工业专用特高芥酸甘蓝型油菜芥酸含量的遗传、杂种优势、分子标记及关键基因克隆分析

发布时间：2018-04-23 16:42

本文选题：甘蓝型油菜 + 特高芥酸含量　；参考：《四川农业大学》2016年博士论文

【摘要】：芥酸是芸苔属(Brassica)植物种子油脂中特有的一种长碳链脂肪酸,作为食用油,其不易被人体消化利用,但作为工业原料,在油田化学、石油化工、日用化学、医药化工等行业有着十分广泛的应用。高芥酸油菜是当今世界上工业芥酸的最重要来源。本研究以3个特高芥酸品系(种)、2个高芥品系(种)、2个中芥品系(种)、2个低芥品系(种),围绕特高芥酸含量性状展开相关的遗传研究。(1)以工业专用特高芥酸、特低油酸甘蓝型油菜新材料703AB-4和低芥酸、高油酸油菜品种中双11为亲本,配制6个遗传世代,采用主基因加多基因混合遗传模型,分析特高芥酸含量和油酸含量的遗传模型,采用F2群体分析芥酸与其它主要脂肪酸含量的相关性；(2)以9个芥酸含量和遗传背景不同的甘蓝型油菜品系(种)为材料,采用完全双列杂交遗传设计,配制获得72个正反交杂交组合和9个自交系亲本后代,在四川成都、绵阳和宜宾三个不同的生态地区进行随机区组试验鉴定,分析芥酸含量的杂种优势表现、配合力及环境效应；(3)通过特高芥酸含量材料703AB-4与低芥酸材料中双11杂交获得F2分离群体,并进行SSR分子标记,筛选能区分芥酸含量的分子标记,以应用于高芥酸辅助选择育种；(4)利用特高芥酸含量材料703AB-4、中芥酸含量材料L155及低芥酸含量材料中双11,进行芥酸合成途径中两个关键调控基因FAE1和FAD2的克隆分析,以分析703AB-4特高芥酸含量的分子遗传机理。主要研究结果如下：1.特高芥酸的最适遗传模型为E-0,即受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制,以主基因效应为主。B1、B2和F2群体的主基因遗传率分别高达94.3%、98.5%和98.2%,多基因效应较弱,2对主基因的加性效应较大,达13.21,均为正效应且相等,累计加性效应值高达26.42。油酸的最适遗传模型为E-1,即受2对加性-显性-上位性主基因+加性-显性多基因控制,B2和F2群体的主基因遗传率较高,分别为98.00%和95.53%,而B1群体的主基因遗传率则较低,为73.71%,说明油酸含量的遗传主要受两对主基因控制,存在多基因效应。2.芥酸与其他主要脂肪酸相关性分析表明,油菜芥酸含量与油酸含量、亚油酸含量之间存在极显著的负相关,但油酸含量变化较大,平均达55个百分点,亚油酸含量变化总量不到10个百分点；芥酸含量与亚麻酸含量关系不显著；芥酸含量与花生烯酸含量关系较复杂,芥酸含量在15以下为正相关,之后为负相关,平均变化约15个百分点。芥酸升高到50%后,继续升高时,主要是以降低花生烯酸含量的库来实现的。3.多数杂交组合芥酸含量都显示出明显的中亲优势,只有少数组合表现出超亲优势,且两亲本间芥酸含量差异越大,其中亲优势越强,超亲优势只表现在特高芥亲本之间,说明高芥酸杂种优势主要以中亲优势为主。环境对亲本及杂交组合的芥酸含量均有影响,但亲本受环境的影响小于杂交组合。芥酸含量杂种优势在不同环境下表现的趋势是一致的,均以中亲优势为主。不同试验点芥酸含量杂种优势的表现存在差异,但出现杂种优势的组合具有一致性,宜宾点更易获得具有芥酸含量杂种优势的组合。不同亲本在提高芥酸含量的作用是不一样的,在选育高芥酸材料的过程中应充分考虑亲本的差异,同时考虑环境因素的影响。4.杂交组合芥酸含量主要由一般配合力决定,在高芥酸油菜组合选配中,要获得高芥酸含量的杂交组合,首先要选芥酸含量高、一般配合力高的亲本,同时考虑反交效应。本研究通过配合力稳定性分析表明,油菜芥酸含量主要受基因遗传控制,但环境因素对油菜芥酸含量有一定的影响。杂交组合芥酸受母性影响,母性效应为正极显著的亲本,其正反交芥酸含量存在显著差异。仅在特定组合间表现的非母性效应,对组合芥酸含量的影响非常大。5.获得2个与芥酸含量紧密连锁的SSR分子标记,CB10364和BRMS-017,单株基因型同时为CB10364-a和BRMS-017-a的芥酸含量57%,其分离比率经χ2检验符合孟德尔分离规律,能较可靠地将群体中特高芥酸单株区分出来。6.低芥材料中双11、中芥材料L155具有两个FAE1基因拷贝,分别位于甘蓝型油菜A8和C3染色体上,特高芥材料703AB-4只有一个FAE1基因拷贝,但均只有一个拷贝可完整编码氨基酸序列。低芥FAE1-1.1、中芥FAE1-2.1和特高芥FAE1-3编码氨基酸序列比对分析结果显示,在第282位氨基酸位点,低芥FAE1-1.1为F(苯丙氨酸),中芥与特高芥均为S(丝氨酸)。在第286、323、395和406位氨基酸位点,特高芥酸含量材料703AB-4的FAE1-3分别编码R(精氨酸)、T(苏氨酸)、K(赖氨酸)和G(甘氨酸),而低芥与中芥基因则均依次编码G(甘氨酸)、Ⅰ(异亮氨酸)、R(精氨酸)和A(丙氨酸)。7.低芥中双11与中芥材料L155均存在4条不同的FAD2基因拷贝,而特高芥酸材料703AB-4存在3条FAD2基因拷贝。同源分析表明,FAD2-1. 1、FAD2-2.1、FAD2-3.1为一类,编码氨基酸序列完全一致；FAD2-1.2, FAD2-2.2、FAD2-3.2为一类,编码氨基酸序列完全一致；FAD2-1.3、FAD2-2.3为一类,但提前出现终止密码；FAD2-1.4、FAD2-2.4、FAD2-3.3为一类,编码氨基酸序列同源性高达99.91%,仅在20位氨基酸位点,中芥酸材料2号材料出现差异,为T苏氨酸,而低芥酸材料(1号材料)和高芥酸材料(3号材料)均为N天冬氨酸,也即低芥酸材料和特高芥酸材料氨基酸序列完全一致。
[Abstract]:Erucic acid is a special kind of long carbon chain fatty acid in the seed oil of the Brassica plant. As a edible oil, it is not easily digested by the human body. But as an industrial raw material, it is widely used in the fields of oil field chemistry, petrochemical, daily chemistry, medicine and chemical industry. High erucic acid is the heaviest in the world. 3 highly erucic acid lines (species), 2 high mustard lines (species), 2 middle mustard lines (species) and 2 low mustard lines (species) were studied in this study. (1) the special high erucic acid, 703AB-4 and low erucic acid of specially low oleic acid Brassica napus, and double 11 in high oleic acid rapeseed were used as parents. 