柠条CkCAD基因的克隆转化及其抗旱功能分析

Cloning and trans format ion of the CkCAD gene in Caragana kor shinski i and analysis of its drought resistance function

干旱胁迫严重影响植物的生长发育甚至生存状态,是限制我国西北荒漠植被恢复的主要非生物胁迫因素之一。实验室前期研究发现随黄土高原由南向北降水减少,柠条苯丙烷生物合成是差异表达最显著的代谢途径。本研究遂以柠条苯丙烷合成途径中木质素合成酶基因CkCAD为研究对象,生信分析表明该基因开放阅读框全长1074 bp,编码357个氨基酸;蛋白序列比对发现柠条CkCAD与非洲相思豆、蒺藜苜蓿、大豆和花生的亲缘关系较近,相似度均在80%以上,其中与非洲相思豆ApCAD最为相似。蛋白质偏酸性且为亲水性蛋白,无跨膜结构域,亚细胞定位于细胞质中。通过农杆菌介导法利用过表达载体pCAMBIA1302将柠条CkCAD转入野生型拟南芥中。在筛选获得T3代纯合阳性植株后进行抗旱性分析。对T3代过表达CkCAD拟南芥进行实时荧光定量PCR(qRTPCR)和Western blot检测发现CkCAD及其酶蛋白在拟南芥中呈稳定表达。相较于野生型拟南芥,T3代过表达CkCAD拟南芥叶脉长度和叶脉密度更大,脉岛长径、脉岛短径更短,脉岛密度更大,叶脉更为发达且木质素含量更高。同时发现干旱处理下T3代过表达植株的叶片萎蔫程度、丙二醛含量、相对电导率均低于野生型植株,而相对含水量则高于野生型植株。从而证实干旱胁迫下柠条木质素合成酶基因CkCAD可以促进木质素合成进而提高过表达拟南芥植株的抗旱性。

Drought stress seriously affects the growth and development and even survival status of plants,which is one of the main abiotic stress factors restricting the restoration of desert vegetation in northwest China. Previous studies in our laboratory have found that phenylpropanoid biosynthesis of Caragana korshinskii was the most significant metabolic pathway for differential expression as precipitation decreases following the Loess Plateau from south to north. This study focused on the lignin synthase gene CkCAD in the phenylpropanoid biosynthesis pathway of C. korshinskii. A bioinformatics analysis showed that open reading frame of the CkCAD gene is 1074 bp,encoding 357 amino acids. Protein sequence alignment showed that CkCAD is closely related to Abrus precatorius,Medicago truncatula,Glycine max and Arachis hypogaea,with a similarity of more than 80%. It is most similar to the A. precatorius ApCAD. The protein is acidic and hydrophilic,with no transmembrane domain,and its subcellular location is in the cytoplasm. C. korshinskii CkCAD was transferred into wild-type Arabidopsis thaliana with overexpression vector pCAMBIA1302 by the Agrobacterium-mediated method to obtain positive plants. The drought resistance was analyzed after screening and obtaining T3 generation homozygous positive plants. It was found by Real-time fluorescence quantitative PCR and Western blot methods that CkCAD and its enzyme protein were stably expressed in T3 Arabidopsis plants overexpressing CkCAD. Compared with wild type A. thaliana,the A.thaliana T3 generation overexpressing CkCAD has longer vein length and greater vein density,shorter vein island length and shorter diameter, greater vein island density, more developed veins and higher lignin content.Simultaneous drought treatment revealed that the degree of leaf wilting,malondialdehyde content,and electrolyte leakage of overexpressed plants were lower than those of the wild type plants,while the relative water content was higher than that of the wild type plants. This study confirmed that the lignin synthase gene CkCAD of C. korshinskii can promote lignin synthesis and improve drought resistance of overexpressed A. thaliana plants under drought stress.