發(fā)布時(shí)間：2018-05-01 13:00

  本文選題：白菜型油菜 + 粒色��； 參考：《青海大學(xué)》2017年博士論文

【摘要】：白菜型油菜(Brassica rapa L,2n=20,AA)為十字花科蕓薹屬作物,屬于栽培油菜基本種。我國是白菜和白菜型油菜的起源中心,與甘藍(lán)型油菜相比,其起源和栽培歷史悠久,遺傳資源豐富,具有天然而穩(wěn)定的黃籽資源。目前,已有大量研究表明,與普通黑褐籽油菜相比,黃籽油菜具有油質(zhì)清澈、脂肪和蛋白質(zhì)含量高、皮殼率低、餅粕飼用價(jià)值高、且單寧等有毒物質(zhì)含量低的優(yōu)點(diǎn)。因此,黃籽作為優(yōu)良油菜品種的一種重要的指示性狀受到人們的重視,黃籽油菜品種的選育一直是重要的育種目標(biāo)。本研究通過全基因組重測序結(jié)合遺傳連鎖圖譜對白菜型黃籽油菜大黃的粒色基因進(jìn)行了精細(xì)定位,并對粒色候選基因進(jìn)行克隆及功能分析,初步鑒定了控制大黃粒色的目標(biāo)基因。此外,研究了黃褐籽種子形成過程中種皮色澤的動(dòng)態(tài)變化規(guī)律及類黃酮代謝中目標(biāo)基因及其他相關(guān)轉(zhuǎn)錄因子與結(jié)構(gòu)基因的差異表達(dá)。揭示了白菜型油菜種皮中色素在種子發(fā)育時(shí)期的積累規(guī)律,為候選基因與類黃酮途徑其他各基因的調(diào)控機(jī)理提供初步線索。主要研究結(jié)果如下:1.精細(xì)定位大黃油菜粒色基因,在前人對大黃油菜粒色基因定位的基礎(chǔ)上,通過新開發(fā)得到的8個(gè)特異性SSR分子標(biāo)記及實(shí)驗(yàn)室已開發(fā)7個(gè)特異性標(biāo)記,進(jìn)一步加密遺傳連鎖圖譜,并得到整合后的物理圖譜,將目標(biāo)基因鎖定在A9染色體上一段1.08Mb(A9:18.26Mb-19.34Mb)區(qū)間。通過全基因組重測序定位大黃油菜粒色基因,結(jié)果顯示三個(gè)候選區(qū)段位于A9染色體上(17.83 Mb-18.93 Mb;20.53 Mb-20.99 Mb;22.57 Mb-26.09 Mb)。其中,重測序定位所得候選區(qū)段之一(17.83 Mb-18.93 Mb)與遺傳連鎖圖譜定位結(jié)果存在大段重疊(overlap),并且該重疊區(qū)段內(nèi)包含5個(gè)與目標(biāo)基因共分離的連鎖標(biāo)記。結(jié)合分子標(biāo)記構(gòu)建遺傳連鎖圖譜的方法和全基因組重測序的定位方法將目標(biāo)基因所在區(qū)間縮短為678 Kb(A9:18.26 Mb-18.93 Mb)。2.大黃油菜粒色候選基因Brtt1的克隆及序列分析,在BRAD數(shù)據(jù)庫中檢索發(fā)現(xiàn)候選區(qū)間(A9:678Kb)共包含46個(gè)候選基因。通過與擬南芥全基因組序列的同源比對,并參考這46個(gè)候選基因同源基因的功能注釋,分析發(fā)現(xiàn)該區(qū)段僅包含一個(gè)與色素合成相關(guān)的候選基因BrTT1(Bra028067),其擬南芥同源基因?yàn)楣δ芤阎�的類黃酮途徑關(guān)鍵基因TT1(At1g34790)。且在擬南芥中TT1基因突變體表現(xiàn)為透明種皮的性狀,推測BrTT1基因?yàn)榇簏S油菜粒色基因的關(guān)鍵候選基因。利用白菜基因組序列,擴(kuò)增獲得BrTT1全長序列4586bp,其中包含啟動(dòng)子片段1796bp,終止子下游序列986bp,編碼區(qū)序列1804bp,該基因包含有2個(gè)外顯子和1個(gè)內(nèi)含子。與白菜基因組序列BrTT1參考序列不同的是,該序列在起始密碼子上游39bp處提前編碼,導(dǎo)致白菜型油菜褐籽中BrTT1序列的第一外顯子比參考序列多39堿基,但未引起移碼突變。等位基因序列分析,結(jié)果發(fā)現(xiàn)黃籽Brtt1與褐籽BrTT1的啟動(dòng)子序列完全一致,與褐籽序列相比,黃籽材料中Brtt1在外顯子區(qū)存在7處堿基替換,內(nèi)含子區(qū)存在13處SNP位點(diǎn),且存在2bp、9bp、114bp片段缺失。進(jìn)一步進(jìn)行氨基酸序列分析,發(fā)現(xiàn)大黃材料Brtt1編碼蛋白序列有4處氨基酸同義突變,有3處氨基酸有義突變,分別為N45H、S294Y、H299L。將BrTT1基因編碼氨基酸序列提交至ExPASy數(shù)據(jù)庫(http://www.expasy.org)進(jìn)行蛋白結(jié)構(gòu)預(yù)測,結(jié)果顯示BrTT1編碼WIP鋅指結(jié)構(gòu)轉(zhuǎn)錄因子蛋白,其分子式為C145C148H161H165,屬于C2H2類型鋅指蛋白。3.大黃油菜粒色候選基因BrTT1的遺傳轉(zhuǎn)化,利用花絮浸染農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法,將BrTT1基因全長轉(zhuǎn)入擬南芥tt1突變體CS82中,共得到20株T1代陽性苗,粒色完全恢復(fù)為野生型種皮顏色褐色,且恢復(fù)率為100%。將T1代陽性苗轉(zhuǎn)化株繼代至T3代苗,粒色性狀表現(xiàn)穩(wěn)定。BrTT1基因能夠完全恢復(fù)tt1突變體粒色性狀,使其表現(xiàn)為野生型粒色性狀。BrTT1基因與擬南芥同源基因TT1有相似的基因功能,參與類黃酮代謝途徑。分別擴(kuò)增黃褐籽BrTT1全長ORF序列,構(gòu)建超量表達(dá)載體p35S::TT1與p35S::tt1,采用農(nóng)桿菌介導(dǎo)的花絮浸染的方法分別轉(zhuǎn)化擬南芥tt1突變體CS82。載體p35S::TT1所得31株T1代陽性苗,且種子粒色完全恢復(fù)至野生型種子粒色,載體p35S::tt1所得39株T1代陽性苗,種子粒色與突變體CS82粒色相近,仍為亮黃色。構(gòu)建BrTT1亞細(xì)胞定位載體,通過農(nóng)桿菌介導(dǎo)的煙草葉片下表皮細(xì)胞和洋蔥內(nèi)皮層細(xì)胞轉(zhuǎn)化法,瞬時(shí)表達(dá)BrTT1:GFP融合蛋白,將BrTT1定位于細(xì)胞核中,這符合轉(zhuǎn)錄因子的特征。4.黃褐籽不同發(fā)育時(shí)期種皮中色素積累規(guī)律與候選基因BrTT1及類黃酮代謝相關(guān)基因的差異表達(dá),通過體視鏡觀察并比較黃褐籽材料種子形成過程中種皮色澤變化,種子發(fā)育中期(授粉后28天、35天)黃褐籽材料的粒色性狀差異較大,褐籽材料種皮色澤明顯變?yōu)楹稚?而黃籽材料在整個(gè)種子發(fā)育過程并未存在粒色性狀的較大變化,表現(xiàn)為透明種皮,呈現(xiàn)種胚的顏色。通過qRT-PCR分析可知,候選基因BrTT1的主要表達(dá)部位在種子形成時(shí)期,且種子形成前期和中期(授粉后7天、14天、21天、28天、35天)黃褐籽材料中BrTT1的表達(dá)量達(dá)到極顯著差異,褐籽表達(dá)量顯著高于黃籽。