陆地棉矮杆突变体与赤霉素相关的分子机理
摘要
中国棉花主产区新疆“矮、密、早”栽培技术的突破,使单产迅速上升。棉花株高性状的研究,特别是寻找控制株高性状的基因资源便成为了与生产紧密联系的主要任务。每年大量缩节胺的使用,给棉农增加了较大的经济负担,培育出纤维产量不受影响而仅株高缩短的品种也是很多株型育种家的梦想。本实验室于1998年在一个种间杂交的F2群体中,发现一株赤霉素敏感的极端矮化的突变体,但是,叶片表现为正常的阔叶,与已经报道的棉花矮化突变体的叶片皱缩有明显区别。经多代自交纯化,已得到纯合突变体。该突变体与野生型杂交能形成中矮(约50cm)紧凑型植株,单铃纤维特点与高株相差不大。本文研究该突变体矮化性状的细胞和组织学特征入手,鉴定和分析了与矮杆突变体矮化有关的基因和调控元件等,为遗传改良和有效利用这一矮杆基因资源奠定基础。
为此,本实验采取的主要试验技术以及获得的结果如下：
1本实验比较和分析了H98及其极矮杆突变体AS98材料的细胞和组织学特征差异,通过显微观察方法得知极矮突变体的细胞膨大生长受到了严重的影响,电镜观察发现AS98细胞壁加厚。外施赤霉素后能够在增大其细胞的同时,细胞壁也变薄。相反,如果利用多效唑使正常的高株细胞生长受抑制以后,细胞壁也变厚。这说明植株茎杆的生长与细胞壁的薄厚关系密切。
2通过RT-PCR和QRT-PCR的技术筛选与细胞壁膨胀有关的基因,最终筛选出3个膨胀素合成基因。利用毕赤酵母分泌表达的膨胀蛋白成功地使棉花悬浮细胞增大。5叶期,赤霉素合成基因的表达在AS98与其H98之间没有差别；膨胀素基因都是在突变体中低表达,在H98中高表达,这说明在突变体中膨胀素基因很可能发生了突变；赤霉素受体基因(6ID-1)和DELLA蛋白基因(6AI-RGA)在第5叶期都是在突变体中高表达,在野生型植株中低表达,而且,在第15叶期都是在突变体中表达量更高；Q-PCR分析结果还表明在主茎伸长的关键时期,突变体中的赤霉素合成过程中的氧化酶基因表达量稍高,可能是由于GA的缺乏造成的反馈调节造成的。
3利用染色体步移技术得到了这3个膨胀蛋白基因的启动子区,通过分析发现这些基因都是与赤霉素有关的基因,在启动区含有TAACAAA元件。利用酵母单杂交的办法筛选到一个能与这个元件互作的GAMYB基因。组织原位杂交显示该基因与膨胀素在相同的细胞中表达；该基因在序列上与其他物种GAMYB同源,烟草体内互补实验证明该基因能够作为miR159的靶标。已有大量的文献证明GAMYB和miR159均受到赤霉素的调控。这说明直接作用到细胞壁的某些膨胀蛋白受到赤霉素的调控,而这种调控由GAMYB直接与其作用,膨胀素便是赤霉素信号转导的终端之一。AS98中赤霉素合成受到影响,导致膨胀素含量降低,细胞壁膨胀受到影响,造成细胞膨大生长困难,引起矮化。
The planting pattern of "dwarf plant、high plant dense and early maturation" is the major cotton planting technique in Xinjiang province of China, which have led to a rapid increase in yield per unit area. Studying on cotton plant height, especially, identification of genetic resources controlled plant height has been becoming one of the most important tasks for the cotton production. The financial burden on farmers increased for the additional aplication of mepiquat chloride every year. Therefore, it is a target for breeders to select the dwarf cotton varieties with excellent fiber quality. A novel cotton dwarf mutant AS98 with extremely short stems but normal leaf shape was discovered in a F2 population of interspecific hybridization in 1998. Compared with the other dwarf cottons with crinkled leaves that had been reported previously, the leaves of it were normal shape. After self pollination for several generations, homozygous mutant have been gained. The mutant crossed with wild-type could produce the short (about 50 cm) and compact plants which had little difference in fiber qualities compared with that of the wild-type. In this study, based on the cell and tissue characters of the dwarf mutant, the related genes and regulation element of dwarf mutant was identified and analyzed. This will lay a foundation for genetic improvement and effiently utilizing this dwarf mutant. To this end, the main results obtained are as follows:
1. Cells and histological features of the dwarf mutant. We found that the enlarge-growth of mutant cells was severely affected by comparison and analysis of different characteristics of cells and tissues of the normal strain and dwarf material through the microscope. Through electron microscopy we found that the cell wall of most dwarf cells was thickening. After treatment with exogenous GA3, the cells can increase with the cell wall thinning. In the contrary, the cells of normal plants were restrained with thicker cell walls after treatment of paclobutrazol. This demonstrated that the growth of the plant stem was tied to thickness of cell walls.
