【摘要】：芥菜紫葉性狀是由于葉片組織中花青苷的積累,紫葉性狀可作為一種苗期形態(tài)學(xué)標(biāo)記應(yīng)用于雜交制種中,且其所含花青苷具有抗逆、抗氧化、抗增殖、抗突變、預(yù)防心腦血管疾病、抑制腫瘤細(xì)胞發(fā)生等多種功能。因此,成為蔬菜基因工程研究和新品種選育的熱點(diǎn)。本研究在劉利艷(2015)構(gòu)建的六世代遺傳群體基礎(chǔ)上,利用紫葉芥菜自交系(ZT-15-P)和綠葉芥菜自交系(ZT-15-G)重新構(gòu)建分離群體,定位并克隆了1個(gè)控制芥菜紫葉性狀的候選基因。主要研究結(jié)果如下:1.紫葉性狀的遺傳模式分析利用六世代群體,重新構(gòu)建了15-F_2A和16-F_2B兩個(gè)F_2定位群體,并回交構(gòu)建了BC5代近等基因系(NIL)。遺傳分析發(fā)現(xiàn),F_2群體及BC各世代群體紫葉、綠葉性狀分離結(jié)果經(jīng)χ2檢驗(yàn)分別符合3:1和1:1分離比,進(jìn)一步證實(shí)芥菜紫葉性狀受單顯性基因控制,命名為Bj.Pur。2.連鎖標(biāo)記篩選與定位以白菜基因組為參考,在劉利艷(2015)對(duì)紫葉芥紫色基因初步定位的基礎(chǔ)上,通過(guò)BSA法分別結(jié)合SSR、InDel及CAPS標(biāo)記技術(shù),利用15-F_2A分離群體中的457個(gè)綠色單株作為定位群體,開(kāi)發(fā)并篩選了453對(duì)引物,未找到表現(xiàn)穩(wěn)定性差異的連鎖標(biāo)記。3.BSA-RNA-seq測(cè)序與分析選取F_2群體中的紫色、綠色單株各30株,分別提取總RNA構(gòu)建兩個(gè)極端池進(jìn)行轉(zhuǎn)錄組測(cè)序,并對(duì)轉(zhuǎn)錄組數(shù)據(jù)先后進(jìn)行了2次分析:(1)芥菜基因組信息公布前,以白菜基因組(http://brassicadb.org/brad/)作為參考基因組進(jìn)行分析,將控制紫葉性狀的基因初略定位到A07染色體19.5-21M區(qū)間內(nèi),并找到1對(duì)與紫葉性狀連鎖的InDel標(biāo)記ID001。(2)2016年9月芥菜基因組信息公布后,基于芥菜參考基因組(https://www.ncbi.nlm.nih.gov/nuccore/LFQT00000000)進(jìn)行了比對(duì)效率統(tǒng)計(jì)、新基因鑒定、基因表達(dá)量及差異表達(dá)基因分析、差異表達(dá)基因功能注釋和富集、BSR關(guān)聯(lián)分析等一系列生物信息學(xué)分析,最終篩選出分別位于B2和B6染色體上的6個(gè)候選區(qū)域:B2:645778-822189,B2:14795886-28411252,B2:30951237-33131066,B6:17982970-18046596,B6:20817254-20923707,B6:22623041-22686844。4.候選基因鑒定、克隆及測(cè)序利用BLAST分析軟件,對(duì)前期篩選到的與紫葉性狀連鎖的標(biāo)記重新定位,將Bj.Pur定位在分子標(biāo)記ID001(染色體位置B2:17.746M)右側(cè),遺傳距離0.6cM。而后篩選出6個(gè)候選區(qū)域中與花青素合成代謝有關(guān)的基因,或光、溫敏感型且表達(dá)量有顯著差異的基因,結(jié)合分子標(biāo)記定位結(jié)果,選取其中45個(gè)基因開(kāi)發(fā)了63對(duì)標(biāo)記,在親本及F1間驗(yàn)證,結(jié)果發(fā)現(xiàn)由B02染色體上的Gglean060401基因(功能注釋為MYB90,染色體位置:B2:17,903,639-17,904,826)開(kāi)發(fā)的1對(duì)標(biāo)記Bj-MYB90在親本間具有片段長(zhǎng)短多態(tài)性。對(duì)親本進(jìn)行Gglean060401基因的gDNA全長(zhǎng)及CDS全長(zhǎng)克隆并測(cè)序,gDNA全長(zhǎng)擴(kuò)增結(jié)果顯示,紫葉芥親本與參考基因組保持一致,而綠葉芥親本相比于紫葉芥親本,在第一個(gè)內(nèi)含子區(qū)存在一段1268bp片段的插入,但兩個(gè)親本CDS序列無(wú)差異。推測(cè)該基因?yàn)榭刂平娌俗先~性狀的候選基因。5.確定Gglean060401(MYB90)為目的基因Bj.Pur將Bj-MYB90標(biāo)記在15-F_2A、16-F_2B兩個(gè)F_2群體共計(jì)2229個(gè)綠色單株中驗(yàn)證,結(jié)果發(fā)現(xiàn)無(wú)交換單株,說(shuō)明該標(biāo)記與目標(biāo)性狀共分離。隨后,又進(jìn)行了紫、綠葉芥親本中Gglean060401基因的q-RT-PCR表達(dá)量分析,結(jié)果表明,Gglean060401基因在紫葉芥中表達(dá)量為綠葉芥的89.6倍。上述結(jié)論進(jìn)一步證實(shí)了Gglean060401基因即為控制芥菜紫葉的目的基因Bj.Pur。綜上所述,葉用芥菜紫葉性狀是由于轉(zhuǎn)錄因子Gglean060401(MYB90)在第一個(gè)內(nèi)含子區(qū)缺失了一段1248bp的片段所造成的,Gglean060401基因即為控制芥菜紫葉的目的基因。
[Abstract]:The purple leaf character of mustard is due to the accumulation of anthocyanin in leaf tissue. The purple leaf character can be used as a seedling morphological marker in hybrid seed production, and the anthocyanins contain antiinverse, antioxidation, anti proliferation, anti mutation, prevention of cardiovascular and cerebrovascular diseases and the inhibition of tumor cell development. Therefore, it has become a vegetable gene engineering research. On the basis of the six generation genetic population constructed by Liu Liyan (2015), this study reconstructs and cloned 1 candidate genes controlling the purple leaf character of mustard leaf mustard inbred line (ZT-15-P) and green leaf mustard self line (ZT-15-G). The main results are as follows: the remains of the 1. purple leaf traits. The six generations of 15-F_2A and 16-F_2B were rebuilt using the six generation group, and the BC5 generation near isogenic line (NIL) was constructed in backcross. The genetic analysis found that the F_2 population and the BC generation group purple leaf, the green leaf character separation results were respectively conformed to the 3:1 and 1:1 separation ratio by the chi 2 test, and further confirmed that the purple leaf character of mustard was single. Dominant gene control, named Bj.Pur.2. linkage marker screening and location for Chinese cabbage genome as reference, on the basis of Liu Li Yan (2015) on purple leaf mustard purple gene preliminary location, by BSA method combined with SSR, InDel and CAPS labeling technology, using 457 green single strains isolated from 15-F_2A as the location group, developed and screened 4. 