大豆叶形突变体的遗传和功能分析

发布时间：2018-04-28 14:50

本文选题：大豆 + 分子标记　；参考：《山东师范大学》2016年博士论文

【摘要】：大豆[Glycine max L.Merr.]属于豆科、蝶形花亚科、大豆属,是世界范围内重要的经济作物,为人类提供了大量的植物蛋白以及油料。大豆是古四倍体,基因组大小为1.1 Gb,编码蛋白质的基因超过46430个,其中大约有75%的基因是以同源基因的形式出现的。由于大豆基因组复杂,遗传转化较为困难,一直以来对大豆基因功能的研究进展较为缓慢。随着大豆基因组序列的公布,大豆遗传学研究迈入了新的时代。本文通过筛选菏豆12突变体库,获得叶皱缩表型的突变体。通过重测序技术获得菏豆12基因组序列信息,并与参考基因组Williams 82进行序列比对得到数量众多的插入缺失片段的物理位置信息,并以此开发新的插入/缺失(INsertion/DELetion,INDEL)分子标记。与此同时构建突变体与Williams82的杂交群体,并通过正向遗传学的手段,对造突变的基因进行图位克隆,利用表型相同的突变体株系之间杂交进行等位鉴定,以及突变体转基因恢复表型等手段确认了该突变体的突变基因。进一步研究发现,该基因参与了大豆叶表皮角质层的发育过程,并进一步影响了突变体应对干旱以及病原菌胁迫。所以该基因对未来培育抗旱以及抗病菌的大豆新品种具有重要的意义。具体实验结果如下:1.大豆品种之间分子标记的筛选1)大豆扩增片段长度多态性(AFLP)的设计以及多态性的筛选对菏豆12,Williams 82,吉林35进行AFLP分析,经过统计这三个大豆品种利用27对不同的选择性扩增引物组合分别获得625条,619条,622条条带,三个品种两两之间共产生了410个差异片段,其中菏豆12与吉林35之间有138个差异性位点,菏豆12与Williams 82之间有145个差异性位点,吉林35与Williams 82之间有127个差异位点。2)大豆SSR分子标记的设计与筛选从BARCSOYSSR Potential SSRs中选取了98个潜在的具有SSR多态性的位点,对菏豆12,Williams 82,吉林35进行了SSR多态性分析。结果表明:菏豆12与Williams 82之间多态性位点有41个,占总数的42%;菏豆12与吉林35之间多态性位点26个,占总数的27%;吉林35与williams82之间多态性位点16个,占总数的16%。菏豆12与williams82之间ssr分子标记设计成功率较高,反映出两者之间的遗传多样性更高,使得两者成为较合适的构建群体组合。3)大豆indel分子标记的设计以及筛选本实验筛选了在染色体上均匀分布的248个indel位点以获得足够数量的锚定分子标记。经pcr验证确定共有169个分子标记具有多态性,预测成功率为69%。169个indel分子标记分布于所有二十条染色体中,其中分布最多的是gm01染色体,共分布有24个,最少的也有4个,平均每条染色体上有8个。indel分子标记的分布基本均匀,很少成簇出现,基本能够满足图位克隆粗定位的要求。运用这些分子标记对其他12个大豆品种进行多态性鉴定发现:这些分子标记在中国和美国品种之间有着良好的多态性。进一步分析实验结果发现:具有多态性的分子标记中有77%pcr产物是单产物,远远高于假阳性indel的41%,这可能表明位于具有同源序列的indel位点可信度较低。生物信息学分析得到近五万个indel位点当中可能会有相当一部分是不真实的,造成这个问题的原因可能是重测序片段错误地比对上了参考基因组相应位置的同源序列,造成了一小段没有比对上的片段被分析成为indel位点。2.大豆皱叶突变体的鉴定在菏豆12伽马射线诱变突变体库中筛选出一个株系的皱叶突变体。皱叶突变体的第一三出复叶叶尖在种植18天之后开始坏死,导致小叶远轴端无法伸长,叶细胞在叶片发育过程中不断堆积,形成皱叶的表型。在纯化突变体遗传背景过程中发现突变体与菏豆12野生型杂交f1代为野生型表型,f2代群体中突变体与野生型的比例为1:3,符合孟德尔遗传定律,证明该突变位点为隐性单位点突变。3.大豆皱叶突变体的图位克隆首先构建了皱叶突变体与williams82的杂交群体。在f2代中选取突变体表型植株做图位克隆。通过粗定位,将突变位点定位于7号染色体1.371mb和2.417mb之间,但再继续向内设计分子标记的时候发现一段区间内扩增不出条带,故怀疑可能存在片段缺失。在缺失边界附近设计inversepcr引物,最终确定缺失范围是7号染色体2118557到2371744bp。内共包含有27个基因涉及脂肪酸代谢,甘油代谢,生长素极性运输等相关功能。4.突变基因的确定随后又在williams82伽马射线诱变,ems诱变突变体库中筛选到表型一致的突变体株系4个,编号分别为:msn7442,sc5591,sc5962,sc7321。菏豆12与msn7442杂交后发现f1代为突变体表型,证明两个株系的突变位点是等位关系。提取msn7442株系突变体的基因组dna,将菏豆12突变体缺失的27个基因进行pcr扩增并测序,发现在glyma.07g028600(soybasev1.1版本的该基因编号为glyma07g03230)基因外显子出存在g到t的碱基颠换,导致编码的氨基酸由天冬氨酸变为酪氨酸。对其余三个株系的glyma.07g028600基因pcr扩增并测序之后发现:sc7321在外显子处缺失tcttttatcc十个碱基并加入a;sc5591在第三个外显子前2个碱基的内含子处存在一个由a到t的碱基颠换,推测可能导致了mrna的错误剪切;sc5962在外显子处有一个g到a的碱基替换,导致编码的氨基酸由甘氨酸变为精氨酸。综上测序的结果表明:这四个株系的突变体在glyma.07g028600基因上存在非同义突变,并且菏豆12皱叶突变与msn7442为等位突变体。构建glyma.07g028600过表载体,并遗传转化msn7442,突变体表型恢复为野生型。以上实验证明glyma.07g028600基因的突变造成了皱叶的表型。5.glyma.07g028600基因的表达模式glyma.07g028600基因编码甘油三磷酸激酶,其在拟南芥中的直系同源基因是nho1/gli1。拟南芥nho1突变体的非寄主性抗性降低导致突变体对病原菌的侵染不耐受。大豆glyma.07g028600基因在全身各组织器官中均有表达,其中果荚中的表达丰度最高,叶片次之。构建glyma.07g028600融合egfp过表载体,转化拟南芥原生质体,结果表明融合蛋白主要定位于细胞膜上。茎尖原位杂交的结果表明该基因在大豆茎尖分生组织,叶原基等处均有表达。6.glyma.07g028600基因的功能分析菏豆12背景的皱叶突变体较其野生型,williams82背景下的4个株系的皱叶突变体较其野生型的离体叶片失水速率均快一倍以上。由此推测突变体叶表皮的角质层可能存在问题。扫描电镜结果显示:突变体叶表面的蜡质晶体堆积不正常。突变体及野生型叶片横切切片的透射电镜结果表明:突变体的表皮层变薄;真角质层亲锇性降低,表明突变体角质的密度变低。对突变体及野生型表层蜡质进行的质谱分析表明:突变体和野生型的蜡质总量没有显著区别,但不同成分含量差别很大。突变体和野生型接种大豆细菌斑点病菌4天后,突变体叶子内的细菌数量是野生型的6倍多,证明突变体应对病原菌侵染的能力变低。7.角质层发育相关基因的表达丰度受Glyma.07G028600基因影响结合扫描电镜,透射电镜,以及蜡质成分质谱分析的结果,对涉及植物蜡质,角质合成,转运,组装,调控的20多个基因在野生型和突变体叶片中的表达丰度进行了分析,结果表明:转运相关基因表达丰度差异不显著。角质合成酶基因(GmLTL1)等合成相关基因在野生型和突变体中表达丰度差异很大,一些转录因子表达丰度差异也很大。推测Glyma.07G028600基因对植物角质层的发育起着重要的作用。本论文的主要创新点:1.开发了一定数量的INDEL分子标记,这些标记在其他大豆品种之间也具有较好的多态性。生物信息学比对得到的近五万个INDEL位点能够为大豆遗传以及分子辅助育种提供丰富的信息。这些数据已经上传至Soybase,并填补了大豆公共数据库中没有INDEL分子标记的空白。2.通过图位克隆,定位到一个参与大豆角质层发育的基因Glyma.07G028600。该基因在拟南芥中的同源基因的研究中发现参与了植物的非寄主性抗性,但没有该基因参与角质层发育的报道。3.进一步揭示了大豆角质层发育的分子调控机制,为下一步培育抗旱,抗病虫的大豆新品种打下基础。
