拟南芥下胚轴向光弯曲不敏感突变体筛选及相关基因克隆
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摘要
植物体为更好适应自然环境,在其长期进化过程中形成诸多适应外界环境的能力。其中向光性反应就是为更多获得光源的一种适应反应。蓝光受体向光素的发现,为深入研究蓝光诱导的向光性信号转导机制提供新的途径。向光素PHTO1、PHOT2感受蓝光后,与其下游信号蛋白NPH3和RPT2相互作用,调节生长素运输载体AUX1和PIN1的活性及定位、从而使拟南芥黄化苗下胚轴向光侧和背光侧中生长素分布不均匀,导致向光弯曲。但是蓝光信号如何调节向光弯曲的机制尚不完全清楚。
本实验以蓝光受体phot1突变体为材料,通过0.3%乙酰甲基磺酸(EMS)诱变,应用100μmol m~(-2)s~(-1)的单侧蓝光处理12 h,对其M2代幼苗进行大规模的筛选。获得3株潜在突变体,分别命名为sph1 (slow phototropic hypocotyl 1)向光性迟缓突变体、lph1(littery phototropic hypocotyl 1)向光性杂乱突变体以及dph1(deletion phototropic hypocotyl 1)向光性缺失突变体.
研究发现,与phot1突变体相比,sph1突变体除在100μmol m~(-2)s~(-1)单侧蓝光刺激下向光弯曲反应迟缓之外,其在25μmol m~(-2)s~(-1)红光下部分失去负向重力性,1μmol m~(-2)s~(-1)的蓝光可以恢复这种现象。将sph1突变体与Ler杂交,观察F1代和F2代的表型分离比,证实sph1为单基因隐性突变。通过图位克隆,将其定位在MSF3 BAC上,经测序发现该突变体在At2G18790.1基因DNA序列的第3482bp发生了点突变,由原来G变为A,蛋白质序列分析后发现,由于三联子密码GCC突变为ACC,因此第1096个氨基酸发生突变,即由丙氨酸突变为苏氨酸,因此可以推测点突变所造成的蛋白质序列的变化会直接导致其向光反应迟缓。
以phot1突变体为对照材料,对sph1突变体进行一系列的表型鉴定,观察它们在生长发育方面的差异。结果显示营养生长时期sph1突变体较phot1突变体相比,其叶片狭小、叶柄较长且逆时针螺旋排列、幼苗茎较长,同时气孔开度和密度较低、失水较慢、叶片温度较高,但其最终结实量同phot1突变体无明显差异。其原因可能是sph1突变体通过较长的叶柄逆时针螺旋排列、相对较长的茎等多种方式调节避荫反应,使其叶片根据光线调整位置从而最大限度获得光能,进行光合作用,积累能量,最终对结实量无影响。
对于lph1和dph1突变体,通过与Ler杂交后F1代和F2代表型分离比,证实lph1和dph1同样为单基因隐性突变。目前已将lph1粗定位在拟南芥第五条染色体的底部,dph1粗定位在第四条染色体的中部。
以上研究结果表明,SPH1、LPH1、DPH1可能参与调控向光素介导的强蓝光向光弯曲的信号转导,但具体机制有待进一步研究。
For the better adaptation to environment and resistance to adverse circumstance, the plant has formed many abilities to adapt external environment in the long-term evolution process. The phototropism is in the case, which enables plant to bend towards light so that they are able to obtain optimum light. The discovery of blue light receptors phototropin (PHTO1,PHOT2), which provided the new ways for us to conduct further studies on the mechanism of phototropism induced by blue light. Now research suggested that PHTO1 and PHOT2 interacted with its downstream signal protein NPH3 and RPT2, regulated the activation and localization of the auxin-efflux and -influx transporters, AUX1 and PIN1, which resulted in asymmetric distribution of the auxin between the illuminated and shaded side of the hypocotyls, and induces phototropic bending. But we are not completely clear how blue light signal regulates the phototropism.
In this study, phot1( a mutant of blue light receptor) was used as experimental materials, through mutagenesis by 0.3% EMS and handled with 100μmol m~(-2)s~(-1) unilateral blue light for 12 hs to screen M2 generation of seedlings on the large scale. Now 3 potential mutants were obtained which were named as sph1 (slow phototropic hypocotyl 1), lph1(littery phototropic hypocotyl 1), dph1(deletion phototropic hypocotyl 1) respectively.
