检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

作物杂志,2016, 第2期: 63–67 doi: 10.16035/j.issn.1001-7283.2016.02.011

• 遗传育种·种质资源·生物科技 • 上一篇    下一篇

普通小麦春化基因VRN3的表达分析及过表达载体构建

张鹏钰1,卫丽2,朱灿灿2,姜玉梅2,张宝娜1,尹钧2,王同朝1   

  1. 1 河南农业大学农学院,450002,河南郑州
    2 河南农业大学/国家小麦工程技术研究中心,450002,河南郑州
  • 收稿日期:2015-10-30 修回日期:2016-02-01 出版日期:2016-04-15 发布日期:2018-08-26
  • 通讯作者: 王同朝
  • 作者简介:张鹏钰,在读硕士研究生,研究方向为小麦发育
  • 基金资助:
    河南省科技攻关项目(132102110044);国家自然科学基金(31471452)

Expression Analysis and Construction of Over-Expression Vector of Vernalization Gene VRN3 in Common Wheat

Zhang Pengyu1,Wei Li2,Zhu Cancan2,Jiang Yumei2,Zhang Baona1,Yin Jun2,Wang Tongchao1   

  1. 1 Agronomy College of Henan Agricultural University,Zhengzhou 450002,Henan, China
    2 Henan Agricultural University/National Engineering Research Centre for Wheat,Zhengzhou 450002,Henan,China
  • Received:2015-10-30 Revised:2016-02-01 Online:2016-04-15 Published:2018-08-26
  • Contact: Tongchao Wang

RichHTML

7

PDF (PC)

191

摘要：

通过实时荧光定量PCR（qRT-PCR）分析了不同发育特性的普通小麦品种VRN3基因的表达,结果表明：VRN3基因在春性品种新春2号叶片中表达呈上升趋势,在雌雄蕊分化期达到最大值,在强冬性品种京841中表达量很低;VRN3在新春2号和京841茎尖中的表达量均极低。以京841的cDNA为模板扩增VRN3基因全长,连接到pCAMBIA3301载体上,构建VRN3基因过表达载体,通过农杆菌茎尖转化法获得了转基因植株,研究结果为春化基因VRN3对小麦春化发育调控的进一步研究提供依据。

关键词: 小麦, 春化基因VRN3, 表达, 载体, 转化

Abstract:

VRN3 expression patterns were analyzed in wheat varieties with different developmental characteristics by qRT-PCR. The results showed that the expression of VRN3 in spring wheat variety Xinchun 2 increased gradually and reached the highest level in pistil and stamen differentiation stage; VRN3 expression level in winter wheat variety Jing 841 was very lower, also in the shoot meristems of both wheat varieties. Using Jing841 cDNA as template and pCAMBIA3301 as expression vector, we amplified the full length of VRN3 gene, constructed VRN3 over-expression vector and transferred into wheat by agro-bacterium mediated shoot apices and obtained transgenic plants by PCR identification. The results provide the basis for further analyzing the function of VRN3 on wheat vernalization regulation.

Key words: Wheat, Vernaliazation gene VRN3, Expression, Vector, Transformation

表1

表达载体构建及转基因后代分子检测所用引物"

引物
Primer		 引物序列（5’-3’）
Primer sequence（5’-3’）		 内切酶或退火温度
Endonuclease or annealing temperature		 用途
Usage		 产物大小
Product size（bp）
VRN3-F1 TTCCGGTAATTTATAGCACAAGC 60℃ VRN3荧光定量PCR
VRN3-R1 GGCTCCAATCGATCAATCAC
WAC-F TTTGAAGAGTCGGTGAAGGG 56℃ 小麦β-Actin荧光定量PCR
WAC-R TTTCATACAGCAGGCAAGCA
VRN3-F AGATCTATGGCCGGTAGGGATAGGGA BglⅡ VRN3过表达片段扩增 534
VRN3-R CACGTGTCAATTGTACATCCTCCTGC PmlⅠ
BAR-F AACCCACGTCATGCCAGTTCC 60℃ BAR基因PCR检测 428
BAR-R GGTCTGCACCATCGTCAACCA
VRN3-F2 ATGGTAGACCCAGATGCTC 58℃ VRN3过表达载体PCR检测 730
VRN3-R2 ACGGATAAACCTTTTCACG

图1

新春2号和京841中VRN3表达的qRT-PCR结果"

图 2

VRN3过表达目的片段的克隆M：DL1000 DNA marker;1、2：VRN3的cDNA全长克隆"

图3

VRN3过表达目的片段的酶切M：DL10000 DNA marker;1、2：pMD19-T和VRN3的重组质粒;3、4：pCAMBIA3301质粒"

图4

VRN3过表达载体测序验证"

图5

VRN3过表达转化T1转基因后代PCR检测M：分量标准DL2000;1-10：T1转基因植株"

