玉米穗发芽突变体和小粒突变体基因克隆及功能验证

发布时间：2018-05-18 08:52

本文选题：玉米 + 穗发芽　；参考：《中国农业大学》2017年博士论文

【摘要】：玉米籽粒突变体是研究玉米种子发育的重要材料,对突变体基因的克隆和功能分析不仅可以了解玉米种子的发育机制,而且具有潜在的应用价值。到目前为止,国内外的研究人员已经对玉米籽粒突变体做了大量的研究工作。本实验室之前已经利用玉米自交系B73与Mutator (Mu)活性系杂交,构建了一个玉米籽粒突变体库,其中有多个符合3:1分离比的隐性突变体,本研究选取其中两个玉米籽粒突变体进行基因克隆和功能分析,其中一个是玉米穗发芽突变体(vp-wl2),另外一个是玉米小粒突变体(smk-wl10)。,穗发芽突变体vp-wl2的种子呈灰白色,突变体种子成熟前在雌穗上萌发,胚乳缺少正常的类胡萝卜素组分,积累ζ-胡萝卜素,ABA合成受阻。利用改进的DLA法克隆到引起vp-wl2突变体发生穗发芽的基因是玉米的ZDS基因(zeta-carotene desaturase gene)。vp-wl2突变体的ZDS基因在第一内含子和第二外显子之间有一个Mu9转座子的插入,导致该基因不能正常转录而发生突变。之前有研究认为ZDS基因可能是vp9突变体的结构基因,也有可能是vp9位点调控了 ZDS基因的表达,但是没有直接的证据能证明二者的关系。为了证明二者的关系,将vp-wl2突变体和三个vp9突变体做了等位测验,结果发现,vp-wl2是vp9的一个新等位突变体,进而证明了vp 的结构基因是ZDS基因。已有研究表明,类胡萝卜素主要在叶绿体中合成,利用玉米原生质体和烟草叶片对ZDS-YFP蛋白进行瞬时表达发现,ZDS蛋白定位在叶绿体中。ZDS蛋白是类胡萝卜素生物合成途径中的一个关键酶,该蛋白在合成后会被运送到叶绿体中催化类胡萝卜素的生物合成,因此,ZDS基因突变后会导致玉米籽粒缺少类胡萝卜素,使ABA的合成受阻,ABA含量不足就不能维持胚在发育过程中的休眠状态,最终导致穗发芽的表型。smk-wll0是一个小粒突变体,大部分小种子的胚发育受阻,只有一小部分小种子能够发芽,少数突变体在田间能散粉,但是不能形成正常的雌穗。通过观察种子发育过程中的切片发现,突变体的胚发育滞后,胚乳发育也不正常,胚乳基部转移层细胞(BETL)不能形成向内生长的细胞壁,进而影响营养物质从母体向胚乳运输。利用图位克隆法将smk-wll0基因定位在玉米2号染色体短臂上433 kb的区间内,区间内有7个开放阅读框,分别是ORF1到ORF7,在突变体中7个候选基因的DNA序列没有发生改变,与B73的参考基因组一致。其中只有ORF2在突变体幼苗和种子中均不表达,其余候选基因在突变体和野生型中均表达,且表达差异不显著。进一步研究发现,ORF2的启动子区CHG甲基化程度较高,经过分离群体的验证发现,启动子区域的CHG甲基化程度与ORF2表达水平呈负相关关系。ORF2编码微管蛋白折叠辅助因子B,拟南芥中该基因的直系同源基因突变会引起胚致死,因此,推测ORF2是smk-wllD的候选基因。以上的实验结果表明,玉米ZDS基因突变后会阻断类胡萝卜素的生物合成,导致ABA含量降低,进而出现穗发芽的表型;玉米微管蛋白折叠辅助因子B基因的不表达很可能会导致玉米种子发育受阻,影响籽粒的正常生长。这些发现为我们解析玉米籽粒的发育过程提供了新的实验证据。
[Abstract]:Maize seed mutants are important materials to study the development of maize seed. Cloning and functional analysis of mutant genes can not only understand the mechanism of maize seed development, but also have potential application value. So far, researchers at home and abroad have done a lot of research on maize seed mutants. A maize seed mutant library was constructed by hybridization between maize inbred line B73 and Mutator (Mu). There were multiple recessive mutants which were in line with 3:1 separation ratio. Two of the maize seed mutants were selected for gene cloning and functional analysis. One of them was the maize ear sprout mutant (vp-wl2), and the other one was the other one. It is a small corn mutant (smk-wl10). The seed of the mutant vp-wl2 of the panicle is gray. The mutant seeds germinate on the female ear before maturity. The endosperm lacks normal carotenoid components, the zeta carotene accumulation is accumulated, and the ABA synthesis is blocked. The ZDS based gene of the maize is cloned by the improved DLA method. The ZDS gene of the (ZETA-Carotene desaturase gene).Vp-wl2 mutant has an insertion of a Mu9 transposon between the first intron and the second exon, causing the gene to be mutated without normal transcription. Previous studies suggest that the ZDS gene may be the structural cause of the VP9 mutant and may also regulate the ZDS gene at the VP9 site. But there is no direct evidence to prove the relationship between the two. In order to prove the relationship between the two, the vp-wl2 mutant and the three VP9 mutants were tested. The results showed that vp-wl2 was a new allele of VP9, and that the structural gene of VP was a ZDS gene. ZDS-YFP protein was instantaneously expressed by maize protoplasts and tobacco leaves. The ZDS protein was located in the chloroplast as a key enzyme in the carotenoid biosynthesis pathway, and the protein was transported to the chloroplast to catalyze the biosynthesis of the catalyzed Hu Luo dopa in the chloroplast. Therefore, the ZDS gene mutation will result in the mutation of the ZDS gene. There is a lack of carotenoid in corn grain, which makes the synthesis of ABA hindered, and the lack of ABA content can not maintain the dormancy state of the embryo during the development of the embryo. Finally, the phenotypic.Smk-wll0 of the bud germination is a small grain mutant, and the embryo development of most small seeds is blocked, only a small fraction of the seed can germinate and a few mutants can be in the field. Powder, but can not form normal female ear. By observing the slicing in the process of seed development, it is found that the embryo development of the mutant is lagging behind and the endosperm development is not normal. The endosperm base transfer layer cells (BETL) can not form the inward cell wall, and then affect the transport of nutrients from the mother to the endosperm. The smk-wll0 base is used by the map cloning method. In the interval of 433 KB on the short arm of chromosome 2, there were 7 open reading frames in the interval, which were ORF1 to ORF7, and the DNA sequence of the 7 candidate genes in the mutant did not change, which was consistent with the reference genome of B73. Only ORF2 was not expressed in the mutant seedlings and seeds, and the other candidate genes were in the mutant and in the mutant. Further studies found that the degree of CHG methylation in the promoter region of ORF2 was higher. It was found that the degree of CHG methylation in the promoter region was negatively related to the level of ORF2 expression,.ORF2 encoded microtubulin folding cofactor B, and the direct homologous of this gene in Arabidopsis thaliana. Gene mutation can cause death of the embryo, so ORF2 is a candidate gene for smk-wllD. The above results show that the mutation of the maize ZDS gene will block the biosynthesis of carotenoid, which leads to the decrease of the ABA content and then the phenotype of the spike germination, and the non expression of the maize microtubulin fold auxiliary factor B gene may lead to the corn. These findings provide new experimental evidence for our understanding of the development of maize seeds.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513

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