Cloning And Functional Analysis Of Yield-related Gene TaSPLs From Common Wheat

Wheat(Triticum aestivum L.) is one of the most important crops in the world, improved yield potential is a long-term task of wheat varieties improvement in China. To excavate and utilize the favourable genetic resources will laid the material foundation for new variety breeding in wheat. The whole growth process of wheat from vegetative growth to reproductive growth stage, will influence the development period of spike and grain, and will influence the final grain yield. Genes of SPL (SQUAMOSA promoter-binding protein-like) encode a plant-specific family of transcription factors, which play an important role in plant development. The presence of miRNA responsive element in many SBP-box gene transcripts indicates the extensive regulation by miR156. Moreover, SBP-box genes have already been characterized as regulating flower and fruit development in crop plants, in other words, they affect the yield.We isolated the SBP-box genes TaSPL17, TaSPL20 and TaSPL21, which showed responses of spike development in wheat. Polymorphism TaSPL17 and TaSPL21 for analysis and the expression vectors of TaSPL20 and TaSPL21-D used for transforming into Arabidopsis thaliana and rice were constructed to reveal the gene functions. The main results are summarized as follows:1. Cloning, subcellular localization and expression analysis of TaSPL20 and TaSPL21 from common wheat. TaSPL20 and TaSPL21 were isolated from common wheat. TaSPL20 in genome is 2028 bp and contains 3 extron and 2 intron. TaSPL21-A in genome is 2699 bp and contains 3 extron and 2 intron. TaSPL21-D in genome is 2414 bp and contains 3 extron and 2 intron. They are belonged to the SBP-box protein family. The sub-cellular location analysis using expression vectors of TaSPL20 and TaSPL21-D constructed showed that they were both located in nucleus. We obtained 28 T1 generation transgenic rice lines, have been planted in Hainan for adding generation.2. The tae-MIR156 precursor gene was cloned, which could form a stem loop after its transcription. Sequence analysis showed that TaSPL3 and TaSPL17 had putative targets of tae-miR156 among the ten wheat SBP-box genes. The diploid donor species of hexaploid common wheat (Triticum aestivum, genome AABBDD), i.e. Triticum urartu (AA) UR209 and Aegilops speltoides (SS, closely related to BB) Y2001 possessed more than one copy of SPL17 (SPL17-A1, SPL17-A2 and SPL17-A3 from Triticum urartu; SPL17-B1, SPL17-B2 and SPL17-B3 from Aegilops speltoides), while Aegilops tauschii (DD) Ae38 only possessed one (SPL17-D). The identities between nucleotide sequences of SPL17-A2 and SPL17-B2, SPL17-A3 and SPL17-B3 or SPL17-D were higher than 99%. They were highly similar with the sequence of TaSPL17 in common wheat cultivars Chinese Spring, Hengguan 35 and Shuangfengshou. These genes might originate from a common ancestor and were highly conserved in the process of evolution. The target site of tae-miR156 in TaSPL17 was also highly conserved in two subgroups consisted of accessions with diverse spike number per plant and genetic background.3. The TaSPL21-A (3463 bp) and TaSPL21-D (4322 bp) sequences consisted of the promoter region and coding region, they were isolated and mapped on chromosomes 6A and 6D. TaSPL21-A was mapped on 6AS in a doubled haploid (DH) population derived from a cross of Hanxuan 10 × Lumai 14, which was flanked by SSR markers CWM306 and Xpsp3071. TaSPL21-D was mapped on 6DS in a recombination inbred line (RIL) population derived from a cross of Opata 85 × W7984, which was flanked by SSR markers Xgwm325-6D and Xcdo270-6D.4. Two SNPs were detected in the promoter region of TaSPL21-D, which formed three haplotypes, Hap-Ⅰ, Hap-Ⅱ and Hap-Ⅲ. The site of TaSPL21-D SNP1 and the haplotype of TaSPL21-D SNP1 and SNP2 were significantly associated with TGW (1000-grain weight). The PVE (Phenotype variety explain) were 5.30%-11.40% and 6.97%-12.22%, respectively. TaSPL21-D-SNP1-C was considered as a favourable allelic variation, and Hapll was considered as an elite haplotype, TaSPL21-D-SNP2-G and HapⅢ were considered as a potentially elite allelic variation and haplotype.