發(fā)布時(shí)間：2017-12-31 14:20

  本文關(guān)鍵詞：紅花草莓MYB轉(zhuǎn)錄因子基因分離及花色基因表達(dá)分析　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 紅花草莓 MYB轉(zhuǎn)錄因子 花色 表達(dá)分析

【摘要】：草莓屬(Fragaria)植物均開白花,紅花草莓為屬間雜種(Fragaria×Potentilla),是利用開白花的8x栽培草莓(F×ananassa,2n=8x=56)與開紅花的6x沼委陵菜(Potentilla palustris,2n=6x=42)進(jìn)行遠(yuǎn)緣雜交,并與草莓栽培品種不斷回交而獲得的。紅花草莓是草莓家族新成員,具有很高的觀賞性和經(jīng)濟(jì)價(jià)值,有關(guān)其花色形成機(jī)理鮮有報(bào)道。MYB轉(zhuǎn)錄因子家族是植物中所含基因數(shù)最多的轉(zhuǎn)錄因子家族之一,通過對(duì)目的基因的轉(zhuǎn)錄水平進(jìn)行調(diào)控,廣泛參與了植物的生長(zhǎng)和代謝過程。本研究以紅花草莓品種'粉佳人'與'俏佳人'雜交后代為試材,檢測(cè)了花瓣中花青素苷的種類和含量;同時(shí),基于紅花草莓紅白花瓣轉(zhuǎn)錄組測(cè)序結(jié)果,克隆出2個(gè)MYB轉(zhuǎn)錄因子基因,對(duì)其進(jìn)行生物信息學(xué)和表達(dá)模式分析,初步解析了 MYB轉(zhuǎn)錄因子在紅花草莓花色代謝中的調(diào)控機(jī)理,主要結(jié)果如下:1.對(duì)不同色系及不同花蕾發(fā)育階段的紅花草莓花瓣進(jìn)行花青素苷種類、含量,以及黃酮/黃酮醇含量進(jìn)行分析。結(jié)果表明,在白色系和未著色的蕾期花瓣中沒有檢測(cè)到花青素苷的積累。在紅、深粉、粉和淺粉色系花瓣中除了檢測(cè)到大量的矢車菊素3-O-葡萄糖苷(Cy3G)外,還檢測(cè)到天竺葵素3-O-葡萄糖苷(Pg3G)、飛燕草素3-0-(阿魏�；�)-葡萄糖苷(Dp3feG)等7種色素,而Cy3G的含量差異是造成不同色系、不同發(fā)育階段間花色深淺變化的主要物質(zhì)基礎(chǔ)。2.根據(jù)不同花色紅花草莓花瓣轉(zhuǎn)錄組測(cè)序結(jié)果,通過RPKM法、同源比對(duì)篩選出2個(gè)在紅白花瓣中差異表達(dá)的MYB類unigene序列,設(shè)計(jì)特異引物,通過RT-PCR法分離并克隆2條MYB轉(zhuǎn)錄因子完整的CDS序列,分別命名為PfaMYB1和PfaMYB86。生物信息學(xué)分析結(jié)果表明,2條序列在N端都具有兩個(gè)典型的MYB結(jié)構(gòu)域,并且與已報(bào)道的部分MYB轉(zhuǎn)錄因子結(jié)構(gòu)域有很高的相似性,推測(cè)PfaMYB1和PfaMYB86均為R2R3-MYB轉(zhuǎn)錄因子。3.實(shí)時(shí)定量PCR結(jié)果表明,PfaMYB1在不同深淺花色、不同花蕾發(fā)育階段的花瓣中,以及果實(shí)、根、莖、葉等組織中均有表達(dá),且在花與果實(shí)中特異表達(dá),在花蕾發(fā)育過程中隨著花瓣顏色加深表達(dá)量升高,在無花青素苷積累的組織中表達(dá)量極低;PfaMYB86在不同花色、不同組織以及不同花蕾發(fā)育階段中的表達(dá)與花色并無顯著相關(guān)。4.對(duì)紅色系不同花蕾發(fā)育階段中的花色合成途徑的結(jié)構(gòu)基因進(jìn)行了定量分析,發(fā)現(xiàn)CHS、CIII、ANS和DFR基因在不同發(fā)育階段花瓣中的表達(dá)量與花青青苷含量顯著相關(guān),可能是造成Cy3G在紅花中大量合成并形成花色差異的關(guān)鍵基因;F3'5'H基因發(fā)育初期的表達(dá)量相對(duì)較高,但花瓣中并未大量積累飛燕草素苷,推測(cè)是ANS、DFR、UFGT1基因的底物特異性導(dǎo)致飛燕草素苷的合成量低或苷元發(fā)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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S668.4

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