Genetic Stability and Disease Resistance Analysis of Hrpzpsta Gene in Transgenic Soybean Lines

Miao Yu,Jie Rong,Jing Qu,Zhuo Zhang,Peiwu Wang,Yang Song,Mo Zhang,Yong Qi Feng

doi:10.5539/jps.v6n2p45

Abstract

This experiment was carried out to evaluate genetic stability and disease resistance in transformed soybean lines with hrpZpsta gene using PCR analysis, southern blotting, real-time quantitative PCR (qRT-PCR) and to analyze the resistance against Phytophthora sojae (P. sojae) and Cercospora sojina (C. sojina) after inoculation. The results obtained using PCR and southern blotting analytical methods showed that exogenous gene functional elements were stably inherited in transgenic soybean and hrpZpsta gene was successfully integrated into the soybean genome in a single copy. Results at high-generation (T7, T8) transgenic lines of hrpZpsta revealed that their relative expression of hrpZpsta gene was the highest in leaves followed by roots, and much lower in stems, flowers, and seeds. Activity change rates of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) showed that transgenic lines significantly enhanced receptor species. The resistance of transgenic strains T7 and T8 generations against P. sojae was significantly increased with artificial inoculation methods, and the resistance against C. sojina was increased from susceptibility to the level of resistance. Under natural conditions in the field, the response of T8 transgenic lines to C. sojina reached disease resistance level. There were no significant differences in transgenic lines and recipient variety in maturing stage, leaf shape, flower color, plant height, 100-grain weight and quality content, and the two years average yield of plots increased to 11.59% and 8.19%, which significantly higher than recipient cultivar. The current results provide data support for the release of transgenic lines.

Highlights

Soybean (Glycine max L.) is a major food and oilseed crop worldwide and provides the largest source of vegetable oil and protein for humans and animals (Li, Zhang, Hao, Hua, Duan, Zhang, & Li, (2013)
Hybridization bands were detected in T7 and T8 transgenic soybean lines, while the receptor was not detected in the control sample
This study analyzes the genetic stability of target genes in T7, T8 transgenic lines; whether the presence of the primary PCR, the number of copies verified by Southern blotting or the expression of the transcriptional level detected by qRT-PCR confirmed that the transformed hrpZpsta gene was stably inherited and expressed in the recipient cultivar

Summary

Introduction

Soybean (Glycine max L.) is a major food and oilseed crop worldwide and provides the largest source of vegetable oil and protein for humans and animals (Li, Zhang, Hao, Hua, Duan, Zhang, & Li, (2013). Harpin protein is a class of nonspecific protein elicitors encoded by the hrp gene in gram-negative plant pathogenic bacteria which can: excite hypersensitive reaction (HR) in plants; make plants gain broad spectrum disease resistance (Alfano, & Collmer, 1997; Strobel, Ji, Goplan, Kuc, & He, 1996); can be induced by different signaling pathways to produce disease, insect and drought resistance and promote plant growth and other beneficial effects (He, Huang, Collmer, 1993; Pandey et al, 2005) It has important theoretical and practical values in the production of better application prospects. The physiological and biochemical mechanisms of resistance in harpin protein shows that it could induce the expression of POD, PPO, PAL and other protective enzymes in tomato, cucumber and eggplant, (Yuan & Meng, 2008), and as a consequence, induce plant defense http://jps.ccsenet.org

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