[1] Yan L, Fu D, Li C , et al. The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proceeding of the National Academy of Sciences, 2006,103:19581-19586.
doi: 10.1073/pnas.0607142103
[2] Iwaki K, Nishida J, Yanagisawa T , et al. Genetic analysis of Vrn-B1 for vernalization requirement by using linked dCAPS markers in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics, 2002,104:571-576.
doi: 10.1007/s00122-001-0769-0
[3] Fu D, Szcs P, Yan L , et al. Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Molecular Genetics and Genomics, 2005,273(1):54-65.
doi: 10.1007/s00438-004-1095-4
[4] Tranquilli G, Dubcovsky J . Epistatic interaction between vernalization genes Vrn-Am1 and Vrn-Am2 in diploid wheat. Journal of Heredity, 2000,91(4):304-306.
doi: 10.1093/jhered/91.4.304
[5] Yan L, Loukoianov A, Dubcovsky J , et al. The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science, 2004,303:1640-1644.
doi: 10.1126/science.1094305
[6] Huang T, Bohlenius H, Eriksson S , et al. The mRNA of the Arabidopsis gene FT moves from leaf to shoot spex and induces flowering. Science, 2005,309:1694-1696.
doi: 10.1126/science.1117768
[7] Corbesier L, Vincent C, Jang S , et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science, 2007,316:1030-1033.
doi: 10.1126/science.1141752
[8] Abe M, Kobayashi Y, Yamamoto S , et al. FD,a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science Signaling, 2005,309(5737):1052.
[9] Bonnin I, Rousset M, Madur D , et al. FT genome A and D polymorphisms are associated with the variation of earliness components in hexaploid wheat. Theoretical and Applied Genetics, 2008,116(3):383-394.
doi: 10.1007/s00122-007-0676-0
[10] 袁秀云, 李永春, 孟凡荣 , 等. 黄淮麦区21个小麦品种中春化基因VRN1的组成分析. 麦类作物学报, 2009,29(5):760-765.
doi: 10.7606/j.issn.1009-1041.2009.05.004
[11] 马丽娟, 王翔, 卫丽 , 等. 不同发育特性小麦品种春化基因VRN2的序列分析. 麦类作物学报, 2012,32(4):603-609.
doi: 10.7606/j.issn.1009-1041.2012.04.002
[12] 张江花, 高曼霞, 许海霞 , 等. 河南小麦新品种的春化基因鉴定. 植物遗传资源学报, 2014,15(5):1040-1045.
[13] 刘文林, 张宏纪, 刘东军 , 等. 黑龙江小麦春化和光周期主要基因组成分析. 植物遗传资源学报, 2014,15(6):1352-1359.
[14] 梁欣欣, 刘录祥, 赵林姝 , 等. 农杆菌介导法向小麦茎尖导入DREB1A基因的研究初报. 麦类作物学报, 2007,27(1):16-19.
doi: 10.7606/j.issn.1009-1041.2007.01.008
[15] Distelfeld A, Li C, Dubcovsky J . Regulation of flowering in temperate cereals. Current Opinion in Plant Biology, 2009,12(2):178-184.
doi: 10.1016/j.pbi.2008.12.010
[16] Li C, Distelfeld A, Comis A , et al. Wheat flowering repressor VRN2 and promoter CO2 compete for interactions with NUCLEAR FACTOR‐Y complexes. The Plant Journal, 2011,67(5):763-773.
doi: 10.1111/j.1365-313X.2011.04630.x
[17] Chen A, Dubcovsky J . Wheat TILLING mutants show that the vernalization gene VRN1 down-regulates the flowering repressor VRN2 in leaves but is not essential for flowering. PLoS Genetics, 2012,8(12):e1003134.
