白菜黄化突变体pylm遗传及突变基因定位

发布时间：2018-07-16 11:23

【摘要】：植物中存在着丰富的叶色变异,迄今已在拟南芥、水稻、玉米、番茄和甘蓝型油菜等许多植物中发现了多种类型的叶色突变体。黄化突变体是最常见的叶色突变体,其是研究叶绿素生物合成、叶绿体结构和发育、光合作用机制、基因功能及核质基因互作等等的理想材料。白菜(.Brassica rapa L.ssp.chinensis)属于十字花科芸薹属芸薹种白菜亚种,是一种重要的叶菜类蔬菜作物。白菜黄化突变体py/m是本课题组利用自日本武藏野种苗公司引进的白菜杂交种'华冠'为试材,通过游离小孢子培养获得的一份全株黄化的双单倍体DH系。本研究在对pylm进行生理生化特性鉴定的基础上,对突变性状的遗传特性进行了分析,利用BSR-Seq方法对黄化突变基因进行了定位。主要研究结果如下:1.突变体pylm形态特征、光合特性及叶绿体解剖特征突变体pylm自种子萌发植株通体黄化,整个生育期黄化表型稳定。植株长势弱小,幼苗下胚轴明显拉长,抽薹开花较早。pylm属于缺总叶绿素型突变体,其气孔导度、蒸腾速率、主要荧光参数以及净光合速率均显著低于对照品系'CK-51',说明光合色素含量降低影响到了植株的光合作用。pylm叶绿体基粒片层稀疏,结构不规整,垛叠程度低,无淀粉粒沉积。2.突变体pylm叶绿素缺乏原因对突变体pylm叶片中8种主要的叶绿素生物合成中间代谢产物含量进行了测定,发现叶片中ALA、PBG、Urogen Ⅲ、Coprogen Ⅲ和Proto Ⅸ的含量明显低于对照'CK-51',Mg-Proto IX、Pchlide和Chlide的含量显著高于对照。结合pylm中Chl a含量降低的情况,推测其叶绿素生物合成受阻发生在由Chlide向Chl a酯化的过程。利用qRT-PCR技术,对pylm和对照'CK-51'的34个叶绿素生物合成相关基因的表达模式进行了分析,发现pylm中绝大多数叶绿素生物合成相关基因下调表达,其中HEMA1,CHLI2,POR C,CHLP,cHLG和c4O表达下调显著,说明突变基因影响到了多数叶绿素生物合成相关基因的表达,尤其是叶绿素生物合成代谢通路下游基因的表达。3.突变体pylm下胚轴异常伸长的机制突变体pylm下胚轴的平均长度为4.41cm,明显超过对照'CK-51'的2.63cm。石蜡切片观察表明,pylm下胚轴细胞纵向异常伸长生长导致了下胚轴拉长。利用液相色谱-质谱联用法(LC-MS)财pylm和对照'CK-51'的下胚轴内源激素含量进行了测定,发现pylm中生长素(IAA)含量为444.50ng/g,高于对照'CK-51'的376.32ng/g;赤霉素(GA3)含量469.00 ng/g,远超对照'CK-51'的173.33 ng/g,说明IAA和GA3含量的增加是导致突变体pylm下胚轴异常伸长的重要原因。4.黄化突变基因py1和py2的定位遗传分析表明,突变体pylm的黄化性状是由2对隐性重叠基因py1和py2互作控制。用py/m与正常绿叶大白菜DH系'FT'杂交构建F2分离群体。从F2中选取野生型和突变体表型的植株各100株,构建两个极端RNA混池进行BSR-Seq分析,预测黄化突变基因相关染色体区域位置。利用F4家系1号株系中的1,520株隐性纯合黄化单株进行SSR分析,将黄化突变基因py1定位于A09染色体的Inde1zk125和SSRzk36标记之间,遗传距离分别为0.13cM和0.2 cM。经与大白菜基因组比对,目标区域约258.3kb,包含34个基因。结合BSR-Seq分析结果,预测BraA09004189为黄化突变基因py1的候选基因。BraA09004189编码血红素加氧酶1(Heme oxygenase 1),参与血红素分解代谢过程。经BraA09004189基因全长测序并与对照'CK-51'比较后发现,二者存在1个SNPs差异。对BraA09004189表达模式分析表明,其在对照'CK-51'中的表达水平显著高于pylm。以F4家系3号株系中的341株隐性纯合黄化单株作为定位群体,将黄化突变基因py2定位在A07染色体SSRzk116和SSRzk133标记之间,遗传距离分别为0.7 cM和1.9 cM。上述研究结果,为最终克隆黄化突变基因py1和py2、探明2对基因互作调控叶绿素生物合成的分子机制奠定了基础。
[Abstract]:A variety of leaf color mutants have been found in many plants, such as Arabidopsis, rice, corn, tomato and Brassica napus. The Yellow mutant is the most common leaf color mutant. It is the study of chlorophyll biosynthesis, the structure and development of leaf green, photosynthesis mechanism, gene function and the function of the plant. .Brassica rapa L.ssp.chinensis belongs to the subspecies of Brassica Brassica in the cruciferous family Brassica, and it is an important leaf vegetable crop. The Yellow mutant py/m of Chinese cabbage is the test material of the Chinese Cabbage Hybrid 'Hua Guan', which was introduced from the Japanese Musashi wild seed company. A double haploid DH lineage obtained from microspore culture. Based on the identification of the physiological and biochemical characteristics of pylm, the genetic characteristics of the mutant traits were analyzed. The mutation gene was identified by the BSR-Seq method. The main results were as follows: 1. the morphological characteristics, photosynthetic characteristics and leaves of the mutant pylm. The mutant pylm of the green body was yellowing from the seed germination. The Yellow phenotype of the plant was stable during the whole growth period. The plant growth was weak, the hypocotyl of the seedlings was obviously elongated, and the early bolting and flowering.Pylm belonged to the total chlorophyll mutant. The stomatal conductance, the transpiration rate, the main fluorescein parameters and the net photosynthetic rate were significantly lower than those of the control strain. CK-51'showed that the decrease of photosynthetic pigment content affected the photosynthesis of the plant, the.Pylm chloroplast lamellae were sparse, the structure was irregular, the