Transgenic Alfalfa with GsPPCK1 and Its Alkaline Tolerance Analysis

Abstract

Environmental stresses, such as drought, high salty and alkali, adversely affect plant growth and productivity. Plants adapt to these environmental stresses by inducing numerous genes at the transcriptional level and by protein phosphorylation. Phosphoenolpyruvate carboxylase kinase (PPCK) is a Ca2+ independent kinase in response to a range of signals in different plant tissues which plays a key role in the control of plant metabolism. As an important extension of our earlier studies summarized above on global transcriptome profiling of wild soybean under NaHCO3 treatment, an alkaline (NaHCO3) related gene GsPPCK1 was identified and subsequently cloned from Glycine soja, which has 99% similarity with PPCK1 of Glycine max (AAQ83695.1), named as GsPPCK1. Expression of GsPPCK1 mRNA was induced by NaHCO3 stress in roots and leaves. GsPPCK1 transcripts increased during 3 hour exposures to NaHCO3 stress. These results indicated that wild soybean PPCK1 was an early responded gene to alkaline stress. We transformed GsPPCK1 gene into alfalfa using a developed method, and transgenic alfalfa showed observably enhanced tolerance to NaHCO3 stress compared with wild-type plants. Transgenic alfalfa grew well in the conditions of 100 mmol L–1 NaHCO3 , while wild type plants exhibited discoloration and stunted growth, or even death. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline-alkaline stress in non-transgenic lines compared to transgenic lines (P0.05). Moreover, compared with non-transgenic, transgenic alfalfa had higher levels of chlorophyll content and root activity under alkali stress conditions. The result indicated that over-expression of GsPPCK1 in alfalfa could enhance alkaline tolerance. All results showed that GsPPCK1 gene could improve the tolerance of transgenic alfalfa to alkali stress; therefore, the study on this field is of significance not only in theory but also in practice.