在授粉后21天褐籽材料中BrTT1的表達(dá)量達(dá)到最高峰值,但在種子形成后期(授粉后49天)BrTT1在黃褐籽材料中的表達(dá)量并未達(dá)到極顯著差異。對大黃油菜粒色性狀候選基因BrTT1及類黃酮代謝途徑其他相關(guān)的8個(gè)結(jié)構(gòu)基因和6個(gè)轉(zhuǎn)錄因子在黃褐籽種子形成不同時(shí)期材料中的表達(dá)量進(jìn)行熱圖分析和表達(dá)譜分析,Br TT3、BrTT18、Br BAN在黃褐籽材料中表達(dá)差異較大,且褐籽材料中檢測到這些基因的大量表達(dá),而在黃籽材料中幾乎不表達(dá)。結(jié)合已有對其他植物中TT1的研究結(jié)果,初步推斷白菜型油菜種皮中類黃酮代謝相關(guān)基因的差異表達(dá)可能是導(dǎo)致黃褐籽種皮粒色差異的根本原因。粒色性狀候選基因BrTT1調(diào)節(jié)因子在大黃油菜黃籽材料中的異常表達(dá),導(dǎo)致靶基因BAN及類黃酮代謝路徑其他結(jié)構(gòu)基因的異常表達(dá)或不表達(dá),阻斷了原花色素的積累,從而使種皮表現(xiàn)為透明種皮形成黃籽。
[Abstract]:Brassica rapa L (2n=20, AA) is a cruciferous Brassica plant and belongs to the basic seed of cultivated rape. China is the origin center of Chinese cabbage and Brassica napus. Compared with Brassica napus, it has a long history of origin and cultivation, rich in genetic resources, and with natural and stable yellow seed resources. Compared with black brown seed rape, yellow seeded rapeseed has the advantages of high oil quality, high fat and protein content, low shell rate, high feeding value and low content of tannin, so yellow seed is regarded as an important indicator character of fine rapeseed, and the breeding of yellow seed rape varieties has been an important breeding. In this study, the whole genome re sequencing and genetic linkage map were used to accurately locate the grain color gene of Rhubarb in Chinese cabbage type yellow seeded rapeseed, and to clone and function analysis of the candidate genes. The target genes controlling the color of rhubarb were preliminarily identified. In addition, the seed color of the seed was studied. The dynamic change rules and the differential expression of target genes and other related transcription factors and structural genes in the flavonoid metabolism reveal the accumulation of pigment in the seed development of Brassica napus, and provide preliminary clues for the regulation mechanism of the candidate genes and other genes in the flavonoid pathway. The main results are as follows: 1. essence On the basis of the location of the grain color gene of rapeseed, 8 specific SSR markers and 7 specific markers developed in the laboratory have been developed to further encrypt the genetic linkage map, and get the integrated physical map, and lock the target gene to a segment of 1.08M on the A9 chromosome. B (A9:18.26Mb-19.34Mb) interval. The results showed that three candidate segments were located on the A9 chromosome (17.83 Mb-18.93 Mb; 20.53 Mb-20.99 Mb; 22.57 Mb-26.09 Mb). Among them, the results of one of the candidate segments (17.83 Mb-18.93 Mb) and the genetic linkage map were large Duan Zhongdie (overlap), and the overlap area contains 5 linkage markers that are co separated with the target genes. The method of constructing genetic linkage map with molecular markers and the location method of whole genome resequencing shorten the region of the target gene to 678 Kb (A9:18.26 Mb-18.93 Mb).2. and the clone of the candidate gene of the candidate gene of the rhubarb rapeseed. In the sequence analysis, 46 candidate genes