大黄油菜粒色性状候选基因的定位克隆及功能分析

发布时间：2018-05-01 13:00

本文选题：白菜型油菜 + 粒色　；参考：《青海大学》2017年博士论文

【摘要】：白菜型油菜(Brassica rapa L,2n=20,AA)为十字花科芸薹属作物,属于栽培油菜基本种。我国是白菜和白菜型油菜的起源中心,与甘蓝型油菜相比,其起源和栽培历史悠久,遗传资源丰富,具有天然而稳定的黄籽资源。目前,已有大量研究表明,与普通黑褐籽油菜相比,黄籽油菜具有油质清澈、脂肪和蛋白质含量高、皮壳率低、饼粕饲用价值高、且单宁等有毒物质含量低的优点。因此,黄籽作为优良油菜品种的一种重要的指示性状受到人们的重视,黄籽油菜品种的选育一直是重要的育种目标。本研究通过全基因组重测序结合遗传连锁图谱对白菜型黄籽油菜大黄的粒色基因进行了精细定位,并对粒色候选基因进行克隆及功能分析,初步鉴定了控制大黄粒色的目标基因。此外,研究了黄褐籽种子形成过程中种皮色泽的动态变化规律及类黄酮代谢中目标基因及其他相关转录因子与结构基因的差异表达。揭示了白菜型油菜种皮中色素在种子发育时期的积累规律,为候选基因与类黄酮途径其他各基因的调控机理提供初步线索。主要研究结果如下:1.精细定位大黄油菜粒色基因,在前人对大黄油菜粒色基因定位的基础上,通过新开发得到的8个特异性SSR分子标记及实验室已开发7个特异性标记,进一步加密遗传连锁图谱,并得到整合后的物理图谱,将目标基因锁定在A9染色体上一段1.08Mb(A9:18.26Mb-19.34Mb)区间。通过全基因组重测序定位大黄油菜粒色基因,结果显示三个候选区段位于A9染色体上(17.83 Mb-18.93 Mb;20.53 Mb-20.99 Mb;22.57 Mb-26.09 Mb)。其中,重测序定位所得候选区段之一(17.83 Mb-18.93 Mb)与遗传连锁图谱定位结果存在大段重叠(overlap),并且该重叠区段内包含5个与目标基因共分离的连锁标记。结合分子标记构建遗传连锁图谱的方法和全基因组重测序的定位方法将目标基因所在区间缩短为678 Kb(A9:18.26 Mb-18.93 Mb)。2.大黄油菜粒色候选基因Brtt1的克隆及序列分析,在BRAD数据库中检索发现候选区间(A9:678Kb)共包含46个候选基因。通过与拟南芥全基因组序列的同源比对,并参考这46个候选基因同源基因的功能注释,分析发现该区段仅包含一个与色素合成相关的候选基因BrTT1(Bra028067),其拟南芥同源基因为功能已知的类黄酮途径关键基因TT1(At1g34790)。且在拟南芥中TT1基因突变体表现为透明种皮的性状,推测BrTT1基因为大黄油菜粒色基因的关键候选基因。利用白菜基因组序列,扩增获得BrTT1全长序列4586bp,其中包含启动子片段1796bp,终止子下游序列986bp,编码区序列1804bp,该基因包含有2个外显子和1个内含子。与白菜基因组序列BrTT1参考序列不同的是,该序列在起始密码子上游39bp处提前编码,导致白菜型油菜褐籽中BrTT1序列的第一外显子比参考序列多39碱基,但未引起移码突变。等位基因序列分析,结果发现黄籽Brtt1与褐籽BrTT1的启动子序列完全一致,与褐籽序列相比,黄籽材料中Brtt1在外显子区存在7处碱基替换,内含子区存在13处SNP位点,且存在2bp、9bp、114bp片段缺失。进一步进行氨基酸序列分析,发现大黄材料Brtt1编码蛋白序列有4处氨基酸同义突变,有3处氨基酸有义突变,分别为N45H、S294Y、H299L。将BrTT1基因编码氨基酸序列提交至ExPASy数据库(http://www.expasy.org)进行蛋白结构预测,结果显示BrTT1编码WIP锌指结构转录因子蛋白,其分子式为C145C148H161H165,属于C2H2类型锌指蛋白。3.大黄油菜粒色候选基因BrTT1的遗传转化,利用花絮浸染农杆菌介导的遗传转化方法,将BrTT1基因全长转入拟南芥tt1突变体CS82中,共得到20株T1代阳性苗,粒色完全恢复为野生型种皮颜色褐色,且恢复率为100%。将T1代阳性苗转化株继代至T3代苗,粒色性状表现稳定。BrTT1基因能够完全恢复tt1突变体粒色性状,使其表现为野生型粒色性状。BrTT1基因与拟南芥同源基因TT1有相似的基因功能,参与类黄酮代谢途径。分别扩增黄褐籽BrTT1全长ORF序列,构建超量表达载体p35S::TT1与p35S::tt1,采用农杆菌介导的花絮浸染的方法分别转化拟南芥tt1突变体CS82。载体p35S::TT1所得31株T1代阳性苗,且种子粒色完全恢复至野生型种子粒色,载体p35S::tt1所得39株T1代阳性苗,种子粒色与突变体CS82粒色相近,仍为亮黄色。构建BrTT1亚细胞定位载体,通过农杆菌介导的烟草叶片下表皮细胞和洋葱内皮层细胞转化法,瞬时表达BrTT1:GFP融合蛋白,将BrTT1定位于细胞核中,这符合转录因子的特征。4.黄褐籽不同发育时期种皮中色素积累规律与候选基因BrTT1及类黄酮代谢相关基因的差异表达,通过体视镜观察并比较黄褐籽材料种子形成过程中种皮色泽变化,种子发育中期(授粉后28天、35天)黄褐籽材料的粒色性状差异较大,褐籽材料种皮色泽明显变为褐色,而黄籽材料在整个种子发育过程并未存在粒色性状的较大变化,表现为透明种皮,呈现种胚的颜色。通过qRT-PCR分析可知,候选基因BrTT1的主要表达部位在种子形成时期,且种子形成前期和中期(授粉后7天、14天、21天、28天、35天)黄褐籽材料中BrTT1的表达量达到极显著差异,褐籽表达量显著高于黄籽。在授粉后21天褐籽材料中BrTT1的表达量达到最高峰值,但在种子形成后期(授粉后49天)BrTT1在黄褐籽材料中的表达量并未达到极显著差异。对大黄油菜粒色性状候选基因BrTT1及类黄酮代谢途径其他相关的8个结构基因和6个转录因子在黄褐籽种子形成不同时期材料中的表达量进行热图分析和表达谱分析,Br TT3、BrTT18、Br BAN在黄褐籽材料中表达差异较大,且褐籽材料中检测到这些基因的大量表达,而在黄籽材料中几乎不表达。结合已有对其他植物中TT1的研究结果,初步推断白菜型油菜种皮中类黄酮代谢相关基因的差异表达可能是导致黄褐籽种皮粒色差异的根本原因。粒色性状候选基因BrTT1调节因子在大黄油菜黄籽材料中的异常表达,导致靶基因BAN及类黄酮代谢路径其他结构基因的异常表达或不表达,阻断了原花色素的积累,从而使种皮表现为透明种皮形成黄籽。
[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.

【学位授予单位】：青海大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.4

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