和丙氨酸黄籽突变后改用于蛋白质合成,而蛋白质含量的提高和细胞壁成分的降低大致相抵,因此含油量无显著变化.另外值得注意的是油菜饼粕赖氨酸含量很高,黄籽油菜赖氨酸含量更高,适合作为谷物饲料的赖氨酸添加来源.