were included in the search candidate region (A9:678Kb) in the BRAD database. By homologous alignment with the whole genome sequence of Arabidopsis, and referring to the functional annotations of the 46 candidate genes, it was found that the segment contained only a candidate gene BrTT1 (Bra028067) related to chromatic synthesis. The southern mustard homologous gene is TT1 (At1g34790), the key gene of the flavonoid pathway known in the Arabidopsis. The TT1 gene mutant in Arabidopsis is characterized by the transparent testa. It is speculated that the BrTT1 base is the key candidate gene of the kernel color gene of the rhubarb rape. Using the genome sequence of the cabbage, the BrTT1 full length sequence 4586bp is amplified, including the promoter slices. Segment 1796bp, terminating the downstream sequence of 986bp and the coding region sequence 1804bp, which contains 2 exons and 1 introns. Unlike the BrTT1 reference sequence of the cabbage genome sequence, the sequence is coded early in the upstream of the initial codon, leading to the first exon of the BrTT1 sequence in the brown seed of Brassica napus, which is more than the 39 base of the reference sequence. However, the sequence analysis of the allele showed that the promoter sequence of the yellow seed Brtt1 and the brown seed BrTT1 was exactly the same. Compared with the brown seed, there were 7 base substitutions in the exons of the yellow seed, 13 SNP loci in the intron, and the absence of 2bp, 9bp and 114bp fragments. Further amino acid sequences were carried out. It was found that there were 4 amino acid synonymous mutations in the Brtt1 encoding protein sequence of rhubarb and 3 amino acid mutations, respectively, N45H, S294Y, and H299L., which submitted the BrTT1 gene encoding amino acid sequence to the ExPASy database (http://www.expasy.org) for protein structure prediction. The results showed that BrTT1 encoded WIP zinc finger structural transcription factor protein. The molecular formula is C145C148H161H165, which belongs to the genetic transformation of the candidate gene BrTT1 of the C2H2 type zinc finger protein.3. of rhubarb rapeseed, and uses the genetic transformation method mediated by Agrobacterium tumefaciens, and the BrTT1 gene is transferred into the TT1 mutant CS82 of Arabidopsis thaliana. A total of 20 T1 generation positive seedlings are obtained, and the grain color is completely restored to the brown color of the wild type seed coat. The recovery rate was 100%., and the T1 generation positive vaccine was replaced by the T3 generation. The grain color character showed that the stable.BrTT1 gene could completely restore the TT1 mutant grain color character, which showed that the.BrTT1 gene of the wild type grain color trait was similar to the Arabidopsis homologous gene TT1, and participated in the flavonoid metabolic pathway. The full length ORF sequence, the construction of the super expression vector p35S:: TT1 and p35S:: TT1, using the method of Agrobacterium tumefaciens mediated dipping to transform the Arabidopsis TT1 mutant CS82. vector p35S:: TT1 