甘蓝型油菜GRAS家族基因BnLAS的分离和功能分析
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摘要
GRAS转录因子作为植物特有的转录因子家族,在植物的生长发育及信号传导途径具有重要作用。但目前在油菜乃至整个芸苔属中对GRAS家族基因的研究还很少。拟南芥LAS基因是GRAS基因家族的成员之一,在植物的腋生分生组织的分化中具有重要地位。为深入了解甘蓝型油菜中GRAS类基因的作用,本研究在克隆芸苔属三个种LAS同源基因的基础上,通过分析过表达甘蓝型油菜BnLAS基因的转化拟南芥,对BnLAS的功能进行了初步分析,重点研究了BnLAS基因在提高植物抗旱性的作用,主要结果如下。
利用拟南芥LAS核苷酸序列设计引物,以甘蓝型油菜华双5号基因组DNA为模板进行PCR扩增获得BnLAS基因的DNA片段。通过TAIL-PCR技术分离到其两侧序列,最终获得全长为1326bp的BnLAS基因序列,同时分离到了BnLAS基因上游1944bp的启动子序列。生物信息学分析表明,BnLAS基因属于GRAS家族成员,Southern杂交显示BnLAS基因在甘蓝型油菜中可能存在2个拷贝。
荧光定量PCR和GUS染色分析表明,BnLAS基因在植物的根部表达最强,其次为花蕾、花、茎顶端。而在腋芽,叶片和茎中仅仅在荧光定量PCR中可以观察到微弱的表达信号。
将BnLAS基因在CaMV35S启动子驱动下,转化到拟南芥植株中进行过量表达分析。结果显示,与野生型拟南芥植株比较,过表达BnLAS对植株形态以及生长发育都有明显影响。过表达植物表现出生长速度延缓、叶绿素含量增加、花期延迟、育性降低等一系列变化。
将转基因植株和野生型植株在等生物量条件下进行抗旱处理15天后,当野生型植物发生萎蔫时,转基因植株生长仍然较为正常。将三种类型植物(野生型植株,las突变体,过表达家系植株OL14和OL18)种植在同一钵环境条件下进行早胁迫处理,经过15天处理后,过量表达植株存活率分别为71.9% (OL14)和91.6% (OL18),而野生型植株和1as突变体植株的存活率分别只为21.9%和11%。离体叶片的失水速率检测结果也表明,转基因植株的叶片离体失水速率显著低于野生型植株。
由于过表达植株的根部没有观察到显著的变异,而离体叶片的失水速率减缓,暗示过表达植株抗旱性的提高可能和叶片结构变化有着直接的关系。扫描电镜和光学显微镜观察发现,过表达植株OL18叶片表皮细胞减小,总气孔数目增加1.7-1.9倍。尽管过表达植株和野生型植株间的开放气孔数目差异不明显,但过表达植株的气孔开度只有野生型植株的18%-40%。
在利用扫描电镜观测到转基因植株叶表面蜡质层增厚的基础上,对几个蜡质合成关键基因在转基因植物中的表达进行了荧光定量PCR分析。蜡质合成关键基因CER1、CER2、KCS1、KCS2等四个基因的表达量在转基因植株中显著上升,表明过表达BnLAS基因影响了叶片表面的蜡质合成。通过叶绿素浸出实验进一步证实了蜡质层的加厚导致了角质层渗透性改变。
综上所述,在拟南芥中过表达BnLAS基因对其生长发育有明显影响,转化植株的抗旱性明显增加,而这种抗旱性的增加主要是叶片表面结构改变所致。
GRAS proteins as a unique transcription factor family to plants were involved in the growth and development and signal transduction pathways. So far there have been very few studies on the functions of GRAS proteins in Brassicasea. Arabidopsis LAS, a member of the GRAS family, has an important role in the initiation of axillary meristems. To study the function of GRAS family in Brassica napus, an Arabidopsis LAS homologous gene was cloned in three Brassica species. The BnLAS gene function was analyzed through overexpressing of BnLAS in Arabidopsis, focusing on the increased drought tolerance. The main results are as follows.
Based on the sequence information of Arabidopsis LAS, BnLAS was amplified from the genomic DNA of Brassica napus cv. Huashuang.5. The full ORF sequence with 1326bp of BnLAS was finally cloned by TAIL-PCR. The upstream region of 1944bp from the start codon of BnLAS was also isolated using TAIL-PCR. Bioinformatic analyses showed that BnLAS gene was one of the members of GRAS family. Southern blotting indicated that there are likely two copies of the gene in Brassica napus genome.