6 Genetic generations were prepared, and the genetic model of the main gene mixed gene was used to analyze the genetic model of high erucic acid content and oleic acid content. The correlation between erucic acid and other main fatty acids was analyzed by F2 population. (2) full diallel was used as the material with 9 erucic acid content and different genetic background of Brassica napus. Genetic design was used to prepare 72 positive and negative cross combinations and 9 progeny of self inbred lines. In three different ecological regions, Chengdu, Mianyang and Yibin, Sichuan, three different ecological regions were tested, and the Heterosis of erucic acid content, combining ability and environmental effect were analyzed. (3) 703AB-4 and low erucic acid material were used as the material of high erucic acid content. Medium double 11 hybrid was used to isolate F2 population, and SSR molecular markers were used to screen molecular markers that could distinguish erucic acid content, which was applied to high erucic acid assisted selection breeding. (4) using high erucic acid content material 703AB-4, medium erucic acid content material L155 and low erucic acid content of 11, the two key regulatory genes of erucic acid synthesis pathway, FAE1, were carried out. The molecular genetic mechanism of 703AB-4 highly erucic acid content was analyzed by cloning and analysis of FAD2. The main results were as follows: 1. the optimum genetic model of high erucic acid was E-0, which was controlled by 2 pairs of additive dominant epistatic main gene + additive dominant epistasis, the main gene effect was mainly.B1, and the main gene heritability of B2 and F2 population was respectively. As high as 94.3%, 98.5% and 98.2%, the effect of polygene is weak, and the additive effect of 2 on the main gene is greater, and the effect is 13.21. The optimum genetic model of the cumulative additive effect value up to 26.42. oleic acid is E-1, that is, it is controlled by 2 additive dominant epistatic main gene + additive dominant gene, and the main gene heritability of B2 and F2 population is higher. The main gene heritability of B1 population was 98% and 95.53%, respectively, and the main gene heritability was 73.71%, indicating that the inheritance of oleic acid content was mainly controlled by two main genes, and the correlation analysis between.2. erucic acid and other main fatty acids showed that there was a very significant negative correlation between the content of erucic acid and the content of oleic acid and the content of linoleic acid. The content of oleic acid changed greatly, with an average of 55 percentage points, the total amount of linoleic acid content was less than 10 percentage points, and the relationship between the content of erucic acid and the content of linolenic acid was not significant. The content of erucic acid was more complex than the content of arachidic acid. The content of erucic acid was positively correlated with the content of erucic acid below 15, and the average change was about 15 percentage points. Erucic acid was raised to 50%. After continuing to rise, most of the.3. hybrid erucic acid content, which was achieved by reducing the content of arachidic acid, showed obvious parent heterosis. Only a few combinations showed superior parent heterosis, and the greater the difference in erucic acid content between the two parents, the stronger the heterosis, the superior parent was only among the highly high mustard parents. The Heterosis of erucic acid was mainly dominated by the middle parent heterosis. The environment had an influence on the erucic acid content of the parent and cross combination, but the parent was less affected by the environment than the hybrid combination. The Heterosis of the erucic acid content