【作者】 李鑫；

【导师】 赵小英；

【作者基本信息】 湖南大学 ， 生物学， 2022， 硕士

【摘要】 长期以来油菜作为我国南方的主要油料作物在农业生产中占据着重要的地位。油菜的种皮颜色是其重要的农艺性状。黄籽油菜的种子有着油质与蛋白质含量较高,纤维含量较低等特征,因此在油菜后续的生产加工过程中有着极大的优势。但是芸苔属中甘蓝型油菜不存在天然的黄籽突变体,现有的甘蓝型黄籽油菜都是通过与其他品种杂交获得且通常存在性状不稳定的问题。油菜黄籽性状的成因一般认为是种皮中缺乏类黄酮合成途径产物黄酮醇和原花青素。类黄酮合成途径合成的原花青素经过氧化积累使种皮呈现褐色或黑色。而类黄酮合成途径的前体酚酰Co A来源于苯丙素途径。为了从遗传水平上解析黄籽性状遗传不稳定的原因,本研究以黄籽性状遗传稳定的黄矮早和黄籽性状遗传不稳定的华黄1号和GH06为材料,对不同发育时期种皮进行了转录组测序及全基因组DNA甲基化测序分析,主要研究结果如下:(1)类黄酮合成途径基因Bn FLS2、Bn FL3c、Bn MYB5b和Bn TT6b在黄矮早种皮中的表达量下降。对授粉后15天,25天和35天3个品种油菜的种皮进行转录组分析发现七个黄酮类化合物合成相关的基因Bn FL3c、Bn TT6b、Bn PAL4a、Bn TT18c、Bn TT19b、Bn MYB5B和Bn FLS2在黄矮早与华黄1号和GH06之间存在表达差异。其中Bn FLS2、Bn FL3c、Bn MYB5B和Bn TT6b在黄矮早不同发育时期种皮中的表达量降低;Bn PAL4a、Bn TT18c和Bn TT19b在黄矮早不同发育时期种皮中的表达量升高。(2)DNA甲基转移酶基因Bn DRM1b和去甲基化酶基因Bn DML4a在黄矮早种皮中的表达量上升,另一去甲基化酶基因Bn DML5b则在黄矮早不同发育时期种皮中表达量下降。编码m6A阅读蛋白的基因Bn CPSF30a,Bn CPSF30e和Bn ECT2c在黄矮早种皮中的表达量明显高于GH06和华黄一号。(3)类黄酮合成途径基因Bn FL3c和Bn TT6b在黄矮早种皮中的甲基化水平升高。通过比较不同发育时期3个品种种皮全基因组DNA甲基化发现,黄矮早全基因组DNA甲基化水平高于华黄1号和GH06。并发现Bn FL3c,Bn TT6b这两个基因在授粉后15天和35天黄矮早种皮中的DNA甲基化水平要明显高于GH06和华黄一号。对具体元件的甲基化进行分析发现Bn FL3c主要在基因下游区域存在CG高甲基化。Bn TT6b在启动子区域存在CHH高甲基化,外显子和内含子区域分别存在CG和CHG高甲基化。因此,推测这两个基因可能由于DNA高甲基化降低了其表达量,从而使黄矮早的黄籽性状稳定。(4)黄矮早转座子表现出高甲基化的特征。根据黄矮早基因组整体高甲基化的特征,我们对样品重复序列的表达量进行了分析,发现在GH06中反转座子的主要成分LTR反转座子的表达量明显高于黄矮早和华黄一号。华黄一号和黄矮早中LTR反转座子表达量基本相同。黄矮早中大多数non-LTR反转座子的表达量明显高于GH06和华黄一号,如-CTA和-TGG家族等。而华黄一号与GH06中non-LTR反转座子的表达量没有明显差异。对基因组转座子区域甲基化进行分析发现华黄一号的转座子甲基化水平高于黄矮早且黄矮早的甲基化水平高于GH06。这为将来深入研究黄籽性状遗传稳定性的调控机制提供了线索。更多还原