The expression pattern of BnLAS was showed highest levels in roots, and lower in bud, flower organs and shoot tips based on qRT-PCR analysis and GUS staining. Lateral meristems, stems and leaves were observed lowest levels in qRT-PCR analysis, but not detectable with GUS staining.
In order to research the function of BnLAS, the vector of overexpression BnLAS was constructed under the CaMV 35S promoter and transformed in Arabidopsis. Compared with wild type Arabidopsis plants,35S::BnLAS transgenic plants exhibited various morphological alterations as well as growth and developmental modifications, such as growth retarded, chlorophyll content increased, flower time delayed, fertility reduced.
Dehydration treatment was conducted with controlled amount of biomass for wild type and transgenic plants. After 15 days, wild type plants were more severe withering than transgenic plants. In a different experiment, three types of plants (wild type, las mutant, transgenic plants from OL14 and OL18 lines) were grown in the same pot and subject to dehydration treatment. After re-watering,71.9%(OL14) and 91.6%(OL18) of transgenic plants recovered, whereas only 21.9% wild type and 11% las mutant plants did. Then water loss rate of leaves was slower in transgenic lines OL14 and OL18, compared to wild type.
The root development in transgenic plants was not significantly different from the wild type. However, the water loss rate in leaves of plants was reduced, indicating that drought resistance of transgenic plants may have a direct relationship to the leaves structural changed. The results of scanning electron microscope (SEM) and light microscopy showed that the leaf stomatal density in line OL18 increased by 1.7-1.9 times compared with the wild type plants. But the number of open stomata was no significant difference between wild type and transgenic plants. However, the stomatal apertures on the leaves of line OL18 reduced to about 18%-40% of wild type plants.
The SEM observations showed a thicker wax in leaf surface, of transgenic plants. Several key wax biosynthetic genes were analyzed by quantitative PCR. The transcription levels of four genes(CER1, CER2, KCS1, KCS2) showed a dramatic increase in transgenic plants, indicating that ovexexpressing BnLAS in Arabidopsis has an important impact on wax biosynthesis. By chlorophyll leaching experiments, the results of thicker wax led to altered cuticular permeability were further confirmed.
In summary, BnLAS play an important role on plant growth and development. The overexpression of the gene resulted in an increased drought tolerance by ovexpressed in Arabidopsis. The increased drought tolerance is mainly caused by the changed leaves structures.
利用拟南芥LAS核苷酸序列设计引物,以甘蓝型油菜华双5号基因组DNA为模板进行PCR扩增获得BnLAS基因的DNA片段。通过TAIL-PCR技术分离到其两侧序列,最终获得全长为1326bp的BnLAS基因序列,同时分离到了BnLAS基因上游1944bp的启动子序列。生物信息学分析表明,BnLAS基因属于GRAS家族成员,Southern杂交显示BnLAS基因在甘蓝型油菜中可能存在2个拷贝。
荧光定量PCR和GUS染色分析表明,BnLAS基因在植物的根部表达最强,其次为花蕾、花、茎顶端。而在腋芽,叶片和茎中仅仅在荧光定量PCR中可以观察到微弱的表达信号。
将BnLAS基因在CaMV35S启动子驱动下,转化到拟南芥植株中进行过量表达分析。结果显示,与野生型拟南芥植株比较,过表达BnLAS对植株形态以及生长发育都有明显影响。过表达植物表现出生长速度延缓、叶绿素含量增加、花期延迟、育性降低等一系列变化。
将转基因植株和野生型植株在等生物量条件下进行抗旱处理15天后,当野生型植物发生萎蔫时,转基因植株生长仍然较为正常。将三种类型植物(野生型植株,las突变体,过表达家系植株OL14和OL18)种植在同一钵环境条件下进行早胁迫处理,经过15天处理后,过量表达植株存活率分别为71.9% (OL14)和91.6% (OL18),而野生型植株和1as突变体植株的存活率分别只为21.9%和11%。离体叶片的失水速率检测结果也表明,转基因植株的叶片离体失水速率显著低于野生型植株。
由于过表达植株的根部没有观察到显著的变异,而离体叶片的失水速率减缓,暗示过表达植株抗旱性的提高可能和叶片结构变化有着直接的关系。扫描电镜和光学显微镜观察发现,过表达植株OL18叶片表皮细胞减小,总气孔数目增加1.7-1.9倍。尽管过表达植株和野生型植株间的开放气孔数目差异不明显,但过表达植株的气孔开度只有野生型植株的18%-40%。
在利用扫描电镜观测到转基因植株叶表面蜡质层增厚的基础上,对几个蜡质合成关键基因在转基因植物中的表达进行了荧光定量PCR分析。蜡质合成关键基因CER1、CER2、KCS1、KCS2等四个基因的表达量在转基因植株中显著上升,表明过表达BnLAS基因影响了叶片表面的蜡质合成。通过叶绿素浸出实验进一步证实了蜡质层的加厚导致了角质层渗透性改变。
综上所述,在拟南芥中过表达BnLAS基因对其生长发育有明显影响,转化植株的抗旱性明显增加,而这种抗旱性的增加主要是叶片表面结构改变所致。
