甘蓝型油菜种子含油量遗传及油脂合成相关基因功能分析
发布时间：2018-09-16 20:56
【摘要】：甘蓝型油菜是世界重要的油料作物之一,在我国其种植面积和总产量均占全球的1/4,居世界首位。高油育种是油菜育种的主要目标之一,提高油菜种子含油量是提高单位面积产油量的关键措施之一。种子含油量是甘蓝型油菜品质特征的重要指标,深入解析种子含油量的遗传基础,将会很大程度上加快高油育种进程。运用完全双列杂交与正反回交设计,全面估算并量化控制甘蓝型油菜种子含油量的母体效应、胚基因效应、花粉直感与细胞质效应等遗传因素。期望通过对甘蓝型油菜种子含油量遗传规律的深入解析,为育种家更加高效快速培育高油新品种打下坚实基础。本研究运用种子含油量不同的甘蓝型油菜品种和高世代纯合自交系,按9×9完全双列杂交设计分析种子含油量的遗传效应及基因与环境互作效应。GoCGm分析结果显示甘蓝型油菜种子含油量主要由遗传效应(VG)和基因与环境互作效应(VGE)共同控制,且二者共占总表型变异的86.19%。因为基因与环境互作效应占总遗传变异的51.68%,在油菜高油育种中基因型与环境互作效应不容忽视。母体效应占总遗传效应的75.03%,胚基因效应和细胞质效应则分别为21.02%和3.95%。F1种子含油量主要由母体效应控制,同时存在较小的花粉直感。配合力方差分析表明,油菜种子含油量的一般配合力和特殊配合力方差均达显著水平,表明加性效应与非加性效应对于该性状而言十分重要。同时,母体效应方差达显著水平而非母体效应则不显著,表明该性状并非严格的受核基因控制,可能同时存在细胞质效应。甘蓝型油菜种子含油量主要由母体效应、胚基因效应、花粉直感、细胞质效应以及相应基因与环境互作效应共同控制。9个亲本中仅高油亲本H1的各遗传成分均为正向,因此该油菜品系在高油育种中更适合做亲本,又因为该品系不存在不良胞质效应亦可做母本。油菜高油育种过程中,亲本的选择特别是母本的选择尤为重要,而提供花粉的父本与来自母本的细胞质效应和胚基因效应均需要关注。为从转录水平阐明调控油菜种子油脂合成的分子机制,本研究运用甘蓝型油菜cDNA芯片差异表达基因分析尝试获得可能参与调控种子油脂合成的基因。cDNA芯片分析结果显示,BnCIPK9在高油单株低表达,然而在低油单株高表达,即该基因可能负调控油菜种子含油量。gDNA测序分析结果显示BnCIPK9基因存在4个拷贝,且氨基酸序列相对比较保守。qRT-PCR分析表明该基因在高油亲本P1的茎、叶片和24DAP角果皮中均高表达,而在花、蕾、24DAP种子中低表达,成熟根部组织中表达水平最低。在胚珠不同发育时期其转录水平不相同,且种子发育中期转录水平相对较高。BnCIPK9启动子GUS活性分析与qRT-PCR结果基本一致,且BnCIPK9的表达模式与拟南芥AtCIPK9类似在光合组织和非光合组织中均普遍表达。从亲本gDNA中分别克隆BnCIPK9不同拷贝的5’非翻译区,并分别命名为BnCIPK9启动子1(3050bp)和BnCIPK9启动子2(3372bp),该启动子区域包含多个顺式调控元件。分析结果显示BnCIPK9基因的两个拷贝的启动子区均包含两个糖抑制顺式调控元件(TATCCA),水稻的α-淀粉酶3基因启动子区最先发现该调控元件。同时,BnCIPK9启动子1和BnCIPK9启动子2分别包含4和6个I-BOX元件。BnCIPK9启动子1和启动子2上则分别含有4和5个E-BOX,该调控元件主要与种子特异性表达有关。甘蓝型油菜种子特异过表达该基因的转基因株系表型分析显示,T2代种子含油量显著低于非转基因对照。即BnCIPK9可能参与调控种子油脂合成,且为负调控因子。GC分析结果表明,拟南芥cipk9突变体种子含油量为26.25%,而野生型则为24.06%。cipk9突变体种子脂肪酸成分分析结果显示,cipk9突变体种子C20:1?11的相对比例显著高于野生型,而C18:2的相对比例则显著低于野生型。互补转基因株系种子含油量与野生型之间不存在差异,同时过表达转基因拟南芥株系种子含油量低于cipk9,表明AtCIPK9可能负调控种子油脂合成。cipk9突变体在无糖培养基上,虽可正常萌发但其幼苗建成严重受抑制,外源添加蔗糖或者葡萄糖亦可恢复该表型。互补转基因株系与过表达转基因株系均可恢复纯合突变体该缺陷表型。CBL2和CBL3是CIPK9上游与之互作的蛋白,仅cbl3突变体与cipk9表型一致,无糖条件下cbl3突变体虽可萌发但幼苗建成受抑制,但是还需做进一步验证。外界环境中糖缺乏时,AtCBL3可能与At CIPK9一起在幼苗建成过程中发挥调控作用。
[Abstract]:Brassica napus is one of the most important oil crops in the world, and its planting area and total yield account for 1/4 of the world in China, ranking first in the world. Important indicators, in-depth analysis of the genetic basis of seed oil content, will greatly speed up the process of high oil breeding. The full diallel crossing and reciprocal backcross design was used to comprehensively estimate and quantify genetic factors such as maternal effect, embryo gene effect, pollen sensitivity and cytoplasmic effect controlling seed oil content in Brassica napus. The genetic analysis of seed oil content in Brassica napus laid a solid foundation for breeders to breed new varieties with high oil content more efficiently and quickly.The genetic effects and genes and rings of seed oil content in Brassica napus with different oil content and high generation homozygous inbred lines were analyzed by 9 *9 complete diallel cross design. The results of GoCGm analysis showed that the seed oil content of Brassica napus was mainly controlled by genetic effect (VG) and gene-environment interaction (VGE), and they accounted for 86.19% of the total phenotypic variation. Maternal effects accounted for 75.03% of the total genetic effects, while embryonic gene effects and cytoplasmic effects accounted for 21.02% and 3.95% respectively. The results indicated that additive and non-additive effects were very important for the trait. Meanwhile, the variance of maternal effects was significant, but not maternal effects, indicating that the trait was not strictly controlled by nuclear genes, and there might be cytoplasmic effects. All the genetic components of only high-oil parent H1 were positive, so the rapeseed strain was more suitable to be a parent in high-oil