Gm4CL2基因对拟南芥和紫花苜蓿耐铝性的影响  

Effects of the Gm4CL2 gene on aluminum tolerance of Arabidopsis and alfalfa

作　　者：林涛[1,2] 张立娇 韩蓉蓉 玉永雄 蒋曹德[1] LIN Tao;ZHANG Li-jiao;HAN Rong-rong;YU Yong-xiong;JIANG Cao-de(College of Animal Science and Technology,Southwest University,Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization,Chongqing 400715,China;Guang’an Feed Industry Management Station,Guang’an 638000,China)

机构地区：[1]西南大学动物科学技术学院,重庆市草食动物资源保护与利用工程研究中心,重庆400715 [2]广安市饲料工业管理站,四川广安638000

出　　处：《草业学报》2022年第10期122-134,共13页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(32171673);西南大学西南山地生态循环农业国家级培育基地项目资助。

摘　　要：4CL(4-coumarate:coenzyme A ligase)是木质素合成途径关键酶,已被证明在生物和非生物胁迫、机械损伤抗性等生物过程中具有重要作用,但与柠檬酸分泌相关的耐铝功能还没有报道。本研究选择丹波黑大豆Gm4CL2,利用RT-PCR技术克隆其全长编码序列,蛋白质序列多重序列比对和进化树分析不同物种间的亲缘关系,农杆菌介导浸花法和叶盘法分别遗传转化拟南芥和紫花苜蓿,qRT-PCR技术检测基因的表达水平。序列分析结果发现,Gm4CL2全长编码序列为1668 bp,该基因编码555个氨基酸,为双子叶植物Ⅰ类4CL。Real-time PCR结果显示,50μmol·L^(-1) AlCl_(3)(pH 4.5)特异诱导Gm4CL2在丹波黑大豆幼苗0~2 cm的根尖组织表达;过表达Gm4CL2拟南芥,在铝处理条件下其根尖AtMATE、AtSTAR1和AtSTAR2表达量显著上调(P<0.05)。Al^(3+)胁迫条件下,过表达Gm4CL2拟南芥根相对伸长量、根尖SOD、POD活性和柠檬酸分泌量显著高于野生型,根尖伊文思蓝和铬天青S染色以及Al^(3+)、ROS、MDA含量显著低于野生型(P<0.05);过表达Gm4CL2紫花苜蓿根相对伸长率、根尖柠檬酸分泌量和生物量显著高于野生型,根尖Al^(3+)含量、伊文思蓝染色显著低于野生型(P<0.05)。细胞壁成分分析表明,Al^(3+)胁迫和非胁迫下,过表达Gm4CL2拟南芥根尖果胶、咖啡酸、阿魏酸比野生型显著降低(P<0.05);Al^(3+)胁迫下,过表达Gm4CL2拟南芥根尖木质素含量显著降低,但是4-香豆酸含量显著升高(P<0.05)。上述结果表明,Gm4CL2为耐铝基因,该基因通过促进细胞壁修饰和柠檬酸分泌提高拟南芥和紫花苜蓿的耐铝性。4CL(4-coumarate:coenzyme A ligase)is a key enzyme in the lignin synthesis pathway,which has been shown to play an important role in biological and abiotic stress,mechanical damage resistance and other biological processes.However,the aluminum tolerance of Gm4CL2 has not been reported.In this work,Gm4CL2 of Glycine max cv.Tamba was chosen,and its full-length coding sequence was cloned by RT-PCR.The genetic relationship among Gm4CL2 protein sequences was analyzed using multiple sequence alignment and phylogenetic tree analysis.Gm4CL2 was transformed into Arabidopsis thaliana and Medicago sativa by Agrobacterium-mediated floral dip and leaf disk method,respectively.Gene expression levels were detected using qRT-PCR.The results of sequence analysis revealed a 1668 bp Gm4CL2 full-length coding sequence,which encodes 555 amino acids and belongs to class I 4CL in dicotyledons.Real-time PCR results showed that Gm4CL2 expression was specifically induced at 50μmol·L^(−1) AlCl_(3)(pH 4.5)in 0-2 cm root tips of G.max cv.Tamba seedlings,and the expression levels of AtMATE,AtSTAR1 and AtSTAR2 were significantly up-regulated in root tips of Arabidopsis overexpressing Gm4CL2 under aluminum treatment(P<0.05).Under Al^(3+) stress,relative root elongation,activities of SOD(superoxide dismutase)and POD(peroxidase)and citrate secretion were significantly elevated,while Evans blue and chrome azure staining and the content of Al^(3+),ROS(reactive oxygen species)and MDA(malondialdehyde)were significantly lower in the roots of Arabidopsis overexpressing Gm4CL2 than in those of the wild type(P<0.05).The relative root elongation,citrate secretion and biomass of alfalfa overexpressing Gm4CL2 were significantly higher,while Al^(3+) content and Evans blue staining were significantly lower than those of the wild type(P<0.05).Cell wall composition analysis showed that pectin,and caffeic and ferulic acid contents in Arabidopsis roots overexpressing Gm4CL2 were significantly lower than those of the wild type under both Al^(3+) and non-Al^(3+)

关 键 词：Gm4CL2 耐铝性 柠檬酸分泌 拟南芥 紫花苜蓿 

分 类 号：S541.9[农业科学—作物学]