The findings demonstrated that compared with the phot1 mutant, sph1 mutant phototropism was slower than that of phot1 under the irritation of 100μmol m~(-2)s~(-1) unilateral blue light. In addition, the sph1 lost the negative gravitropism under the25μmol m~(-2)s~(-1)red light, and this phenomenon restored to normal under 1μmol m~(-2)s~(-1) blue light. sph1 and Ler were hybridized, segregation ratio statistics of backcross between F1 and F2 showed that sph1 was recessive single gene mutation.
We located the gene on MSF3 BAC by map-based cloning. Every gene’s sequence of this genomic region was examined. 3482bp point mutation occurred At2G18790.1 was found to be mutated in sphl by sequence analysis. Compared with wild type, sphl contained an G-to- A change at 3482bp in the gene DNA. Protein sequence analysis indicated that GCC was mutated to ACC, and 1096 lactamine mutated to threonine. So we assumed that the point mutations resulted in protein sequence change, which directly leads to the slow phototropic bending.
Using phot1 mutant as the control materials, sph1 mutant was conducted a series of identification, the difference in their growth and development was observed. The results showed that there existed the remarkable difference between sph1 mutant and phot1 mutant. sph1 mutant was similar to phot1 mutant during nourishing development, the leaves were narrow and long, petioles were longer, appearing the counter clockwise screw arrangement, the stems are long, simultaneously the stomata was open, the density of stomata was low, water was losing slowly, the temperature on the surface of leaves is high, however there was no obvious difference between sph1 mutant and phot1 mutant in the fruit capacity. Its reason possibly is that the sph1 mutant can regulate the shade avoidance syndrome(SAS)by many ways such as the long petiole anti-clockwise reverse-acting spiral arrangement and the relatively long stem. Based on the light, each leaf blade of sph1 mutant adjusts its position to obtain the luminous energy to the utmost, to conduct photosynthesis, accumulate energy, and eventually it has no influence on its fruit capacity.
As for dph1and iph1 mutants, after they were hybridized with Ler, segregation ratio statistics of backcross between F1 and F2 showed that dph1 and iph1 were recessive single gene mutants as well. At present, we has roughly located dph1 at the fifth chromosome base end, while located iph1 in the middle of the fourth chromosome in Arabidopsis thaliana.
Above results suggest that SPH1,DPH1,IPH1 probably involved in strong BL-induced phototropic bending in Arabidopsis etiolated seedlings. And the exact mechanisms will be tested by complementary experiment further.
本实验以蓝光受体phot1突变体为材料,通过0.3%乙酰甲基磺酸(EMS)诱变,应用100μmol m~(-2)s~(-1)的单侧蓝光处理12 h,对其M2代幼苗进行大规模的筛选。获得3株潜在突变体,分别命名为sph1 (slow phototropic hypocotyl 1)向光性迟缓突变体、lph1(littery phototropic hypocotyl 1)向光性杂乱突变体以及dph1(deletion phototropic hypocotyl 1)向光性缺失突变体.
