光皮桦miR166及其靶基因HD-ZIP Ⅲ转录因子家族成员鉴定与分析

发布时间：2018-05-27 07:29

本文选题：光皮桦 + miR166　；参考：《浙江农林大学》2016年硕士论文

【摘要】：光皮桦(Betula luminifera)是主要分布在我国南方的珍贵用材树种,材性优良,是林木遗传育种研究的良好材料。miR166在植物顶端分生组织发育、维管组织的形成、胚的发育中具有重要作用,其对靶基因HD-ZIP Ⅲ转录因子的调控是维管组织发育的重要途径。鉴定miR166及HD-ZIP Ⅲ转录因子基因家族成员,并研究其功能,可为光皮桦木材形成机制和遗传改良提供重要的理论基础。本研究以光皮桦为材料,对miR166及其靶基因HD-ZIP Ⅲ的家族成员进行了鉴定,并通过表达及转基因分析对其功能进行了研究,获得以下主要结果。1.根据光皮桦转录组及部分基因组数据,鉴定并克隆得到8个miR166前体序列,命名为BlmiR166a~BlmiR166g,序列长为0.3Kb~2.4Kb。对8个miR166和靶基因Bl HDZ Ⅲ进行了基因结构、顺式作用元件与系统进化等生物信息学分析,结果显示BlmiR166b、BlmiR166c与BlmiR166e含有1个内含子,其它miR166在基因组上则不含内含子。5个靶基因除了Bl HDZ1有19个外显子,其他Bl HD-ZIP Ⅲ基因都由18个外显子组成。对所有基因的顺式元件统计显示,光启动元件出现次数最多。系统进化分析表明,光皮桦8个miR166成熟体全部在同一个分支,而在前体序列比对中,8个pre-miR166分布在3个不同的亚组。2.组织表达特性分析表明,在所有miR166中,只有BlmiR166a和BlmiR166h在韧皮部表达量高,可能与其含有维管束发育相关元件有关;而BlmiR166d、BlmiR166e的表达十分相似,都是在芽中有高丰度的转录水平;BlmiR166b、BlmiR166c和BlmiR166f的表达较为相似,在雄花中表达量高;BlmiR166g则是在芽、雌花、雄花等组织都有表达。我们发现所有Bl HD-ZIP Ⅲ基因在韧皮部、种子和根中表达量都很低。Bl HDZ2和Bl HDZ5的表达情况相似,在木质部中的表达量远高于其他组织,暗示其参与了木质部的发育。Bl HDZ3和Bl HDZ4在茎中的表达丰度很高,且成熟茎段的明显高于幼嫩茎段。Bl HDZ1则在顶端分生组织中表达丰度高,表明其参与到了分生组织的发育中。此外,发现部分miR166与HD-ZIP Ⅲ间的表达呈现负调控。如BlmiR166a和BlmiR166h在木质部中几乎不表达而在韧皮部中表达量高,而靶基因Bl HDZ2和Bl HDZ5的表达正好相反。这表明这些基因在木质部与韧皮部的生长发育中存在负调控关系。3.Bl HD-ZIP Ⅲ基因在不同激素处理下的表达情况进行研究发现,5个Bl HD-ZIP Ⅲ基因在0.1m M浓度的IAA处理下都明显的受到诱导调控,其表达量与对照相比显著上调。这暗示Bl HDZⅢ基因与激素IAA参与了调控木质部的发育。Bl HDZ1、Bl HDZ3和Bl HDZ4分别在IAA、ET和GA的诱导下上调明显,可能与所含激素类相关顺式元件有关。Bl HDZ2和Bl HDZ5只有在0.1m M IAA处理下表达量有所增加,而在其他激素处理下变化不大。4.构建了5个Bl HDZⅢ基因的超表达载体并利用农杆菌介导法转化拟南芥,在分子水平进行了目的基因和报告基因的PCR检测,成功将Bl HDZ1、Bl HDZ2、Bl HDZ4和Bl HDZ5转到拟南芥中,其中Bl HDZ1和Bl HDZ5得到T2代各8个株系,Bl HDZ2和Bl HDZ4得到T2代各2个株系。发现与拟南芥野生型相比,Bl HDZ4和Bl HDZ5在表型性状上有一定的差异,这些基因的具体功能还有待于后续的深入研究。
[Abstract]:Betula luminifera is a valuable timber tree species mainly distributed in the south of China. It is a good material in wood. It is a good material for the study of tree genetics and breeding..miR166 plays an important role in the development of the apical meristem, the formation of vascular tissue and the development of embryo, and its regulation of the target gene HD-ZIP III transcription factor is the development of vascular tissue. The identification of miR166 and HD-ZIP III transcription factor gene family members, and the study of their functions, can provide an important theoretical basis for the formation and genetic improvement of birch wood. In this study, the family members of miR166 and its target gene HD-ZIP III were identified by Betula birch, and the expression and transgene analysis were used. The following main results were studied, and the following main results,.1., were identified and cloned to obtain 8 miR166 precursors based on the birch transcription group and some genomic data, named BlmiR166a~BlmiR166g, and the sequence length was 0.3Kb~2.4Kb. to 8 miR166 