[Abstract]:Soybean [Glycine Max L.Merr.] belongs to leguminous, spoenoy and soybean, which is an important economic crop in the world. It provides a large amount of plant protein and oil for mankind. The soybean is an ancient tetraploid, the genome size is 1.1 Gb, and the gene encoding protein is more than 46430, of which about 75% of the genes are in the form of homologous genes. Because of the complexity of the soybean genome and the difficulty of genetic transformation, the research of soybean gene function has been progressing slowly. With the publication of the soybean genome sequence, the research of soybean genetics has entered a new era. In this paper, the mutant library of the 12 mutants of hedou was screened and the mutant of the leaf shrinkage phenotype was obtained. The genomic sequence information of hedou 12 was obtained, and the sequence alignment of the reference genome Williams 82 was compared to obtain the physical location information of a large number of inserted deletion fragments, and to develop a new insertion / deletion (INsertion/DELetion, INDEL) molecular marker. At the same time, the hybrid population of the mutant and Williams82 was constructed and passed through forward inheritance. By means of the study, the mutant gene was cloned, the mutant gene was identified by the allele identification of the mutant lines with the same phenotype, and the mutant transgenic recovery phenotype. The further study found that the gene was involved in the development of the cuticle of the soybean leaf and further shadow The mutants respond to drought and pathogen stress. Therefore, the gene is of great significance to the future cultivation of drought resistant and anti pathogenic soybean varieties. The specific results are as follows: 1. the screening of molecular markers between soybean varieties 1) the design of the amplified fragment length polymorphism of Soybean (AFLP) and the selection of polymorphism in hedou 12, William S 82, Jilin 35 performed AFLP analysis. After statistics, the three soybean varieties obtained 625, 619, 622 bands using 27 different selective amplification primers, and 410 differences were produced between three varieties and 22, of which there were 138 difference sites between hedou and Jilin 35. Difference loci, Jilin 35 and Williams 82 have 127 difference sites.2) the design and screening of soybean SSR molecular markers, 98 potential SSR polymorphic loci were selected from BARCSOYSSR Potential SSRs, and SSR polymorphism analysis was carried out on hedou 12, Williams 82, and 35. The results showed that the polymorphism between hedou 12 and Williams 82 was polymorphic. There were 41 loci, accounting for 42% of the total, 26 polymorphic loci between hedou 12 and Jilin 35, 27% of the total, and 16 polymorphic loci between Jilin 35 and williams82, which accounted for a higher success rate of SSR molecular markers between the total and williams82, reflecting the higher genetic diversity between the two and making the two more appropriate. Construction of group combination.3) the design of soybean indel molecular markers and screening the 248 indel loci evenly distributed on the chromosome to obtain a sufficient number of anchoring molecular markers. A total of 169 molecular markers were identified by PCR, and the prediction success rate was 69%.169 indel molecular markers distributed in all twenty. Among the chromosomes, the most distributed is the gm01 chromosome, with a total distribution of 24, the least and 4. The average distribution of 8.Indel markers on each chromosome is basically uniform and rarely appears in clusters. It can basically meet the requirements of the rough location of the clones. These molecular markers are used for the polymorphism of the other 12 soybean varieties. It is found that these molecular markers have good polymorphism between Chinese and American varieties. Further analysis of the