Cloning and Salt Tolerance Validation of AhLea-D Gene in Peanut (Arachis hypogaea)
JIANG Ping-Ping1,2, PAN Lei-Lei1,2, HUANG Jian-Bin1, JI Hong-Chang1, TANG Yan-Yan1, YU Ming-Yang3, ZHU Hong1, SUI Jiong-Ming1, WANG Jing-Shan1, QIAO Li-Xian1,*
1 College of Agronomy, Qingdao Agricultural University / Shandong Provincial Peanut Industry Cooperative Innovation Center, Qingdao 266109, China; 2 College of Life Science, Qingdao Agricultural University/Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao 266109, China; 3 Foodlink Food Company Ltd., Pingdu 266706, China
Abstract：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 CO2 concentration (Ci), and transpiration rate (Tr) were analyzed and determined, which showed that the Pn, Gs and Tr of transgenic plants were higher than those of the non-transgenic plants, and the Ci was lower in transgenic plants than non-transgenic plants. The superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities and malonic dialdehyde (MDA) content of the plants were also analyzed and determined, which showed that the activities of SOD, POD and CAT of transgenic plants were significantly higher than those of control plants (P<0.05), and the content of MDA was lower than that of the control plants. Based on the above results, it was speculated that over-expression of the AhLea-D gene increased the salt tolerance of the transgenic peanut plants by maintaining Pn and photosynthetic efficiency, and by keeping the activities of antioxidant enzymes to eliminate free radicals. The results might provide theoretical basis for developing a new stress-resistant breeding approach in peanut, and also provide a new salt tolerance candidate gene.
[1] 陈娜, 程果, 陈明娜, 等. 2018. 花生胚胎发育晚期丰富蛋白基因AhLEAL的克隆及其在非生物胁迫下的表达分析[J]. 花生学报, 47(2): 6-12. (Chen N, Cheng G, Chen M N.2018. Cloning and expression analysis of late embryo genesis abundant protein gene a AhLeaL under abiotics stress conditions in peanut (Arachis hypogaea L.)[J]. Journal of Peanut Science, 47(2): 6-12. [2] 刘莉娜, 张卫强, 黄芳芳, 等. 2019. 盐胁迫对银叶树幼苗光合特性与叶绿素荧光参数的影响[J]. 森林与环境学报, 39(6): 601-607. (Liu L N, Zhang W Q, Huang F F, et al.2019. Effects of NaCl stress on the photosynthesis and cholorophyll fluorescence of Heritiera littoralis seedlings[J]. Journal of Forest and Environment, 39(6): 601-607. [3] 刘新. 2015. 植物生理学实验指导[M]. 中国农业出版社, 北京. pp. 59-68. (Liu X, 2015. Plant Physiology Experiment Guide[M]. China Agricultural Press, Beijing. pp. 59-68.) [4] 聂利珍, 刘红葵, 李晓东, 等. 2017. 沙冬青脱水素基因提高转基因紫花苜蓿的耐旱性[J]. 基因组学与应用生物学, 36(7): 2947-2953. (Nie L Z, Liu H K, Li X D, et al.2017. AmDHN gene from Ammopiptanothus mongolicus improved the drought tolerance of transgenic Alfalfa (Medicago Sativa)[J]. Genomics and Applied Biology, 36(7): 2947-2953.) [5] 宋汝涛, 陈新利, 于明洋, 等. 2015. 以木糖作为筛选剂的花生子叶外植体遗传转化[J]. 农业生物技术学报, 23(9): 1141-1148. (Song R T, Chen X L, Yu M Y, et al.2015. The genetic transformation of peanut (Arachis hypogaea L.) cotyledon explants using xylose as screening reagent[J]. Journal of Agricultural Biotechnology, 23(9): 1141-1148. [6] 孙守文, 李宏, 郑朝晖, 等. 2011. 6种新疆主栽树种耐盐能力及盐胁迫下光合特性分析[J]. 西南林业大学学报, 31(05): 10-14. (Sun S W, Li H, Zheng C H, et al.2011, Study on salt tolerance of six main tree species in Xinjiang and their photosynthetic characteristics under salt stress[J]. Journal of Southwest Forestry University, 31(05): 10-14.) [7] 张冠初, 张智猛, 慈敦伟, 等. 2018. 干旱和盐胁迫对花生渗透调节和抗氧化酶活性的影响[J]. 华北农学报, 33(3): 176-181. (Zhang G C, Zhang Z M, Ci D W, et al.2018. Effects of drought and salt sstress on osmotic regulator and antioxidase activities[J]. Acta Agriculturae Boreali-Sinica. 33(3): 176-181.) [8] 张亨, 刘盈盈, 陈云, 等. 2018. 戈壁异常球菌Dgl5蛋白生物学功能研究[J]. 生物技术通报, 34(03): 177-184. (Zhang H, Liu Y Y, Chen Y, et al.2018. Biological identification of Dgl5 in Deinococcus gobiensis I-0[J]. Biotechnology Bulletin, 34(03): 177-184.) [9] 张宁, 孙敏善, 刘露露, 等. 2013.