玉米中ZmPIFs基因的克隆与功能分析
发布时间：2018-08-13 16:22
【摘要】：光对植物的生长发育起着重要的作用,一方面它不仅为植物带来了能量,使植物通过光合作用完成了同化物的积累;另一方面光又扮演了信号分子的角色,使植物通过感应光的变化调整自身的生长发育。植物通过体内的光敏色素(phytochrome,PHY)感知光信号,并通过光敏色素互作因子(phytochrome-interacting factors,PIFs)与光敏色素的相互作用调节植物的生长过程。PIFs对拟南芥的生长发育起着至关重要的作用,促进下胚轴的伸长,并抑制子叶的张开,还有抑制光形态建成。目前对于PIFs的研究主要集中于模式植物,而对一些高等作物尤其是玉米中ZmPIFs的研究还较少。因此,对玉米中ZmPIFs基因家族的研究有着重要的意义。在本实验中,我们从玉米基因数据库MaizeGDB中确定了玉米ZmPIFs基因家族的成员,探究了该基因家族的表达模式,分离得到了10个玉米基因。将其中4个基因转入拟南芥,结果发现bHLH44基因在抑制光形态建成和影响植株响应重力方面具有重要作用。主要结果如下:(1)根据NCBI上查询的拟南芥AtPIFs基因家族序列,将其在MaizeGDB上进行BLAST分析,获得了17个玉米ZmPIFs同源基因。通过进化树比对、基因序列分析、氨基酸序列分析和蛋白结构域预测,初步分析、预测了玉米ZmPIFs基因的结构与功能。最终确定了10个与拟南芥AtPIF1,AtPIF3,AtPIF4和AtPIF5同源且起主要作用的玉米ZmPIFs基因:ZmbHLH12、ZmbHLH30、ZmbHLH37、ZmbHLH40、ZmbHLH44、ZmbHLH51、ZmbHLH74、ZmbHLH88、ZmbHLH113和ZmbHLH180。由于PIFs基因属于bHLH基因家族,所以我们对玉米ZmPIFs基因的命名使用了其在bHLH基因家族中的编号。实时荧光定量PCR和RNA-seq高通量测序分析表明,我们发现玉米ZmPIFs基因的表达模式存在特异性:在不同的组织部位、不同的发育时期、不同基因对遮阴后的响应;并且发现bHLH24和bHLH74两个基因在短日照下的表达存在昼夜节律。(2)克隆出10个玉米ZmPIFs基因:bHLH1、bHLH12、bHLH24、bHLH30、bHLH40、bHLH44、bHLH51、bHLH113、bHLH130和bHLH180。结合基因序列的正确性、结构域的完整性和是否在玉米中起主要作用,将其中的部分玉米ZmPIFs基因转入到拟南芥pifq缺失突变体和野生型Col中。通过进一步验证,发现bHLH44基因对于抑制光形态建成的作用最明显:在黑暗环境中,bHLH44促进了35S:bHLH44-3FLAG/pifq和35S:bHLH44-3FLAG/Col的下胚轴长度,改变了35S:bHLH44-3FLAG/pifq的子叶张开程度。其抑制光形态建成的功能类似于AtPIFs基因。(3)bHLH44基因影响了植株的向重力性,在黑暗环境中,缺失突变体pifq的下胚轴不存在负向地性,而35S:bHLH44-3FLAG/pifq和野生型Col的下胚轴响应重力。我们通过I2-KI染色实验检测植株体内的淀粉合成情况解释了转基因幼苗可以感应重力的原因,bHLH44基因像拟南芥AtPIF1基因一样,阻止了植株体内胚层造粉体向白色体的转换。
[Abstract]:Light plays an important role in the growth and development of plants. On the one hand, it not only brings energy to plants and makes them accumulate assimilates through photosynthesis; on the other hand, light plays the role of signal molecule. Make plants adjust their growth and development through the change of induced light. Plants perceive light signals through Guang Min pigments in vivo, and regulate the growth process of Arabidopsis thaliana by the interaction of Guang Min pigments with Guang Min pigments. PIFs play an important role in Arabidopsis thaliana growth and development. Promote the elongation of hypocotyls, inhibit cotyledon opening, and inhibit photomorphogenesis. At present, the research on PIFs is mainly focused on model plants, but the research on ZmPIFs in some high crops, especially in maize, is still less. Therefore, it is of great significance to study the ZmPIFs gene family in maize. In this experiment, we identified the members of maize ZmPIFs gene family from maize gene database MaizeGDB, explored the expression pattern of the gene family, and isolated 10 maize genes. Four of the genes were transferred into Arabidopsis thaliana. The results showed that bHLH44 gene plays an important role in inhibiting light morphogenesis and affecting plant response to gravity. The main results are as follows: (1) according to the AtPIFs gene family sequence of Arabidopsis thaliana queried on NCBI, 17 ZmPIFs homologous genes of maize were obtained by BLAST analysis on MaizeGDB. The structure and function of maize ZmPIFs gene were predicted by evolutionary tree alignment, gene sequence analysis, amino acid sequence analysis and protein domain prediction. Finally, ten maize ZmPIFs genes, ZmbHLH12, ZmbHLH30, ZmbHLH37, ZmbHLH40, ZmbHLH74, ZmbHLH78, ZmbHLH83, ZmbHLH113 and ZmbHLH180, which are homologous to Arabidopsis thaliana AtPIF1, ATPIF3 and AtPIF5, have been identified, and their main roles are ZmbHLH12, ZmbHLH30, ZmbHLH40, ZmbHLH40, ZmbHLH74, ZmbHLH88, ZmbHLH113 and ZmbHLH180. Because the PIFs gene belongs to the bHLH gene family, we use its number in the bHLH gene family to name the ZmPIFs gene in maize. Real-time fluorescence quantitative PCR and RNA-seq high throughput sequencing analysis showed that the expression pattern of ZmPIFs gene in maize was specific: the response of different genes to shading was different in different tissues and different developmental stages. It was also found that there was a circadian rhythm in the expression of bHLH24 and bHLH74 genes in short sunlight. (2) Ten maize ZmPIFs genes: bHLH1, bHLH12, bHLH24, bHLH30, bHLH40, bHLH4H4BHLH113bHLH130 and bHLH180were cloned. Combined with the correctness of the gene sequence, the integrity of the domain and whether or not it plays a major role in maize, some of the maize ZmPIFs genes were transferred into Arabidopsis thaliana pifq deletion mutant and wild-type Col. Through further verification, we found that bHLH44 gene had the most obvious effect on inhibiting photomorphogenesis: in dark environment, bHLH44 promoted the length of hypocotyls of 35S:bHLH44-3FLAG/pifq and 35S:bHLH44-3FLAG/Col, and changed the opening degree of cotyledons of 35S:bHLH44-3FLAG/pifq. The inhibition of photomorphogenesis was similar to that of AtPIFs gene. (3) bHLH44 gene affected plant gravity. In dark environment, the hypocotyls of deletion mutant pifq had no negative ground, while 35S:bHLH44-3FLAG/pifq and wild-type Col Hypocotyl responded to gravity. The starch synthesis in transgenic seedlings was detected by I2-KI staining. The reason why transgenic seedlings can induce gravity is that the gene of bHLH44, like Arabidopsis AtPIF1 gene, prevents the transformation of the plantlets from endosperm to white bodies in the same way as in Arabidopsis thaliana.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S513
