Function Analysis Of RING Finger Gene, And Creation Of Pre-harvest Sprouting Resistant Transgenic Wheat

Based on the RING finger RHAs transcript factor, two novel RHA2b type genes, TaRHA2b and RsRHA2b were cloned by RT-PCR along with RACE amplification from wheat and radish, respectively. The characterization structure and expression pattern of TaRHA2b and RsRHA2b were analysized. To elucidate the function of RHA2b, three type plant expression vectors, p35S::RsRHA2b, p35S::GluCP1::RsRHA2b and p35S::GluCP2::RsRHA2b were constructed, and transgenic rice and wheat plants were abtained respectively. The results were as followings:1. Clone expression, and characterization of TaRHA2b and RsRHA2bAfter RT-PCR amplification along with 5' and 3' RACE, a full-length RING finger RHAs gene cDNA, named TaRHA2b was cloned. TaRHA2b was 845 bp in full length, including 5' untranslated region of 79 bp, 3' untranslated region of 301 bp, and an open reading frame (ORF) of 465 bp encoding 155 amino acid with the molecular weight about 16.9 kDa, and the isoelectric point 7.77. RsRHA2b gene cDNA was cloned by RT-PCR, sequence analysis showed that RsRHA2b was 697 bp in full length, including 5' untranslated region of 86 bp, 3' untranslated region of 152 bp, and an open reading frame (ORF) of 459 bp encoding 152 amino acid with the molecular weight about 16.4 kDa, and the isoelectric point 7.65. Homology analysis showed that TaRHA2b and RsRHA2b protein shared 38.8% - 53.1% and 38.8% -91.8% identities on the amino acid level with the known RING-H2 motif from Arabidopsis.The expression of TaRHA2b was the highest in endosperm followed by that in panicles, lemma, roots, stems, and the least in leaf. qRT-PCR assays indicated that transcripts of TaRHA2b were abundant in dry seeds but were dramatically reduced to a barely detectable level when seeds were stratified at 30℃for 12 h, and then increased slowly until 48 h, the longest time in our study. The expression profiling also showed that TaRHA2b expression was up-regulated by ABA treatment during wheat seeds germination and early stages of seed germination.2. Construction of RsRHA2b over-expression rice plantsTo elucidate the function of RsRHA2b, three type constructs, p35S::RsRHA2b, p35S::GluCP1::RsRHA2b and p35S::GluCP2::RsRHA2b were constructed, and the three constructs along with the empty vector were transformed into rice (Oriza sativa. japannica) variety Kitaake by Agrobacterium-mediated, respectively. After identified with special PCR, 26, 28, 22 and 20 independent transgenic lines were obtained, and the total positive percents were about 100%.3. ABA response of RsRHA2b over-expression plants during seed germination and early seedling developmentSeed germination study showed that the rate of different transgenic lines were much prolonged than that of wild type seeds both in presence and absent of ABA. However, in the presence of ABA pretreatment, seed germination of various transgenic lines seeds, especially bivalent promoter transgenic seeds showed increased sensitivity to ABA, whereas seed germination of the wild type was less sensitive to ABA. The fact of transgenic rice seed germination rate was significantly slower than that of non-transgenic control suggests that RsRHA2b can serve as excellent resistance gene to create new pre-harvest sprouting wheat genetic resources.4. Generation of pre-harvest sprouting resistant transgenic wheatTo create pre-harvest sprouting transgenic wheat, immature embryos of Yumai No.18 were bombarded with the above three type plasmids containing bar and RsRHA2b gene respectively. A total of 8 independent lines were obtained, confirmed by special PCR analysis. The above transgenic wheat lines were reproduced in the greenhouse, and the identification of anti-sprouting ability to be carried out follow-up.