stacking degree was low, and the chlorophyll deficiency of the.2. mutant pylm without starch grain deposition was determined by the content of the intermediate metabolites of 8 main chlorophyll biosynthesis in the mutant pylm leaves. The contents of ALA, PBG, Urogen III, Coprogen III and Proto IX were significantly lower than those of the control'CK-51', Mg-Proto IX, Pchlide and Chlide were significantly higher than those of the control. The expression pattern of 34 chlorophyll biosynthesis related genes in 1'was analyzed. Most of the chlorophyll biosynthesis related genes were downregulated in pylm, of which the expression of HEMA1, CHLI2, POR C, CHLP, cHLG and c4O downregulated significantly, indicating that the mutant genes affected most of the expression of chlorophyll biosynthesis related genes, especially leaf green. The average length of the Hypocotyl in the hypocotyl extension of the hypocotyl of the.3. mutant pylm was 4.41cm, which was obviously higher than the 2.63cm. paraffin section of the control'CK-51'. The observation showed that the lengthwise elongation of the hypocotyl cells in the hypocotyl cells of the hypocotyl of pylm led to the elongation of the hypocotyl. The liquid chromatography-mass spectrometry (GC-MS) was used in the pylm hypocotyl cells of the hypocotyl. The content of endogenous hormone in hypocotyl of pylm and control'CK-51'was measured by LC-MS, and the content of auxin (IAA) in pylm was 444.50ng/g, higher than that of the 376.32ng/g of the control'CK-51', and the content of gibberellin (GA3) was 469 ng/g, which was far beyond the control'CK-51'173.33 ng/g. The location genetic analysis of.4. yellow mutant gene py1 and py2 showed that the Yellow character of the mutant pylm was controlled by 2 recessive overlapping genes py1 and py2. F2 separation group was constructed with py/m and normal green leaf cabbage DH system'FT'hybridization. 100 plants of wild type and mutant body surface type were selected from F2, and two extremes were constructed. The RNA mixed pool was used for BSR-Seq analysis to predict the location of chromosome related chromosomes in the mutant gene. Using 1520 recessive homozygous yellows in F4 family 1 line, SSR analysis was used to locate the yellow mutation gene py1 between the Inde1zk125 and SSRzk36 markers on the A09 chromosome, and the genetic distance was 0.13cM and 0.2 cM., respectively, and the Chinese cabbage gene. The target area is about 258.3kb and contains 34 genes. Combined with the results of BSR-Seq analysis, it is predicted that BraA09004189 is the candidate gene for the mutation of the yellow mutation gene py1,.BraA09004189 encoding heme oxygenase 1 (Heme oxygenase 1), and participates in the heme catabolism process. After BraA09004189 based full length sequencing and compared with the control'CK-51', two persons are found. There were 1 SNPs differences. The analysis of BraA09004189 expression patterns showed that the expression level in the control'CK-51'was significantly higher than that of pylm. with the 341 recessive homozygous yellows in the F4 family 3 line as the location group, and the mutation gene py2 was located between the A07 chromosome SSRzk116 and the SSRzk133 markers, and the genetic distance was 0.7 cM and 1, respectively. .9 cM. these findings laid the foundation for the final cloning of the yellowing mutant genes py1 and py2, and exploring the molecular mechanism of 2 pairs of gene interactions regulating chlorophyll biosynthesis.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S634.3

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