Crops ›› 2016, Vol. 32 ›› Issue (2): 63-67.doi: 10.16035/j.issn.1001-7283.2016.02.011

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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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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

Table 1

Primers for vector construction and transgenic plants molecular detection"

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

Fig.1

Expression analysis of VRN3 by qRT-PCRin Xinchun 2 and Jing 841"

Fig.2

VRN3 gene cloningM：DL1000 DNA marker;1,2：VRN3 gene cloning"

Fig.3

VRN3 enzyme digestion by BglⅡ and PmlⅠM：DL10000 DNA marker;1,2：recombinant plasmid of pMD19-T-VRN3;3,4：pCAMBIA3301"

Fig.4

The identification of VRN3 overexpression vector by sequences alignment"

Fig.5

PCR detecting of transgenic plantsM：DL2000 DNA marker;1-10：DNA of T1 transgenic plants"

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