doi: 10.1371/journal.pgen.1003134
[18] 黎冬华, 廖玉才, 李和平 . 根癌农杆菌介导的大麦茎尖转化研究.麦类作物学报, 2002(1):44-47.
[19] Park S H , Pinson S R M,Smith R H.T-DNA integration into genomic DNA of rice following agrobacterium inoculation of isolated shoot apices. Plant Molecular Biology, 1996,32(6):1135-1148.
doi: 10.1007/BF00041397
[20] Zhang S, Cho M J, Koprek T , et al. Genetic transformation of commercial of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings, Plant Cell Reports, 1999,18:959-966.
doi: 10.1007/s002990050691
[1] 王汉霞 单福华 田立平 马巧云 赵昌平 张风廷. 北部冬麦区冬小麦区试品种（系）的#br# 稳定性和适应性分析[J]. 作物杂志, 2018, (5): 40–44
[2] 安 霞 张海军 蒋方山 吕连杰 陈 军. 播期播量对不同穗型冬小麦群体及子粒产量的影响[J]. 作物杂志, 2018, (5): 132–136
[3] 王嘉楠 李小艳 魏石美 赵会杰 赵明奇 汪月霞. 5-ALA 对干旱胁迫下小麦幼苗#br# 光合作用及D1 蛋白的调节作用[J]. 作物杂志, 2018, (5): 121–126
[4] 赵广才,常旭虹,王德梅,陶志强,王艳杰,杨玉双,朱英杰. 小麦生产概况及其发展[J]. 作物杂志, 2018, (4): 1–7
[5] 陆梅,孙敏,任爱霞,雷妙妙,薛玲珠,高志强. 喷施叶面肥对旱地小麦生长的影响及与产量的关系[J]. 作物杂志, 2018, (4): 121–125
[6] 温辉芹,程天灵,裴自友,李雪,张立生,朱玫. 山西省近年审定小麦品种的综合性状分析[J]. 作物杂志, 2018, (4): 32–36
[7] 罗海斌, 蒋胜理, 黄诚梅, 曹辉庆, 邓智年, 吴凯朝, 徐林, 陆珍, 魏源文. 甘蔗ScHAK10基因克隆及表达分析[J]. 作物杂志, 2018, (4): 53–61
[8] 杨飞,马文礼,陈永伟,张战胜,王昊. 匀播、滴灌对春小麦幼穗分化进程及产量的影响[J]. 作物杂志, 2018, (4): 84–88
[9] 王君婵,高致富,李东升,朱冬梅,吴宏亚. 农业信息技术在小麦育种中的应用研究[J]. 作物杂志, 2018, (3): 37–43
[10] 张彬,李金秀,王震,冯浩,李金榜. 小麦主要农艺性状的相关性及聚类分析[J]. 作物杂志, 2018, (3): 57–60
[11] 王拯,陈兆波,张胜全,任立平,高新欢,叶志杰,张风廷. BS系列杂交小麦在陕西渭南地区制种可行性分析[J]. 作物杂志, 2018, (3): 174–179
[12] 石晓华,杨海鹰,康文钦,秦永林,樊明寿,贾立国. 不同施氮量对马铃薯-小麦轮作体系产量及土壤氮素平衡的影响[J]. 作物杂志, 2018, (2): 108–116
[13] 黄少辉,杨云马,刘克桐,孙彦铭,杨军芳,贾良良. 河北省小麦产量潜力、产量差与效率差分析[J]. 作物杂志, 2018, (2): 118–122
[14] 宋敏,张海鹏,路兴涛,吴翠霞,张勇. 不同除草剂对冬小麦田宝盖草的防除效果[J]. 作物杂志, 2018, (2): 161–165
[15] 张帅,庞玉辉,王征宏,王黎明,陈春燕,曾占奎,王春平. 小麦种质资源农艺性状变异及其遗传多样性分析[J]. 作物杂志, 2018, (2): 44–51
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] 赵广才,常旭虹,王德梅,陶志强,王艳杰,杨玉双,朱英杰. 小麦生产概况及其发展[J]. 作物杂志, 2018, (4): 1 –7 .
[2] 权宝全,白冬梅,田跃霞,薛云云. 不同源库关系对花生光合特性及产量的影响[J]. 作物杂志, 2018, (4): 102 –105 .
[3] 黄学芳,黄明镜,刘化涛,赵聪,王娟玲. 覆膜穴播条件下降水年型和群体密度对张杂谷5号分蘖成穗及产量的影响[J]. 作物杂志, 2018, (4): 106 –113 .
[4] 黄文辉, 王会, 梅德圣. 农作物抗倒性研究进展[J]. 作物杂志, 2018, (4): 13 –19 .
[5] 赵云,徐彩龙,杨旭,李素真,周静,李继存,韩天富,吴存祥. 不同播种方式对麦茬夏大豆保苗和生产效益的影响[J]. 作物杂志, 2018, (4): 114 –120 .
[6] 陆梅,孙敏,任爱霞,雷妙妙,薛玲珠,高志强. 喷施叶面肥对旱地小麦生长的影响及与产量的关系[J]. 作物杂志, 2018, (4): 121 –125 .
[7] 王晓飞,徐海军,郭梦桥,肖宇,程薪宇,刘淑霞,关向军,吴耀坤,赵伟华,魏国江. 播期、密度及施肥对寒地油用型紫苏产量的影响[J]. 作物杂志, 2018, (4): 126 –130 .
[8] 朱鹏锦,庞新华,梁春,谭秦亮,严霖,周全光,欧克维. 低温胁迫对甘蔗幼苗活性氧代谢和抗氧化酶的影响[J]. 作物杂志, 2018, (4): 131 –137 .
[9] 高杰,李青风,彭秋,焦晓燕,王劲松. 不同养分配比对糯高粱物质生产及氮磷钾利用效率的影响[J]. 作物杂志, 2018, (4): 138 –142 .
[10] 商娜,杨中旭,李秋芝,尹会会,王士红,李海涛,李彤,张晗. 鲁西地区常规棉聊棉6号留叶枝栽培的适宜密度研究[J]. 作物杂志, 2018, (4): 143 –148 .