花生AhLea-D基因的克隆及耐盐性验证  

Cloning and Salt Tolerance Validation of AhLea-D Gene in Peanut(Arachis hypogaea)

作　　者：姜平平 潘雷雷[1,2] 黄建斌 纪红昌 唐艳艳 于明洋 朱虹 隋炯明 王晶珊 乔利仙 JIANG Ping-Ping;PAN Lei-Lei;HUANG Jian-Bin;JI Hong-Chang;TANG Yan-Yan;YU Ming-Yang;ZHU Hong;SUI Jiong-Ming;WANG Jing-Shan;QIAO Li-Xian(College of Agronomy,Qingdao Agricultural University/Shandong Provincial Peanut Industry Cooperative Innovation Center,Qingdao 266109,China;College of Life Science,Qingdao Agricultural University/Key Lab of Plant Biotechnology in Universities of Shandong Province,Qingdao 266109,China;Foodlink Food Company Ltd.,Pingdu 266706,China)

机构地区：[1]青岛农业大学农学院/山东省花生产业协同创新中心,青岛266109 [2]青岛农业大学生命科学学院/山东省高校植物生物技术重点实验室,青岛266109 [3]青岛福德隆食品有限公司,平度266706

出　　处：《农业生物技术学报》2020年第5期811-822,共12页Journal of Agricultural Biotechnology

基　　金：国家自然基金(31872875);农业部油料作物生物学与遗传育种重点实验室开放课题基金资助(KF2018008)。

摘　　要：花生(Arachis hypogaea)属于中度耐盐作物,可在0.3%的盐碱地上正常生长,但产量会因盐胁迫造成的伤害而减产,挖掘花生耐盐基因、创制耐盐种质、选育耐盐品种成为花生在盐碱地有效利用的关键问题。本研究利用已构建的花生耐盐突变体及其诱变亲本转录组数据库,对花生中的胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins, Lea)基因进行差异表达筛选,获得盐胁迫差异表达基因AhLeaD。为了进一步研究AhLea-D基因与花生耐盐性的关系,从花生耐盐突变体中克隆AhLea-D基因(GenBank No. MN393179)全长c DNA序列,该序列全长534 bp,编码的AhLea-D蛋白含有177个氨基酸,分子量为17.89 kD,等电点为5.78,为酸性蛋白。亲水性平均系数为-0.983,蛋白的不稳定指数小于40,属于稳定的亲水性蛋白。构建AhLea-D基因的过表达载体PCAMBIA1301-AhLea-D,并利用花粉管通道法转入花生受体品种’花育23’。收获所注射花蕾上的荚果,利用PCR扩增法对收获的籽粒进行分子鉴定,结果表明转基因籽粒阳性率为52%。q RT-PCR检测结果表明,转基因植株中AhLea-D基因的表达量是非转基因对照植株的7~12倍。利用250 mmol/L NaCl对转基因植株和非转基因对照植株进行盐胁迫处理,1周后对照植株叶片出现明显的萎蔫、发黄现象,而转基因植株无明显变化。转基因植株的净光合速率、气孔导度、蒸腾速率等均高于对照植株,而胞间CO2浓度低于对照植株。转基因植株中超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)、过氧化氢酶(catalase, CAT)的酶活性均显著高于非转基因对照植株,而丙二醛(malonic dialdehyde, MDA)含量则显著低于非转基因对照植株(P<0.05)。本研究结果表明,AhLea-D基因的过量表达提高了转基因花生的耐盐性。本研究结果将为在花生中开拓新的抗逆育种途径提供理论依据,并为花生抗逆耐盐育种提供耐盐新�Peanut(Arachis hypogaea)was regarded to have the characteristic of moderate salt tolerance.It has become an important way during the full utilization of saline and alkali soil by searching salt-tolerant related genes,creating salt-tolerant germplasms and breeding salt-tolerant varieties in peanut.The peanut transcriptome database of salt tolerant mutant and mutagenic parent was used to screen the differentially expressed genes of late embryogenesis abundant proteins(Lea)family,and AhLea-D was obtained successfully.In order to make a further study on the role of the AhLea-D in peanut salt tolerance,the full length cDNA sequence of AhLea-D(GenBank No.MN393179)containing 534 bp was obtained by PCR amplification,which encoded an acidic protein with 177 amino acids,molecular weight 17.89 kD,theoretical isoelectric point 5.78.AhLea-D protein had average coefficient of hydrophilicity-0.983,instability index less than 40,and it belonged to the stable hydrophilic protein.AhLea-D was ligated to plasmid PCAMBIA1301 to construct the over-expression recombinant vector PCAMBIA1301-AhLea-D,which was then transferred to peanut variety'Huayu23'by Agrobacterium-mediated pollen tube channel method.The pods from the infected flowers were harvested and the seeds were identified by PCR amplification method.The result showed the positive rate of transformation was about 52%.The expression level of AhLea-D gene was detected by qRT-PCR amplification,and the expression level of the transgenic plants were found to be 7~12 times higher than that of the non-transformed control plants.Transgenic plants and controls were treated with salt stress by irrigating 250 mmol/L NaCl.After a week of stress treatment,the control leaves showed obvious wilting and yellowing,while the transgenic plants showed no obvious changes.The photosynthesis parameter including photosynthetic net rate(Pn),stomatal conductance(Gs),intercellular CO 2 concentration(Ci),and transpiration rate(Tr)were analyzed and determined,which showed that the Pn,Gs and Tr of transgenic

关 键 词：花生 花生胚胎发育晚期丰富蛋白基因(AhLea-D) 过量表达 遗传转化 耐盐性 

分 类 号：S565.2[农业科学—作物学] Q785[生物学—分子生物学]