發(fā)布時間：2018-09-16 20:56

【摘要】：甘藍(lán)型油菜是世界重要的油料作物之一,在我國其種植面積和總產(chǎn)量均占全球的1/4,居世界首位。高油育種是油菜育種的主要目標(biāo)之一,提高油菜種子含油量是提高單位面積產(chǎn)油量的關(guān)鍵措施之一。種子含油量是甘藍(lán)型油菜品質(zhì)特征的重要指標(biāo),深入解析種子含油量的遺傳基礎(chǔ),將會很大程度上加快高油育種進(jìn)程。運用完全雙列雜交與正反回交設(shè)計,全面估算并量化控制甘藍(lán)型油菜種子含油量的母體效應(yīng)、胚基因效應(yīng)、花粉直感與細(xì)胞質(zhì)效應(yīng)等遺傳因素。期望通過對甘藍(lán)型油菜種子含油量遺傳規(guī)律的深入解析,為育種家更加高效快速培育高油新品種打下堅實基礎(chǔ)。本研究運用種子含油量不同的甘藍(lán)型油菜品種和高世代純合自交系,按9×9完全雙列雜交設(shè)計分析種子含油量的遺傳效應(yīng)及基因與環(huán)境互作效應(yīng)。GoCGm分析結(jié)果顯示甘藍(lán)型油菜種子含油量主要由遺傳效應(yīng)(VG)和基因與環(huán)境互作效應(yīng)(VGE)共同控制,且二者共占總表型變異的86.19%。因為基因與環(huán)境互作效應(yīng)占總遺傳變異的51.68%,在油菜高油育種中基因型與環(huán)境互作效應(yīng)不容忽視。母體效應(yīng)占總遺傳效應(yīng)的75.03%,胚基因效應(yīng)和細(xì)胞質(zhì)效應(yīng)則分別為21.02%和3.95%。F1種子含油量主要由母體效應(yīng)控制,同時存在較小的花粉直感。配合力方差分析表明,油菜種子含油量的一般配合力和特殊配合力方差均達(dá)顯著水平,表明加性效應(yīng)與非加性效應(yīng)對于該性狀而言十分重要。同時,母體效應(yīng)方差達(dá)顯著水平而非母體效應(yīng)則不顯著,表明該性狀并非嚴(yán)格的受核基因控制,可能同時存在細(xì)胞質(zhì)效應(yīng)。甘藍(lán)型油菜種子含油量主要由母體效應(yīng)、胚基因效應(yīng)、花粉直感、細(xì)胞質(zhì)效應(yīng)以及相應(yīng)基因與環(huán)境互作效應(yīng)共同控制。9個親本中僅高油親本H1的各遺傳成分均為正向,因此該油菜品系在高油育種中更適合做親本,又因為該品系不存在不良胞質(zhì)效應(yīng)亦可做母本。油菜高油育種過程中,親本的選擇特別是母本的選擇尤為重要,而提供花粉的父本與來自母本的細(xì)胞質(zhì)效應(yīng)和胚基因效應(yīng)均需要關(guān)注。為從轉(zhuǎn)錄水平闡明調(diào)控油菜種子油脂合成的分子機(jī)制,本研究運用甘藍(lán)型油菜cDNA芯片差異表達(dá)基因分析嘗試獲得可能參與調(diào)控種子油脂合成的基因。cDNA芯片分析結(jié)果顯示,BnCIPK9在高油單株低表達(dá),然而在低油單株高表達(dá),即該基因可能負(fù)調(diào)控油菜種子含油量。gDNA測序分析結(jié)果顯示BnCIPK9基因存在4個拷貝,且氨基酸序列相對比較保守。qRT-PCR分析表明該基因在高油親本P1的莖、葉片和24DAP角果皮中均高表達(dá),而在花、蕾、24DAP種子中低表達(dá),成熟根部組織中表達(dá)水平最低。在胚珠不同發(fā)育時期其轉(zhuǎn)錄水平不相同,且種子發(fā)育中期轉(zhuǎn)錄水平相對較高。BnCIPK9啟動子GUS活性分析與qRT-PCR結(jié)果基本一致,且BnCIPK9的表達(dá)模式與擬南芥AtCIPK9類似在光合組織和非光合組織中均普遍表達(dá)。從親本gDNA中分別克隆BnCIPK9不同拷貝的5’非翻譯區(qū),并分別命名為BnCIPK9啟動子1(3050bp)和BnCIPK9啟動子2(3372bp),該啟動子區(qū)域包含多個順式調(diào)控元件。分析結(jié)果顯示BnCIPK9基因的兩個拷貝的啟動子區(qū)均包含兩個糖抑制順式調(diào)控元件(TATCCA),水稻的α-淀粉酶3基因啟動子區(qū)最先發(fā)現(xiàn)該調(diào)控元件。同時,BnCIPK9啟動子1和BnCIPK9啟動子2分別包含4和6個I-BOX元件。BnCIPK9啟動子1和啟動子2上則分別含有4和5個E-BOX,該調(diào)控元件主要與種子特異性表達(dá)有關(guān)。甘藍(lán)型油菜種子特異過表達(dá)該基因的轉(zhuǎn)基因株系表型分析顯示,T2代種子含油量顯著低于非轉(zhuǎn)基因?qū)φ�。即BnCIPK9可能參與調(diào)控種子油脂合成,且為負(fù)調(diào)控因子。GC分析結(jié)果表明,擬南芥cipk9突變體種子含油量為26.25%,而野生型則為24.06%。cipk9突變體種子脂肪酸成分分析結(jié)果顯示,cipk9突變體種子C20:1?11的相對比例顯著高于野生型,而C18:2的相對比例則顯著低于野生型�；パa轉(zhuǎn)基因株系種子含油量與野生型之間不存在差異,同時過表達(dá)轉(zhuǎn)基因擬南芥株系種子含油量低于cipk9,表明AtCIPK9可能負(fù)調(diào)控種子油脂合成。cipk9突變體在無糖培養(yǎng)基上,雖可正常萌發(fā)但其幼苗建成嚴(yán)重受抑制,外源添加蔗糖或者葡萄糖亦可恢復(fù)該表型。互補轉(zhuǎn)基因株系與過表達(dá)轉(zhuǎn)基因株系均可恢復(fù)純合突變體該缺陷表型。CBL2和CBL3是CIPK9上游與之互作的蛋白,僅cbl3突變體與cipk9表型一致,無糖條件下cbl3突變體雖可萌發(fā)但幼苗建成受抑制,但是還需做進(jìn)一步驗證。外界環(huán)境中糖缺乏時,AtCBL3可能與At CIPK9一起在幼苗建成過程中發(fā)揮調(diào)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S565.4

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