2. Three Expansin genes associated with cell wall expansion of the dwarf mutant were identified and screened by RT-PCR and QRT-PCR techniques. The expansion protein expressed by the Pichia succeeded in expanding suspension cells of the normal plant type in cotton. There is no transcription difference between the mutant and the wild type on the GA synthesis genes like KO, KAO. Transcripts of the Expansins in the mutant was less than that in the wild-type at both 5th and 15th-leaf time point. Reversely, the mRNA of the GA receptor gene GID and DELLA were more in the dwarf, especially, the GID which was 60 times more at the 15th-leaf time point. Meanwhile the oxidase genes in the GA synthesis pathway were slightly high in the mutant, maybe it was caused by feedback regulation.
3. The promoter region of the three Expansin genes was obtained by using genome walking technique. These genes were related to GA, and element of TAACAAA was found in the promoter region of these genes. A MYB gene interaction with this element was screened by using yeast one-hybrid screening approach. The in situ hybridization showed that this gene expressed in the same cell as that of expansion gene. The MYB gene sequence was homology with the GAMYB of other species. And the complementary experiments in vivo in tobacco show that the gene can be used as miR159 targets. Abundant studies have showed that the gibberellin regulated GAMYB and miR159 expression. The above shows that some expansin which directly role in the cell wall were regulated by gibberellins and GAMYB. The Expansin gene family was one of the gibberellin signal transduction terminal protein. Gibberellin synthesis was hampered in AS98, resulting in Expansin was reduced, and then, cell wall expansion was affected, causing dwarf finally.
为此,本实验采取的主要试验技术以及获得的结果如下：
1本实验比较和分析了H98及其极矮杆突变体AS98材料的细胞和组织学特征差异,通过显微观察方法得知极矮突变体的细胞膨大生长受到了严重的影响,电镜观察发现AS98细胞壁加厚。外施赤霉素后能够在增大其细胞的同时,细胞壁也变薄。相反,如果利用多效唑使正常的高株细胞生长受抑制以后,细胞壁也变厚。这说明植株茎杆的生长与细胞壁的薄厚关系密切。
2通过RT-PCR和QRT-PCR的技术筛选与细胞壁膨胀有关的基因,最终筛选出3个膨胀素合成基因。利用毕赤酵母分泌表达的膨胀蛋白成功地使棉花悬浮细胞增大。5叶期,赤霉素合成基因的表达在AS98与其H98之间没有差别；膨胀素基因都是在突变体中低表达,在H98中高表达,这说明在突变体中膨胀素基因很可能发生了突变；赤霉素受体基因(6ID-1)和DELLA蛋白基因(6AI-RGA)在第5叶期都是在突变体中高表达,在野生型植株中低表达,而且,在第15叶期都是在突变体中表达量更高；Q-PCR分析结果还表明在主茎伸长的关键时期,突变体中的赤霉素合成过程中的氧化酶基因表达量稍高,可能是由于GA的缺乏造成的反馈调节造成的。
3利用染色体步移技术得到了这3个膨胀蛋白基因的启动子区,通过分析发现这些基因都是与赤霉素有关的基因,在启动区含有TAACAAA元件。利用酵母单杂交的办法筛选到一个能与这个元件互作的GAMYB基因。组织原位杂交显示该基因与膨胀素在相同的细胞中表达；该基因在序列上与其他物种GAMYB同源,烟草体内互补实验证明该基因能够作为miR159的靶标。已有大量的文献证明GAMYB和miR159均受到赤霉素的调控。这说明直接作用到细胞壁的某些膨胀蛋白受到赤霉素的调控,而这种调控由GAMYB直接与其作用,膨胀素便是赤霉素信号转导的终端之一。AS98中赤霉素合成受到影响,导致膨胀素含量降低,细胞壁膨胀受到影响,造成细胞膨大生长困难,引起矮化。
The planting pattern of "dwarf plant、high plant dense and early maturation" is the major cotton planting technique in Xinjiang province of China, which have led to a rapid increase in yield per unit area. Studying on cotton plant height, especially, identification of genetic resources controlled plant height has been becoming one of the most important tasks for the cotton production. The financial burden on farmers increased for the additional aplication of mepiquat chloride every year. Therefore, it is a target for breeders to select the dwarf cotton varieties with excellent fiber quality. A novel cotton dwarf mutant AS98 with extremely short stems but normal leaf