GRAS proteins as a unique transcription factor family to plants were involved in the growth and development and signal transduction pathways. So far there have been very few studies on the functions of GRAS proteins in Brassicasea. Arabidopsis LAS, a member of the GRAS family, has an important role in the initiation of axillary meristems. To study the function of GRAS family in Brassica napus, an Arabidopsis LAS homologous gene was cloned in three Brassica species. The BnLAS gene function was analyzed through overexpressing of BnLAS in Arabidopsis, focusing on the increased drought tolerance. The main results are as follows.
Based on the sequence information of Arabidopsis LAS, BnLAS was amplified from the genomic DNA of Brassica napus cv. Huashuang.5. The full ORF sequence with 1326bp of BnLAS was finally cloned by TAIL-PCR. The upstream region of 1944bp from the start codon of BnLAS was also isolated using TAIL-PCR. Bioinformatic analyses showed that BnLAS gene was one of the members of GRAS family. Southern blotting indicated that there are likely two copies of the gene in Brassica napus genome.
The expression pattern of BnLAS was showed highest levels in roots, and lower in bud, flower organs and shoot tips based on qRT-PCR analysis and GUS staining. Lateral meristems, stems and leaves were observed lowest levels in qRT-PCR analysis, but not detectable with GUS staining.
In order to research the function of BnLAS, the vector of overexpression BnLAS was constructed under the CaMV 35S promoter and transformed in Arabidopsis. Compared with wild type Arabidopsis plants,35S::BnLAS transgenic plants exhibited various morphological alterations as well as growth and developmental modifications, such as growth retarded, chlorophyll content increased, flower time delayed, fertility reduced.
Dehydration treatment was conducted with controlled amount of biomass for wild type and transgenic plants. After 15 days, wild type plants were more severe withering than transgenic plants. In a different experiment, three types of plants (wild type, las mutant, transgenic plants from OL14 and OL18 lines) were grown in the same pot and subject to dehydration treatment. After re-watering,71.9%(OL14) and 91.6%(OL18) of transgenic plants recovered, whereas only 21.9% wild type and 11% las mutant plants did. Then water loss rate of leaves was slower in transgenic lines OL14 and OL18, compared to wild type.
The root development in transgenic plants was not significantly different from the wild type. However, the water loss rate in leaves of plants was reduced, indicating that drought resistance of transgenic plants may have a direct relationship to the leaves structural changed. The results of scanning electron microscope (SEM) and light microscopy showed that the leaf stomatal density in line OL18 increased by 1.7-1.9 times compared with the wild type plants. But the number of open stomata was no significant difference between wild type and transgenic plants. However, the stomatal apertures on the leaves of line OL18 reduced to about 18%-40% of wild type plants.
The SEM observations showed a thicker wax in leaf surface, of transgenic plants. Several key wax biosynthetic genes were analyzed by quantitative PCR. The transcription levels of four genes(CER1, CER2, KCS1, KCS2) showed a dramatic increase in transgenic plants, indicating that ovexexpressing BnLAS in Arabidopsis has an important impact on wax biosynthesis. By chlorophyll leaching experiments, the results of thicker wax led to altered cuticular permeability were further confirmed.
In summary, BnLAS play an important role on plant growth and development. The overexpression of the gene resulted in an increased drought tolerance by ovexpressed in Arabidopsis. The increased drought tolerance is mainly caused by the changed leaves structures.
引文
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