breeding, because there was no bad cytoplasmic effect and it could also be a female parent in high-oil breeding. In order to elucidate the molecular mechanism of regulating oil synthesis in rapeseed seeds at transcriptional level, differential expression gene analysis of Brassica napus cDNA microarray was used to try to obtain the seeds that might be involved in regulation. The results of cDNA microarray analysis showed that BnCIPK9 was low expressed in high oil plants, but high expressed in low oil plants, indicating that the gene may negatively regulate oil content in rapeseed seeds. The GUS activity of BnCIPK9 promoter was similar to that of qRT-PCR, and the expression level of BnCIPK9 was the lowest in flower, bud and 24DAP seeds. The expression pattern of BnCIPK9 was similar to that of AtCIPK9 in both photosynthetic and non-photosynthetic tissues. The 5'untranslated regions of different copies of BnCIPK9 were cloned from gDNA and named BnCIPK9 promoter 1 (3050bp) and BnCIPK9 promoter 2 (3372bp), respectively. The promoter region contained several cis regulatory elements. The promoter regions of two copies of BnCIPK9 gene contain two sugar-suppressing cis-regulatory elements (TATCCA), which were first found in the promoter region of rice alpha-amylase 3 gene. At the same time, BnCIPK9 promoter 1 and BnCIPK9 promoter 2 contain four and six I-BOX elements, respectively. BnCIPK9 promoter 1 and 2 contain four and five E-BOX elements, respectively. Phenotypic analysis of transgenic lines with seed-specific overexpression of the gene showed that the oil content in seeds of T2 generation was significantly lower than that of non-transgenic control, i.e. BnCIPK9 may be involved in regulating seed oil synthesis and is a negative regulator. The results of fatty acid composition analysis showed that the relative proportion of C20:1?11 in cipk9 mutant seed was significantly higher than that of wild type, while that of C18:2 was significantly lower than that of wild type. The seed oil content of transgenic Arabidopsis thaliana was lower than that of cipk9, suggesting that AtCIPK9 may negatively regulate seed oil synthesis. Although the mutant could germinate normally in sugar-free medium, its seedling formation was severely inhibited, and the phenotype could be restored by the addition of sucrose or glucose. CBL2 and CBL3 are interacting proteins upstream of CIPK9. Only the cbl3 mutant is consistent with the cipk9 phenotype. Although the cbl3 mutant can germinate but the seedling formation is inhibited under sugar-free condition, further verification is needed. AtCBL3 may be associated with At CIPK9 in the process of seedling formation in the absence of sugar in the external environment. Play a regulatory role.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.4
本文编号：2244762
[Abstract]:Brassica napus is one of the most important oil crops in the world, and its planting area and total yield account for 1/4 of the world in China, ranking first in the world. Important indicators, in-depth analysis of the genetic basis of seed oil content, will greatly speed up the process of high oil breeding. The full diallel crossing and reciprocal backcross design was used to comprehensively estimate and quantify genetic factors such as maternal effect, embryo gene effect, pollen sensitivity and cytoplasmic effect controlling seed oil content in Brassica napus. The genetic analysis of seed oil content in Brassica napus laid a solid foundation for breeders to breed new varieties with high oil content more efficiently and quickly.The genetic effects and genes and rings of seed oil content in Brassica napus with different oil content and high generation homozygous inbred lines were analyzed