and target gene Bl HDZ III gene structure, cis acting element and phylogenetic evolution. The results of information analysis showed that BlmiR166b, BlmiR166c and BlmiR166e contained 1 introns, while other miR166 had no intron.5 target genes with 19 exons in addition to Bl HDZ1, and the other Bl HD-ZIP III genes were composed of 18 exons. Phylogenetic analysis showed that 8 miR166 mature bodies were in the same branch. In the alignment of the precursors, 8 pre-miR166 distribution in 3 different subgroups of.2. tissue showed that, in all miR166, only BlmiR166a and BlmiR166h were high in the phloem, and may be related to the vascular bundle development components. The expression of BlmiR166d, BlmiR166e is very similar, with high abundance of transcription in buds; BlmiR166b, BlmiR166c and BlmiR166f are similar in expression, high in male flowers; BlmiR166g is expressed in buds, female flowers, male flowers and other tissues. We found that all the Bl HD-ZIP III genes are in the phloem, seed and root table. The expression of.Bl HDZ2 and Bl HDZ5 is similar. The expression in xylem is much higher than that of other tissues, suggesting that the expression of.Bl HDZ3 and Bl HDZ4 in the xylem is very high, and the mature stem segment is obviously higher than the young stem segment.Bl HDZ1 in the top end meristem, indicating its participation in the xylem. In addition, the expression of some miR166 and HD-ZIP III shows negative regulation. Such as BlmiR166a and BlmiR166h are almost non expressed in the xylem and are highly expressed in the phloem, while the expression of the target gene Bl HDZ2 and Bl HDZ5 is the opposite. This indicates that these genes are stored in the growth and development of the xylem and phloem. The expression of.3.Bl HD-ZIP III gene in the negative regulatory relationship under the treatment of different hormones showed that 5 Bl HD-ZIP III genes were obviously induced under the IAA treatment of 0.1M M, and the expression of the gene was significantly up-regulated compared with the control. This suggests that Bl HDZ III gene and irritable IAA participate in the regulation of.Bl HDZ1. Bl HDZ3 and Bl HDZ4 are up regulated under the induction of IAA, ET and GA, which may be associated with the CIS associated elements associated with the hormone related.Bl HDZ2 and Bl HDZ5 only under the 0.1M process. Bl HDZ1, Bl HDZ2, Bl HDZ4 and Bl HDZ5 were transferred to Arabidopsis by PCR detection at molecular level, and Bl HDZ1 and Bl HDZ5 were obtained from 8 strains of each generation. There are some differences. The specific functions of these genes need to be further studied.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.15

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