experimental results found that 77%pcr products with polymorphic molecular markers are single products, far higher than 41% of false positive indel, which may indicate that the indel loci with homologous sequence are lower in reliability. A considerable portion of the nearly fifty thousand indel loci may be untrue, and the cause of this problem may be that the sequenced fragment is wrongly compared to the homologous sequence on the corresponding position of the reference genome, causing a small segment to be identified as the identification of the indel site.2. soybean wrinkle mutant. A leaf mutant of a plant line was screened in the 12 gamma ray mutants Library of hedou. The first three leaves of the mutant leaf mutant began to die after 18 days of planting, causing the apex of the lobule far axis to grow, the leaf cells accumulated during the leaf development and formed the phenotypes of the leaves, and the genetic background process of the mutant was purified. The mutant and hedou 12 wild type hybrid F1 generation is a wild type, and the proportion of the mutant and the wild type in the F2 generation group is 1:3, which conforms to the Mendel's law of inheritance. It is proved that the mutation site is a recessive unit point mutation of the.3. soybean leaf mutant and first constructs a hybrid population of the wrinkled leaf mutant and williams82. In the F2 generation. The mutant phenotypic plants were cloned. The mutant loci were located between 1.371mb and 2.417mb on chromosome 7 by rough location, but the deletion bands were found in a section of the interval when the molecular markers were continuously designed. Therefore, the deletion of fragments might exist. The design of inversepcr primers near the missing boundary was finally determined. The deletion range is 2118557 to 2371744bp. of chromosome 7, including 27 genes involved in fatty acid metabolism, glycerol metabolism, auxin polar transport and other related.4. mutation genes, followed by williams82 gamma ray mutagenesis, and EMS mutants library screening 4 phenotypic mutant lines, respectively, msn74 42, sc5591, sc5962, and sc7321. hedou 12 were hybridized with msn7442 and found that the F1 generation was the mutant phenotype. It was proved that the mutation site of the two strains was allele. The genomic DNA of the mutant msn7442 strain was extracted and 27 genes missing from the 12 mutant of hedou were amplified and sequenced by PCR, and now the glyma.07g028600 (soybasev1.1 version number) was issued. The exons of glyma07g03230) exon exon from G to t, causing the encoded amino acid to change from aspartic acid to tyrosine. After amplification and sequencing of the glyma.07g028600 gene PCR of the remaining three lines, it was found that sc7321 was missing the ten bases of tcttttatcc at exon and added to a; sc5591 was within the 2 bases of the third exons. There is a base change from a to t in the subregion, which may lead to the wrong shear of mRNA; sc5962 has a base substitution of G to a at exons, causing the encoded amino acid to be transformed from glycine to arginine. The results of the sequencing show that the mutants of these four strains have a non synonymous mutation on the glyma.07g028600 gene, and The mutation of 12 leaf of hedou and msn7442 was a allelic mutant. The glyma.07g028600 overtable vector was constructed and msn7442 was transformed into the wild type. The mutation of the mutant body surface was in the wild type. The above experiment proved that the mutation of the glyma.07g028600 gene resulted in the expression pattern of the.5.glyma.07g028600 gene of the phenotypic.5.glyma.07g028600 gene of the wrinkle, which encodes the glycerol three phosphate kinase. The direct homologous gene in Arabidopsis thaliana is the non