红花草莓MYB转录因子基因分离及花色基因表达分析

发布时间：2017-12-31 14:20

本文关键词：红花草莓MYB转录因子基因分离及花色基因表达分析　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 红花草莓 MYB转录因子 花色 表达分析

【摘要】：草莓属(Fragaria)植物均开白花,红花草莓为属间杂种(Fragaria×Potentilla),是利用开白花的8x栽培草莓(F×ananassa,2n=8x=56)与开红花的6x沼委陵菜(Potentilla palustris,2n=6x=42)进行远缘杂交,并与草莓栽培品种不断回交而获得的。红花草莓是草莓家族新成员,具有很高的观赏性和经济价值,有关其花色形成机理鲜有报道。MYB转录因子家族是植物中所含基因数最多的转录因子家族之一,通过对目的基因的转录水平进行调控,广泛参与了植物的生长和代谢过程。本研究以红花草莓品种'粉佳人'与'俏佳人'杂交后代为试材,检测了花瓣中花青素苷的种类和含量;同时,基于红花草莓红白花瓣转录组测序结果,克隆出2个MYB转录因子基因,对其进行生物信息学和表达模式分析,初步解析了 MYB转录因子在红花草莓花色代谢中的调控机理,主要结果如下:1.对不同色系及不同花蕾发育阶段的红花草莓花瓣进行花青素苷种类、含量,以及黄酮/黄酮醇含量进行分析。结果表明,在白色系和未着色的蕾期花瓣中没有检测到花青素苷的积累。在红、深粉、粉和浅粉色系花瓣中除了检测到大量的矢车菊素3-O-葡萄糖苷(Cy3G)外,还检测到天竺葵素3-O-葡萄糖苷(Pg3G)、飞燕草素3-0-(阿魏酰基)-葡萄糖苷(Dp3feG)等7种色素,而Cy3G的含量差异是造成不同色系、不同发育阶段间花色深浅变化的主要物质基础。2.根据不同花色红花草莓花瓣转录组测序结果,通过RPKM法、同源比对筛选出2个在红白花瓣中差异表达的MYB类unigene序列,设计特异引物,通过RT-PCR法分离并克隆2条MYB转录因子完整的CDS序列,分别命名为PfaMYB1和PfaMYB86。生物信息学分析结果表明,2条序列在N端都具有两个典型的MYB结构域,并且与已报道的部分MYB转录因子结构域有很高的相似性,推测PfaMYB1和PfaMYB86均为R2R3-MYB转录因子。3.实时定量PCR结果表明,PfaMYB1在不同深浅花色、不同花蕾发育阶段的花瓣中,以及果实、根、茎、叶等组织中均有表达,且在花与果实中特异表达,在花蕾发育过程中随着花瓣颜色加深表达量升高,在无花青素苷积累的组织中表达量极低;PfaMYB86在不同花色、不同组织以及不同花蕾发育阶段中的表达与花色并无显著相关。4.对红色系不同花蕾发育阶段中的花色合成途径的结构基因进行了定量分析,发现CHS、CIII、ANS和DFR基因在不同发育阶段花瓣中的表达量与花青青苷含量显著相关,可能是造成Cy3G在红花中大量合成并形成花色差异的关键基因;F3'5'H基因发育初期的表达量相对较高,但花瓣中并未大量积累飞燕草素苷,推测是ANS、DFR、UFGT1基因的底物特异性导致飞燕草素苷的合成量低或苷元发生降解。
[Abstract]:Strawberry (Fragaria) plants were white and red flowered strawberry as intergeneric hybrids (Fragaria * Potentilla), is the use of 8x white flowers cultivated strawberry (F x ananassa, 2n=8x=56) and saffron 6x (Potentilla palustris 2n=6x=42, marsh cinquefoil) of distant hybridization, and strawberry cultivars obtained by continuous backcross strawberry strawberry. Safflower is a new member of the family, with high ornamental and economic value, the color forming mechanism is rarely reported in the.MYB family of transcription factors is one of the genes contained in the plant number, through the transcription of target gene regulation, widely involved in growth and metabolism of plants in this study, safflower varieties of strawberry. The 'Pink Lady' and 'woman' hybrids as test materials, kinds and content of anthocyanin in the detection; at the same time, red flowered strawberry red white petals node based on transcriptome sequencing Fruit, cloned 2 MYB transcription factor gene, bioinformatics analysis and expression pattern of the preliminary analysis of the regulation mechanism of MYB transcription factor in the metabolism of red flowered strawberry variety, the main results are as follows: 1. the different colors and different developmental stages of the flower buds of red flowered strawberry flap anthocyanin content types and flavone / flavonol content were analyzed. The results showed that no detectable anthocyanin accumulation in the bud stage, petals white and unpigmented. Deep in the red powder, powder, and light pink petals in addition to detect large amounts of cyanidin 3-O- glucoside (Cy3G), also detected pelargonidin 3-O- glucoside (Pg3G), 3-0- (delphinidin feruloyl) - glucoside (Dp3feG) and other 7 kinds of pigment, and the differences in the content of Cy3G is caused by the different color, color depth change in different developmental stages of the main material basis of.2. according to different Flower petals of red flowered strawberry transcriptome sequencing results by RPKM method, and selected 2 homology in red and white petals of MYB differentially expressed UniGene sequences, specific primers were designed by RT-PCR, a method for the separation and cloning of 2 MYB transcription factor CDS complete sequence, named PfaMYB1 and PfaMYB86. bioinformatics analysis showed that 2 sequences with two typical MYB domain in N terminal, has a very high similarity to MYB transcription factor domain and has been reported, suggesting that PfaMYB1 and PfaMYB86 are R2R3-MYB transcription factor.3. real-time quantitative PCR results showed that the PfaMYB1 in different shades of color, different stages of bud development in the petals. As well as the fruit, roots, stems, leaves and other tissues were expressed, and the specific expression of flower and fruit, in the developing process of flower buds with petals color expression elevated in flowers green Sugan accumulation organization The expression is very low; PfaMYB86 in different colors, makes a quantitative analysis of gene expression, structure and no color significantly related to.4. in different tissues and different developmental stages in the buds of red flower bud development in different stage in the synthesis pathway found in CHS, CIII, ANS and DFR gene expression was significantly correlated in different developmental stages. The amount of flowers and green was the highest, may be caused by the key gene Cy3G in the synthesis of a large number of safflower and the formation of color difference; F3'5'H gene expression at the early stage of development is relatively high, but not the accumulation of large petals fly Larkspur glycoside, that is ANS, DFR, the substrate specificity of UFGT1 gene leads to production of Delphinium glycoside aglycones low or degraded.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S668.4

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