was consistent in different environment, and the dominant parent was the middle parent. The Heterosis of the erucic acid content in different test points was present. However, the combination of heterosis is consistent, and the combination of Heterosis with erucic acid content is more easily obtained in Yibin point. The effect of different parents on erucic acid content is different. In the process of breeding high erucic acid, the difference of parents should be taken into consideration, and the effect of environmental factors on the erucic acid content of.4. hybrid combinations should be considered. It is mainly determined by the general combining ability. In the selection of high erucic acid rapeseed combination, the high erucic acid content should be obtained by the combination of high erucic acid. First, the parent with high erucic acid content and high common strength should be selected and the anti cross effect is considered. Erucic acid content had a certain influence. The combination of erucic acid was influenced by maternal sex, maternal effect was a positive parent, and there was a significant difference in the content of erucic acid. Only the non maternal effect between the specific combinations, the effect of the combination of the erucic acid on the combination of erucic acid content was very large.5. obtained 2 SSR molecular markers linked with the erucic acid content, CB10364 And BRMS-017, single plant genotype was 57% of erucic acid in CB10364-a and BRMS-017-a, and its separation ratio was conformed to Mendel separation law by chi 2 test. It could be more reliable to distinguish high erucic acid from group.6. low mustard material in 11, and L155 of medium mustard material with two copies of FAE1 gene, which were located in A8 and C3 of Brassica napus, respectively. On the body, there is only one copy of FAE1 gene in 703AB-4, but only one copy can fully encode amino acid sequence. Low mustard FAE1-1.1, middle mustard FAE1-2.1 and highly high mustard FAE1-3 coding amino acid sequence alignment analysis results show that at the 282nd bit amino acid site, low mustard FAE1-1.1 is F (phenylalanine), medium mustard and highly high mustard are S (silk ammonia). At 286323395th and 406th amino acid sites, FAE1-3 of 703AB-4, T (threonine), K (lysine) and G (glycine) were encoded in the highly erucic acid content material, respectively, while the low mustard and middle mustard genes encode G (glycine), I (ISO) (ISO), R (arginine) and A (alanine).7. low mustard and medium mustard material L155 were 4 There are 3 copies of different FAD2 genes, while the highly erucic acid material 703AB-4 has 3 copies of FAD2 gene. Homology analysis shows that FAD2-1. 1, FAD2-2.1, FAD2-3.1 are a class, the sequence of encoded amino acids is identical; FAD2-1.2, FAD2-2.2, FAD2-3.2 are a class, the sequence of encoded amino acids is all consistent; FAD2-1.3, FAD2-2.3 is a class, but appears in advance to terminate. Ciphers, FAD2-1.4, FAD2-2.4, FAD2-3.3 are a class of encoded amino acid sequence homology up to 99.91%, only at 20 amino acid sites, medium erucic acid material 2 material difference, T threonine, and low erucic acid material (1 material) and high erucic acid material (3 material) are N aspartic acid, that is, low erucic acid material and high erucic acid material amino acids. The sequence is exactly the same.

【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

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