Abstract:

【Objective】The objective of the study is to develop a transgenic barley germplasm with low protein content under high nitrogen dosage by RNA interference (RNAi) to specially down-regulate the expression of grain B-hordein genes.【Method】The conserved sequence of B-hordein gene (gi|18928) in barley was cloned based on recombinant PCR and confirmed by sequence analysis. The RNAi expression vector pBract207-zz-gp4, aiming at silencing barley grain B-hordein gene, was reconstructed by Gateway technology and transformed into barley cultivar Golden Promise by Agrobacterium-mediated method using immature embryo. Transgenic plants were obtained after resistance selection, differentiation and regeneration, and then the transgenic progeny was screened by PCR, Southern blotting, and semi-quantitative RT-PCR. Protein content were determined by near-infrared spectroscopy analyzer, SDS-PAGE analysis and nitrogen management.【Result】Two B-hordein fragments (Gp4 and Gp5 ) were identified in this study. Sequence analysis revealed that the length of the sequence was 349 bp. Cluster analysis showed that the highest degree of identity (92%) was between Gp4/Gp5 and other known B-hordein. And also the highest degree of identity (98%) was between conserved sequence and the mRNA of B-hordein. The hpRNA silencing fragment was designed on the basis of Gp4 in sense and antisense orientation with the sequence of the i18 and iv2 intron as spacer region between the repeats and driven by Ubi promoter. Twenty-three T₁ transgenic lines were obtained by Agrobacterium-mediated method and eleven transgenic plants of T₁ generation were confirmed by PCR of selectable marker gene and sense and antisense reaction of B-hordein. Southern blot analysis showed that RNAi constructs were successfully integrated into the plant genomes in T₂/T₃ generations. Further, six transgenic barley positive lines of T₃ progeny were selected by random and analyzed by semi-quantitative RT-PCR after flowering. The results showed that the development expression of B-hordein genes of six T₃ transgenic plants was significantly lower than that of non-transgenic plant, especially on the 20 DAF (Day after flowering), which was not only reduced by 28.19%-55.19% compared with the control, but also was the lowest one among the developing periods after flowering. Then protein content analysis of eight lines of T₁/T₃ selected by random demonstrated that the total protein content decreased significantly compared with the control. At the same time, SDS-PAGE revealed that the ration of B-hordein of different transgenic barley plants or lines decreased compared with that of non-transgenic plants, that of three lines of them reduced significantly and the degree of decreasing was 49.42% on average. In addition, RNAi-20, which had low protein content, was used to carry out nitrogen management analysis. The result revealed that the protein content of the line significantly decreased compared with that of control in high nitrogen dosage (HN₂ and HN₃) treatment, and which gradually lower than the control with the change of nitrogen application from HN₂ to HN₃. SDS-PAGE demonstrated that the B-hordein content of transgenic barleys decreased and electrophoretic band changed.【Conclusion】The RNAi technology can effectively inhibit the expression of B-hordein gene in barley grain, decrease the B-hordein content of RNAi lines, reduce the protein content under high nitrogen dosage, improve the quality of barleys, and create new superordinary germplasm resources of barley.

Key words: barley, B-hordein, protein content, RNA interference