甘蓝型油菜紫叶基因BnaA.PL1定位和候选基因分析

发布时间：2018-04-20 07:18

本文选题：甘蓝型油菜 + 紫叶性状　；参考：《华中农业大学》2016年博士论文

【摘要】：甘蓝型油菜紫叶性状是由于花色苷在叶片中的积累所产生的形态学变异。紫叶除可作为一种重要的指示性状运用于杂交制种外,其所富含的花色苷具有光保护、耐干旱、耐寒、减少病虫害以及抗氧化、抗肿瘤、改善心血管等诸多的生物学保健功能。正因如此,与花色苷相关的性状成为近年来的研究热点之一。本研究对甘蓝型油菜紫叶突变体(6-]029)进行了如下研究：(1)全生育期表型观察、花色苷分布的解剖学观察：(2)利用紫叶突变体与绿叶野生型(6-1025)建立了定位群体和近等基因系(NIL);(3)分析了6-1029、6-1025及其F1花色苷含量差异以及NIL中3种叶色(紫叶、浅紫叶、绿叶)单株叶绿素、花色苷含量、光合作用特性：(4)在利用定位群体分析紫叶性状遗传模式的基础上,依据公布的甘蓝型油菜和白菜基因组数据库对紫叶基因进行了精细定位；(5)在确定同源的甘蓝型油菜和白菜候选区段后,进行了候选基因预测、候选基因表达量分析、候选基因比较测序以及NIL中3种叶色单株的转录组测序及花色苷相关基因表达量分析。本研究的结论如下：16-1029全生育期叶色形态学观察通过对6-1029全生育期的叶色形态学观察发现,6-1029的子叶在初生时呈绿色,6天后开始显浅紫色；6-1029的真叶在初生时呈绿色,生成3天后叶脉处开始显浅紫色；随着生长时间的延长,6-1029叶片紫色逐渐加深,第35天开始呈现典型的紫色性状：自抽薹期开始,6-1029叶片紫色开始变淡,至终花期完全恢复绿色。从上述结果来看,6-1029的紫叶性状与其所处生长发育时期有关。26-1029花色苷分布的解剖学观察对6-1029和6-1025及两者杂交后代F1叶片的显微观察发现,6-1029和F1叶片上表皮细胞均有花色苷分布,但F1叶片中花色苷的密度要低于6-1029;6-1025叶片则没有花色苷分布。此结果与表型观察相符,即6-1029叶片正面呈紫色、F1叶片正面呈浅紫色、6-1025叶片为绿色。3叶绿素和花色苷含量比较对6-1029、6-1025及F1进行的花色苷含量分析表明,6-1029花色苷含量(64.33±0.162 mg/100g),显著高于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)。对于NIL紫叶、浅紫叶和绿叶单株的叶绿素和花色苷含量比较分析表明,3种叶色单株叶绿素a含量相同；叶绿素b和总叶绿素含量差异达到显著水平,其中绿叶单株叶绿素b(0.40±0.037 mg/g)及总叶绿素(0.75±0.036 mg/g)含量最高而紫叶单株叶绿素b(0.26±0.016 mg/g)及总叶绿素(0.62±0.017 mg/g)含量最低；3种叶色单株叶绿素a/b差异达到极显著水平,其中紫叶单株叶绿素a/b(1.39±0.085)最高,绿叶单株叶绿素a/b(0.91±0.080)最低；3种叶色单株花色苷含量差异达到极显著水平,其中紫叶单株花色苷(62.55±0.159mg/100g)含量最高而绿叶单株花色苷(9.19±0.067 mg/100g)含量最低。4 NIL中光合作用比较利用LI-6400光合作用系统测定了NIL中3种叶色单株的光响应曲线(A-Q Curve)及CO2响应曲线(A-Ci Curve)。分析表明,3种叶色单株之间光响应曲线和CO2响应曲线变化趋势基本相同；紫叶单株的净光合速率高于相同光照强度或相同CO2浓度条件下浅紫叶或绿叶单株的净光合速率,但差异未达到显著水平：浅紫叶单株与绿叶单株的净光合速率在相同光照强度或相同CO2浓度条件下没有差异。说明6-1029的紫色性状与绿色性状相比,在光合作用特性方面没有负面效应,可以将其作为特异种质资源应用于油菜新品种选育。5 BnaA.PLl的遗传分析及精细定位对定位群体的遗传分析发现,6-1029紫叶性状受1对不完全显性基因控制；通过BSA法分别结合SSR、AFLP及SNP技术,发现及开发了一批与紫叶性状连锁的SSR、SCAR及SNP标记,最终将该性状定位在甘蓝型油菜A03 random序列(对应于白菜基因组Scaffold000096),根据染色体的共线性关系蒋BnaA.PL1定位于A3染色体的末端。在初定位的基础上,利用定位BnaA.PL1的2646单株的群体继续缩小候选基因范围至Scaffold000096起始位置99kb。遗憾的是所有的标记均位于BnaA.PLl的同一侧。6候选基因的确定根据白菜和甘蓝型油菜基因功能注释,发现Scaffold000096候选区段中共有11个注释基因。在对NIL 3种叶色单株进行的半定量及定量PCR分析的结果中,仅有BnaA03g59800D高表达且在3种叶色单株中的表达量变化趋势与花色苷含量变化一致,即紫叶浅紫叶绿叶；对BnaA03g59800D进行比较测序发现,两个亲本间的基因序列存在3个InDel和18个SNP位点差异,其中1个InDel和7个SNP位于编码区,使得6-1029外显子区域产生了3个氨基酸的缺失和1个氨基酸的替换。对NIL中3种叶色单株进行了RNA-seq分析,结果表明有22个与花色苷合成相关的基因高表达且符合不同叶色单株中花色苷含量差异规律(紫叶浅紫叶绿叶),其中仅BnaA03g4S610D位于A3染色体,但远离精细定位区段,不作为候选基因；在Scaffold000096候选区段中仅有BnaA03g59800D高表达同时符合NIL中差异表达规律,该结果与qRT-PCR结果相同,初步将BnaA03g59800D确定为候选基因。
[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.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4;Q943.2

【参考文献】