53 pairs of primers, which did not find the difference of the stability of the linkage marker.3.BSA-RNA-seq sequencing and analysis, selected the F_2 population purple, 30 green single plant each, respectively extracted the total RNA to construct two extreme pools to sequence the transcriptional group, and analyzed the transcriptional data of the transcriptional group 2 times: (1) before the publication of the genomic information of mustard, with the cabbage genome (htt P://brassicadb.org/brad/) as a reference genome, the genes that control the purple leaf traits were initially located in the 19.5-21M interval of the A07 chromosome, and 1 pairs of InDel markers linked to the purple leaf traits were found, ID001. (2) of the mustard genome information published in September 2016, based on the reference genome of the mustard (https://www.ncbi.nlm.nih.gov/nuccore/L) FQT00000000) carried out a series of bioinformatics analysis, such as comparison efficiency statistics, new gene identification, gene expression and differential expression gene analysis, differentially expressed gene function annotation and enrichment, BSR association analysis, and finally screened 6 candidate regions on B2 and B6 chromosomes, B2:645778-822189, B2:14795886-28411252, B2:3095, respectively. 1237-33131066, B6:17982970-18046596, B6:20817254-20923707, B6:22623041-22686844.4. candidate genes identification, cloning and sequencing use BLAST analysis software to relocate the early screened markers linked to the purple leaf traits, and locate Bj.Pur on the right side of the molecular marker ID001 (chromophore location B2:17.746M), genetic distance 0.6cM. and then sifting In 6 candidate regions, genes related to anthocyanin anabolism were selected, or light, temperature sensitive and significant differentially expressed genes, combined with molecular marker localization results, 45 of them were selected to develop 63 pairs of markers, which were verified between parents and F1. The results were found by the Gglean060401 gene of the B02 chromophore (functional annotation MYB90, staining). Body position: B2:17903639-17904826) 1 pairs of labeled Bj-MYB90 had fragment length polymorphism between parents. GDNA full length and CDS full-length clone of Gglean060401 gene were cloned and sequenced. The result of gDNA full length amplification showed that the parent of purple leaf mustard was consistent with the reference genome, and the leaf mustard parent was compared to the parent of purple leaf mustard. There was a segment of 1268bp fragment in the first intron, but there was no difference in the CDS sequence of the two parents. It was speculated that the base was determined by the candidate gene.5. to control the purple leaf character of the mustard leaf, and Gglean060401 (MYB90) was the target gene Bj.Pur to mark Bj-MYB90 in 15-F_2A and 16-F_2B two F_2 populations in 2229 green single plants. Then, the q-RT-PCR expression of Gglean060401 gene in purple and green leaf mustard parent was then analyzed. The results showed that the expression of Gglean060401 gene in mustard was 89.6 times as high as that of green leaf mustard. The conclusion further confirmed that the Gglean060401 gene was the base for controlling the purple leaf of mustard. As described in Bj.Pur., the leaf mustard purple leaf character is caused by a fragment of the transcription factor Gglean060401 (MYB90) missing a segment of 1248bp in the first intron, and the Gglean060401 gene is the target gene for controlling the purple leaf of mustard.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S637

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