甘蓝型油菜矮秆基因定位、克隆及功能分析

发布时间：2018-03-19 12:19

本文选题：甘蓝型油菜　切入点：矮秆突变体　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：株高是株型的重要组成部分,也是影响作物产量的重要基础性状。小麦和水稻半矮秆基因的应用是第一次“绿色革命”成功的关键。半矮秆基因的利用显著降低了水稻和小麦植株的高度,增强了植株的倒伏抗性、耐肥性,同时还提高了收获指数,从而显著地增加了水稻和小麦的产量。油菜是我国重要的油料作物之一,现有的油菜品种植株高大,易因倒伏而导致产量降低、品质下降。油菜矮秆突变体较少,对油菜株型调控的分子机理研究有限。因此,筛选矮秆突变体,克隆相关基因,阐明油菜株高的形成机制,对于油菜的矮化育种具有重要意义。本研究以甘蓝型油菜(Brassica napus,AACC,2n=38)矮秆突变体ds-3为材料,对株高基因DS-3进行了遗传定位,并最终克隆了控制矮秆性状的矮秆基因ds-3,该基因编码赤霉素(GA)信号转导抑制因子DELLA蛋白,命名为Bna C07.rga-ds。本研究还对Bna C07.rga-ds及其同源基因进行了功能分析。此外,本研究对油菜矮秆突变体ds-4的矮秆基因进行了精细定位。主要结果如下:1.矮秆基因DS-3的定位、克隆以及功能验证突变体ds-3苗期叶片暗绿,植株匍匐生长。成熟期,植株株高及其各构成因子:第一分枝高度,节间数目,节间长度以及主花序长度均显著降低。此外,ds-3下胚轴的伸长对外源GA3处理的敏感性减弱。F1杂种的株高介于ds-3与野生型“华双5号”之间;BC1和F2群体株高分离比分别符合1:1和1:2:1的预期值,表明矮秆性状受单个不完全显性基因控制。BSA分析将目标基因DS-3定位在C07染色体两个SSR标记(Bo GMS4033和Bo GMS4044)之间,两个标记之间的遗传距离为6 c M。DS-3基因一侧4个连锁的SSR标记对应的C07染色体区段与A06染色体上株高基因Bna A06.RGA区段存在很好的共线性。在C07染色体目标区间内,存在与Bna A06.RGA同源的Bna C07.RGA,因此后续研究中将Bna C07.RGA作为DS-3的候选基因。比较测序发现,突变体ds-3的Bna C07.rga-ds基因编码区第272位核苷酸发生了C?T突变,该突变导致DELLA蛋白N端保守结构域VHYNP的脯氨酸(P)被替换成亮氨酸(L)。位点特异性标记分析结果表明,该点突变与F2和BC1群体株高共分离。在拟南芥中分别表达Bna C07.RGA和Bna C07.rga-ds,Bna C07.RGA的转基因植株株高与野生型没有显著差异,而Bna C07.rga-ds能显著降低植株高度。酵母双杂交试验结果显示:GA能介导Bna C07.RGA与受体GID1a的结合,但不能介导Bna C07.rga-ds与GID1a的互作。2.甘蓝型油菜RGA的拷贝数、进化分析及功能鉴定以拟南芥At RGA的氨基酸序列,进行双向BLASTp比对,在白菜、甘蓝以及甘蓝型油菜基因组中分别鉴定到2个Bra RGA基因,2个Bol RGA基因以及4个Bna RGA基因。基于核苷酸序列的进化树以及氨基酸序列一致性的分析结果均发现,油菜的4个Bna RGA基因来源于白菜的2个Bra RGA基因以及甘蓝的2个Bol RGA基因,构成4个直系同源基因对;RT-PCR分析发现,ds-3的4个Bna RGAs基因在茎秆与叶片中均有表达;GA处理能增强Bna RGA基因表达,但其响应GA的模式不尽相同。酵母双杂交试验结果发现,GA能介导各Bna RGA蛋白与GID1a互作。此外,通过比较ds-1与ds-3两矮秆突变位点对株高的影响发现,DS-3(Bna C07.RGA)对茎秆伸长的调控力要弱于DS-1(Bna A06.RGA)。3.矮秆基因DS-4的精细定位矮秆突变体ds-4幼苗期叶片皱缩、向下卷曲,植株匍匐生长,成熟期植株矮小。此外,ds-4的种子萌发率较低,下胚轴变短明显,开花前期茎顶端表现出轻微的正向地性,但暗形态建成表现正常。将矮秆突变体ds-4和野生型ZY821杂交,比较发现,F1的株高远低于中亲值,更接近矮秆亲本ds-4;F2和BC1群体植株可以明显地划为矮秆类型和正常类型,而且矮秆类型和正常类型的分离比分别符合3:1和1:1。BSA分析将目标基因DS-4定位于油菜C05染色体介于In Del标记ID-147与ID-162之间25.6 c M的区间内。随后,基于双亲重测序的数据在目标区间开发了4个连锁的CAPS标记,利用这些标记从F2群体高秆表型的植株中筛选交换单株,进而将DS-4定位在SNP2CAPS-1和SNP2CAPS-4之间4.1 M的物理区间。随后,在目标区间内开发了多个SNP标记,利用SNPseq?分型技术筛选BC1F2分离群体中在SNP2CAPS-4和SNP2CAPS-39之间的交换单株。最终,将DS-4锁定在SNP32和SNP40之间,对应于甘蓝型油菜“Darmor-bzh”基因组C05染色体上约为475 kb的区间内。
[Abstract]:Plant height is an important part of the plant, is also the important foundation of crop yield. The application of semi dwarf gene of wheat and rice is the key to the first "green revolution" successful. By using the semi dwarf gene significantly reduced the rice and wheat plant height, enhanced plant fertilizer tolerance and lodging resistance. At the same time also increased the harvest index, which significantly increased the yield of rice and wheat. Rape is one of the most important oil crops in China, rapeseed plants existing tall, easily lead to lower production, quality decline due to lodging. Rape dwarf mutant of rape plant is less, the molecular mechanism of regulation is limited. Therefore, screening of dwarf mutant, gene cloning, clarify the formation mechanism of plant height, has important significance for breeding dwarf rapeseed. In this study, Brassica napus (Brassica napus, AACC, 2n=38) dwarf Mutant ds-3 as material, the plant height gene DS-3 genetic location, and finally cloned the dwarf gene ds-3 controlling dwarf phenotype, the gene encoding gibberellin (GA) inhibitory factor DELLA protein signal transduction, named Bna C07.rga-ds., the study of Bna C07.rga-ds and its homologous genes were functional analysis. In addition, the study of dwarf the gene of rape dwarf mutant DS-4 in the fine mapping. The main results are as follows: 1. dwarfing genes DS-3, cloning and functional verification of ds-3 mutant seedling leaves dark green, plants prostrate growth. Maturity, plant height and its various components: the first branch height, internode number and internode length and the length of main inflorescence were significantly decreased. In addition, between ds-3 on Hypocotyl Elongation sensitivity to exogenous GA3 treatment reduced plant height of.F1 hybrids