研究发现,与phot1突变体相比,sph1突变体除在100μmol m~(-2)s~(-1)单侧蓝光刺激下向光弯曲反应迟缓之外,其在25μmol m~(-2)s~(-1)红光下部分失去负向重力性,1μmol m~(-2)s~(-1)的蓝光可以恢复这种现象。将sph1突变体与Ler杂交,观察F1代和F2代的表型分离比,证实sph1为单基因隐性突变。通过图位克隆,将其定位在MSF3 BAC上,经测序发现该突变体在At2G18790.1基因DNA序列的第3482bp发生了点突变,由原来G变为A,蛋白质序列分析后发现,由于三联子密码GCC突变为ACC,因此第1096个氨基酸发生突变,即由丙氨酸突变为苏氨酸,因此可以推测点突变所造成的蛋白质序列的变化会直接导致其向光反应迟缓。
以phot1突变体为对照材料,对sph1突变体进行一系列的表型鉴定,观察它们在生长发育方面的差异。结果显示营养生长时期sph1突变体较phot1突变体相比,其叶片狭小、叶柄较长且逆时针螺旋排列、幼苗茎较长,同时气孔开度和密度较低、失水较慢、叶片温度较高,但其最终结实量同phot1突变体无明显差异。其原因可能是sph1突变体通过较长的叶柄逆时针螺旋排列、相对较长的茎等多种方式调节避荫反应,使其叶片根据光线调整位置从而最大限度获得光能,进行光合作用,积累能量,最终对结实量无影响。
对于lph1和dph1突变体,通过与Ler杂交后F1代和F2代表型分离比,证实lph1和dph1同样为单基因隐性突变。目前已将lph1粗定位在拟南芥第五条染色体的底部,dph1粗定位在第四条染色体的中部。
以上研究结果表明,SPH1、LPH1、DPH1可能参与调控向光素介导的强蓝光向光弯曲的信号转导,但具体机制有待进一步研究。
For the better adaptation to environment and resistance to adverse circumstance, the plant has formed many abilities to adapt external environment in the long-term evolution process. The phototropism is in the case, which enables plant to bend towards light so that they are able to obtain optimum light. The discovery of blue light receptors phototropin (PHTO1,PHOT2), which provided the new ways for us to conduct further studies on the mechanism of phototropism induced by blue light. Now research suggested that PHTO1 and PHOT2 interacted with its downstream signal protein NPH3 and RPT2, regulated the activation and localization of the auxin-efflux and -influx transporters, AUX1 and PIN1, which resulted in asymmetric distribution of the auxin between the illuminated and shaded side of the hypocotyls, and induces phototropic bending. But we are not completely clear how blue light signal regulates the phototropism.
In this study, phot1( a mutant of blue light receptor) was used as experimental materials, through mutagenesis by 0.3% EMS and handled with 100μmol m~(-2)s~(-1) unilateral blue light for 12 hs to screen M2 generation of seedlings on the large scale. Now 3 potential mutants were obtained which were named as sph1 (slow phototropic hypocotyl 1), lph1(littery phototropic hypocotyl 1), dph1(deletion phototropic hypocotyl 1) respectively.
The findings demonstrated that compared with the phot1 mutant, sph1 mutant phototropism was slower than that of phot1 under the irritation of 100μmol m~(-2)s~(-1) unilateral blue light. In addition, the sph1 lost the negative gravitropism under the25μmol m~(-2)s~(-1)red light, and this phenomenon restored to normal under 1μmol m~(-2)s~(-1) blue light. sph1 and Ler were hybridized, segregation ratio statistics of backcross between F1 and F2 showed that sph1 was recessive single gene mutation.
We located the gene on MSF3 BAC by map-based cloning. Every gene’s sequence of this genomic region was examined. 3482bp point mutation occurred At2G18790.1 was found to be mutated in sphl by sequence analysis. Compared with wild type, sphl contained an G-to- A change at 3482bp in the gene DNA. Protein sequence analysis indicated that GCC was mutated to ACC, and 1096 lactamine mutated to threonine. So we assumed that the point mutations resulted in protein sequence change, which directly leads to the slow phototropic bending.
Using phot1 mutant as the control materials, sph1 mutant was conducted a series of identification, the difference in their growth and development was observed. The results showed that there existed the remarkable difference between sph1 mutant and phot1 mutant. sph1 mutant was similar to phot1 mutant during nourishing development, the leaves were narrow and long, petioles were longer, appearing the counter clockwise screw arrangement, the stems are long, simultaneously the stomata was open, the density of stomata was low, water was losing slowly, the temperature on the surface of leaves is high, however there was no obvious difference between sph1 mutant and phot1 mutant in the fruit capacity. Its reason possibly is that the sph1 mutant can regulate the shade avoidance syndrome(SAS)by many ways such as the long petiole anti-clockwise reverse-acting spiral arrangement and the relatively long stem. Based on the light, each leaf blade of sph1 mutant adjusts its position to obtain the luminous energy to the utmost, to conduct photosynthesis, accumulate energy, and eventually it has no influence on its fruit capacity.
As for dph1and iph1 mutants, after they were hybridized with Ler, segregation ratio statistics of backcross between F1 and F2 showed that dph1 and iph1 were recessive single gene mutants as well. At present, we has roughly located dph1 at the fifth chromosome base end, while located iph1 in the middle of the fourth chromosome in Arabidopsis thaliana.
Above results suggest that SPH1,DPH1,IPH1 probably involved in strong BL-induced phototropic bending in Arabidopsis etiolated seedlings. And the exact mechanisms will be tested by complementary experiment further.
引文
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