Setaria italica),核轉錄因子A亞基,功能分析,低氮脅迫," /> 福彩3d论坛

中國農業科學 ?? 2015, Vol. 48 ?? Issue (20): 3989-3997.doi: 10.3864/j.issn.0578-1752.2015.20.001

? 作物遺傳育種·種質資源·分子遺傳學 ?    下一篇

谷子轉錄因子SiNF-YA6的過表達提高轉基因植株對低氮脅迫的抗性

方廣寧1,2,胡利芹2,王二輝1,薛飛洋1,馬有志2,徐兆師2,李連城2,周永斌2,刁現民2,賈冠清2,陳明2,閔東紅1   

  1. 1西北農林科技大學農學院/旱區作物逆境生物學國家重點實驗室,陜西楊凌 712100
    2中國農業科學院作物科學研究所/國家農作物基因資源與基因改良重大科學工程/農業部麥類生物學與遺傳育種重點實驗室,北京 100081
  • 收稿日期:2015-03-25 出版日期:2015-10-20 發布日期:2015-10-20
  • 通訊作者: 閔東紅,E-mail:[email protected]。陳明,Tel:010-82108750;E-mail:[email protected] E-mail:[email protected]; [email protected]
  • 作者簡介:方廣寧,E-mail:[email protected]。胡利芹,E-mail:[email protected]。方廣寧和胡利芹為同等貢獻作者。
  • 基金資助:
    國家轉基因新品種生物培育重大專項(2014ZX08002-002)、國家轉基因生物新品種培育重大專項(2014ZX08002-003B)、陜西省農業科技攻關項目(2013K02-01)

Overexpression of a Transcription Factor Gene SiNF-YA6 from Millet (Setaria italica) Enhanced the Resistance of Transgenic Plants to Nitrogen Starvation

FANG Guang-ning1,2, HU Li-qin2, WANG Er-hui1, XUE Fei-yang1, MA You-zhi2, XU Zhao-shi2, LI Lian-cheng2, ZHOU Yong-bin2, DIAO Xian-min2, JIA Guan-qing2, CHEN Ming2, MIN Dong-hong1   

  1. 1College of Agronomy, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
     2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS)/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081
  • Received:2015-03-25 Online:2015-10-20 Published:2015-10-20

摘要: 【目的】核轉錄因子Y(nuclear transcription factor Y,NF-Y)屬于一類廣泛存在于真核生物中的轉錄因子基因家族,參與植物生長發育、脅迫響應等過程,但其參與低氮脅迫響應的研究尚未報道。從抗旱及耐瘠薄的谷子中分離出一個新的NF-Y類轉錄因子基因SiNF-YA6,對其表達模式和生物學功能進行分析。【方法】通過對谷子低氮處理前后的轉錄組差異表達譜分析,鑒定出一個受低氮誘導表達明顯上調的的NF-Y類轉錄因子A亞基基因SiNF-YA6。利用DNAMAN和MEGA6.0軟件對SiNF-YA6進行系統進化樹分析;通過Real-time PCR方法分析SiNF-YA6在不同處理條件下的表達模式;并將SiNF-YA6蛋白進行亞細胞定位;通過SiNF-YA6轉化擬南芥,對SiNF-YA6的功能進行鑒定。【結果】SiNF-YA6全長714 bp,編碼237個氨基酸,包含一個由62個氨基酸組成的CBF保守域。系統進化樹分析結果表明,NF-YA類蛋白家族一共分為6個組,SiNF-YA6屬于第5組。SiNF-YA6與一個水稻NF-YA類蛋白(Os03g07880)同源性最高(77.4%)。SiNF-YA6表達模式分析結果顯示,SiNF-YA6在受到低氮脅迫時,其表達量顯著上調,在處理5 d時,其表達量達到最高,較處理前表達量上調約5倍。同時SiNF-YA6在高鹽、干旱、ABA處理條件下的表達量也呈現上升趨勢。SiNF-YA6對于SA、GA處理無明顯響應。組織特異性表達分析顯示,SiNF-YA6主要在谷子的根部表達。蛋白亞細胞定位結果顯示,SiNF-YA6主要定位于植物的細胞核及細胞膜上。低氮脅迫抗性試驗結果顯示,在未處理條件下,SiNF-YA6轉基因擬南芥與野生型擬南芥長勢無明顯差異,但在低氮(總氮含量為1 mmol·L-1)處理條件下,SiNF-YA6轉基因擬南芥的總根長及主根長均明顯高于野生型擬南芥,鮮重也明顯大于野生型植株,并達到顯著性差異(P<0.01),表明過表達SiNF-YA6顯著提高轉基因植物耐低氮脅迫的能力。下游基因表達分析結果顯示在SiNF-YA6轉基因擬南芥中氮轉運相關基因(NRT2.1NRT2.4)的表達量顯著提高。同時,SiNF-YA6轉基因擬南芥對于高鹽與干旱脅迫的抗性與野生型擬南芥無明顯差異。【結論】過表達谷子NF-Y類轉錄因子基因SiNF-YA6通過提高氮轉運基因的表達顯著提高轉基因植株在低氮條件下的抗性。