host resistance of nho1/gli1. Arabidopsis nho1 mutant, which leads to the infection intolerance of the mutant to the pathogen. The soybean glyma.07g028600 gene is expressed in all tissues and organs of the whole body, among which the expression in the fruit pods is the highest and the leaves are the second. The glyma.07g028600 fusion EGFP has been constructed. The results showed that the fusion protein was mainly located on the cell membrane. The results of the stem tip in situ hybridization showed that the gene expressed.6.glyma.07g028600 gene in the shoot apex meristem, leaf primordium and other parts of the leaf primordium. The leaf mutants of the 12 background were compared with the wild type and 4 in the background of williams82. The leaf epidermis of the mutant leaf mutant was more than twice as fast as that of the wild type. Therefore, it is suggested that the cuticle of the mutant leaf epidermis may have problems. The scanning electron microscope shows that the wax crystal accumulation in the mutant leaf surface is not normal. The transmission electron microscope results of the mutant and the wild type leaf cross section show that the mutant is a mutant. The epidermis of the epidermis was thinner, the true osmium in the true stratum corneum decreased, indicating the low density of the mutant horniness. The mass spectrometric analysis of the mutants and wild type surface wax showed that there was no significant difference between the mutants and the wild type, but the content of different components was very different. The mutant and the wild type inoculated with soybean bacterial speckles were 4 days after the mutant and wild type inoculation. The number of bacteria in the variant leaves is more than 6 times that of the wild type. It is proved that the ability of the mutant to cope with the infection of the pathogen is less than that of the pathogen. The expression abundance of the.7. cuticle related genes is affected by the Glyma.07G028600 gene combined with scanning electron microscopy, transmission electron microscopy, and the fruit of the wax composition mass spectrometry analysis, which involves plant wax, horniness synthesis, transport and assembly. The expression abundance of more than 20 genes regulated in the wild and mutant leaves was analyzed. The results showed that there was no significant difference in the expression abundance of the transporter related genes. The expression abundance difference between the horny synthase gene (GmLTL1) and other genes in the wild type and mutant was very large, and the expression abundance difference of some transcription factors was also great. The Glyma.07G028600 gene plays an important role in the development of the plant cuticle. The main innovation of this paper is: 1. a certain number of INDEL markers have been developed, and these markers also have good polymorphism among other soybean varieties. The nearly fifty thousand INDEL loci obtained by bioinformatics comparison can be genetic and molecular for soybeans. Auxiliary breeding provides rich information. These data have been uploaded to Soybase and fill the blank.2. without INDEL molecular markers in the soybean public database by mapping the gene to a gene involved in the development of the soybean cuticle, which is found to be involved in the plant's homologous gene in Arabidopsis. Non host resistance, but no part of the gene involved in the development of cuticle,.3. further revealed the molecular regulation mechanism of the development of soybean cuticle, which lays the foundation for the next breeding of drought resistant and disease resistant soybean varieties.

【学位授予单位】：山东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.1

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