拟南芥AtCPK30基因的表达模式和功能分析
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摘要
钙是真核生物信号转导中普遍存在的第二信使。植物中各种各样的信号都调节细胞内钙离子的水平,如激素、光、协迫等。关于钙离子介导的信号是如何特异地传递是一个重要的生物学问题,目前研究表明,似乎是不同的刺激引起不同的钙识别物,而钙信号中的传递因子能够识别这些特异的钙识别物,并将这种信号传递到下游从而发挥作用,包括改变蛋白的磷酸化作用和基因的表达模式。
钙依赖蛋白激酶(CDPKs)广泛存在于植物体内,目前发现,大多的钙信号传递都与钙依赖蛋白激酶有关。作为钙信号途径中一个传递因子,它参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥的一个钙依赖蛋白激酶基因(AtCPK30)进行了深入的研究。具体研究结果如下:
为了便于实验中T-DNA插入突变体的筛选,首先制作了一种简易可行的拟南芥水培装置。这套装置主要由两部分组成:播放种子的塑料管和作为支撑物的不锈钢丝网,盛溶液的容器。实验表明:T-DNA插入突变体在这种水培装置中生长良好,且培养的费用低,易于管理等。由于有钢丝网作支撑,植株能够顺利完成整个生长周期并且结实饱满,种子萌发率高。并采用“双引物法”筛选到了多个T-DNA插入突变体纯合子,为进行下一步实验做好准备。
接下来进行了AtCPK30基因的表达模式研究。收集野生型拟南芥根、茎、叶、花、果,通用半定量RT-PCR分析组织表达特异性。结果表明:AtCPK30在植物根中有很高的表达量,而在其它组织表达较低。将生长三周的野生型幼苗分别用植物激素脱落酸(ABA)、吲哚乙酸(IAA)、2,4-二氯苯氧乙苯酸(2,4-D)、赤霉素(GA3)和6-苄氨基嘌呤(BA)处理,然后采用半定量RT-PCR(逆转录PCR)分析AtCPK30基因在幼苗中的转录水平的变化情况。实验发现AtCPK30基因的表达受ABA、IAA、2,4-D、GA3和BA等激素的诱导。
又通过基因克隆构建了AtCPK30基因的植物超表达载体。并将此质粒利用基因枪法转入洋葱表皮细胞中,进行瞬时表达实验,了解AtCPK30基因的亚细胞定位情况。结果证明AtCPK30蛋白定位在细胞壁和细胞膜上。
同时将超表达载体通过浸花蕾法转入野生型拟南芥中,得到了AtCPK30基因超表达的转基因株系纯合子。测量在同一MS培养基上生长的野生型和转基因植株幼苗主根长,结果表明转基因植株主根在幼苗生长前期比野生型的长,但在第11天时,这种差异不明显。同时发现转基因植株幼苗的根在缺钙的MS培养基上长势较野生型植株好,表明缺钙对转基因幼苗影响较小。以野生型为对照,用植物激素ABA和IAA、GA3、BA处理转基因植株时,结果发现转基因植株幼苗的根对激素更敏感,具体表型为:ABA对转基因植株根的抑制更强烈,IAA和GA3、BA三种激素在低浓度时更能促进转基因植株根的生长。而当野生型和转基因植株生长在含有生长素抑制剂萘丙酸(NPA)的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。综合以上实验结果,AtCPK30蛋白可能作为一个正调节因子参与了植物激素信号途径。
Calcium is a ubiquitous second messenger in eukaryotic signal transduction cascades. In plants, intracellular Ca2+ levels are modulated in response to various signals, including hormones, light, and abiotic stress. How response specificity is regulated during Ca2+-mediated signal transduction is an important biological issue. It appears that different stimuli elicit specific calcium signatures, different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. CDPKs, calcium-dependent protein kinases, are wildly distributed in plants and algae, and also in some protistas. Currently, most of the known calcium-stimulated protein kinase activities in plants are associated with CDPKs. As calcium sensors, CDPKs mediate numerous responses including hormone signaling. Basing on previous studies, we made additional functional analysis of the gene AtCPK30 encoding a calcium-dependent protein kinase in Arabidopsis. The results of this study were listed as follows:
In order to screen homozygous seedlings of T-DNA insertion, we had prepared a simple and feasible hydroponic device. This device is made up of two parts: some seed-holders and stainless steel net as support, a tank with fit cover. Results showed that T-DNA insertion of AtCPK30 grew well in this hydroponic system. Furthermore, this system has the advantage of its low cost of planting and easy maintenance. Owing to the support of steel net, Arabidopsis Plants can finish their whole life cycle successfully and can bear lots of normal seeds with characteristic of high germination rate. Moreover, multi-homozygous of T-DNA insertion was identified by PCR through double primer.
