紫花苜蓿蓝光受体CRY2B过表达载体构建及转化

doi:10.11733/j.issn.1007-0435.2017.05.017

草地学报 ›› 2017, Vol. 25 ›› Issue (5): 1029-1036.DOI: 10.11733/j.issn.1007-0435.2017.05.017

紫花苜蓿蓝光受体CRY2B过表达载体构建及转化

樊文娜¹, 李润林¹, 王占彬¹, 杜红旗², 赵凌平¹, 王成章²

1. 河南科技大学动物科技学院, 河南洛阳 471003;
2. 河南农业大学牧医工程学院, 河南郑州 450002

收稿日期:2016-04-26 修回日期:2017-09-14 出版日期:2017-10-15 发布日期:2018-01-25
通讯作者: 王成章,E-mail:wangchengzhang@263.net
作者简介:樊文娜(1981-),女,河南许昌人,博士,讲师,E-mail:chou0516@163.com
基金资助:
河南科技大学博士科研启动基金（13480078）；国家牧草产业技术体系（CARS-35）资助

Construction of Blue Light Receptor CRY2B over Expression Vector and Genetic Transformation in Medicago Sativa

FAN Wen-na¹, LI Run-lin¹, WANG Zhan-bin¹, DU Hong-qi², ZHAO Ling-ping¹, WANG Cheng-zhang²

1. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan Province 471003, China;
2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province 450002, China

Received:2016-04-26 Revised:2017-09-14 Online:2017-10-15 Published:2018-01-25

摘要/Abstract

摘要：

短日照是影响苜蓿秋眠性的主要因素，即光周期效应。根据转录组测序的结果，对主要的蓝光受体隐花色素CYR2B基因和苜蓿秋眠性之间关系的研究可以从光受体途径来揭示苜蓿秋眠性调控机制，本试验首先克隆紫花苜蓿1级品种‘驯鹿’隐花色素CRY2B基因的完整开放阅读框（696~2553 bp），设计1对含有酶切位点的特异性引物CRY2B-F，CRY2B-R，通过多次酶切和连接，将克隆目的片段连接到植物表达载体pCAMBIA3301上得到重组载体pCAMBIA3301-CRY2B，成功地构建了紫花苜蓿CRY2B的过表达载体，并利用农杆菌介导pCAMBIA3301-CRY2B成功转化苜蓿植株，为进一步研究CRY2B调控苜蓿秋眠性分子机制奠定基础。

关键词: 苜蓿, CRY2B, 秋眠性, 载体构建, 转化

Abstract:

The short-day is one of the main factors affected alfalfa fall dormancy, which is called as the photoperiodic effect. Based on the results of alfalfa transcriptome sequencing, the relationship between the main photoreceptors-CRY2B gene and alfalfa fall dormancy may reveal the regulation mechanism of alfalfa fall dormancy. In this experiment, cryptochrome gene——CRY2B of Reindeer (the variety of fall dormancy class NO.1) with complete open reading frame (696~2553 bp) was cloned. For the design of specific primers with the restriction sites (CRY2B-F and CRY2B-R), the cloned fragment was connected to the plant expression vector pCAMBIA3301 to get a recombinant vector pCAMBIA3301-CRY2B after enzymes digestion, clean up and ligation. Agrobacterium-mediated genetic transformation using alfalfa cotyledonary node was explored, it laid the foundation for the further research of CRY2B regulating fall dormancy molecular mechanism.

Key words: Medicago sativa, CRY2B, Fall dormancy, Vector construct, Transformation

中图分类号:

S541

樊文娜, 李润林, 王占彬, 杜红旗, 赵凌平, 王成章. 紫花苜蓿蓝光受体CRY2B过表达载体构建及转化[J]. 草地学报, 2017, 25(5): 1029-1036.

FAN Wen-na, LI Run-lin, WANG Zhan-bin, DU Hong-qi, ZHAO Ling-ping, WANG Cheng-zhang. Construction of Blue Light Receptor CRY2B over Expression Vector and Genetic Transformation in Medicago Sativa[J]. Acta Agrestia Sinica, 2017, 25(5): 1029-1036.

0
/ / 推荐

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://manu40.magtech.com.cn/Jweb_cdxb/CN/10.11733/j.issn.1007-0435.2017.05.017

