芥菜紫叶基因Bj.Pur定位及候选基因分析

发布时间：2018-07-27 15:48

【摘要】：芥菜紫叶性状是由于叶片组织中花青苷的积累,紫叶性状可作为一种苗期形态学标记应用于杂交制种中,且其所含花青苷具有抗逆、抗氧化、抗增殖、抗突变、预防心脑血管疾病、抑制肿瘤细胞发生等多种功能。因此,成为蔬菜基因工程研究和新品种选育的热点。本研究在刘利艳(2015)构建的六世代遗传群体基础上,利用紫叶芥菜自交系(ZT-15-P)和绿叶芥菜自交系(ZT-15-G)重新构建分离群体,定位并克隆了1个控制芥菜紫叶性状的候选基因。主要研究结果如下:1.紫叶性状的遗传模式分析利用六世代群体,重新构建了15-F_2A和16-F_2B两个F_2定位群体,并回交构建了BC5代近等基因系(NIL)。遗传分析发现,F_2群体及BC各世代群体紫叶、绿叶性状分离结果经χ2检验分别符合3:1和1:1分离比,进一步证实芥菜紫叶性状受单显性基因控制,命名为Bj.Pur。2.连锁标记筛选与定位以白菜基因组为参考,在刘利艳(2015)对紫叶芥紫色基因初步定位的基础上,通过BSA法分别结合SSR、InDel及CAPS标记技术,利用15-F_2A分离群体中的457个绿色单株作为定位群体,开发并筛选了453对引物,未找到表现稳定性差异的连锁标记。3.BSA-RNA-seq测序与分析选取F_2群体中的紫色、绿色单株各30株,分别提取总RNA构建两个极端池进行转录组测序,并对转录组数据先后进行了2次分析:(1)芥菜基因组信息公布前,以白菜基因组(http://brassicadb.org/brad/)作为参考基因组进行分析,将控制紫叶性状的基因初略定位到A07染色体19.5-21M区间内,并找到1对与紫叶性状连锁的InDel标记ID001。(2)2016年9月芥菜基因组信息公布后,基于芥菜参考基因组(https://www.ncbi.nlm.nih.gov/nuccore/LFQT00000000)进行了比对效率统计、新基因鉴定、基因表达量及差异表达基因分析、差异表达基因功能注释和富集、BSR关联分析等一系列生物信息学分析,最终筛选出分别位于B2和B6染色体上的6个候选区域:B2:645778-822189,B2:14795886-28411252,B2:30951237-33131066,B6:17982970-18046596,B6:20817254-20923707,B6:22623041-22686844。4.候选基因鉴定、克隆及测序利用BLAST分析软件,对前期筛选到的与紫叶性状连锁的标记重新定位,将Bj.Pur定位在分子标记ID001(染色体位置B2:17.746M)右侧,遗传距离0.6cM。而后筛选出6个候选区域中与花青素合成代谢有关的基因,或光、温敏感型且表达量有显著差异的基因,结合分子标记定位结果,选取其中45个基因开发了63对标记,在亲本及F1间验证,结果发现由B02染色体上的Gglean060401基因(功能注释为MYB90,染色体位置:B2:17,903,639-17,904,826)开发的1对标记Bj-MYB90在亲本间具有片段长短多态性。对亲本进行Gglean060401基因的gDNA全长及CDS全长克隆并测序,gDNA全长扩增结果显示,紫叶芥亲本与参考基因组保持一致,而绿叶芥亲本相比于紫叶芥亲本,在第一个内含子区存在一段1268bp片段的插入,但两个亲本CDS序列无差异。推测该基因为控制芥菜紫叶性状的候选基因。5.确定Gglean060401(MYB90)为目的基因Bj.Pur将Bj-MYB90标记在15-F_2A、16-F_2B两个F_2群体共计2229个绿色单株中验证,结果发现无交换单株,说明该标记与目标性状共分离。随后,又进行了紫、绿叶芥亲本中Gglean060401基因的q-RT-PCR表达量分析,结果表明,Gglean060401基因在紫叶芥中表达量为绿叶芥的89.6倍。上述结论进一步证实了Gglean060401基因即为控制芥菜紫叶的目的基因Bj.Pur。综上所述,叶用芥菜紫叶性状是由于转录因子Gglean060401(MYB90)在第一个内含子区缺失了一段1248bp的片段所造成的,Gglean060401基因即为控制芥菜紫叶的目的基因。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S637

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