转OvBAN/bar双价基因的紫花苜蓿对虫蚀及除草剂的耐受性分析

doi:10.11686/cyxb2019152

摘要/Abstract

摘要： 以“甘农3号”紫花苜蓿为受体,利用农杆菌介导法导入双价基因OvBAN/bar,通过PCR检测和Southern杂交分析表明,4个转基因株系含有目的基因且均出现杂交条带,其中2个株系为单拷贝,1个株系为双拷贝,1个株系为三拷贝;对获得的4株转基因苜蓿进行qRT-PCR分析表明,具有三拷贝数的1个转基因株系OvBAN基因的表达量最高,其次为双拷贝数的2个转基因株系,并且这些株系的花青素还原酶活性及缩合单宁含量均显著高于野生型苜蓿。以过量表达OvBAN基因的3株转基因苜蓿为试验材料,进行抗蚜性和除草剂耐受性分析。结果表明,与野生型苜蓿相比,转OvBAN/bar基因苜蓿对苜蓿蚜都具有较好的抗性,蚜虫抑制率为78%~93%;用0.5%的市售草铵膦喷施各株系,10 d后各转基因株系除个别叶片枯萎死亡,其他叶片及茎秆生长状况良好,而野生型苜蓿全部枯萎死亡,表明bar基因已整合到转基因苜蓿基因组中并稳定表达。综上所述,转OvBAN/bar基因紫花苜蓿表现出良好的抗虫性和除草剂耐受性。

关键词: 转基因苜蓿, OvBAN基因, bar基因, 抗虫性, 除草剂耐受性

Abstract: The bifunctional OvBAN/bar gene was inserted into cell lines of the alfalfa cultivar ‘Gannong No.3’ using Agrobacterium-mediated transformation. PCR and southern blot analysis showed that four transgenic lines that contained the target gene were obtained, and all showed hybridization bands; two lines had single copies, one had two copies, and one had three copies. Expression of OvBAN in the four transgenic plants was analyzed by quantitative RT-PCR, and confirmed to be most highly expressed in the line with 3 copies, and also highly expressed in the two lines with two copies. In these transgenic alfalfa plants, anthocyanin reductase activity and condensed tannin content were significantly higher than in untransformed alfalfa. These three lines of transgenic alfalfa over-expressing the OvBAN/bar gene were tested for resistance to aphids, and the transgenic material demonstrated stronger aphid resistance than non-transgenic plants, with a 78%-93% reduction in aphid numbers per plant. Transgenic plants were then sprayed 0.5% glufosinate to test for herbicide resistance. After 10 days, the transgenic plants were still healthy, while the untransformed plants withered and eventually died, indicating that the bar gene had been integrated into the genome and stably expressed. In summary, the study indicated that introduction of the OvBAN/bar bifunctional genes significantly enhanced insect resistance and herbicide tolerance in transgenic alfalfa plants.

Key words: transgenic alfalfa, OvBAN, bar, insect resistance, herbicide tolerance

董文科, 陈春艳, 马晖玲. 转OvBAN/bar双价基因的紫花苜蓿对虫蚀及除草剂的耐受性分析[J]. 草业学报, 2019, 28(7): 159-167.

DONG Wen-ke, Chen Chun-yan, MA Hui-ling. Analysis of insect resistance and herbicide resistance in transgenic alfalfa plants over-expressing the OvBAN/bar gene[J]. Acta Prataculturae Sinica, 2019, 28(7): 159-167.

