發(fā)布時(shí)間：2018-04-20 07:18

  本文選題：甘藍(lán)型油菜 + 紫葉性狀��； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：甘藍(lán)型油菜紫葉性狀是由于花色苷在葉片中的積累所產(chǎn)生的形態(tài)學(xué)變異。紫葉除可作為一種重要的指示性狀運(yùn)用于雜交制種外,其所富含的花色苷具有光保護(hù)、耐干旱、耐寒、減少病蟲害以及抗氧化、抗腫瘤、改善心血管等諸多的生物學(xué)保健功能。正因如此,與花色苷相關(guān)的性狀成為近年來的研究熱點(diǎn)之一。本研究對甘藍(lán)型油菜紫葉突變體(6-]029)進(jìn)行了如下研究：(1)全生育期表型觀察、花色苷分布的解剖學(xué)觀察：(2)利用紫葉突變體與綠葉野生型(6-1025)建立了定位群體和近等基因系(NIL);(3)分析了6-1029、6-1025及其F1花色苷含量差異以及NIL中3種葉色(紫葉、淺紫葉、綠葉)單株葉綠素、花色苷含量、光合作用特性：(4)在利用定位群體分析紫葉性狀遺傳模式的基礎(chǔ)上,依據(jù)公布的甘藍(lán)型油菜和白菜基因組數(shù)據(jù)庫對紫葉基因進(jìn)行了精細(xì)定位；(5)在確定同源的甘藍(lán)型油菜和白菜候選區(qū)段后,進(jìn)行了候選基因預(yù)測、候選基因表達(dá)量分析、候選基因比較測序以及NIL中3種葉色單株的轉(zhuǎn)錄組測序及花色苷相關(guān)基因表達(dá)量分析。本研究的結(jié)論如下：16-1029全生育期葉色形態(tài)學(xué)觀察通過對6-1029全生育期的葉色形態(tài)學(xué)觀察發(fā)現(xiàn),6-1029的子葉在初生時(shí)呈綠色,6天后開始顯淺紫色；6-1029的真葉在初生時(shí)呈綠色,生成3天后葉脈處開始顯淺紫色；隨著生長時(shí)間的延長,6-1029葉片紫色逐漸加深,第35天開始呈現(xiàn)典型的紫色性狀：自抽薹期開始,6-1029葉片紫色開始變淡,至終花期完全恢復(fù)綠色。從上述結(jié)果來看,6-1029的紫葉性狀與其所處生長發(fā)育時(shí)期有關(guān)。26-1029花色苷分布的解剖學(xué)觀察對6-1029和6-1025及兩者雜交后代F1葉片的顯微觀察發(fā)現(xiàn),6-1029和F1葉片上表皮細(xì)胞均有花色苷分布,但F1葉片中花色苷的密度要低于6-1029;6-1025葉片則沒有花色苷分布。此結(jié)果與表型觀察相符,即6-1029葉片正面呈紫色、F1葉片正面呈淺紫色、6-1025葉片為綠色。3葉綠素和花色苷含量比較對6-1029、6-1025及F1進(jìn)行的花色苷含量分析表明,6-1029花色苷含量(64.33±0.162 mg/100g),顯著高于6-1025花色苷含量(7.73±0.088 mg/100g)及F1花色苷含量(20.11±0.262 mg/100g)。對于NIL紫葉、淺紫葉和綠葉單株的葉綠素和花色苷含量比較分析表明,3種葉色單株葉綠素a含量相同；葉綠素b和總?cè)~綠素含量差異達(dá)到顯著水平,其中綠葉單株葉綠素b(0.40±0.037 mg/g)及總?cè)~綠素(0.75±0.036 mg/g)含量最高而紫葉單株葉綠素b(0.26±0.016 mg/g)及總?cè)~綠素(0.62±0.017 mg/g)含量最低；3種葉色單株葉綠素a/b差異達(dá)到極顯著水平,其中紫葉單株葉綠素a/b(1.39±0.085)最高,綠葉單株葉綠素a/b(0.91±0.080)最低；3種葉色單株花色苷含量差異達(dá)到極顯著水平,其中紫葉單株花色苷(62.55±0.159mg/100g)含量最高而綠葉單株花色苷(9.19±0.067 mg/100g)含量最低。4 NIL中光合作用比較利用LI-6400光合作用系統(tǒng)測定了NIL中3種葉色單株的光響應(yīng)曲線(A-Q Curve)及CO2響應(yīng)曲線(A-Ci Curve)。分析表明,3種葉色單株之間光響應(yīng)曲線和CO2響應(yīng)曲線變化趨勢基本相同；紫葉單株的凈光合速率高于相同光照強(qiáng)度或相同CO2濃度條件下淺紫葉或綠葉單株的凈光合速率,但差異未達(dá)到顯著水平：淺紫葉單株與綠葉單株的凈光合速率在相同光照強(qiáng)度或相同CO2濃度條件下沒有差異。說明6-1029的紫色性狀與綠色性狀相比,在光合作用特性方面沒有負(fù)面效應(yīng),可以將其作為特異種質(zhì)資源應(yīng)用于油菜新品種選育。5 BnaA.PLl的遺傳分析及精細(xì)定位對定位群體的遺傳分析發(fā)現(xiàn),6-1029紫葉性狀受1對不完全顯性基因控制；通過BSA法分別結(jié)合SSR、AFLP及SNP技術(shù),發(fā)現(xiàn)及開發(fā)了一批與紫葉性狀連鎖的SSR、SCAR及SNP標(biāo)記,最終將該性狀定位在甘藍(lán)型油菜A03 random序列(對應(yīng)于白菜基因組Scaffold000096),根據(jù)染色體的共線性關(guān)系蔣BnaA.PL1定位于A3染色體的末端。在初定位的基礎(chǔ)上,利用定位BnaA.PL1的2646單株的群體繼續(xù)縮小候選基因范圍至Scaffold000096起始位置99kb。遺憾的是所有的標(biāo)記均位于BnaA.PLl的同一側(cè)。6候選基因的確定根據(jù)白菜和甘藍(lán)型油菜基因功能注釋,發(fā)現(xiàn)Scaffold000096候選區(qū)段中共有11個(gè)注釋基因。在對NIL 3種葉色單株進(jìn)行的半定量及定量PCR分析的結(jié)果中,僅有BnaA03g59800D高表達(dá)且在3種葉色單株中的表達(dá)量變化趨勢與花色苷含量變化一致,即紫葉淺紫葉綠葉；對BnaA03g59800D進(jìn)行比較測序發(fā)現(xiàn),兩個(gè)親本間的基因序列存在3個(gè)InDel和18個(gè)SNP位點(diǎn)差異,其中1個(gè)InDel和7個(gè)SNP位于編碼區(qū),使得6-1029外顯子區(qū)域產(chǎn)生了3個(gè)氨基酸的缺失和1個(gè)氨基酸的替換。對NIL中3種葉色單株進(jìn)行了RNA-seq分析,結(jié)果表明有22個(gè)與花色苷合成相關(guān)的基因高表達(dá)且符合不同葉色單株中花色苷含量差異規(guī)律(紫葉淺紫葉綠葉),其中僅BnaA03g4S610D位于A3染色體,但遠(yuǎn)離精細(xì)定位區(qū)段,不作為候選基因；在Scaffold000096候選區(qū)段中僅有BnaA03g59800D高表達(dá)同時(shí)符合NIL中差異表達(dá)規(guī)律,該結(jié)果與qRT-PCR結(jié)果相同,初步將BnaA03g59800D確定為候選基因。
[Abstract]:The purple leaf character of Brassica napus is the morphological variation caused by the accumulation of anthocyanins in the leaves. The purple leaf can be used as an important indicator for hybrid seed production. The anthocyanins rich in anthocyanins are protected by light, drought resistance, cold tolerance, disease and insect pests, anti oxidation, anti-tumor, and cardiovascular biology. Because of this, the characters associated with anthocyanins have become one of the hotspots of research in recent years. This study has studied the purple leaf mutant of Brassica napus (6-]029) as