https://manu40.magtech.com.cn/Jweb_cdxb/CN/Y2017/V25/I5/1029

参考文献

[1] Wang C, Ma B, Yan X, et al. Yields of alfalfa varieties with different fall-dormancy levels in a temperate environment[J]. Agronomy Journal,2009,101(5):1146-1152
[2] Wang C, Ma B, Han J, et al. Photoperiod effect on phytochrome and abscisic acid in alfalfa varieties differing in fall dormancy[J]. Journal of Plant Nutrition,2008,31(7):1257-1269
[3] Lang G, Early J, Martin G, et al. Endodormancy, Paradormancy, Ecodormancy-Physiological Terminology and Classification for Dormancy Research[J]. Hortscience,1987,22(3):371-377
[4] Wang C, Ma B L, Han J, et al. Photoperiod effect on phytochrome and abscisic acid in alfalfa varieties differing in fall dormancy[J]. Journal of Plant Nutrition,2008,31(7):1257-1269
[5] 李向林,万里强. 苜蓿秋眠性及其与抗寒性和产量的关系[J]. 草业学报,2004,13(3):57-61
[6] Shih S, Jung G, Shelton D. Effects of temperature and photoperiod on metabolic changes in alfalfa in relation to cold hardiness[J]. Crop Science,1967,7(4):385-389
[7] Barnes D K, Smith D M, Stucker R E, et al. Fall dormancy in alfalfa:A valuable predictive tool[J]. Agricultural Reviews and Manuals, Science and Education Administration,1979,34
[8] Staub J M, Wei n, Deng X-W. Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis[J]. The Plant Cell Online,1996,8(11):2047-2056
[9] Jarillo J A, Capel J, Tang R-H, et al..An Arabidopsis circadian clock component interacts with both CRY1 and phyB[J]. Nature,2001,410(6827):487-490
[10] S Zhang, Y Shi, N Cheng, et al. De novo characterization of fall dormant and nondormant alfalfa (Medicago sativa L.) leaf transcriptome and identification of candidate genes related to fall dormancy[J]. PloS one,2015:e0122170
[11] Storey J D. The positive false discovery rate:A Bayesian interpretation and the q-value[J]. Annals of statistics,2003:2013-2035
[12] 韩利芳,张玉发. 烟草MnSOD基因在保定苜蓿中的转化[J]. 生物技术通报,2004,(1):39-46
[13] 李志亮,黄丛林,张秀海,等. 利用基因枪法向高羊茅导入P5CS基因的研究[J]. 园艺学报,2005,32(4):653-657
[14] 吴关庭,陈锦清,胡张华,等. 根癌农杆菌介导转化获得耐逆性增强的高羊茅转基因植株[J]. 中国农业科学,2005,38(12):2395-2402
[15] 张亚军,严学兵,朱见明,等. 紫花苜蓿光敏色素A基因片段的克隆及其RNA干扰载体的构建[J]. 草地学报,2010,18(6):897-901
[16] 郑宝仁,王洪军,卢宝伟,等. 苜蓿子叶节离体培养体系的研究[J]. 东北农业大学学报,2008,39(10):27-32
[17] Kao K N, Michayluk M R. Plant regeneration from mesophyll protoplasts of alfalfa[J]. Zeitschrift für Pflanzenphysiologie,1980,96(2):135-141
[18] 刘明志. 大叶紫花苜蓿愈伤组织原生质体再生植株[J]. 植物科学学报,1996,14(4):329-333
[19] Gilmour D M, Davey M R, Cocking E C. Isolation and culture of heterokaryons following fusion of protoplasts from sexually compatible and sexually incompatible Medicago species[J]. Plant Science,1987,53(3):263-270
[20] Jin T, Chang Q, Li W, et al. Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa[J]. Plant Cell, Tissue and Organ Culture (PCTOC),2010,100(2):219-227
[21] Li W, Wang D, Jin T, et al..The vacuolar Na+/H+ antiporter gene SsNHX1 from the halophyte Salsola soda confers salt tolerance in transgenic alfalfa (Medicago sativa L.)[J]. Plant Molecular Biology Reporter,2011,29(2):278-290
[22] Ahmad M, Cashmore A R. The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana[J]. The Plant Journal,1997,11(3):421-427
[23] 樊文娜. 调控苜蓿秋眠的光信号转导、糖代谢途径相关基因及酶的研究[D]. 郑州:河南农业大学,2015:60-77
[24] Chen M. Phytochrome nuclear body:an emerging model to study interphase nuclear dynamics and signaling[J]. Current opinion in plant biology,2008,11(5):503-508
[25] Horvath D. Common mechanisms regulate flowering and dormancy[J]. Plant Science,2009,177(6):523-531
[26] Clack T, Mathews S, Sharrock R A. The phytochrome apoprotein family inArabidopsis is encoded by five genes:the sequences and expression ofPHYD andPHYE[J]. Plant molecular biology,1994,25(3):413-427
[27] 王成章,韩锦峰,胡喜峰,等. 不同光周期条件下PhyB和ABA对不同苜蓿品种的秋眠性调控[J]. 草业学报,2006,15(6):56-63
[28] 李燕,康俊梅,张铁军,等. 紫花苜蓿MsZIP基因超表达载体的构建及烟草转化[J]. 草地学报,2012,20(04):735-740
[29] 张亚军,严学兵,朱见明,等. 紫花苜蓿光敏色素A基因片段的克隆及其RNA干扰载体的构建[J]. 草地学报,2010,18(06):897-901
[30] 乌艳红,米福贵,李志明,等. 紫花苜蓿GDP解离抑制蛋白基因cDNA的克隆、分析及烟草转化[J]. 草地学报,2011,19(01):157-163
[31] 白永琴,康俊梅,孙彦,等. 紫花苜蓿LEA蛋白基因ihpRNA表达载体构建及烟草转化[J]. 作物学报,2010,36(9):1484-1489
[32] 郑宝仁,王洪军,卢宝伟,等. 苜蓿子叶节离体培养体系的研究[J]. 东北农业大学学报,2008,39(10):27-32
[33] Kumar S, Tiwari R, Chandra A, et al. In vitro direct plant regeneration and Agrobacterium-mediated transformation of lucerne (Medicago sativa L.)[J]. Grass and Forage Science,2013,68(3):459-468