Next, Expression pattern of AtCPK30 had been studied under different conditions. Results of semi-quantitative reverse transcription PCR (RT-PCR) analysis indicated that AtCPK30 was highly expressed in root and induced by ABA, IAA, 2,4-D, GA and BA treatment.
Then cDNA of AtCPK30 was subcloned into the pEGAD binary vector with a GFP gene and a Bar gene under the cauliflower mosaic virus (CaMV) 35S promoter, and the recombinant was transformed into onion epidermis cells by gene gun. GFP-CPK30 fusion gene transient transformation of onion epidermis cells revealed the localization of CPK30 in cell wall and cell membrane.
The physiological roles of AtCPK30 were studied using a gain-of-function approach. Seedlings of AtCPK30 transgenic lines had longer primary roots than those plants of wild-type at the early stages. Interestingly, when these plants grew on MS lack of Ca2+ including wild-type and transgenic lines, the roots of transgenic line were more sensitive to calcium, lack of Ca2+ had less effect on roots of transgenic lines than those of wild-type. Treated with several plant hormones, such as ABA, IAA, GA and BA, the roots of seedlings of transgenic line developed abnormally because they were more sensitive to hormones. Furthermore, NPA relatively less inhibited emergency of lateral roots of transgenic line than those of the wild-type. These results suggest that AtCPK30, as a positive regulator, involved in the hormone-signaling pathways.
钙依赖蛋白激酶(CDPKs)广泛存在于植物体内,目前发现,大多的钙信号传递都与钙依赖蛋白激酶有关。作为钙信号途径中一个传递因子,它参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥的一个钙依赖蛋白激酶基因(AtCPK30)进行了深入的研究。具体研究结果如下:
为了便于实验中T-DNA插入突变体的筛选,首先制作了一种简易可行的拟南芥水培装置。这套装置主要由两部分组成:播放种子的塑料管和作为支撑物的不锈钢丝网,盛溶液的容器。实验表明:T-DNA插入突变体在这种水培装置中生长良好,且培养的费用低,易于管理等。由于有钢丝网作支撑,植株能够顺利完成整个生长周期并且结实饱满,种子萌发率高。并采用“双引物法”筛选到了多个T-DNA插入突变体纯合子,为进行下一步实验做好准备。
接下来进行了AtCPK30基因的表达模式研究。收集野生型拟南芥根、茎、叶、花、果,通用半定量RT-PCR分析组织表达特异性。结果表明:AtCPK30在植物根中有很高的表达量,而在其它组织表达较低。将生长三周的野生型幼苗分别用植物激素脱落酸(ABA)、吲哚乙酸(IAA)、2,4-二氯苯氧乙苯酸(2,4-D)、赤霉素(GA3)和6-苄氨基嘌呤(BA)处理,然后采用半定量RT-PCR(逆转录PCR)分析AtCPK30基因在幼苗中的转录水平的变化情况。实验发现AtCPK30基因的表达受ABA、IAA、2,4-D、GA3和BA等激素的诱导。
又通过基因克隆构建了AtCPK30基因的植物超表达载体。并将此质粒利用基因枪法转入洋葱表皮细胞中,进行瞬时表达实验,了解AtCPK30基因的亚细胞定位情况。结果证明AtCPK30蛋白定位在细胞壁和细胞膜上。
同时将超表达载体通过浸花蕾法转入野生型拟南芥中,得到了AtCPK30基因超表达的转基因株系纯合子。测量在同一MS培养基上生长的野生型和转基因植株幼苗主根长,结果表明转基因植株主根在幼苗生长前期比野生型的长,但在第11天时,这种差异不明显。同时发现转基因植株幼苗的根在缺钙的MS培养基上长势较野生型植株好,表明缺钙对转基因幼苗影响较小。以野生型为对照,用植物激素ABA和IAA、GA3、BA处理转基因植株时,结果发现转基因植株幼苗的根对激素更敏感,具体表型为:ABA对转基因植株根的抑制更强烈,IAA和GA3、BA三种激素在低浓度时更能促进转基因植株根的生长。而当野生型和转基因植株生长在含有生长素抑制剂萘丙酸(NPA)的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。综合以上实验结果,AtCPK30蛋白可能作为一个正调节因子参与了植物激素信号途径。