by 9 *9 complete diallel cross design. The results of GoCGm analysis showed that the seed oil content of Brassica napus was mainly controlled by genetic effect (VG) and gene-environment interaction (VGE), and they accounted for 86.19% of the total phenotypic variation. Maternal effects accounted for 75.03% of the total genetic effects, while embryonic gene effects and cytoplasmic effects accounted for 21.02% and 3.95% respectively. The results indicated that additive and non-additive effects were very important for the trait. Meanwhile, the variance of maternal effects was significant, but not maternal effects, indicating that the trait was not strictly controlled by nuclear genes, and there might be cytoplasmic effects. All the genetic components of only high-oil parent H1 were positive, so the rapeseed strain was more suitable to be a parent in high-oil breeding, because there was no bad cytoplasmic effect and it could also be a female parent in high-oil breeding. In order to elucidate the molecular mechanism of regulating oil synthesis in rapeseed seeds at transcriptional level, differential expression gene analysis of Brassica napus cDNA microarray was used to try to obtain the seeds that might be involved in regulation. The results of cDNA microarray analysis showed that BnCIPK9 was low expressed in high oil plants, but high expressed in low oil plants, indicating that the gene may negatively regulate oil content in rapeseed seeds. The GUS activity of BnCIPK9 promoter was similar to that of qRT-PCR, and the expression level of BnCIPK9 was the lowest in flower, bud and 24DAP seeds. The expression pattern of BnCIPK9 was similar to that of AtCIPK9 in both photosynthetic and non-photosynthetic tissues. The 5'untranslated regions of different copies of BnCIPK9 were cloned from gDNA and named BnCIPK9 promoter 1 (3050bp) and BnCIPK9 promoter 2 (3372bp), respectively. The promoter region contained several cis regulatory elements. The promoter regions of two copies of BnCIPK9 gene contain two sugar-suppressing cis-regulatory elements (TATCCA), which were first found in the promoter region of rice alpha-amylase 3 gene. At the same time, BnCIPK9 promoter 1 and BnCIPK9 promoter 2 contain four and six I-BOX elements, respectively. BnCIPK9 promoter 1 and 2 contain four and five E-BOX elements, respectively. Phenotypic analysis of transgenic lines with seed-specific overexpression of the gene showed that the oil content in seeds of T2 generation was significantly lower than that of non-transgenic control, i.e. BnCIPK9 may be involved in regulating seed oil synthesis and is a negative regulator. The results of fatty acid composition analysis showed that the relative proportion of C20:1?11 in cipk9 mutant seed was significantly higher than that of wild type, while that of C18:2 was significantly lower than that of wild type. The seed oil content of transgenic Arabidopsis thaliana was lower than that of cipk9, suggesting that AtCIPK9 may negatively regulate seed oil synthesis. Although the mutant could germinate normally in sugar-free medium, its seedling formation was severely inhibited, and the phenotype could be restored by the addition of sucrose or glucose. CBL2 and CBL3 are interacting proteins upstream of CIPK9. Only the cbl3 mutant is consistent with the cipk9 phenotype. Although the cbl3 mutant can germinate but the seedling formation is inhibited under sugar-free condition, further verification is needed. AtCBL3 may be associated with At CIPK9 in the process of seedling formation in the absence of sugar in the external environment. Play a regulatory role.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.4
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