關鍵詞: 谷子(Setaria italica), 核轉錄因子A亞基, 功能分析, 低氮脅迫

Abstract: 【Objective】Nuclear transcription factor Y (NF-Y) belongs to a transcription factor gene family widely existing in eukaryotic. NF-Y-like transcription factors are involved in the process of gene expression regulation of plant growth and development and stress responses. So far, it is never been reported that NF-Y-like transcription factors are involved in plant resistance to low nitrogen stress. In this study, a NF-Y-like transcription factor gene SiNF-YA6 was isolated from millet, and its characteristics and biological function were identified.【Method】A NF-Y-like transcription factor gene SiNF-YA6 highly up-regulated by low nitrogen stress was selected from the results of transcriptome sequencing of millet treated by low nitrogen. DNAMAN and MEGA6.0 software were used to make the phylogenetic tree of NF-YA6. The Real-time PCR was used to analyze expression patterns of SiNF-YA6 under different treatments. SiNF-YA6 was transformed into Arabidopsis protoplast to observe its subcellular localization. SiNF-YA6 was overexpressed in Arabidopsis to identify gene function.【Result】SiNF-YA6 is 714 bp in length and encoding 237 amino acids. SiNF-YA6 contains a CBF conservative domain with 62 amino acids. Phylogenetic tree analysis showed that NF-YA was fell into 6 classes, and SiNF-YA6 belongs to the fifth class. SiNF-YA6 was the highest homology with a rice NF-YA protein (Os03g07880) (77.4%). Expression pattern analysis showed that NF-YA6 was induced by low nitrogen stress, and its expression reach peak after treatment for 5 d that raised about 5 times compared with the expression level at the beginning of treatment. While transcription of SiNF-YA6 under high salt, drought, ABA treatment also showed a trend of increasing. SiNF-YA6 under SA and GA treatments was no obvious different. Tissue specific expression analysis showed that SiNF-YA6 mainly expressed in millet roots. Subcellular localization indicated that SiNF-YA6 was mainly located on the plasmalemma and nucleus in plant cell. Results of low nitrogen stress resistance test showed that the main root length and root surface areaof SiNF-YA6 transgenic Arabidopsis were significantly higher than in wild type Arabidopsis, and the fresh weight of transgenic plants was also significantly greater than the wild type plants (P<0.01) under low nitrogen stress (total nitrogen content of 1 mmol·L-1), and the growthof SiNF-YA6 transgenic Arabidopsis showed no obvious difference with wild-type Arabidopsis under SA and GA treatments. Those results indicated that overexpression of SiNF-YA6 gene significantly enhanced transgenic plants resistance to low nitrogen stress. The downstream genes, including some nitrogen transporter genes (NRT2.1 and NRT2.4), were up-regulated in SiNF-YA6 transgenic Arabidopsis. However, SiNF-YA6 transgenic Arabidopsis showed no obvious difference with wild-type Arabidopsis under high salt and drought stresses.【Conclusion】Overexpression of SiNF-YA6 enhanced transgenic plants tolerance to low nitrogen stress by up-regulated expression of several nitrogen transporter genes, which will give new candidate for improvement of the nitrogen use efficiency in crops.

Key words:  foxtail millet (Setaria italica), NF-YA transcription factor, function analysis, low nitrogen stress

福利彩票3d开奖结果