摘要
青葙(Celosia argentea L.)是苋科一年生草本植物,同时也是一种高钾植物。随着分子生物学的发展,高亲和钾转运体基因的研究已越来越深入,但主要集中在一些模式植物中,如拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、大麦(Hordeum uhulgare)等。本试验针对作物钾素营养特性改良方面的迫切需要和利用钾素营养相关基因的现状,以野生高钾植物青葙为材料,对其钾营养特性进行了研究,克隆到青葙高亲和钾转运体基因,并对其表达和序列进行了分析。主要研究结果如下：
1.在不同供钾水平下,青葙Km值随着外界K+浓度的升高而升高,且在高于0.20mmol/L时,其值急剧上升,很可能在高于此浓度时K+的吸收机制发生了改变。在低钾条件下,青葙对K+的亲和力更大,说明青葙是高亲和钾的植物。同时青葙低钾条件下根冠比值要大于在高钾条件下的比值,说明在低钾条件下青葙根系机能活性强。外界钾对青葙叶绿素含量影响不大,但是K+浓度过低或过高都会影响青葙的光合作用。
2.正常供钾条件下青葙各部位钾含量大小为茎>根>叶,且含量都达50mg/g以上；外界K+浓度直接影响青葙体内钾含量的分布。
3.利用RT-PCR和RACE法克隆得到青葙一个HAK基因,其全长cDNA长2498bp,共编码777个氨基酸,将该基因提交到GenBank,登录号为JF719837,并命名为CaHAK1。
4.对CaHAK1进行Real-Time PCR的表达分析,结果显示其主要在根中表达,且在较低钾浓度下其相对表达量较正常钾浓度下的要高。说明低钾对青葙HAK基因具有诱导作用。
5.进行同源性分析,结果显示CaHAK1核苷酸序列与其他植物中的HAK基因都具有较高的相似性,且其所编码的氨基酸序列与其他植物如冰叶日中花、商陆、棉花的HAK的相似性分别达87%、86%、79%。推测的蛋白质分子量为87.5518kD,等电点pI为7.89。经功能保守域和系统进化分析发现CaHAK1与其它植物的HAK基因的保守性高,亲缘关系很近,说明CaHAK1是青葙的高亲和钾转运体基因。
Celosia argentea L. is an amaranthaceae annual herb. And it is also a typical K-rich plant. More about genes of high affinity potassium transporters has been known with the development of molecular biology. But those reseaarch mainly focus on some model plants such as arabidopsis, rice, barley and other crops. In this study, in order to meet the need of potassium nutrition characteristic improvement for crop and use of potassium nutrition related gene. Celosia argentea as the material has been investigated about its k nutrition characteristics, and a high affinity potassium transporter gene has been cloned. And also CaHAKl gene expression and sequence have been analyzed. The main research results were as follows:
1. Under the different potassium level, with K+ concentration increasing, the Km value of Celosia argentea was also increasing. And under K+ concentration higher than 0.20 mmol/L, its value has risen sharply. Probably in this concentration the K+absorption mechanism of Celosia argentea has been changed. Under low potassium condition, the affinity of K+ was greater than other plant, indicated that Celosia argentea was a high affinity potassium plant. Meanwhile under low potassium condition, the root/shoot ratio of Celosia argentea was higher than the ratio under high potassium condition, this showed that its root activity was strong in low potassium condition. The chlorophyll content of Celosia argentea did not change significantly in different K+ concentration. But the K+ concentration of solution would influence the photosynthesis when concentration was too low or too high.
2. Under the normal potassium condition, the content of potassium in stem was higher than root and leaf. And the contents were 50 mg/g above. Potassium supply directly affects distribution of potassium in plants.
3. A HAK gene from Celosia argentea has been cloned using RT-PCR and RACE. Its cDNA consisted 2498 bp and could encode 777 amino acids. This gene was submitted to GenBank with an accession number JF719837, and was designated as CaHAKl.
4. Real-Time PCR showed that the CaHAK1 was detected in all tissue of Celosia argentea, but it was main expressed in roots. And under lower potassium concentration the relative expression of CaHAKl was higher than the relative expression under normal potassium concentration. These showed that CaHAKl could be induced expression under lower potassium concentration.
5. For homology analysis, the results indicated that the nucleotides sequence of CaHAK1 has high similarity with other HAK genes. And the homology of amino acid sequence was high identity with other plants such as Mesembryanthemum crystallinum with 87%, Phytolacca acinosa with 86%, Gossypium hirsutum with 79%. Molecular weight of putative CaHAK1 protein is 87.5518 kD, and isoelectric pI is 7.89. By function conservative domain and phylogenetic analysis, CaHAKl has high conservation and closely relationship with other HAK genes, the results indicated that the CaHAKl gene was the high affinity potassium transporter gene of Celosia argentea.
引文
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