玉米CPS1功能分析与AtLOS5调控玉米抗盐机制的研究

发布时间：2018-01-20 09:03

本文关键词： 玉米抗倒伏 CPS1 AtLOS5 抗盐 ABA　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：我国是世界上玉米第二大生产国,玉米在我国国民经济发展中起到非常重要的作用。目前由于气候条件变化以及灌溉不合理等原因,使得玉米倒伏现象越来越严重,同时大面积的土壤盐渍化导致耕地质量下降,因此提高玉米抗倒伏与抗盐性是目前急需解决的问题。一方面,在玉米栽培中,提高玉米抗倒伏目前应用最多的生长延缓剂是乙烯利,但是乙烯利的使用会造成玉米严重减产。赤霉素在调节节间生长过程中起到很关键的作用,且CPS是合成赤霉素的第一个关键酶。因此本论文对玉米CPS1进行研究,为通过调控CPS1来塑造玉米理想株型奠定坚实的基础;另一方面,ABA是重要的逆境信号转导植物激素,对玉米响应盐渍化等非生物胁迫具有重要的调节作用。在盐胁迫下,超表达AtLOS5基因能调控转基因植株一系列的生理生化反应,从而使其表现出抗盐性。主要结果如下:(1)本研究成功从玉米花粉中克隆ZmCPS1基因,全长为2568bp,编码855个氨基酸,包含DXDD底物催化位点,与其它物种CPSs的氨基酸序列进行比对,同源性大约为50%;该基因在拟南芥和烟草中都定位于质体;本研究构建了超表达35S::ZmCPS1表达载体,同时转化到野生型拟南芥和cps突变体拟南芥ga1-1和ga1-5中,RT-PCR分析表明ZmCPS1均能可在野生型拟南芥和突变体拟南芥中正常表达,Ler::ZmCPS1株系与野生型Ler相比表型没有显著差异,ga1-1::ZmCPS1和ga1-5::ZmCPS1株系均能完全恢复突变体到野生型表型;本研究利用体外细胞工程的方法成功表达并鉴定出ZmCPS1蛋白信号肽为前面的112aa,表达纯化出切掉信号肽且具有活性的ZmCPS1蛋白,进行timecoures 和 kinetics 的研究,最终得出 Kcat=0.25405S-1、KM=0.60173μM 与 Ki=162.74μM;本研究通过CRISPR基因编辑技术成功获得了玉米cps1突变体,得知CPS1在调节玉米节间伸长起到关键作用,cps1突变体表现出严重矮化的表型,能被外源的GA恢复表型。(2)本研究明确了超表达AtLOS5转基因玉米株系的抗盐性:在盐胁迫下各转基因株系中AtLOS5均能上调ABA合成基因的表达与关键酶AO的活性,从而促进ABA的积累;在盐胁迫下超表达AtLOS5能通过调控根中K+、Na+、H+净流速进而使植株体内维持较高的K+/Na+,并且调控其相关基因的表达从而表现出抗盐性;在盐胁迫下超表达AtLOS5能够维持细胞内较高的水势和渗透势并缓和根导水率的下降,调控ZmPIP家族基因的表达使植株体内保持较好的水分状况从而表现出抗盐性。
[Abstract]:China is the second largest producer of maize in the world, maize plays a very important role in the development of our national economy. The phenomenon of maize lodging is becoming more and more serious, and a large area of soil salinization leads to the decline of cultivated land quality. Therefore, it is urgent to solve the problem of improving the lodging and salt resistance of maize. On the one hand, in maize cultivation. Ethephon is the most widely used growth retarder to improve the lodging resistance of maize. However, the use of ethephon will lead to a serious reduction in maize production. Gibberellin plays a key role in regulating the growth of internode. CPS is the first key enzyme to synthesize gibberellin. Therefore, the study of maize CPS1 in this paper will lay a solid foundation for shaping the ideal plant type of maize by regulating CPS1. On the other hand, ABA is an important stress signal transduction plant hormone, which plays an important role in regulating maize response to abiotic stress, such as salinization. Overexpression of AtLOS5 gene can regulate a series of physiological and biochemical responses of transgenic plants. The main results are as follows: 1) the ZmCPS1 gene was cloned successfully from maize pollen. The total length of ZmCPS1 gene was 2568 BP, encoding 855 amino acids. The amino acid sequence of CPSs of other species was compared with the catalytic site of DXDD substrate, and the homology was about 50; The gene is located in plastids in Arabidopsis thaliana and tobacco. In this study, the overexpression vector of 35s:: ZmCPS1 was constructed and transformed into wild-type Arabidopsis thaliana and cps mutants Arabidopsis thaliana ga1-1 and ga1-5. RT-PCR analysis showed that ZmCPS1 could be expressed normally in wild type Arabidopsis thaliana and mutant Arabidopsis thaliana. There was no significant difference in phenotype between wild type Ler and wild type Ler. Both ga1-1::ZmCPS1 and ga1-5::ZmCPS1 lines could completely restore the mutant to wild-type phenotype. In this study, we successfully expressed and identified the signal peptide of ZmCPS1 protein as 112aa in vitro by cell engineering in vitro, and expressed and purified the ZmCPS1 protein with the activity of cut off signal peptide. Timecoures and kinetics were studied and finally Kcat=0.25405S-1 was obtained. KM=0.60173 渭 M and Ki=162.74 渭 M; In this study, cps1 mutants of maize were successfully obtained by CRISPR gene editing technique, and it was found that CPS1 plays a key role in regulating internode elongation of maize. Cps1 mutants showed severe dwarfing phenotype. Phenotypic recovery by exogenous GA. In this study, we confirmed the salt tolerance of transgenic maize lines with overexpression of AtLOS5: under salt stress, AtLOS5 could up-regulate the expression of ABA synthesis gene and the activity of key enzyme AO. In order to promote the accumulation of ABA; The overexpression of AtLOS5 under salt stress could maintain a high K / Na in the plant by regulating the net flow rate of K ~ (2 +) Na in the root. And the expression of its related genes was regulated to show salt resistance. Under salt stress, overexpression of AtLOS5 could maintain high water potential and osmotic potential in the cells and alleviate the decrease of root water conductivity. Regulating the expression of ZmPIP family genes made the plants maintain good water status and showed salt resistance.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513

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