拟南芥发育异常突变体的基因克隆与表型分析

发布时间：2018-06-06 14:19

本文选题：拟南芥 + 衰老　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：植物发育在器官、组织和细胞等不同层次上受到复杂的遗传互作网络调控。本实验室的研究方向是利用正向遗传学方法探索植物非生物逆境响应相关发育性状形成的遗传基础和分子机制。实验室在前期工作中,在激活标签突变体库中筛选获得一个拟南芥显性突变体abs3-1D(abnormal shoot3-1 dominant)。abs3-1D突变体呈现株型小,叶柄短,叶片紧凑,颜色偏黄,开花明显提前,次生花序簇生,主花序茎矮化,短角果且种子的育性下降。在黑暗诱导条件下,abs3-1D表现出叶片衰老加速的特征。为进一步研究ABS3在植物发育过程的功能,以abs3-1D为遗传背景进行EMS(乙基磺酸甲酯,ethyl-methane sulphonate)诱变。在诱变体库中筛选到能够逆转abs3-1D早花表型的突变体F08-69。F08-69表现出晚花表型,并且F08-69single营养生长期延长,黑暗条件下叶片衰老速度减慢。全基因组重测序鉴定出At3g10390基因+678bp处G变成A,导致甘氨酸(G)替换为丝氨酸(S)。另外还发现叶色、叶形和表皮毛发育缺陷的突变,其中一株隐性突变体abt3-1(aberrantly branched trichome 3),其突变表型为植株小,叶形宽,叶色浓绿,尤其是其出现了高比例的多分支的表皮毛,并且根的生长存在缺陷。abt3-1根的伸长速率仅为Col-0的三分之一。通过对根的分生组织中皮层细胞数目和细胞长度的测定,表明abt3-1根的分生区变短。该研究利用图位克隆技术发现abt3-1中At1g66510的第8个外显子的+33bp处G突变为A,提前引入终止密码子。回复验证表明At1g66510(ABT3)的突变导致了abt3-1的表型。ABT3基因编码一个目前功能未知的AAR2(a1-?2 repression 2)家族蛋白。我们同时对ABT3蛋白的亚细胞定位进行了观察,发现ABT3蛋白定位于细胞质和细胞核中。
[Abstract]:Plant development is regulated by complex genetic interaction networks at different levels, such as organs, tissues and cells. The research direction of our laboratory is to explore the genetic basis and molecular mechanism of plant abiotic stress response related developmental traits by means of forward genetics. In previous work, a dominant mutant abs3-1D(abnormal shoot3-1 dominant).abs3-1D mutants of Arabidopsis thaliana were screened from activated tag mutants. The mutant was small in type, short in petiole, compact in leaf, yellow in color, early in flowering and clustered in secondary inflorescences. The stem of the main inflorescence was dwarfed, the short pod and the fertility of the seed decreased. Under the condition of dark induction, abs3-1D showed the characteristics of accelerated leaf senescence. In order to further study the function of ABS3 in plant development, abs3-1D (ethyl methyl sulfonate methyl methyl-methane sulphonate) was used as genetic background. The mutant F08-69.F08-69 which could reverse the early flowering phenotype of abs3-1D showed late flowering phenotype, and the vegetative growth period of F08-69single was prolonged, and the senescence rate of leaves was decreased under dark conditions. The whole genome re-sequencing identified that G changed to A at 678bp of At3g10390 gene, which resulted in the substitution of GG with serine. In addition, mutations in leaf color, leaf shape and epidermal development defects were also found. One of the recessive mutants, abt3-1(aberrantly branched trichome 3, had a small, wide leaf shape, thick green leaf color, especially a high proportion of multi-branched epidermal hairs. The elongation rate of root is only 1/3 of that of Col-0. The number and length of cortical cells in the meristem of abt3-1 root were measured. The results showed that the meristem region of abt3-1 root became shorter. In this study, the G mutation in the 33bp of the eighth exon of At1g66510 in abt3-1 was found to be A by map cloning, and the termination codon was introduced in advance. The response test showed that the mutation of At1g66510- ABT3) led to the phenotype of abt3-1. ABT3 gene encodes a AAR2(a1-?2 repression 2) family protein with unknown function. We also observed the subcellular localization of ABT3 protein and found that ABT3 protein was located in cytoplasm and nucleus.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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