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Transformation and Functional Analysis of Tatoc1-a, TaTOC1-B and TaTOC1-D Genes in Wheat

Author: PanYanYou
Tutor: ZhangXianSheng；ZhaoXiangYu
School: Shandong Agricultural University
Course: Developmental Biology
Keywords: Wheat Photoperiod Circadian clock TOC1 Transgene Flowering
CLC: S512.1
Type: Master's thesis
Year: 2011
Downloads: 32
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Abstract


The timing of the change from vegetative to reproductive growth is critical for higher plant. Photoperiodic pathway is an important flowering regulatory pathway that is one of flowering regulatory pathways. In previous studies, three nearly full-length cDNA of homolog genes TaTOC1-A, TaTOC1-B and TaTOC1-D were isolated from homologous hexaploid wheat. Here, to investigate their function, we constructed overexpression vectors and transformated them into Arabidopsis. Under short-day conditions, the transgenic plant overexprssing TaTOC1 genes delayed flowering. But it had no visible effect on flowering under long-day conditions. The results of genetic complementation experiments indicated that the early flowering phenotype of toc1-1 were recovered by overexprssion of TaTOC1 genes. Then we analyzed the expression patterns of key regulate flowering genes such as GI, CO, FT and SOC1 in toc1-1 mutant. Compared with the wild-type control, we found that the expression of GI, CO and FT genes increased in toc1-1 mutant, while SOC1 gene’s expression was not significantly changed. To further study their function in wheat (Triticum astivum L.), an RNAi vector was constructed to knock down there genes including TaTOC1-A, TaTOC1-B and TaTOC1-D. Then it was transformed into the wheat (cv. CB037) mediated Agrobacterium tumefaciens. And we obtained 1053 regenerate seedings. After molecular identification, we selected 13 positive transgenic plants from the resistant seedlings. And the heading time of transgenic plants is 5-7 days earlier than that of control. Semi-quantitative reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction results showed that the expression of TaTOC1-A, TaTOC1-B and TaTOC1-D genes were reduced in transgenic plants. This suggests that the reduction of TaTOC1-A, TaTOC1-B and TaTOC1-D gene in transcript levels promote heading earlier in wheat. Further, we analyzed the expression patterns of key regulate flowering genes such as TaGI1, TaHd1, TaFT and TaSOC1. Compared with the wild-type wheat, we found that the expression of TaGI1, TaHd1 and TaFT genes increased in transgenic wheat under long-days. TaGI1 and TaFT genes’ expression had a higher peak in transgenic wheat under short-day conditions. And the other gene’s expression was not significantly changed. These genes’ expressions were changed by reducing the expression of TaTOC1 gene. Meanwhile, semi-quantitative reverse transcription PCR and real-time quantitative PCR results showed that TaTOC1-A, TaTOC1-B and TaTOC1-D gene had different expression in detected tissues and organs. And their expression had an higher levels in the leaves. In the leaves, TaTOC1 mRNA was localized in the leaf vascular bundle tissue and the most obvious hybridization signals was detected in the phloem.

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CLC: > Agricultural Sciences > Crop > Cereal crops > Wheat > Wheat
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