shape was discovered in a F2 population of interspecific hybridization in 1998. Compared with the other dwarf cottons with crinkled leaves that had been reported previously, the leaves of it were normal shape. After self pollination for several generations, homozygous mutant have been gained. The mutant crossed with wild-type could produce the short (about 50 cm) and compact plants which had little difference in fiber qualities compared with that of the wild-type. In this study, based on the cell and tissue characters of the dwarf mutant, the related genes and regulation element of dwarf mutant was identified and analyzed. This will lay a foundation for genetic improvement and effiently utilizing this dwarf mutant. To this end, the main results obtained are as follows:
1. Cells and histological features of the dwarf mutant. We found that the enlarge-growth of mutant cells was severely affected by comparison and analysis of different characteristics of cells and tissues of the normal strain and dwarf material through the microscope. Through electron microscopy we found that the cell wall of most dwarf cells was thickening. After treatment with exogenous GA3, the cells can increase with the cell wall thinning. In the contrary, the cells of normal plants were restrained with thicker cell walls after treatment of paclobutrazol. This demonstrated that the growth of the plant stem was tied to thickness of cell walls.
2. Three Expansin genes associated with cell wall expansion of the dwarf mutant were identified and screened by RT-PCR and QRT-PCR techniques. The expansion protein expressed by the Pichia succeeded in expanding suspension cells of the normal plant type in cotton. There is no transcription difference between the mutant and the wild type on the GA synthesis genes like KO, KAO. Transcripts of the Expansins in the mutant was less than that in the wild-type at both 5th and 15th-leaf time point. Reversely, the mRNA of the GA receptor gene GID and DELLA were more in the dwarf, especially, the GID which was 60 times more at the 15th-leaf time point. Meanwhile the oxidase genes in the GA synthesis pathway were slightly high in the mutant, maybe it was caused by feedback regulation.
3. The promoter region of the three Expansin genes was obtained by using genome walking technique. These genes were related to GA, and element of TAACAAA was found in the promoter region of these genes. A MYB gene interaction with this element was screened by using yeast one-hybrid screening approach. The in situ hybridization showed that this gene expressed in the same cell as that of expansion gene. The MYB gene sequence was homology with the GAMYB of other species. And the complementary experiments in vivo in tobacco show that the gene can be used as miR159 targets. Abundant studies have showed that the gibberellin regulated GAMYB and miR159 expression. The above shows that some expansin which directly role in the cell wall were regulated by gibberellins and GAMYB. The Expansin gene family was one of the gibberellin signal transduction terminal protein. Gibberellin synthesis was hampered in AS98, resulting in Expansin was reduced, and then, cell wall expansion was affected, causing dwarf finally.
引文
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