参考文献

[1] Hong F Z.Alfalfa science. Beijing: China Agriculture Press, 2009.
洪绂曾. 苜蓿科学. 北京:中国农业出版社, 2009.
[2] Bai Y, Gao X K, Wang Y C, et al. Field comparison of the resistance of 33 alfalfa varieties to thrips. Acta Prataculturae Sinica, 2015, 24(3): 187-194.
白宇, 高兴珂, 王业臣, 等. 33个苜蓿品种对蓟马的田间抗性比较. 草业学报, 2015, 24(3): 187-194.
[3] Zhang B, Zhou M Q, Wang J, et al. Species checklist and research status of alfalfa insect pests reported in China. Prataculturae Science, 2016, 33(4): 785-812.
张奔, 周敏强, 王娟, 等. 我国苜蓿害虫种类及研究现状. 草业科学, 2016, 33(4): 785-812.
[4] Geng H Z.Chinese alfalfa. Beijing: China Agriculture Press, 1995: 1-7.
耿华珠. 中国苜蓿. 北京:中国农业出版社, 1995: 1-7.
[5] Luo L, Yang G F, Liu Z L, et al. Control efficacy of acetamiprid and pymetrozine on alfalfa aphides and thrips and the residues in alfalfa. Chinese Journal of Grassland, 2017, 39(6): 21-25.
罗兰, 杨国锋, 刘兆良, 等. 啶虫脒和吡蚜酮对苜蓿蚜虫和蓟马的防效及残留. 中国草地学报, 2017, 39(6): 21-25.
[6] Liu Y F, He L, Wang Q, et al. Effect of and ecological safety insect-resistant Cry1Ac/sck transgenic rice on key non-target pests in paddy fields. Acta Entomologica Sinica, 2006, 49(6): 955-962.
刘雨芳, 贺玲, 汪琼, 等. 转Cry1Ac/sck基因抗虫水稻对稻田寄生蜂群落影响的评价. 昆虫学报, 2006, 49(6): 955-962.
[7] Zhou X, Xie X, Zuo J, et al. The advance on the effects of transgenic crops on natural enemies. Journal of Environmental Entomology, 2018, 40(5): 1021-1026.
周霞, 谢翔, 左娇, 等. 转基因作物对天敌的影响研究进展. 环境昆虫学报, 2018, 40(5): 1021-1026.
[8] Dong J.Isolation and transformation of DFR and ANR genes of tannins synthetic way in Medicago sativa L. Beijing: Chinese Academy of Agricultural Science Dissertation, 2012.
董洁. 紫花苜蓿浓缩单宁合成途径中DFR和ANR基因的分离及遗传转化. 北京: 中国农业科学院, 2012.
[9] Lees G L, Hinks C F, Suttill N H.Condensed tannins in some forage legumes their rolein the improvement of ruminant pasture bloat. Basic Life Science, 1992, 59: 915-934.
[10] Mangan J.Nutritional effects of tannins in animal feeds. Nutrition Research Reviews, 1988, 1(1): 209-231.
[11] Wu C Z, Zhong L, Liang P, et al. Effects of ethomyl and imidacloprid on contents of condensed tannin in cotton seedlings. Acta Phytophylacica Sinica, 2010, 37(1): 62-66.
武传志, 仲丽, 梁沛, 等. 多灭威和吡虫啉对棉苗缩合单宁含量的影响. 植物保护学报, 2010, 37(1): 62-66.
[12] Zhao W J, Zhang D, Ma L J, et al. Biosynthetic pathway, functional genes and metabolic engineering of proanthocyanidins. Plant Physiology Journal, 2009, 45(5): 509-519.
赵文军, 张迪, 马丽娟, 等. 原花青素的生物合成途径、功能基因和代谢工程. 植物生理学通讯, 2009, 45(5): 509-519.
[13] Xie D Y, Sharma S B, Paiva N L, et al. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 2003, 299: 396-399.
[14] Nesi N, Lucas M O, Auger B, et al. The promoter of the Arabidopsis thaliana BAN gene is active in proanthocyanidin-accumulating cells of the Brassica napus seed coat. Plant Cell Reports, 2009, 28(4): 601-617.
[15] Xie D Y, Sharma S B, Dixon R A.Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana. Archives of Biochemistry and Biophysics, 2004, 422(1): 91-102.
[16] Paolocci F, Robbins M P, Madeo L, et al. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiology, 2007, 143(1): 504-516.
[17] Zhu Y, Peng Q Z, Li K G, et al. Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula. Planta, 2014, 240(2): 381-398.
[18] Goplen B P, Howarth R E, Sarkar S K, et al. A search for condensed tannis in annual and perennial species of Medicago, Trigone and Onobrychis. Crop Science, 1980, 20: 801-804.
[19] Tan Y, Liu J X, Fu L Y, et al. Species identification and damage investigation of weeds in alfalfa field. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 2016, 37(6): 59-64.
谭瑶, 刘嘉鑫, 付丽媛, 等. 紫花苜蓿田杂草种类及危害调查. 内蒙古农业大学学报(自然科学版), 2016, 37(6): 59-64.
[20] Duan F P, Liang C Y, Li Y Q, et al. Research advances of Bar gene and its transgenic crops. Guihaia, 2001, 21(2): 166-172.
段发平, 梁承邺, 黎垣庆, 等. Bar基因和转Bar基因作物的研究进展. 广西植物, 2001, 21(2): 166-172.
[21] Dong W K.The transformation of BAN genes by Agrobacterium mediate and its expression analysis in alfalfa. Lanzhou: Gansu Agricultural University, 2017.
董文科. 农杆菌介导的BAN基因在紫花苜蓿中的转化和表达的研究. 兰州: 甘肃农业大学, 2017.
[22] Chen C Y.Cloning and functional analysis of two key genes in the procyanidins synthesis in Gansu sainfoin. Lanzhou: Gansu Agricultural University, 2016.
