Abstract
Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.
Highlights
Soybean is rich of protein and oil and has high economic value
Jack and all seeds harvested from T0 plants obtained through CRISPR/Cas9 and Agrobacteriummediated transformation were planted on June 22, 2017 at the Shunyi Experimental Station of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences
Two targets for the E1 gene were designed (Figure 2), and the CRISPR/Cas9 expression vector were transferred into the soybean cultivar Jack by Agrobacterium-mediated genetic transformation to knock out soybean endogenous gene E1
Summary
Soybean is rich of protein and oil and has high economic value. With the increasing demand for soybean globally, it is urgent to clarify gene function, and accelerate functional gene research and breeding speed for increasing yield and improving quality. CRISPR/Cas system includes gene knock out, knock in, multiple genes and sites editing, large fragment deletion and replacement (Gratz et al, 2013; Shan et al, 2013; Feng et al, 2014; Gratz et al, 2014; Zhou et al, 2014). Feng et al (2013) and Mao et al (2013) firstly proved that CRISPR/Cas system could be used to target genome editing in crop by introducing site-directed mutations in specific genes in Arabidopsis and rice. Jacobs et al (2015) first used CRISPR/Cas technology to knock out the green fluorescent protein gene (GFP), and produced targeted editing for nine soybean endogenous genes. CRISPR/Cas is an effective tool for soybean targeted genome editing, providing a theoretical and technical basis for further research on soybean genome, as well as improving the breeding efficiency and accelerating the breeding process
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