Calcium is a ubiquitous second messenger in eukaryotic signal transduction cascades. In plants, intracellular Ca2+ levels are modulated in response to various signals, including hormones, light, and abiotic stress. How response specificity is regulated during Ca2+-mediated signal transduction is an important biological issue. It appears that different stimuli elicit specific calcium signatures, different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. CDPKs, calcium-dependent protein kinases, are wildly distributed in plants and algae, and also in some protistas. Currently, most of the known calcium-stimulated protein kinase activities in plants are associated with CDPKs. As calcium sensors, CDPKs mediate numerous responses including hormone signaling. Basing on previous studies, we made additional functional analysis of the gene AtCPK30 encoding a calcium-dependent protein kinase in Arabidopsis. The results of this study were listed as follows:
In order to screen homozygous seedlings of T-DNA insertion, we had prepared a simple and feasible hydroponic device. This device is made up of two parts: some seed-holders and stainless steel net as support, a tank with fit cover. Results showed that T-DNA insertion of AtCPK30 grew well in this hydroponic system. Furthermore, this system has the advantage of its low cost of planting and easy maintenance. Owing to the support of steel net, Arabidopsis Plants can finish their whole life cycle successfully and can bear lots of normal seeds with characteristic of high germination rate. Moreover, multi-homozygous of T-DNA insertion was identified by PCR through double primer.
Next, Expression pattern of AtCPK30 had been studied under different conditions. Results of semi-quantitative reverse transcription PCR (RT-PCR) analysis indicated that AtCPK30 was highly expressed in root and induced by ABA, IAA, 2,4-D, GA and BA treatment.
Then cDNA of AtCPK30 was subcloned into the pEGAD binary vector with a GFP gene and a Bar gene under the cauliflower mosaic virus (CaMV) 35S promoter, and the recombinant was transformed into onion epidermis cells by gene gun. GFP-CPK30 fusion gene transient transformation of onion epidermis cells revealed the localization of CPK30 in cell wall and cell membrane.
The physiological roles of AtCPK30 were studied using a gain-of-function approach. Seedlings of AtCPK30 transgenic lines had longer primary roots than those plants of wild-type at the early stages. Interestingly, when these plants grew on MS lack of Ca2+ including wild-type and transgenic lines, the roots of transgenic line were more sensitive to calcium, lack of Ca2+ had less effect on roots of transgenic lines than those of wild-type. Treated with several plant hormones, such as ABA, IAA, GA and BA, the roots of seedlings of transgenic line developed abnormally because they were more sensitive to hormones. Furthermore, NPA relatively less inhibited emergency of lateral roots of transgenic line than those of the wild-type. These results suggest that AtCPK30, as a positive regulator, involved in the hormone-signaling pathways.
引文
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