obtained 31 T1 generation positive seedlings, and the seed color is completely restored to the wild type seed color, the carrier p35S:: TT1 obtained 39 strains positive seedlings, seed color and process. The variant CS82 is similar in color and still bright yellow. Construction of BrTT1 subcellular location vector, through agrobacterium mediated tobacco leaf epidermis cells and onion endothelial cell transformation, instantaneously express BrTT1:GFP fusion protein and locate BrTT1 in the nucleus, which conforms to the characteristics of the transcription factor of.4. yellow brown seed pigments in different developmental stages. The differential expression of BrTT1 and flavonoid metabolism related genes of the candidate gene was observed and the seed color and lustre changes in the seed formation of Huang He seed were observed and compared with the stereoscopy. The grain color characters of the Huang He seed materials in the middle period of the seed development (28 days after pollination, 35 days after pollination) were different, and the color and lustre of the brown seed material changed to brown, and Huang Zicai was obviously brown. In the whole seed development process, there is no big change in the color character of the seed, showing the transparent seed skin and showing the color of the embryo. By qRT-PCR analysis, the main expression of the candidate gene BrTT1 is in the period of seed formation, and the BrTT1 table of the yellow brown seed material is in the early and middle stage of seed formation (7 days after pollination, 14 days, 21 days, 28 days, 35 days). The expression of BrTT1 in brown seed material reached the highest peak value at 21 days after pollination, but the expression of BrTT1 in yellow brown seed material was not significantly different at the late stage of seed formation (49 days after pollination). The candidate gene BrTT1 and flavonoids metabolism of rhubarb rapeseed The expression of 8 other related genes and 6 transcription factors in the material of different period of seed formation of yellow brown seed was analyzed by thermography and expression analysis. The expression of Br TT3, BrTT18, Br BAN in the yellow brown seed material was very different, and the brown seed material detected a large number of these genes, but not in the yellow seed material. Combining with the results of TT1 in other plants, it was preliminarily deduced that the differential expression of flavonoid metabolism related genes in the seed coat of Brassica napus may be the root cause of the difference in the skin color of the yellow brown seed. The abnormal expression of the candidate gene BrTT1 regulator in the yellow seed material of the rapeseed, leading to the target gene BAN, The abnormal expression or non expression of other structural genes in flavonoid metabolic pathway blocked the accumulation of proanthocyanidins, thus making the testa transparent seed coat forming yellow seeds.

【學(xué)位授予單位】：青海大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S565.4

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