紫花苜蓿蓝光受体CRY2B过表达载体构建及转化

Construction of Blue Light Receptor CRY2B over Expression Vector and Genetic Transformation in Medicago Sativa

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王静, 刘晓静, 程甜甜, 童长春, 汪雪. 不同氮效率紫花苜蓿叶特征及其产量效应研究[J]. 草地学报, 2021, 29(9): 1941-1949.
[2]	孟宣辰, 马鹏程, 马杰, 段珍, 韩阳阳, 张吉宇. 黄土高原半干旱区紫花苜蓿覆膜垄沟和施肥效应研究[J]. 草地学报, 2021, 29(9): 2098-2106.
[3]	孙伟, 刘玉玲, 王德平, 赵敏, 杨合龙, 宋倩, 肖红, 王开丽, 曹婧, 戎郁萍. 补播羊草和黄花苜蓿对退化草甸植物群落特征的影响[J]. 草地学报, 2021, 29(8): 1809-1817.
[4]	陈雷, 暴雪艳, 郭刚, 霍文婕, 李清宏, 许庆方, 王聪, 刘强. 单宁酸和乳酸菌对紫花苜蓿青贮品质和体外瘤胃发酵的影响[J]. 草地学报, 2021, 29(8): 1853-1858.
[5]	李春萍, 邱轶辉, 夏厚胤, 谭璐娜, 许环宇, 张瀚文, 高景慧. 紫花苜蓿多父本F₁代低蒸腾及产量性状的隶属函数分析[J]. 草地学报, 2021, 29(7): 1416-1422.
[6]	李天琦, 林志玲, 卢轩, 黄佳媛, 杨宁, 沃野, 高凯, 柳小妮, 王显国. 灌溉量对科尔沁沙地紫花苜蓿土壤速效养分分布及淋失的影响[J]. 草地学报, 2021, 29(7): 1454-1461.
[7]	李红, 李波, 方志坚, 杨曌, 张超, 冯成龙, 金姗姗, 王思琦, 石婉莹. 冻融和冻融+苏打盐碱复合胁迫下苜蓿幼苗的生理响应[J]. 草地学报, 2021, 29(7): 1469-1476.
[8]	李鹏珍, 赵得琴, 邓波, 杨军, 赵国华, 赵亮. 生物炭与干旱胁迫对接种紫花苜蓿光合效率及生长的影响[J]. 草地学报, 2021, 29(6): 1257-1264.
[9]	王文静, 麻冬梅, 赵丽娟, 马巧利. 2,4-表油菜素内酯对盐胁迫下紫花苜蓿生理指标及根系离子积累的影响[J]. 草地学报, 2021, 29(6): 1363-1368.
[10]	潘新怡, 史昆, 龚攀, 韦宝, 吴欣明, 王赞. 紫花苜蓿花青苷合成的转录组分析[J]. 草地学报, 2021, 29(5): 866-875.
[11]	孙雷雷, 赵桂琴, 柴继宽, 刘富渊, 聂秀美, 王军. 贮藏方式和贮藏时间对苜蓿干草霉菌数量和种类的影响[J]. 草地学报, 2021, 29(5): 884-893.
[12]	陈彩锦, 张尚沛, 师尚礼, 曾燕霞, 吴娟, 尚继红, 高婷, 张蓉. 基于GGE双标图对苜蓿品种丰产性和稳定性综合评价[J]. 草地学报, 2021, 29(5): 912-918.
[13]	黄荣, 姚博, 张玉娟, 贾倩英, 李向阳, 张宏, 张振粉. 成团泛菌CQ10脂多糖对紫花苜蓿种子萌发及苗期生长的影响[J]. 草地学报, 2021, 29(5): 965-971.
[14]	陈卫东, 张玉霞, 丛百明, 阿玛尔, 田永雷, 张庆昕, 杜晓艳. 钾肥对紫花苜蓿根颈丙二醛、可溶性蛋白含量与抗氧化系统的影响[J]. 草地学报, 2021, 29(4): 717-723.
[15]	徐洪雨, 甄莉丽, 李钰莹, 董宽虎, 李向林. 低温干旱环境对紫花苜蓿根颈耐寒性的影响[J]. 草地学报, 2021, 29(4): 724-733.