小麦脱水素基因TaDHN-1的特征及其对非生物胁迫响应[J].中国农业科学, 46(04): 849-858. (Zhang N, Sun M S, Liu L L, et al.2013. Characterization of a dehydrin gene TaDHN-1 and its response to abiotic stresses in Wheat[J]. Scientia Agricultura Sinica, 46(04): 849-858.) [10] 赵小波, 张廷婷, 闫彩霞, 等. 2016. 花生中三个LEA基因的克隆与表达分析[J]. 花生学报, 4: 14-19. (Zhao X B, Zhang T T, Yan C X, et al.2016. Cloning and expression analysis of three LEA genes in peanut[J]. Journal of Peanut Science, 4: 14-19) [11] Akanksha S, Kumari S, Jitendra K, et al.2017. Effects of drought, heat and their interaction on the growth, yield and photosynthetic function of Lentil (Lens culinaris Medikus) genotypes varying in heat and drought sensitivity[J]. Frontiers in Plant Science, 8: 1776. [12] Charfeddine S, Saidi M N, Charfeddine S, et al.2015. Genome wide identification and expression profiling of the late embryogenesis abundant genes in potato with emphasis on dehydrins[J]. Molecular Biology Reports, 42(7): 63-74. [13] Chen Z J, Zhao W S, Ge D F, et al.2018. LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.)[J]. Plant Journal, 95(3): 516-528 [14] Close T J.1996. Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins[J]. Physiologia Plantarum, 97(04): 795-803. [15] Du D, Zhang Q, Cheng T, et al.2013. Genome wide identification and analysis of late embryogenesis abundant (LEA) genes in Prunus mume[J]. Molecular Biology Reports, 40(2): 37-46. [16] Dure L, Greenway S C, Galau G A.1981. Developmental biochemistry of cotton seed embryogenesis and germination: Changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J]. Biochemistry, 20(14): 4162-4168. [17] Dure L, Crouch M, Harada J Thd Ho, et al.1989. Common amino acid sequence demains among the Lea proteins of higher plants[J]. Plant Molecular Biology, 12(5): 475-486. [18] Gao J, Lan T.2016. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli[J]. Science Report, 6(1): 19467. [19] Magwanga R O, Lu P, Kirungu J N, et al.2018. Cotton late embryogenesis abundant (LEA2) genes promote root growth and confer drought stress tolerance in transgenic Arabidopsis thaliana[J]. Genes Genomes Genetics, (8): 2781-2802. [20] Park S C, Kim Y H, Jeong J C, et al.2011. Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli[J]. Planta, 233(3): 621-634. [21] Reddy P S, Reddy G M, Pandey P, et al.2012. Cloning and molecular characterization of a gene encoding late embryogenesis abundant protein from Pennisetum glaucum: Protection against abiotic stresses[J]. Molecular Biology Reports, 39(6): 63-74. [22] Tiwari P, Indoliya Y, Singh P K, et al.2018. Role of dehydrin-FK506-bindingproteins complex in enhancing drought tolerance through ABA-mediated signaling pathway[J]. Environmental and Experimental Botany, (158): 136-149 [23] Tiwari V, Chaturvedi A K, Mishra A, et al.2015. Introgression of the SbASR-1 gene cloned from a halophyte Salicornia brachiata enhances salinity and drought endurance in transgenic groundnut (Arachis hypogaea L.) and acts as a transcription factor[J]. PLOS ONE, 10(7): e0131567. [24] Wang L, Li X, Chen S, et al.2009. Enhanced drought tolerance in transgenic Leymus chinensis plants with constitutively expressed wheat TaLEA3[J]. Biotechnology Letters, 31(2): 313-319 [25] Yang Z, Sheng J Y, Lv K, et al.2019. Y2SK2 and SK3 type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants[J]. Plant Science, 284:143-160. [26] Zheng J, Su H, Lin R, et al.2019. Isolation and characterization of an atypical LEA gene (IpLEA) from Ipomoea pes-caprae conferring salt/drought and oxidative stress tolerance[J]. Scientific Reports, 9(1): 14838.
[1]
刘阳杰, 何美敬, 崔顺立, 杨鑫雷, 穆国俊, Charles Y CHEN, 刘立峰. 美国花生种质资源果腐病抗性分子标记的筛选及分析[J]. 农业生物技术学报, 2019, 27(4): 743-751.
