中国农业科学 ›› 2015, Vol. 48 ›› Issue (20): 3989-3997.doi: 10.3864/j.issn.0578-1752.2015.20.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

谷子转录因子SiNF-YA6的过表达提高转基因植株对低氮胁迫的抗性

方广宁1,2,胡利芹2,王二辉1,薛飞洋1,马有志2,徐兆师2,李连城2,周永斌2,刁现民2,贾冠清2,陈明2,闵东红1   

  1. 1西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室,陕西杨凌 712100
    2中国农业科学院作物科学研究所/国家农作物基因资源与基因改良重大科学工程/农业部麦类生物学与遗传育种重点实验室,北京 100081
  • 收稿日期:2015-03-25 出版日期:2015-10-20 发布日期:2015-10-20
  • 通讯作者: 闵东红,E-mail:mdh2493@nwsuaf.edu.cn。陈明,Tel:010-82108750;E-mail:chenming02@caas.cn E-mail:mdh2493@nwsuaf.edu.cn; chenming02@caas.cn
  • 作者简介:方广宁,E-mail:451298574@qq.com。胡利芹,E-mail:1069423177@qq.com。方广宁和胡利芹为同等贡献作者。
  • 基金资助:
    国家转基因新品种生物培育重大专项(2014ZX08002-002)、国家转基因生物新品种培育重大专项(2014ZX08002-003B)、陕西省农业科技攻关项目(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】核转录因子Y(nuclear transcription factor Y,NF-Y)属于一类广泛存在于真核生物中的转录因子基因家族,参与植物生长发育、胁迫响应等过程,但其参与低氮胁迫响应的研究尚未报道。从抗旱及耐瘠薄的谷子中分离出一个新的NF-Y类转录因子基因SiNF-YA6,对其表达模式和生物学功能进行分析。【方法】通过对谷子低氮处理前后的转录组差异表达谱分析,鉴定出一个受低氮诱导表达明显上调的的NF-Y类转录因子A亚基基因SiNF-YA6。利用DNAMAN和MEGA6.0软件对SiNF-YA6进行系统进化树分析;通过Real-time PCR方法分析SiNF-YA6在不同处理条件下的表达模式;并将SiNF-YA6蛋白进行亚细胞定位;通过SiNF-YA6转化拟南芥,对SiNF-YA6的功能进行鉴定。【结果】SiNF-YA6全长714 bp,编码237个氨基酸,包含一个由62个氨基酸组成的CBF保守域。系统进化树分析结果表明,NF-YA类蛋白家族一共分为6个组,SiNF-YA6属于第5组。SiNF-YA6与一个水稻NF-YA类蛋白(Os03g07880)同源性最高(77.4%)。SiNF-YA6表达模式分析结果显示,SiNF-YA6在受到低氮胁迫时,其表达量显著上调,在处理5 d时,其表达量达到最高,较处理前表达量上调约5倍。同时SiNF-YA6在高盐、干旱、ABA处理条件下的表达量也呈现上升趋势。SiNF-YA6对于SA、GA处理无明显响应。组织特异性表达分析显示,SiNF-YA6主要在谷子的根部表达。蛋白亚细胞定位结果显示,SiNF-YA6主要定位于植物的细胞核及细胞膜上。低氮胁迫抗性试验结果显示,在未处理条件下,SiNF-YA6转基因拟南芥与野生型拟南芥长势无明显差异,但在低氮(总氮含量为1 mmol·L-1)处理条件下,SiNF-YA6转基因拟南芥的总根长及主根长均明显高于野生型拟南芥,鲜重也明显大于野生型植株,并达到显著性差异(P<0.01),表明过表达SiNF-YA6显著提高转基因植物耐低氮胁迫的能力。下游基因表达分析结果显示在SiNF-YA6转基因拟南芥中氮转运相关基因(NRT2.1NRT2.4)的表达量显著提高。同时,SiNF-YA6转基因拟南芥对于高盐与干旱胁迫的抗性与野生型拟南芥无明显差异。【结论】过表达谷子NF-Y类转录因子基因SiNF-YA6通过提高氮转运基因的表达显著提高转基因植株在低氮条件下的抗性。

关键词: 谷子(Setaria italica), 核转录因子A亚基, 功能分析, 低氮胁迫

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

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