follows: (1) the phenotypic observation and anatomic observation of anthocyanin distribution during the whole growth period: (2) the location population was established by using the purple leaf mutant and the green leaf wild type (6-1025). And near isogenic lines (NIL); (3) analysis of the differences in the content of anthocyanins in 6-1029,6-1025 and its F1 and the single plant chlorophyll, anthocyanin content and photosynthetic characteristics of 3 leaf colors (purple leaves, light purple leaves and green leaves) in NIL: (4) based on the analysis of the genetic model of the purple leaf traits by using the location population, according to the published genomic data of Brassica napus and Chinese Cabbage The library made a fine location of the purple leaf gene; (5) after the identification of the homologous Brassica napus and the candidate sections of cabbage, the candidate gene prediction, candidate gene expression analysis, candidate gene comparison sequencing, and the transcriptional sequence of 3 leaf color single strains in NIL and the expression of anthocyanin phase gene expression were analyzed. The conclusions of this study are as follows: 16- 1029 the morphological observation of leaf color in the whole growth period showed that 6-1029 of the cotyledon was green at the beginning of birth and light purple at the beginning of birth in the 6-1029 whole growth period. 6-1029 of the true leaves were green at the beginning of birth, and the leaf veins began to show light purple at the beginning of birth, and the 6-1029 leaf purple gradually deepened with the lengthening of the length of life. The thirty-fifth days began to present a typical purple character: from the beginning of the bolting period, the 6-1029 leaf purple began to fade, and the final flowering period completely restored green. From the above results, the anatomical observation of the distribution of.26-1029 anthocyanins related to the 6-1029 purple leaf traits and their growth and development period showed that 6-1029 and 6-1025 and the F1 leaves of the two hybrid offspring were obvious. The micro observation showed that anthocyanins were distributed in the epidermis of 6-1029 and F1 leaves, but the density of anthocyanins in F1 leaves was less than 6-1029, and the 6-1025 leaves did not distribute anthocyanins. The results were in accordance with the phenotypic observation, that is, 6-1029 leaves are purple, F1 leaves are light purple, and the 6-1025 leaves are green.3 chlorophyll and anthocyanin content ratio Analysis of anthocyanin content of 6-1029,6-1025 and F1 showed that the content of 6-1029 anthocyanins (64.33 + 0.162 mg/100g) was significantly higher than 6-1025 anthocyanins (7.73 + 0.088 mg/100g) and F1 anthocyanins (20.11 + 0.262 mg/100g). For NIL purple leaves, chlorophyll and anthocyanins content of light purple leaves and green leaves were compared and analyzed in 3 leaves. Chlorophyll a content of single plant was the same, chlorophyll b and total chlorophyll content were significantly different. The contents of chlorophyll b (0.40 + 0.037 mg/g) and total chlorophyll (0.75 + 0.036 mg/g) in green leaves were the highest, while the contents of chlorophyll b (0.26 + 0.016 mg/g) and total chlorophyll (0.62 + 0.017 mg/g) were the lowest, and 3 leaf color single plant chlorophyll a/. The difference of B reached a very significant level, of which the chlorophyll a/b (1.39 + 0.085) was the highest, the chlorophyll a/b (0.91 + 0.080) of the green leaves was the lowest, and the difference of anthocyanins in the single plant color single plant was extremely significant. The content of