between ds-3 and wild type "Huashuang No. 5"; the BC1 and F2 groups The height of separation with 1:1 and 1:2:1 respectively than the expected value, showed that the dwarf trait controlled by a single incompletely dominant gene control.BSA analysis target gene DS-3 located on chromosome C07 two SSR markers (Bo GMS4033 and Bo GMS4044), the genetic distance between the two markers was 6 C M.DS-3 gene on the side of 4 SSR markers the corresponding C07 chromosome region on the A06 chromosome of plant height gene Bna A06.RGA section there exist good linear. C07 chromosome in the target range, there are Bna A06.RGA and homologous Bna C07.RGA, Bna C07.RGA, so further research as a candidate gene of DS-3. Comparison was found, the C mutant ds-3 Bna C07.rga-ds the 272nd gene encoding region nucleotide? T mutation, the mutation causes a proline DELLA N terminal protein conserved domain VHYNP (P) is replaced by leucine (L). Site specific marker analysis results show that the Point mutations with F2 and BC1 groups were isolated respectively. The height of the expression of Bna C07.RGA and Bna C07.rga-ds in Arabidopsis thaliana, there was no significant difference between the Bna C07.RGA transgenic plant height and wild type, Bna and C07.rga-ds can significantly reduce the plant height. The yeast two hybrid experiment results show that GA can bind to C07.RGA receptor mediated Bna and GID1a C07.rga-ds, but not Bna mediated by interaction with GID1a copy number.2. RGA of Brassica napus, phylogenetic analysis and functional characterization of the amino acid sequence of the Arabidopsis At RGA, two-way BLASTp comparison in Chinese cabbage, cabbage and Brassica napus genome were identified to 2 Bra 2 Bol RGA gene, RGA gene and 4 Bna of RGA gene were found. Phylogenetic tree of nucleotide sequence and amino acid sequence analysis of the consistency of the results based on the 2 rape of the 4 Bna RGA gene from Chinese cabbage RGA gene and 2 Bra of cabbage Bol RGA gene, 4 orthologous gene pairs; RT-PCR analysis showed that 4 Bna RGAs of ds-3 gene in the stem and leaf tissues; GA can enhance the expression of Bna RGA gene, but the response of GA model is not the same. The yeast two hybrid results showed that GA could mediate the Bna RGA protein interaction with GID1a. In addition, site effect on plant height was found by comparing the DS-1 and ds-3 two DS-3 dwarf mutation, (Bna C07.RGA) on the regulation of stem elongation is weaker than DS-1 (Bna A06.RGA) fine mapping of dwarf mutant DS-4 in seedling stage.3. dwarf gene DS-4 in the leaves of shrinkage, downward curling, creeping plants the growth and maturity of dwarf DS-4. In addition, the seed germination rate was low, hypocotyl shortening significantly, early stem apex showed positive to slight flowering, but skotomorphogenesis showed normal. The dwarf mutant DS-4 and wild type ZY821 hybrid, comparison, F 1 lines below the high parent value, closer to the dwarf parent DS-4; F2 and BC1 populations were significantly classified as dwarf type and normal type, and dwarf type and normal type separation ratio are in line with the analysis of 3:1 and 1:1.BSA target gene DS-4 is located on chromosome C05 between In rape Del markers ID-147 and ID-162 interval 25.6 C M. Then, the parents re sequencing data developed 4 CAPS markers linked to the target range based on the screening exchange plant from high plants in F2 population culm phenotype using these markers, and the location of DS-4 in between SNP2CAPS-1 and SNP2CAPS-4 4.1 M physical interval. Then, a number of SNP mark, developed in the target range by SNPseq? Exchange plant between SNP2CAPS-4 and SNP2CAPS-39 genotyping screening BC1F2 populations. Finally, DS-4 will be locked in between SNP32 and SNP40, corresponding to Brassica oil The genomic C05 chromosome of the vegetable "Darmor-bzh" is within the interval of about 475 KB.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.4

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