本文编号：2181531
[Abstract]:Light plays an important role in the growth and development of plants. On the one hand, it not only brings energy to plants and makes them accumulate assimilates through photosynthesis; on the other hand, light plays the role of signal molecule. Make plants adjust their growth and development through the change of induced light. Plants perceive light signals through Guang Min pigments in vivo, and regulate the growth process of Arabidopsis thaliana by the interaction of Guang Min pigments with Guang Min pigments. PIFs play an important role in Arabidopsis thaliana growth and development. Promote the elongation of hypocotyls, inhibit cotyledon opening, and inhibit photomorphogenesis. At present, the research on PIFs is mainly focused on model plants, but the research on ZmPIFs in some high crops, especially in maize, is still less. Therefore, it is of great significance to study the ZmPIFs gene family in maize. In this experiment, we identified the members of maize ZmPIFs gene family from maize gene database MaizeGDB, explored the expression pattern of the gene family, and isolated 10 maize genes. Four of the genes were transferred into Arabidopsis thaliana. The results showed that bHLH44 gene plays an important role in inhibiting light morphogenesis and affecting plant response to gravity. The main results are as follows: (1) according to the AtPIFs gene family sequence of Arabidopsis thaliana queried on NCBI, 17 ZmPIFs homologous genes of maize were obtained by BLAST analysis on MaizeGDB. The structure and function of maize ZmPIFs gene were predicted by evolutionary tree alignment, gene sequence analysis, amino acid sequence analysis and protein domain prediction. Finally, ten maize ZmPIFs genes, ZmbHLH12, ZmbHLH30, ZmbHLH37, ZmbHLH40, ZmbHLH74, ZmbHLH78, ZmbHLH83, ZmbHLH113 and ZmbHLH180, which are homologous to Arabidopsis thaliana AtPIF1, ATPIF3 and AtPIF5, have been identified, and their main roles are ZmbHLH12, ZmbHLH30, ZmbHLH40, ZmbHLH40, ZmbHLH74, ZmbHLH88, ZmbHLH113 and ZmbHLH180. Because the PIFs gene belongs to the bHLH gene family, we use its number in the bHLH gene family to name the ZmPIFs gene in maize. Real-time fluorescence quantitative PCR and RNA-seq high throughput sequencing analysis showed that the expression pattern of ZmPIFs gene in maize was specific: the response of different genes to shading was different in different tissues and different developmental stages. It was also found that there was a circadian rhythm in the expression of bHLH24 and bHLH74 genes in short sunlight. (2) Ten maize ZmPIFs genes: bHLH1, bHLH12, bHLH24, bHLH30, bHLH40, bHLH4H4BHLH113bHLH130 and bHLH180were cloned. Combined with the correctness of the gene sequence, the integrity of the domain and whether or not it plays a major role in maize, some of the maize ZmPIFs genes were transferred into Arabidopsis thaliana pifq deletion mutant and wild-type Col. Through further verification, we found that bHLH44 gene had the most obvious effect on inhibiting photomorphogenesis: in dark environment, bHLH44 promoted the length of hypocotyls of 35S:bHLH44-3FLAG/pifq and 35S:bHLH44-3FLAG/Col, and changed the opening degree of cotyledons of 35S:bHLH44-3FLAG/pifq. The inhibition of photomorphogenesis was similar to that of AtPIFs gene. (3) bHLH44 gene affected plant gravity. In dark environment, the hypocotyls of deletion mutant pifq had no negative ground, while 35S:bHLH44-3FLAG/pifq and wild-type Col Hypocotyl responded to gravity. The starch synthesis in transgenic seedlings was detected by I2-KI staining. The reason why transgenic seedlings can induce gravity is that the gene of bHLH44, like Arabidopsis AtPIF1 gene, prevents the transformation of the plantlets from endosperm to white bodies in the same way as in Arabidopsis thaliana.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S513
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