【正文】 ..... 34 基因預(yù)測及克隆 .................................................................................................... 34 參考文獻(xiàn) ........................................................................................................................... 37 附錄 .................................................................................................................................. 45 致謝 .................................................................................................................................. 47 發(fā)表論文、參加課題及 獲獎情況一覽表 ............................................................................. 49 摘要 I 玉米籽粒花色苷含量主效 QTLAC6 的精細(xì)定位及基因預(yù)測 研究生：王 旭 專業(yè)：作物遺傳育種 指導(dǎo)老師：蔡一林 教授 摘 要 隨著生活水平的提高以及飲食結(jié)構(gòu)的調(diào)整，人們對玉米的營養(yǎng)品質(zhì)要求越來越高。玉米籽粒中色素的成分和含量與玉米的營養(yǎng)品質(zhì)密切相關(guān)，不同顏色的玉米所含的色素成分不同，其功能性品質(zhì)作為重要的營養(yǎng)品質(zhì)之一，對增進(jìn)人體健康、防治疾病起著非常重要的作用，是發(fā)展天然保健食品的重要原材料，同時也廣泛應(yīng)用于化妝品、醫(yī)藥 等行業(yè)中。黑玉米富含花色苷，對人體具有多種生理生化功能。所以，黑玉米是研究花色苷的良好材料。認(rèn)識花色苷的遺傳規(guī)律，可以為玉米的功能性品質(zhì)在天然保健食品 、 化妝品、醫(yī)藥等領(lǐng)域中的應(yīng)用提供新的參考和借鑒，為玉米中花色苷相關(guān)基礎(chǔ)研究及玉米花色苷的分子標(biāo)記輔助育種提供參考。 黑玉米材料 SDM (super dark maize)是西南大學(xué)玉米研究所 自主選育 的一個特異玉米自交系，本研究使用 SDM 為共同父本，分別與黃玉米自交系 Mo17 和白玉米自交系木 6 雜交，構(gòu)建兩套相關(guān)分離群體，即 MoS 群體和 MuS 群體，對花色苷的遺傳規(guī) 律進(jìn)行研究。前人已將控制花色苷含量的主效 QTLAC6 定位在第 6 染色體的分子標(biāo)記 S8umc1014 之間。本研究將延續(xù)前人研究結(jié)果，利用 AC6 的近等基因系的分離群體 BF 4C3 進(jìn)一步對花色苷含量的主效QTLAC6 進(jìn)行定位，并利用該近等基因系上一世代的交換單株自交的后代群體對 AC6 進(jìn)行精細(xì)定位。分析定位的區(qū)間附近的基因組序列，對該段區(qū)域上的基因進(jìn)行注釋。主要研究結(jié)果如下： 群體精細(xì)遺傳圖譜的構(gòu)建 選取目標(biāo) QTL 區(qū)域附近已有的 SSR 多態(tài)性標(biāo)記以及開發(fā)新的多態(tài)性標(biāo)記，分 別 用 8 對和 7 對 SSR 多態(tài)性標(biāo)記 對 MuSBC4F3 群體和 MoSBC4F3 群體構(gòu)建精細(xì) 的 連鎖圖譜。在MuSBC4F3 群體的 精細(xì) 的 遺傳圖譜中，每個分子標(biāo)記之間的平均遺傳距離是 cM， 在MoSBC4F3 群體的 精細(xì) 的 遺傳圖譜中，每個分子標(biāo)記之間的平均遺傳距離是 cM。 西南大學(xué)碩士學(xué)位論文 II QTLAC6 的精細(xì)定位 利用 MuSBC4F3 和 MoSBC4F3 兩個近等基因系的分離群體將主效 QTLAC6 分別限定于分子標(biāo)記 S8umc1014 和 umc1014mmc0523 之間，兩個區(qū)間緊 密連 接一起，具有共同分子標(biāo)記 umc1014。利用上一世代的交換單株自交后代 BC4F3 對 AC6 進(jìn)行精細(xì)定位，在 MuS 群體和 MoS 群體中，分別篩選到 10 株交換單株。根據(jù)交換單株標(biāo)記的基因型和表型，將 AC6 界定在分子標(biāo)記 ssr9umc1014 和 S73umc1014 之間，且兩個區(qū)間重合。其中 ssr9 與 umc1014的物理距離為 255KB， S73 與 umc1014 的物理距離 為 114KB。 利用 、 b2ghome 等生物信息 學(xué) 網(wǎng)站對鎖定區(qū)域進(jìn)行分析，對 目標(biāo) 序列上的基因進(jìn)行注釋。該片段上共有 49 個轉(zhuǎn)座子， 29 個 還原 轉(zhuǎn) 座 子 ， 8 個 假設(shè) 蛋 白 ， 31 個 基 因。 通 過NCBI( 信息比對分析后發(fā)現(xiàn)在這定位區(qū)域內(nèi)， pl transcription factor 是一個血紅素加氧酶基因，參與玉米的色素調(diào)節(jié)。從其他研究者已經(jīng)分離的花色苷含量相關(guān)基因在染色體中的分布情況來看，在第 6 染色體上也正好是這個基因，所以， pl transcription factor 極有可能就是候選基因。但對該基因克隆 過程中發(fā)現(xiàn)，使用多種擴(kuò)增條件都無法將該基因克隆出來。分析其主要原因，可能是在突變材料 SDM 中， pl transcription factor 這一區(qū)域插入了一段未知長度的 DNA 序列，導(dǎo)致在 SDM 中無法克隆出該基因。 關(guān)鍵詞：玉米；花色苷含量； QTL 精細(xì)定位；基因預(yù)測。 