the single plant anthocyanins (62.55 + 0.159mg/100g) of the purple leaves was the highest and the single plant anthocyanin (9.19 + 0.067 mg/100g) content of the green leaf was the most. Light response curve (A-Q Curve) and CO2 response curve (A-Ci Curve) of 3 single leaves of NIL were measured by LI-6400 photosynthesis system in low.4 NIL. The analysis showed that the change trend of light response curve and CO2 response curve between 3 leaf colors was basically the same; the net photosynthetic rate of single plant of purple leaf was higher than that of the same Guang Zhaoqiang. The net photosynthetic rate of light purple leaf or green leaf single plant under the condition of degree or same CO2 concentration, but the difference was not significant: the net photosynthetic rate of light purple leaf single plant and green leaf single plant was not different under the same light intensity or the same CO2 concentration condition. It showed that the 6-1029 purple character compared with the green character, it did not have the photosynthetic characteristics. The negative effect can be used as a specific germplasm resource for genetic analysis of.5 BnaA.PLl in the breeding of new varieties of rape and the genetic analysis of fine location for the location population. It is found that 6-1029 purple leaf traits are controlled by 1 pairs of incomplete dominant genes, and a batch of purple leaf traits connected with SSR, AFLP and SNP technology are discovered and developed by BSA method respectively. The SSR, SCAR and SNP markers were labeled, and the character was finally located in the A03 random sequence of Brassica napus (corresponding to the genomic Scaffold000096 of cabbage). Based on the Colosse of chromosomes, Chiang BnaA.PL1 was located at the end of the A3 chromosome. On the basis of the initial location, the population of the 2646 single plant locates BnaA.PL1 to continue to narrow the range of the candidate genes. To the beginning of the Scaffold000096, 99kb. regrets that all the.6 candidate genes located on the same side of the BnaA.PLl are determined on the basis of the functional annotation of cabbage and Brassica napus, and there are 11 annotated genes in the Scaffold000096 candidate section. In the results of semi quantitative and quantitative PCR analysis of 3 NIL leaf colors, only the results of quantitative and quantitative PCR analysis are carried out. There was a high expression of BnaA03g59800D and the variation trend of expression in 3 leaf color single plants was the same as anthocyanin content, that is, purple leaf green leaf green leaf. BnaA03g59800D sequencing showed that there were 3 InDel and 18 SNP loci differences between the gene sequences of two parents, of which 1 InDel and 7 SNP were located in the coding region, making 6-1029 outside. The deletion of 3 amino acids and the substitution of 1 amino acids were produced in the exon region. The RNA-seq analysis of 3 leaf colors in NIL showed that 22 genes related to anthocyanin synthesis were highly expressed and conformed to the difference law of anthocyanin content in different leaf colors (violet Ye Lvye), only BnaA03g4S610D was located on the A3 chromosome. But far from the fine location, it is not a candidate gene; only the high expression of BnaA03g59800D in the Scaffold000096 candidate section conforms to the differential expression in NIL. The result is the same as the result of qRT-PCR, and BnaA03g59800D is preliminarily identified as a candidate gene.

【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】：2016
【分類號】：S565.4;Q943.2

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