中科院張愛民團隊發現抑制小麥種子貯藏蛋白合成的轉錄因子

種子貯藏蛋白(SSP)賦予小麥種子彈性和延展性,是小麥品質的決定因素之一。研究發現,一些轉錄因子能與SSP基因啟動子中的順式元件發生協同作用,在轉錄水平上調控SSP合成。目前已知的調控小麥SSP合成的轉錄調控因子很少,都是通過玉米、大麥等谷類作物的同源克隆鑒定出來的。二倍體烏拉爾圖小麥(Triticum urartu)是六倍體普通小麥(Triticum aestivum)的基因組供體,兩者同源性很高。因此,挖掘烏拉爾圖小麥中新的SSP合成轉錄調控因子,對進一步分析普通小麥SSP合成機理具有重要意義。

最近,中國科學院遺傳發育生物學研究所張愛民團隊發現R2R3 MYB轉錄因子TuODORANT1在籽粒灌漿期的烏拉爾圖小麥胚乳中優先表達。SSP是在小麥未成熟胚乳中合成的,所以發育胚乳中優先表達的轉錄因子極有可能是SSP合成的調控因子。鑒于烏拉爾圖小麥沒有成熟的遺傳轉化體系,研究人員將過表達TuODORANT1的載體Glu-1Bx14pro:TuODORANT1轉入普通小麥品種科農199中,成功獲得了3個過表達系OE34、OE38和OE59。RNA‐Seq檢測結果顯示,過表達TuODORANT1的普通小麥中SSP基因的轉錄水平在整個灌漿期間降低了49.71%,這導致成熟籽粒SSP的總表達量下降了13.38%‐35.60%。qRT-PCR分析表明,在體外瞬時超表達TuODORANT1的胚乳中SSP基因的啟動子活性和基因轉錄效率均降低了1-13倍。電泳遷移率轉移分析(EMSA)和芯片定量PCR分析表明,TuODORANT1在體外和體內都與SSP基因啟動子中的順式元件5"-T/CAACCA-3"和5"-T/CAACT/AG-3"結合。

與此同時,研究人員找到了TuODORANT1在普通小麥中的同源基因TaODORANT1。同樣的,TaODORANT1在體外過表達使SSP啟動子活性和基因轉錄效率下降了1-112倍。為了研究TaODORANT1在普通小麥SSP合成過程中的抑制作用,研究人員利用RNAi技術敲除了科農199中TaODORANT1基因,獲得了3個沉默株系RNAi#1、RNAi#2和RNAi#3。研究結果表明,RNAi株系的SSP基因轉錄效率增加了14.73%-232.78%,基因表達量升高了11.43%-19.35%。

以上研究數據表明,新發現的TuODORANT1和TaODORANT1都是小麥SSP合成過程中的負調控因子,會在小麥胚乳發育時抑制種子貯藏蛋白的合成。

 

Plant Biotechnology Journal, 5 May 2021

The MYB family transcription factor TuODORANT1 from Triticum urartu and the homolog TaODORANT1 fromTriticum aestivum inhibit seed storage protein synthesis in wheat

Author

Guangbin Luo, Lisha Shen, Yanhong Song……Caixia Gao, Dongcheng Liu, Aimin Zhang*

*: State Key Laboratory of Plant Cell and Chromosome Engineering, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology/Innovative Academy of Seed Design, Chinese Academy of Sciences, China

Summary

Seed storage proteins (SSPs) are determinants of wheat end‐product quality. SSP synthesis is mainly regulated at the transcriptional level. Few transcriptional regulators of SSP synthesis have been identified in wheat and this study aims to identify novel SSP gene regulators. Here, the R2R3 MYB transcription factor TuODORANT1 from Triticum urartu was found to be preferentially expressed in the developing endosperm during grain filling. In common wheat (Triticum aestivum) overexpressing TuODORANT1, the transcription levels of all the SSP genes tested by RNA-Seq analysis were reduced by 49.71% throughout grain filling, which contributed to 13.38%-35.60% declines in the total SSP levels of mature grains. In in vitro assays, TuODORANT1 inhibited both the promoter activities and the transcription of SSP genes by 1‐13‐fold. The electrophoretic mobility shift assay (EMSA) and ChIP‐qPCR analysisdemonstrated that TuODORANT1 bound tothe cis‐elements 5"‐T/CAACCA‐3"and 5"‐T/CAACT/AG‐3" in SSP gene promoters both in vitro and in vivo. Similarly, the homolog TaODORANT1 in common wheat hindered both the promoter activities and the transcription of SSP genes by 1‐112‐fold in vitro. Knockdown of TaODORANT1 in common wheat led to 14.73%‐232.78% increases in the transcription of the tested SSP genes, which contributed to 11.43%‐19.35% elevation in the total SSP levels. Our >TuODORANT1 and TaODORANT1 are repressors of SSP synthesis.

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