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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

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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"

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