陈春艳. 甘肃红豆草原花青素合成途径的两个关键酶基因的克隆和功能分析. 兰州: 甘肃农业大学, 2016.
[23] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-delta/delta (CT) method. Methods, 2001, 25(4): 402-408.
[24] Zhang X L.Study on anthocyanin reductase in Camellia sinensis. Hefei: Anhui Agricultural University, 2009.
张宪林. 茶树中花青素还原酶的研究. 合肥: 安徽农业大学, 2009.
[25] Terrill T H, Rowan A M, Douglas G B, et al. Determination of extractable and bound condensed tannin concentration in forage plants, protein concentrate meals and cereal grains. Journal of the Science of Food and Agriculture, 1992, 58(3): 321-329.
[26] Wang M F, Yuan G H, Chen J L, et al. Aphids-resistance of wheat varieties (lines) in winter wheat region of China. Journal of Henan Agricultural Sciences, 2008, 37(8): 87-90.
王美芳, 原国辉, 陈巨莲, 等. 我国冬麦区小麦品种抗蚜性鉴定. 河南农业学报, 2008, 37(8): 87-90.
[27] Deng Y M, Chen S M, Lu A M, et al. Production and characterisation of the intergeneric hybrids between Dendranthema morifolium and Artemisia vulgaris exhibiting enhanced resistance to chrysanthemum aphid (Macrosiphoniella sanbourni). Planta, 2010, 231(3): 693-703.
[28] Han L Z, Wu K M, Peng Y F, et al. Research advances in ecological safety of insect-resistant transgenic rice. Chinese Journal of Applied & Environmental Biology, 2006, 12(3): 431-436.
韩兰芝, 吴孔明, 彭于发, 等. 转基因抗虫水稻生态安全性研究进展. 应用与环境生物学报, 2006, 12(3): 431-436.
[29] Stoger E, Williams S, Christou P, et al. Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: Effects on predation by the grain aphid Sitobion avenae. Molecular Breeding, 1999, 5(1): 65-77.
[30] Gatehouse A M, Down R E, Powell K S, et al. Transgenic potato plants with enhanced resistance to the peach potato aphid (Myzus persicae). Entomologia Experimentalis et Applicata, 1996, 79(3): 295-307.
[31] Birch A N, Geoghegan I E, Majerus M E, et al. Tri-trophic interactions involving pest aphids, predatory 2-spot ladybirds and transgenic potatoes expressing snow-drop lectin for aphid resistance. Molecular Breeding, 1999, 5(1): 75-83.
[32] Wu Y Q, Guo Y Y.Potential resistance of tannins-flavonoids in upland cotton against Helicpverpa armigera. Acta Ecologica Sinica, 2001, 21(2): 286-289.
武予清, 郭予元. 棉花单宁-黄酮类化合物对棉铃虫的抗性潜力. 生态学报, 2001, 21(2): 286-289.
[33] Chen J L, Ni H X, Sun J R, et al. Effects of major secondary chemicals of wheat plants on enzyme activity in Sitobion avenae. Acta Entomologica Sinica, 2003, 46(2): 144-149.
陈巨莲, 倪汉祥, 孙京瑞, 等. 小麦几种主要次生物质对麦长管蚜几种酶活力的影响. 昆虫学报, 2003, 46(2): 144-149.
[34] Liu X P, Ge F, Chen C P, et al. Progress in induced resistance of pines. Scientia Silvae Sinicae, 2003, 39(5): 119-128.
刘兴平, 戈峰, 陈春平, 等. 我国松树诱导抗虫性研究进展. 林业科学, 2003, 39(5): 119-128.
[35] Lin F M, Wu D, Lu Y H, et al. The relationship between the main secondary metabolites and the resistance of cotton to Apolygus lucorum. Acta Phytophylacica sinica, 2011, 38(3): 202-208.
林凤敏, 吴敌, 陆宴辉, 等. 棉花主要抗虫次生物质与其对绿盲蝽抗性的关系. 植物保护学报, 2011, 38(3): 202-208.
[36] Shen F F, Yu Y J, Zhang X K, et al. Insect resistant stock selected by introducing dogbane DNA into cotton. Acta Agriculturae Boreali-Sinica, 1999, 14(4): 34-38.
沈法富, 于元杰, 张学坤, 等. 罗布麻DNA导入棉花选育抗虫种质系. 华北农学报, 1999, 14(4): 34-38.
[37] Mo J C, Wang W X, Zhou L J, et al. Effect of quality of tung tree leaf on the growth and development of Buasra suppressaria Guenee. Entomological Knowledge, 1995, 32(2): 100-102.
莫建初, 王问学, 周丽君, 等. 油桐叶品质对油桐尺蠖生长发育的影响. 昆虫知识, 1995, 32(2): 100-102.
[38] Wu D G, Wang S S, Liu C Z, et al. Effects of herbivore stress by Acyrthosiphon pisum on the contents of tannin and physiological activity in different alfalfa cultivars. Acta Agrestia Sinica, 2011, 19(2): 351-355.
武德功, 王森山, 刘长仲, 等. 豌豆蚜刺吸胁迫对不同苜蓿品种体内单宁含量及生理活性的影响. 草地学报, 2011, 19(2): 351-355.
[39] Zou X W, Jiang Z S.Advances in studies on the insecticidal plant and its applications and prospects. Chinese Journal of Pesticides, 2004, 43(11): 481-486.
邹先伟, 蒋志胜. 杀虫植物的研究新进展及应用发展前景. 农药, 2004, 43(11): 481-486.
[40] Yang Z Q, Chen H, Tan J H, et al. Cloning of three genes involved in the flavonoid metabolic pathway and their expression during insect resistance in Pinus massoniana Lam. Genetics & Molecular Research, 2016, 15(4): 1-12.