Cloning and Expression Analysis of &amp;lt;i&amp;gt;GmCYP78A5&amp;lt;/i&amp;gt; Promoter

Xiaofeng Chen,Chunmei Zhao,Zhaoyong Lv,Qiuli Du,Ren-Gao Xue

doi:10.11648/j.ajpb.20190401.12

Abstract

CYP78A5 promoter was isolated from soybean (Glycine max L. Merrill) plant by using PCR technology. DNA sequence alignment indicated that the amplified fragment (1650bp) was 99.21% homologous to the correspondent regions of the reported sequences. Bioinformatics analysis showed that the GmCYP78A5 promoter contains a lot of inducible or tissue-specific expression elements. RT-PCR results indicated that the gene GmCYP78A5 highly expressed in immature seed, weekly expressed in stem of soybean, but no expressed in root, leaf and flower. To further study the tissue expression patterns of GmCYP78A5 gene, the promoter of the gene GmCYP78A5 was fused with GUS reporter gene to construct a plant expression vector and the vector was transformed into tobacco (Nicotiana tabacum) by Agrobacterium-meditated method. The expression of the GUS gene in the transgenic tobacco plants indicated that the GmCYP78A5 promoter could drive the GUS reporter gene to express highly in the leaf, stem, sepal, pedicel, seeds of the transgenic tobacco plants, demonstrating that the expression patterns of the GmCYP78A5 promoters in soybean and tobacco were inconsistent.

Highlights

The size of plant organs is regulated by genes
Plant expression vector pCAMBIA1301S, E.coli DH5a and Agrobacterium EHA105 strains; clone vector pMD19-T purchased from TaKaRa Company (Dalian); PCR Product Recovery Kit purchased from Omega Company; PCR primers were synthesized by Beijing Sunbiotech co., Ltd. and sequenced in TaKaRa Company (Dalian)
The expression level of GmCYP78A5 gene was higher in immature seed, weakly expressed in epicotyl and hypocotyl, but not in root, leaf and flower (Figure 1)

Summary

Introduction

The size of plant organs is regulated by genes. The CYP78A5 is a member of the CYP78 family encoding cytochrome P450 monooxygenase [1,2,3]. The expression patterns of the CYP78A5 was varied at different stages of growth and development in Arabidopsis. The stem of Arabidopsis was distorted and the floral organs were defected when the CYP78A5 was overexpressed in transgenic Arabidopsis [4], but the cell division terminated earlier that resulted in smaller floral, leaf, and stem when the gene was knocked out [5]. Adamski et al (2009) found that the overexpression of the CYP78A5 in Arabidopsis caused organ enlargement, otherwise, the organ became smaller when the gene expression was inhibited, determining the yield of Arabidopsis thaliana [6]. It is obvious that the CYP78A5 encodes a class of transcription regulators, which is an important gene regulating the size of plant organs and plays a very important role in plant growth and development. The upstream regulatory sequence of the CYP78A5 is seldom studied

Methods

Results

Discussion

Conclusion