【Abstract】 Rape has long been the main oil crop in southern my country and occupies an important position in my country’s agricultural production.The seed coat color of rape is an important agronomic trait.The seeds of yellow-seed rape have the characteristics of high oil and protein content and low fiber content,which have great advantages in the subsequent production and processing of rape.However,there is no natural yellow-seed mutant in Brassica oleracea,and the existing Brassica napus yellow-seed rape is obtained by crossing with other varieties,and usually has the problem of unstable characters.The origin of the traits of rape yellow seed is generally considered to be the lack of flavonols and procyanidins in the seed coat of flavonoid synthesis pathway products.The proanthocyanidins synthesized by the flavonoid synthesis pathway undergo oxidation to synthesize anthocyanins,which makes the seed coat appear brown or black.The precursor phenolic Co A in the flavonoid synthesis pathway is derived from the phenylpropanoid pathway.In order to analyze the reasons for the genetic instability of the yellow seed trait from the genetic level,this study used the genetically stable yellow seed trait Huang Ai Zao and the yellow seed trait genetically unstable Huahuang 1 and GH06 as the materials.Transcriptome sequencing and whole-genome DNA methylation sequencing analysis were performed.The main findings are as follows:(1)The expression of flavonoid synthesis pathway genes Bn FLS2,Bn FL3 c,Bn MYB5 b and Bn TT6 b decreased in seed coat of Huang Ai Zao.Transcriptome analysis of the seed coats of three cultivars at 15,25 and 35 days after pollination revealed that seven flavonoid synthesis-related genes Bn FL3 c,Bn TT6 b,Bn PAL4 a,Bn TT18 c,Bn TT19 b,Bn MYB5 B and Bn FLS2 were found in Huangaizao and Huaihuai.There are differences between Hua Huang 1 and GH06.Among them,the expression levels of Bn FLS2,Bn FL3 c,Bn MYB5 B and Bn TT6 b decreased in the seed coat of Huang Ai Zao at different developmental stages;the expression levels of Bn PAL4 a,Bn TT18 c and Bn TT19 b increased in the different development stages of Huang Ai Zao.(2)The expression levels of DNA methyltransferase gene Bn DRM1 b and demethylase gene Bn DML4 a increased in the seed coat of Huang Ai Zao,and another demethylase gene Bn DML5 b was expressed in the seed coat of Huang Ai Zao at different developmental stages expression decreased.The expression levels of genes Bn CPSF30 a,Bn CPSF30 e and Bn ECT2 c encoding three different m6 A reading proteins were significantly higher than those of GH06 and Huahuang 1 in seed coat of Huang Ai Zao(3)The methylation levels of the flavonoid synthesis pathway genes Bn FL3 c and Bn TT6 b were increased in the seed coat of Huang Ai Zao.By comparing the wholegenome DNA methylation of the seed coats of the three varieties at different developmental stages,it was found that the whole-genome DNA methylation level of Huang’aizao was higher than that of Huahuang 1 and GH06.It was found that the DNA methylation levels of Bn FL3 c and Bn TT6 b in the seed coat of Huang Ai Zao at15 and 35 days after pollination were significantly higher than those of GH06 and Huahuang 1.The methylation of specific elements was analyzed,and it was found that Bn FL3 c mainly had CG hypermethylation in the downstream region of the gene.Bn TT6 b has CHH hypermethylation in the promoter region,and CG and CHG hypermethylation in the exon and intron regions,respectively.Therefore,it is speculated that these two genes may reduce their expression levels due to DNA hypermethylation,thus making the yellow seed traits of Huang Aizao stable.(4)The Huang Ai Zao transposon showed the characteristics of hypermethylation.According to the overall hypermethylation characteristics of Huang’aizao’s genome,we analyzed the expression levels of repetitive sequences in the samples,and found that the expression level of the LTR retrotransposon,the main component of retrotransposons,was significantly higher in GH06 than in Huang’aizao and Huahuang.Number one.The expression levels of LTR retrotransposon in Huahuang No.1 and Huang’aizao were basically the same.The expression levels of most nonLTR retrotransposons in Huangaizao were significantly higher than those in GH06 and Huahuang 1,such as-CTA and-TGG families,while the non-LTR transposons between Huahuang 1 and GH06 were reversed.There was no significant difference in the expression levels of transposons.Analysis of methylation 3 in the transposon region of the genome showed that the methylation level of the transposon in Huahuang 1 was higher than that in Huang’aizao,and the methylation level of Huang’aizao was higher than that in GH06.This provides clues for the future in-depth study of the regulatory mechanism of the genetic stability of yellow seed traits.更多还原