Abstract III Fine mapping of the main effect QTLAC6 and gene prediction for Anthocyanin content in kernel of maize. Candidate: Wang Xu Major: Crop Geics and Breeding Supervisor: Professor Cai Yilin Abstract With the improvement of living standard and the adjustment of the diet, the nutritional quality of maize is increasingly high required. Composition and content of the pigment are closely related to the nutritional quality of maize kernel. The pigment position changes with the different colors of maize kernel. As one of the important nutritional quality, anthocyanin plays an important role in improving human health and preventing disease. Maize is not only an important raw material for the development of natural health food, but also is widely used in cosmetics, medicine and industries. Black maize is rich in anthocyanins, which possesses a variety of physiological and biochemical functions to the human body. Hence, black maize is a good material for anthocyanins study. The understanding of the geic rule of anthocyanins provide a new reference for the application of the functional quality in natural health food, cosmetics, medicine and other fields. Meanwhile, it gives some reference for maize anthocyanins relative basic research and molecular marker assisted breeding. Black maize SDM (super dark maize) is a specific maize inbred line created by maize research institute of southwest university. In this study, SDM was used as mon male parent to create two populations MoS from the cross Mo17 SDM and MuS from Mu6 SDM for studying the geic rule of anthocyanin. QTLAC6, the main effect QTL controlling of anthocyanin content, located in the mon interval of S8umc1014 on chromosome 6. To continue the previous research, BF 4C3 was used for further positioning QTLAC6. The progeny from one plant of the last generation of near isogenic line was used to fine mapping AC6. Then, the genome sequences of the nearby positioning area were analysised and genes within this region were annotated. The main results were as follows: 西南大學(xué)碩士學(xué)位論文 IV 1. The construction of fine geic map of BC4F3 population By selecting the existing SSR polymorphism markers and the new developed polymorphism markers nearby the target QTL regions, seven pairs and eight pairs of SSR polymorphism markers were used to constract fine linkage map for MuSBC4F3 population and MoSBC4F3 population, respectively. The average geic distance between each molecular marker was cM in the fine geic map of MuS BC4F3 population and cM. in MoSBC4F3 population. 2. Fine mapping of the main effect QTLAC6 of Anthocyanins content Two near isogenic line segregation populations of MuSBC4F3 and MoSBC4F3 were used to restrict the main effect QTLAC6 in the interval of S8umc1014 and umc1014mmc0523 respectively, which were close to each other, sharing the mon umc1014 molecular marker. Selfing generation BC4F3 of single plant of last generation was used to fine mapping AC6. In MuS population and MoS population, 10 single plants were screened respectively. According to the marker genotype and phenotype of the single plant, AC6 was defined in molecular marker ssr9umc1014 and S73umc1014, and the two regions overlapped. The physical distance between ssr9 and umc1014 was 255 KB, while the distance between S73 and umc1014 was 114 KB. 3. Gene annotation and prediction of the positioning region , b2ghome were used for analysis of locking region and the annotation of