Mutant Identification And Characterization Of Starch Synthesis Key Genes In Wheat Based On TILLING

TILLING populations of wheat have been constructed by EMS treatment, and starch synthesis key genes Agp2 B, Wx-A1, SSII-A, SSII-B and SBE1 etc., had been identified previously. In this study, point mutations of starch synthesis gene SSâ…£ were screened by TILLING; the effects of some mutations on gene expression in developing endosperm and leaves and on starch content were also evaluated in M3 generation. The main results were as following:1. 54 point mutations in SSâ…£ gene were obtained by screening the TILLING populations. Mutation density was 1/198.82 kb. The transition mutation frequency was 96.3%. There were 1 nonsense mutation, 2 splice junction mutations and 22 missense mutations. A nonsense mutation, T2188 C, leaded to the polypeptide produced a premature stop codon at the 269 th amino acid. 2 splicing mutations located at the first base of intron 7 in the splice donor site, where G was replaced by A. Among 22 missense mutations, 4 of them, T1991 C, C2254 Y, C4061 T and G6438 A, might severely impact on the protein function.2. M3 of 13 lines of SSâ…£ mutations which were predicted to impact on the protein function were identified, of which 1 nonsense mutation, 2 splice junction mutations and 10 missense mutations. Expression level of SSâ…£ of M3 homozygous nonsense mutants was very low, which might result from the degradation of m RNA mediated by premature translational termination. The m RNAs produced from SSâ…£ of the M3 homozygous splice junction mutants were aberrantly spliced in five ways: remaining intron 7 or intron 7 and intron 8 or intron 6 to intron 8, splicing out exon 7 or exon 7 and exon 8. In each of these five ways, incorrect splicing was predicted to change the open reading frame and to give rise to premature stop codons in the translated sequence. The corresponding polypeptides subsequently lost ADP-binding sites at C-terminal would have no function.3. Six missense mutant lines in Agp2 B were obtained from M3 generation, expression of 3 mutants were significantly lower than that of the wild-type during endosperm development. Compared with wild-type, starch contents in the seeds of E3-1-3 droped 9.1%. The high expression level of Agp2 B at 24 th day after flowering suggested that this gene might play an important role in maintaining AGPase enzyme activity and promoting the grain starch synthesis at late filling stage.4. We identified 6 mutations in Wx-A1, 12 mutations in SSâ…¡-A, 3 mutations in SSâ…¡-B and 3 mutations in SBE1. All of mutations in Wx-A1 and SSâ…¡-B, 3 missense mutations in SSâ…¡-A could stably inherit in progeny. But other 9 mutations in SSâ…¡-A and all of mutations in SBE1 were not idenfied. The non-identifed mutations might be of low ratio or be lost.Point mutations identified by TILLING could stably inherit in progeny, and mutation of these genes could result in their expression and related phenotypes. These mutant materials would be used for gene function analysis and crop breeding programs.